Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session

86 Pages·2010·3.61 MB·English

Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
140 - A313 141 - A314
Aqueous Humor Cytokine Levels in Glaucoma Aqueous PGE Inhibition of Ketorolac Tromethamine 0.45%, Bromfenac 0.09%,
2
and Nepafenac 0.1% in Patients Undergoing Phacoemulsification
G. Fuchsjaeger-Mayrl1, M. Funk1, H. Resch1, C. Vass1, A. Luksch2, C. Hirn3, L. Rozanowa1,
U. Schmidt-Erfurth1. 1Ophthalmology, Medical University of Vienna, Vienna, F.A. Bucci, Jr.1, D. Waterbury2, R. Evans3, L. Amico3. 1Bucci Laser Vision Institute,
Austria; 2Ophthalmology, Sanatorium Hera, Vienna, Austria; 3Ophthalmology, Wilkes-Barre, PA; 2Raven Biosolutions, LLC, San Carlos, CA; 3Bucci Laser Vision
University Hospital of Zurich, Vienna, Austria. Institute, Wilkes Barre, PA.
Purpose: Glaucoma has been associated with low grade inflammation. The exact role Purpose: To compare the PGE inhibition of three topical NSAIDs - ketorolac 0.45%
2
of inflammatory processes in the pathophysiology of various types of glaucoma is, (Acuvail), bromfenac 0.09% (Xibrom), and nepafenac 0.1% (Nevanac) in phaco patients
however, unclear. In addition, few in vivo studies have provided direct evidence for at peak dosing levels.
inflammatory processes in glaucoma. Methods: A single center, double-masked study randomized 121 pts to receive one of
Methods: In this prospective open cross-sectional study 4 groups of patients scheduled three treatment arms. Pts were instructed to administer each NSAID on label at BID
for cataract surgery were included. The population consisted of 16 control subjects for ketorolac and bromfenac, and TID for nepafenac for 1 day preop. Each patient was
without glaucoma, 16 patients with primary open angle glaucoma (POAG), 11 patients instructed to instill one drop the morning of surgery. The patients received 4 additional
with normal tension glaucoma (NTG) and 14 patients with pseudexfoliation glaucoma doses 1 hour prior to phaco. After completion of the paracentesis site with a super
(PEXG). Aqueous humor samples were taken in all patients during cataract surgery. blade, aqueous (0.15cc) was collected through the peripheral clear cornea with a 30
Luminex (Luminex Inc., Austin, TX) multiplex assays were used for measurement of gauge needle attached to a TB syringe. Following collection, aqueous samples were
cytokines including Interleukin 1α (IL1α), Interleukin 1β (IL1β), Interleukin 6 (IL6), stored at -60° C prior to analysis. The aqueous was diluted 1:10 with diluent and assays
Matrix metallopeptidase-9 (MMP9), Plasminogen activator inhibitor-1 (PAI1), Inter- were conducted on multiple plates in duplicate with 7 standards per plate. Assay was
Cellular Adhesion Molecule 1 (ICAM-1) and C-reactive protein (CRP). Data analysis conducted using a Parameter PGE Assay kit which is a 3.5 hour sequential competitive
2
was done using an ANOVA model with planned comparisons for post hoc analysis. enzyme immunoassay designed to measure PGE2 in cell culture supernatans, serum,
Results: IL1α was significantly reduced in PEXG (3.0 pg/ml, p = 0.011) and tended to plasma, and urine. PGE values observed below the level of detection were assigned
2
be reduced in POAG (6.8 pg/ml) and NTG (4,8 pg/ml) as compared to controls (10,5 a value of 50pg/ml. An independent samples Student’s t test was used to test for
pg/ml, p = 0.011). IL1β, IL6, MMP9, PAI1, ICAM-1 and CRP aqueous humor levels were between group differences.
not significantly different between groups. Results: Ketorolac 0.45% produced the greatest inhibition of PGE. The mean PGE
2 2
Conclusions: The present study does not provide evidence for an increase in cytokine values were 224.8 +/-164.87 pg/ml for ketorolac (n=42), 288.7 +/-226.05 pg/ml for
levels in the aqueous humor of glaucoma patients. The reduction in IL1α levels in bromfenac (n=41), and 320.4 +/-205.6 pg/ml for nepafenac (n=38). The difference
PEXG may either be a chance finding or related to the reduced aqueous turnover in between ketorolac and nepafenac was statistically significantly in favor of ketorolac
these patients. (p=0.025). The comparison of ketorolac to bromfenac approached a trend in favor
CR: G. Fuchsjaeger-Mayrl, None; M. Funk, None; H. Resch, None; C. Vass, None; A. of ketorolac (p=0.14). The difference between bromfenac and nepafenac was not
Luksch, None; C. Hirn, None; L. Rozanowa, None; U. Schmidt-Erfurth, None. significantly different (p=0.516).
Support: None Conclusions: Ketorolac 0.45% (Acuvail) has a lower pH(6.8), higher concentration
than previous ketorolac compounds, and contains carboxymethylcellulose with no
preservatives. This new ketorolac compound achieved the greatest inhibition of PGE
2
compared to nepafenac 0.1% and bromfenac 0.09%. Ketorolac was significantly more
effective than nepafenac while bromfenac was not significantly more effective than
nepafenac. These results suggest that ketorolac 0.45% may be more efficacious in
controlling inflammation at the time of cataract surgery vs. nepafenac and bromfenac.
CR: F.A. Bucci, Jr., None; D. Waterbury, Allergan, Inc., C; R. Evans, Allergan, Inc.,
R; L. Amico, Allergan, Inc., R.
Support: Grant CT: www.clinicaltrials.gov, NCT01021761
142 - A315 143 - A316
Aqueous Outflow Increase Resulting From Transient Blink-Induced IOP Non-Invasive Assessment of Aqueous Outflow in Collector Channels by
Elevation Spectral Domain Optical Coherence Tomography (SD-OCT)
E. Martin1, M.A. Johnstone2, A. Jamil2. 1University of Washington School of Medicine, L. Kagemann1,2, G. Wollstein1, H. Ishikawa1,2, R.A. Bilonick1, P.M. Brennen1,
Seattle, WA; 2Glaucoma Consultants Northwest, Seattle, WA. M.L. Gabriele2,1, L.S. Folio1, J.S. Schuman1,2. 1UPMC Eye Center, Eye and Ear
Institute, Ophthalmology and Visual Science Research Center, Department of
Purpose:To observe and measure how transient blink-induced changes in intraocular
Ophthalmology, University of Pittsburgh School of Medicine, Pittsburgh, PA;
pressure cause changes in aqueous flow into the aqueous and episcleral veins in
2Department of Bioengineering, University of Pittsburgh, Swanson School of
normal subjects.
Engineering, Pittsburgh, PA.
Methods:Research microscope (80-power magnification), micrometer scale,
videography. Five eyes, 5 normal subjects, mean age 33.2 (R24-54), F/M 4/1, race C4, Purpose: To compare collector channel (CC) flow with Schlemm’s Canal (SC) morphology.
O1. Aqueous veins contain separate strata; the clear aqueous strata originates from Methods: SC ostia and CC’s distal to the ostia were imaged with a modified Bioptigen
Schlemm’s canal while the blood strata originates from the episcleral veins . The (Bioptigen Inc, USA; SuperLum LTD., Ireland) SD-OCT in cross-section in both eyes of
diameter of the aqueous strata oscillates in synchrony with the ocular pulse. Blinking 14 healthy subjects. Ten frame raster scans (700 1.3mm A-scans x 0.6 mm) containing SC
occurs about every 20 seconds and induces a transient IOP increase of ~10 mm Hg. centered at CC ostia were recorded. Contrast and magnification were adjusted to maximize
It is well-documented that the diameter of the aqueous strata increases transiently visualization. SC ostia cross-sectional area was measured by manually tracing in ImageJ
following a blink1. However, a quantitative study to document the transient blink- (ImageJ 1.40g, http://rsb.info.nih.gov/ij/). Mean Doppler shift was calculated as ½ of measured
induced increase in aqueous strata diameter has not previously been undertaken. maximum within the adjoined CC. Velocity angle and CC diameter at the point of Doppler
Twenty measurements were made of the maximum aqueous strata diameter (ASD) measurement were used to calculate volumetric aqueous outflow within the CC. Flow, CC
immediately following a blink and the measurements made again once the aqueous diameter, and velocity were compared with ostia area by linear regression.
vein returned to their preblink appearance. Results: Aqueous outflow (mean ± SD) in individual CC’s was 0.042 ± 0.05 μl/min, with
Results:An observable increase in flow into the aqueous veins became manifest velocity and diameters of 3.1 ± 2.0 mm/s and 0.015 ± 0.008 μm respectively. No significant
immediately following a blink. The ASD once stabilization occurred following recovery correlations between ostia area and CC flow parameters were detected (Figure).
from a blink was 35.0+/- 19.6 microns (R = 0-66.2microns). Mean ASD immediately Conclusions: Individual CC flows measured using our non-invasive
following a blink was 56.6+/- 19.2 microns (R = 0-104.7microns) representing a 62% technique were in excellent agreement with published values using
increase. The difference between ASD immediately following a blink and ASD once invasive techniques. Aqueous outflow within individual CC’s appears
stabilization occurred following recovery from the blink was significant in each subject to be independent of the cross-sectional area of the ostia of its origin.
(p=<.0001(3), .003(1), .0198(1)). There was no relationship detected between collector channel
Conclusions:Our study describes a technique that allows direct observation and diameter (A), aqueous outflow velocity with the collector channel
measurement of changes in the ASD in aqueous veins that result from blinking. (B), or aqueous outflow (C) and Schlemm’s canal ostia area.
The increased ASD is a reflection of increased blink-induced aqueous entry to the Reference: Moses RA: Circumferential flow in Schlemm’s canal. Am
aqueous veins from SC. The physiologic blink reflex appears to induce regularly J Ophthalmol 88:585, 1979.
recurring transient increases in aqueous flow through the conventional outflow system.
1. Ascher KW. The Aqueous Veins Vol 1. Springfield: Charles C. Thomas; 1961 CR: L. Kagemann, None; G. Wollstein, Carl Zeiss Meditec, F;
CR: E. Martin, None; M.A. Johnstone, None; A. Jamil, None. Optovue, F; Bioptigen, P; H. Ishikawa, Bioptigen, P; R.A. Bilonick,
Support: Charles Applegate Research Fund None; P.M. Brennen, None; M.L. Gabriele, None; L.S. Folio, None;
J.S. Schuman, Bioptigen, P; Carl Zeiss Meditec, P; Carl Zeiss Meditec,
R; Heidelberg Engineering, R; Pfizer, R.
Support: NIH RO1-EY013178; P30-EY008098; Eye and Ear Foundation (Pittsburgh, PA);
Research to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
140-143
Monday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1382 - 1406 / B121 - B145
219. Functional Retinal Imaging Organizing Section: MOI
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
144 - A317 145 - A318
Observational Study of Aqueous Dynamic Effects of Selective Laser Abnormal-Cannabidiol-Induced Increase in Aqueous Humor Outflow
Trabeculoplasty (SLT) and Topical Latanoprost
Z.-H. Song, Z. Qiao. Pharmacology and Toxicology, University of Louisville,
E.C. Jones, P. Alaghband, L. Beltran-Agullo, S. Goyal, K. Lim. Ophthalmology, St Louisville, KY.
Thomas’ Hospital, London, United Kingdom.
Purpose: To study the effects of abnormal-cannabidiol (aCBD), a non-CB1/CB2
Purpose: SLT is thought to increase trabecular outflow and prostaglandins are beleived cannabinoid agonist, on aqueous humor outflow via the trabecular meshwork pathway.
to increase uveoscleral outflow. We compared the aqueous dynamics changes following Methods: The effects of aCBD on aqueous humor outflow via the trabecular meshwork
SLT and latanoprost treatment in a group of previously untreated patients. pathway were measured using a porcine anterior segment perfused organ culture
Methods: Retrospective observational report. Since June 2007, all untreated primary model. Different concentrations of aCBD were administered to the perfusion medium
open angle glaucoma (POAG) and ocular hypertension (OHT) patients in our clinic and the aqueous humor outflow facility was monitored for 5 hours. To investigate
are invited to have aqueous dynamic measurements, at baseline and 3 month post whether non-CB1/CB2 cannabinoid receptors are involved in the aCBD-induced
treatment: intraocular pressure (IOP) using pneumotonometry, tonography using the outflow effects, O-1918, a cannabidiol analog that acts as a selective antagonist at
electronic Schiotz and fluorophotometry for aqueous flow. Patients were free to chose the non-CB/CB receptors, was administered to the perfused anterior chamber 30
1 2
between primary 360 degree SLT or medical treatment. minutes prior to the application of aCBD. CB1 antagonist SR141716A or CB2 antagonist
Results: 60 patients were identified from our database with baseline and 3 month data. SR144528 was also used to investigate the possible involvement of CB1 or CB2 receptors,
16 patients were excluded as they were treated with either timolol or bimatoprost. respectively, in the outflow effects induced by aCBD.
20 had SLT and 24 had latanoprost, of which 5 SLT and 10 latanoprost patients were Results: Administration of aCBD caused a concentration-dependent enhancement of
excluded due to poor measurements. 16 eyes of 15 SLT treated patients and 17 eyes of aqueous humor outflow facility, with the maximum effect (155 ± 11.7 % of basal outflow
14 latanoprost treated patients were included in this report (Table 1). Mean IOP was facility) achieved at 5 hour after the administration of 30 nM of aCBD. Pretreatment with
reduced in each treatment group: 24.2mmHg pre and 18.9mmHg post SLT (p<0.001), 1 μM of O-1918 produced a full antagonism on the aCBD-induced increase of aqueous
24.5mmHg pre and 19.8mmHg post latanoprost (p<0.001) (Table 2). Tonographic outflow humor outflow facility. Pretreatment with either 1 μM of SR141716A or 1 μM SR144528
facility improved from 0.095 to 0.156 in SLT (P=0.003), but did not change significantly had no effect on aCBD-induced enhancement of aqueous humor outflow facility.
in latanoprost (0.166 pre to 0.160 post). Both treatments did not have significant effect Conclusions: The results from this study demonstrate that abnormal-cannabidiol
on aqueous flow rate. increases aqueous humor outflow through the trabecular meshwork pathway, and
Conclusions: SLT and latanoprost have slightly different effects on aqueous dynamics these effects are mediated by non-CB1/CB2 cannabinoid receptors.
in that only SLT appeared to enchance tonographic outflow facility significantly. CR: Z.-H. Song, None; Z. Qiao, None.
Support: NIH Grants EY13632 and DA11551
CR: E.C. Jones, None; P. Alaghband, None; L. Beltran-Agullo, None; S. Goyal, Ellex
for travel grant, R; K. Lim, Ellex for travel grants and lecture fees, R.
Support: UK National Institute for Health Research (KSL)
146 - A319 147 - A320
Day and Night Differences in Aqueous Humor Dynamics and IOP in Ocular Comparison of the Effects of Timolol on Aqueous Humor Dynamics During the
Hypertensive Patients Treated With Dorzolamide Day and the Night
S. Rai, S. Fan, V. Gulati, M. Zhao, H. Liu, C.B. Camras, C.B. Toris. Ophthalmology and S.L. Galata, S. Fan, C. Gangahar, M. Zhao, J.J. Hejkal, C.B. Camras, C.B. Toris.
Visual Sciences, University of Nebraska Medical Center, Omaha, NE. Ophthalmology and Visual Sciences, Univ of Neb Med Center, Omaha, NE.
Purpose: To evaluate the effect of topical dorzolamide on intraocular pressure (IOP) Purpose: The purpose of this study was to compare the daytime and nighttime
and aqueous humor dynamics during the day and night in patients with ocular effects of timolol on aqueous humor dynamics in patients with ocular hypertension.
hypertension. Methods: Thirty ocular hypertensive volunteers were enrolled in this study after
Methods: Thirty patients were enrolled in the 4-visit randomized, double-masked washout of all ocular drugs. Study measurements were made during the day and night,
study following screening and 6 weeks of washout. On the two daytime visits from at baseline and after twice-daily timolol (0.5%) for two weeks. Daytime measurements
9 AM to 3 PM (baseline and following two weeks of twice daily dosing of drug), included blood pressure, seated intraocular pressure (IOP) central corneal thickness
intraocular pressure (IOP) was determined by pneumatonometry, aqueous flow (CCT), anterior chamber depth, episcleral venous pressure (Pev), aqueous flow
(Fa) and outflow facility by fluorophotometry (Cfl), episcleral venous pressure (Pev) (Fa) and outflow facility by fluorophotometry (Cfl) and tonography (Cton). These
by venomanometry and outflow facility again by two minute tonography (Cton), measurements were repeated at night (starting at 56 hours after completion of the
central corneal thickness (CCT) and anterior chamber depth (ACd) by pachymetry daytime measurements) with the exception of Pev and Cfl. Additionally, IOP at night
and blood pressure by sphygmomanometry. Two days after each daytime visit, all was taken in the supine position. Uveoscleral outflow was calculated mathematically
measurements except for Cfl and Pev were repeated between the hours of 10 PM and using either Cfl or Cton in the Goldmann equation.
5 AM. Uveoscleral outflow was calculated by the Goldmann equation. Baseline and Results:Compared to baseline, timolol significantly lowered IOP by at least 24% at
dorzolamide treatment were compared using two-tailed paired t-tests. 9AM and 18% at noon (p<0.001 for both), but at no time at night. During the day timolol
Results: Dorzolamide significantly (p<0.001) reduced IOP by 16% at 11AM (two hours reduced aqueous flow by 25% (p=0.001), but at night the aqueous flow reduction
after the previous dose) but not at 9 AM (12 hours after the previous dose, 7% drop, was no different than at baseline. There was a significant (p=0.035) increase (0.86
p=0.07) or at any IOP overnight (10 PM, midnight, 2 AM, 5 AM). Compared to baseline, μl/min) in daytime uveoscleral outflow calculated with tonographic data but not
dorzolamide during the day reduced aqueous flow by 16% (p=0.03). Dorzolamide fluorophotometric data. No other parameter was significantly changed by timolol
did not change any of the day versus night differences in IOP and aqueous humor treatment.
dynamics found at baseline (reduced Fa and Fu and increased CCT). Physiological Conclusions:Timolol effectively lowers IOP during the day by decreasing aqueous
nighttime suppression of Fa seen at baseline was not decreased further by dorzolamide flow. It is ineffective at night because it cannot reduce aqueous flow below its normally
use (47%, p<0.001). low nocturnal rate. The finding that IOP is reduced at 9AM, 12 hours after the previous
Conclusions: Dorzolamide reduced IOP during the day in ocular hypertensive patients dose, suggests that timolol blocks the normal aqueous flow rise in the morning. The
by reducing aqueous flow. It had no effect on IOP or aqueous humor dynamics at night effect of timolol on uveoscleral outflow is an inconsistent finding that should be
nor did it affect CCT, ACd or blood pressure day or night. viewed with caution.
CR: S. Rai, None; S. Fan, None; V. Gulati, None; M. Zhao, None; H. Liu, None; C.B. CR: S.L. Galata, None; S. Fan, None; C. Gangahar, None; M. Zhao, None; J.J. Hejkal,
Camras, None; C.B. Toris, None. None; C.B. Camras, None; C.B. Toris, None.
Support: Supported by an unrestricted grant from Pfizer; Research to Prevent Support: Unrestricted grant from Pfizer; Research to Prevent Blindness CT: www.
Blindness CT: www.clinicaltrials.gov, NCT00572936 clinicaltrials.gov, NCT00572936
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
144-147
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
148 - A321 149 - A322
Aqueous Humor Dynamics During the Day and Night in Ocular Hypertensive Time-Dependent Correlation Between Nitric Oxide-Induce Decreases in
Patients Schlemm’s Canal Cell Volume and Nitric Oxide -Induced Increases in Outflow
Facility
S. Fan, J.J. Hejkal, H. Liu, V. Gulati, G. Zhan, C.B. Camras, C.B. Toris. Ophthalmology,
Univ of Nebraska Medical Ctr, Omaha, NE. D.Z. Ellis1,2, W.M. Dismuke1, N.A. Sharif2. 1Pharmacodynamics, University of Florida,
Gainesville, FL; 2Core Pharmacology & Imaging, Alcon Research. Ltd., Ft. Worth,
Purpose: This study evaluates the differences in aqueous humor dynamics between
TX.
daytime and nighttime in patients with ocular hypertension (OHT).
Methods: Thirty patients (58.9±11.0 years) with OHT were enrolled in the study. Time-dependent correlation between nitric oxide-induced decreases in Schlemm’s
Subjects were studied at one daytime and one nighttime visit. Measurements canal cell volume and nitric oxide -induced increases in outflow facility. Dorette
include central cornea thickness (CCT) by pachymetry, intraocular pressure (IOP) Z. Ellis,1 William M. Dismuke1 and Najam A. Sharif2 1From the Department of
by pneumatonometry, aqueous flow (Fa) by fluorophotometry, outflow facility by Pharmacodynamics, College of Pharmacy, University of Florida, Gainesville, Florida;
tonography (C ), and seated systolic (SBP) and diastolic (DBP) blood pressure by and 2Alcon Research, Ltd., Fort Worth, Texas.
ton
sphygmomanometry at both visits. Daytime measurements only were made of Purpose. We previously demonstrated a time-course correlation between nitric
episcleral venous pressure (P ) by venomanometry, anterior chamber depth (AC) oxide (NO)-induced decreases in trabecular meshwork cell volume and NO-induced
ev d
by pachymetry, and outflow facility by fluorophotometry (C). Nighttime IOP increases in outflow facility. Because the Schlemm’s canal (SC) cells also provide
fl
measurements were made both in the seated and supine positions. Uveoscleral outflow resistance to aqueous humor outflow, this study was conducted to test the involvement
was calculated mathematically during the day and at night (Fu ). One-way repeated of the NO/soluble guanylyl cyclase (sGC) / protein kinase G (PKG) pathway and the
ton
measures ANOVA was used to compare daytime seated IOPs versus nighttime seated BK -channel in mediating SC cell volume decreases.
Ca
or supine IOPs. All other comparisons were made by two-tailed paired t-tests. P Method. Low passage human SC cells were grown to confluency and cell volume was
values< 0.05 were considered statistically significant. measured using Calcein AM fluorescent dye; images were captured with a confocal
Results: Compared with daytime seated IOP (21.3±3.5 mmHg), nighttime seated microscope, and data quantified using NIH ImageJ software.
IOP (17.2±3.7 mmHg) was significantly reduced (p<0.001) and nighttime supine IOP Results. Exposure of SC cells to DETA-NO (50 -100 μM) resulted in a 12% decrease
(22.7±4.6 mmHg, habitual position) was increased (p=0.03). CCT was increased at in cell volume: mean voxels, 9972.9 ± SEM, 469.8; n = 143 cells. These decreases were
night from 570±39 to 585±46 μm (p<0.001). There was a 47% nocturnal reduction in abolished by the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 5
aqueous flow from 2.09±0.71 to 1.11±0.38 μL/min (p<0.001). Uveoscleral outflow was μM; the PKG inhibitor, (RP)-8-Br-PET-cGMP-S, (25 μM); and the BK channel inhibitor,
Ca
significantly reduced (p=0.02) by145% at night when using supine IOP and P adjusted iberiotoxin (IBTX), (100 nM). Furthermore, hypertonic media significantly decreased SC
ev
for postural changes in the Goldmann equation. Tonographic outflow facility and all cell volume (14 %) while hypotonic media significantly increased cell volume (11.2 %).
other measurements were unchanged. Conclusions. These data suggest the involvement of the sGC/cGMP/PKG pathway
Conclusions: The study shows that significant ocular changes occur at night in and the BK -channel in mediating the NO-induced decreases in SC cell volume.
Ca
patients with ocular hypertension including thickening of CCT, decrease of Fa and These decreases correlated with the time course for NO-induced increases in outflow
Fu , reduction in seated IOP but increase in supine IOP. Compared to our previous facility, suggesting that the NO-induced decreases in SC cell volume may also influence
ton
study of ocular normotensive volunteers of similar age, OHT patients had decreased outflow facility.
uveoscleral outflow but unchanged outflow facility at night. CR: D.Z. Ellis, Alcon Research, Ltd., F; W.M. Dismuke, None; N.A. Sharif, Alcon
CR: S. Fan, None; J.J. Hejkal, None; H. Liu, None; V. Gulati, None; G. Zhan, None; C.B. Research, Ltd., E.
Camras, None; C.B. Toris, None. Support: Alcon Research, Ltd. & University of Florida Start-UP Funds
Support: Supported by an unrestricted grant from Pfizer; Research to Prevent
Blindness CT: NCT00572936
150 - A323 151 - A324
Pressure-Facility Relationship and Washout in the Enucleated Mouse Eye Prostaglandin EP Receptor Activation Increases Outflow Facility in Perfused
4
Human Eyes
Y. Lei, D.R. Overby, C.R. Ethier. Bioengineering, Imperial College London, London,
United Kingdom. L.H. Millard1A, D.F. Woodward2, W.D. Stamer1B. APharmacology, BOphthalmology,
1University of Arizona, Tucson, AZ; 2Bioll Sci RD-2C, Allergan, Inc, Irvine, CA.
Purpose: Mice are now widely used in glaucoma research yet the physiology of
murine aqueous humour drainage is incompletely understood. Here we determine Lindsey H. Millard, David F. Woodward and W. Daniel Stamer
the relationship between intraocular pressure (IOP) and aqueous outflow resistance,
Purpose: Prostaglandin EP receptors are expressed by many human ocular tissues
and measure ocular compliance and washout rate. 4
including both trabecular meshwork (TM) and Schlemm’s canal (SC) cells of the
Methods: Eyes from adult mice of either sex (C57BL/6 background) were enucleated,
conventional outflow pathway. Activation of EP receptors in monkey eyes efficaciously
cannulated with a 33G Nanofil needle (WPI), and attached to a perfusion system 4
lowers intraocular pressure, while having no effect on aqueous humor secretion or
consisting of a pressure transducer and a computer-controlled syringe pump that
uveoscleral outflow. We hypothesize that prostaglandin EP receptors regulate human
delivered a variable flow rate to the anterior chamber to maintain a desired IOP. 4
conventional outflow.
Eyes were perfused with phosphate buffered saline following one of two regimens:
Methods: Paired post-mortem human eyes were received within 24 hours, prepared
1) at 8 mmHg constant pressure for 2-3 hours, corresponding to ~15 mmHg in vivo;
for organ-culture and were perfused at 8 mmHg constant pressure. After establishing
2) at constant pressures of 8, 15 and 4 mmHg for 20 min each, with stepwise changes
a stable baseline, anterior chamber contents were exchanged with either drug (10 nM
between each pressure level. Washout rate was estimated according to Erickson-
3,7 dithia PGE) or media (control). After 4 hours, eyes were perfused-fixed with 4%
Lamy et al. (IOVS,1990). Needle resistance, perfusion system compliance and ocular 1
PFA at 8 mmHg for 30 min. Corneo-scleral region of eyes were embedded in plastic,
compliance were also measured.
sectioned and stained with toluidine blue.
Results: At 8 mmHg, eyes had a stable baseline outflow resistance of 59.1 ± 9.9 mmHg
Results: Baseline outflow facility for control (0.19±0.067 ul/min/mmHg; mean ±SEM)
min/uL (facility = 0.017 ± 0.003 uL/min/mmHg; mean ± SD, n = 22). Outflow resistance
and drug-treated eyes (0.21±0.02 ul/min/mmHg) were similar. Outflow facility
was 35 ± 6 and 81 ± 24 mmHg/uL/min at 4 mmHg and 15 mmHg respectively,
increased by 51±18% over baseline levels in drug-treated eyes after exchange (n=6
increasing with IOP (p<0.005, linear regression, r2=0.97). At 8 mmHg, washout was
eyes; p=0.005). When compared to contralateral eyes, outflow facility in drug treated
not observed (insignificant facility decrease of 0.4 ± 8 % of starting facility per hour;
eyes increased by 69±23% (p<0.01). Histological analysis of perfused eyes showed no
n=7). Initial studies revealed that outflow resistance measurements were sensitive to
gross abnormalities in tissue architecture or discernable differences in cell abundance
the compliance of the perfusion system, and even when using high pressure tubing,
or morphology.
the system compliance (0.10 uL/mmHg) was comparable to the ocular compliance
Conclusion: Results suggest that the EP receptor activation significantly increases
(0.078 ± 0.007 uL/mmHg; n = 4). In contrast, the resistance of the perfusion system 4
outflow facility in human eyes in organ culture and is likely responsible for changes
was negligible (0.77 ± 0.05 mmHg min/uL) compared with the eye.
in IOP observed in 3,7 dithia PGE-treated monkey eyes.
Conclusions: Similar to human eyes, mouse eyes show a pressure-dependent outflow 1
CR: L.H. Millard, None; D.F. Woodward, Allergan, Inc., E; W.D. Stamer, Allergan,
facility and no detectable washout. Mouse seems to be the only species (other than
Inc., F.
human) where washout is absent. Our measured outflow facility is ~3-fold greater
Support: Allergan
than previous in vivo reports (Aihara et al, IOVS, 2003, Crowston et al, IOVS, 2004 and
Zhang et al, JGP, 2002), which may be due to differences in measurement techniques
or mouse strain.
CR: Y. Lei, None; D.R. Overby, None; C.R. Ethier, None.
Support: Royal Society Wolfson Foundation Research Excellence Award (CRE)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
148-151
Monday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1382 - 1406 / B121 - B145
219. Functional Retinal Imaging Organizing Section: MOI
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
152 - A325 153 - A326
Sphingosine-1-Phosphate Receptor Antagonist Increases Outflow Facility Patterned Expression of LM-511 and Integrin α6 in Human Schlemm’s Canal
Endothelia
G.M. Sumida1A, W. Stamer1B. APhysiological Sciences Graduate Program,
BOphthalmology & Vision Science, 1University of Arizona, Tucson, AZ. S.S. VanderWyst1A, A.T. Read2, D.R. Overby3, W.D. Stamer1B. ABiomedical Engineering
Graduate Inter-Disciplinary Program, BOphthalmology & Vision Science,
Purpose: Sphingosine 1-phosphate (S1P) decreases outflow facility in both porcine
1University of Arizona, Tucson, AZ; 2Ophthalmology & Vision Science, University
and human perfused eyes and the S1P and S1P receptors are expressed by cells that
1 3 of Toronto, Toronto, ON, Canada; 3Bioengineering, Imperial College London,
populate the conventional outflow pathway, namely trabecular meshwork (TM) and
London, United Kingdom.
Schlemm’s canal (SC) endothelial cells. Using S1P receptor antagonists, the aim of
this study was to determine the relative involvement of the receptor subtypes in the Purpose: Aqueous humor outflow resistance is generated near the juxtacanalicular
S1P-mediated effects on outflow facility. connective tissue and the inner wall endothelium of Schlemm’s canal (SC). Due to a
Methods: Porcine eyes in organ-culture were perfused at 15 mmHg constant pressure basal-to-apical directed pressure drop across these tissues, we hypothesize that SC cells
to obtain a baseline facility. Anterior chamber contents were then exchanged with must strongly adhere to their basement membrane through function-specific laminin
50 μM antagonist or antagonist + 5 μM S1P and further perfused. Control eyes were heterotrimers and their cognate integrins. The purpose of this study was to characterize
exchanged with media and S1P alone and normalized facilities (facility /baseline the laminin and laminin-specific integrins in Schlemm’s canal basement membrane.
facility) were used to compare the treatment groups. 10 μM antagonists pre-incubated Methods: Frozen sagittal sections through conventional outflow tissues from human
with TM and SC primary cells prior to S1P treatment (0.1, 1, and 10 μM) were used to donor eyes were probed with antibodies recognizing several monomers of laminin
block S1P-induced MLC phosphorylation. Phospho-MLC was detected via immunoblot (laminin α5, β1, γ1, α3, β3, γ2) and laminin-specific integrin pairs (integrin α3, α6, β1, β4).
analysis with β-actin used as a loading control. En-face preparations of SC’s inner wall from perfusion-fixed eyes were immunostained
Results: W146 (S1P receptor antagonist) failed to block the S1P-induced decrease in for laminin and integrin co-localization. All tissue samples were examined with
1
outflow facility. Similarly, VPC23019 (S1P receptor antagonist) also did not block the confocal microscopy and compared to controls. Mature SC cell monolayers were
1,3
decrease in outflow facility, but interestingly, anterior chamber exchange with media prepared for immunoprecipitation and Western blot to examine specific laminin
containing VPC23019 (1.52 ± 0.23, n=4) increased facility compared to media exchange heterotrimers.
(1.17 ± 0.21, n=7) after 1 hr of perfusion (mean normalized facility ± SD, p < 0.05). In Results: In situ immunofluorescence microscopy showed the presence of LM α5,
both cultured TM and SC cells, W146 enhanced S1P-induced phosphorylation of MLC but not LM β3, and laminin-specific integrin α6 in SC basement membrane. LM-511
in all experiments (n=3). Responses with the inclusion of VPC23019 were variable. expression, but not LM-332, was localized to SC endothelia. The en-face labeling of
Conclusions: The VPC23019-induced increase in outflow facility indicates the presence the SC inner wall displayed LM α5 staining, often co-localizing with integrin α6 and
of a basal S1P receptor activation level in the outflow pathway. W146 effects on the PECAM-1 in a distinctive interwoven pattern. Reverse co-immunoprecipitation of
phosphorylation status of MLC indicate that the S1P and S1P receptors may have SC lysates with LM α5 and LM γ1 showed that while LM γ1 associates with LM α5,
1 3
opposing roles in regulating cell contractility. The inability of the antagonists tested LM α5’s primary association in vitro is with another gamma monomer (i.e. γ2 or γ3).
to block S1P effects may suggest involvement of a separate S1P receptor subtype. Conclusions: Immunofluorescence, en-face imaging and Western blot show SC
CR: G.M. Sumida, None; W. Stamer, None. expression of LM-511 and integrin α6 in situ and in vitro. The patterned expression
Support: NIH Grant EY17007, Research to Prevent Blindness Foundation suggests that inner wall endothelia utilize integrin α6 to adhere to LM-511 in opposition
to the force of basal-apical pressure gradient in vivo.
CR: S.S. VanderWyst, None; A.T. Read, None; D.R. Overby, None; W.D. Stamer, None.
Support: NIH Cardiovascular Biomedical Engineering Training Grant (HL007955),
NEI (E717007)
154 - A327 155 - A328
Markers That Differentiate Human Schlemm’s Canal From Trabecular Protein Expression Pattern of Trabecular Meshwork Cell Exosomes
Meshwork Cells in Culture
E.A. Hoffman1, K.L. Schey2, W.D. Stamer1. 1Ophthalmology, University of Arizona,
K.M. Perkumas1A, S.S. VanderWyst1B, R.L. Heimark1C, W.D. Stamer1A. AOphthalmology, Tucson, AZ; 2Department of Biochemistry, Vanderbilt University, Nashville, TN.
BBiomedical Engineering Graduate Inter-Disciplinary Program, CDepartment of
Purpose:To define the protein expression pattern of purified exosomes from
Surgery, 1University of Arizona, Tucson, AZ.
conditioned media of human trabecular meshwork (HTM) cells.
Purpose: Schlemm’s canal (SC) and trabecular meshwork (TM) cells are the two cell Methods:Exosome samples were purified by differential centrifugation from
types that populate the human conventional outflow pathway and regulate intraocular conditioned media of mature HTM cell monolayers (two cell strains from two
pressure. SC and TM cells in culture have been useful toward understanding their role individual donor eyes) or from human urine (as control), solubilized in SDS sample
in conventional outflow homeostasis. Currently available markers that distinguish SC buffer and run into an SDS-PAGE gel. Following in-gel tryptic digestion of excised
and TM cells in culture are limited and motivated the present study. gel regions, extracted peptides were subjected to two dimensional HPLC separation
Methods: SC and TM cells were isolated from human cadaver eyes and cultured in (strong cation exchange and reverse phase) and tandem mass spectrometry analysis
vitro as described previously. Cell lysates were collected from multiple cell strains (MS/MS) using the multidimensional protein identification technology (MudPIT)
from each cell type and were analyzed after a period of at least a week at confluence strategy. MS/MS spectra were searched against a human protein database using
(n=4 SC strains and n=3 TM strains). Lysates were subjected to SDS-PAGE followed by Sequest for protein identification. Protein expression was confirmed by Western blot
western blot analyses using antibodies that recognize different vascular endothelial analyses in exosomes from 5 different HTM cell strains.
markers as probes. Candidate marker proteins included: ERG1/2/3, Robo1, Robo4, Results:HTM cell and urine exosomes contained many of the same proteins, including
alpha-6 integrin, Slug, Snail and TEK/TIE2. previously characterized exosome markers such as membrane organizers (annexins1,
Results: SC and TM cells did not differ appreciably in their expression of many of 2, 4-7, 11, flotillin and myoferlin), tetraspanins (CD9, CD81, syntenin-1) as well as
the candidate proteins: ERG1/2/3, Robo1, Robo4 or TEK/TIE2. In contrast, SC cells integral membrane proteins and ESCRT proteins involved in vesicular trafficking.
expressed two proteins differentially from TM cells, the laminin-specific integrin Interestingly, Myocilin was uniquely expressed by HTM cell exosomes.
subunit, alpha-6, and the transcriptional repressor, Slug. Differential integrin alpha-6 Conclusions: While myocilin expression appears distinctive, exosomes from HTM
expression was confirmed by immunohistochemistry in human ocular tissues and cells contain proteins that characterize exosomes in other cell types.
Slug by RT-PCR in multiple cell strains. CR: E.A. Hoffman, None; K.L. Schey, None; W.D. Stamer, None.
Conclusions: While TM and SC both form monolayer structures, some endothelial Support: NIH EY12797
responsibilities and several endothelial markers, SC cells differentially express at
least two proteins which likely reflect a distinction in cellular responsibilities in vivo.
CR: K.M. Perkumas, None; S.S. VanderWyst, None; R.L. Heimark, None; W.D.
Stamer, None.
Support: NIH Grant EY17007
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
152-155
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
156 - A329 157 - A330
Prolonged Dexamethasone Exposure Reduces Adenosine A1 Agonist Signaling Downregulation of Glucocorticoid Receptor Beta Increases Glucocorticoid
in the Trabecular Meshwork Cells Sensitivity in Trabecular Meshwork Cells
C.E. Crosson, P.W. Yates, J.A. Fant. Ophthalmology, Medical Univ of South Carolina, M. Jiang1A, R. Krishnamoorthy1B, T. Yorio1A. APharmacol & Neuroscience, BCell Biology
Charleston, SC. and Genetics, 1UNT Health Science Center, Fort Worth, TX.
Purpose: The chronic administration of steroids can increase IOP; however, the cellular Purpose: Previously studies from our laboratory have shown that glaucomatous
mechanisms responsible for this rise in IOP are not understood. Previous studies trabecular meshwork (TM) cells have lower expression of glucocorticoid receptor beta
have provided evidence that the activation of adenosine A receptors in the trabecular (GR-beta), compared to normal TM cells. Overexpression of glucocorticoid receptor
1
meshwork by endogenous purines can regulate outflow facility and IOP. The current beta was found to attenuate glucocorticoid sensitivity in glaucomatous TM cells.
studies investigate if dexamethasone exposure of these cells alters purinergic signaling. The purpose of this study was to determine if downregulating GR-beta expression
Methods: Cultured primary human trabecular meshwork (HTM) cells were incubated could increase glucocorticoid sensitivity in cultured transformed normal trabecular
in the presence or absence of dexamethasone (10-6 mol/L) for 14 days. The activation (i.e., meshwork cells (NTM5 cells).
phosphorylation) of ERK 1/2 pathway was induced by the addition of the adenosine Methods: siRNA duplexes specific for GR beta were designed and custom synthesized.
A agonist, CHA (10-7 mol/L). The activation of ERK 1/2 and the expressions of dual- NTM5 cells were transfected with either GR beta siRNA duplexes or a non-target siRNA
1
specificity phosphatase, MKP-1 and the adenosine receptor were determined by duplex (control) and cotransfected with a GRE-luciferase reporter and a SV40 promoter-
Western blot analysis and/or quantitative RT-PCR. beta galactosidase construct to normalize for efficiency of transfection. Promoter-
Results: In primary HTM culture, the addition of CHA produced a rapid increase in reporter assays were carried out to determine the effect of GR beta knockdown on
ERK 1/2 activation that peaked within 10 minutes. This increase in ERK 1/2 activation glucocorticoid-mediated luciferase reporter activity.
remained elevated for 30 minutes and then slowly returned to basal levels between 60 Results: NTM5 cells transfected with the non-targeting siRNA duplex showed a 7- to
and 120 minutes post-administration. Exposure of trabecular cells to DEX for 14 days did 8- fold increase in luciferase activity after dexamethasone treatment. After knock
not produce any significant change in peak ERK activation; however, dexamethasone down of GRbeta expression using siRNA duplexes specific for GR beta, there was 18-
treatment significantly accelerated the deactivation (i.e., dephosphorylation) of ERK 1/2 to 23- fold increase in luciferase activity after dexamethasone treatment, compared
by over 70%. In cells treated with dexamethasone, no change in the expression of ERK to non-targeting siRNA controls .
1/2 or adenosine A receptor levels was measured. However, dexamethasone treatment Conclusions: Decreased expression of glucocorticoid receptor beta in trabecular
1
significantly increase the expression of dual-specificity phosphatase, MKP-1, by more meshwork could be a key mechanism accounting for glucocorticoid sensitivity that
than 8-fold. In cells treated with dexamethasone, the addition of doxorubicin, a known is observed in most primary open angle glaucoma patients.
repressor of MKP expression, for 4 hours prior to CHA administration reversed the CR: M. Jiang, None; R. Krishnamoorthy, None; T. Yorio, None.
effects of dexamethasone treatment. Support: EY71150
Conclusions: These studies demonstrate that dexamethasone treatment can suppress
adenosine-A1 agonist activation of ERK 1/2 in HTM cells. The suppression appears
to be due to the upregulation of MKP-1 increasing the rate of ERK 1/2 inactivation.
Thus, reduced ERK 1/2 activation in the trabecular meshwork may play a role in
steroid-induced glaucoma.
CR: C.E. Crosson, None; P.W. Yates, None; J.A. Fant, None.
Support: NIH Grant EY009741
158 - A331 159 - A332
The Effects of Age on Aqueous Humor Dynamics and Intraocular Pressure in a Effects of Sildenafil and Tadalafil on Intraocular Pressure in Sheep;
Monkey Model of Glaucoma Implications on Aqueous Humor Dynamics
M.A. Maslonka1, S. Fan2, L.S. Stapp1, C.B. Toris2. 1Ophthalmology and Visual Sciences, O.A. Candia1, R. Gerometta2. 1Ophthalmology, Mount Sinai School of Medicine,
University of Nebraska Medical Center, Omaha, NE; 2Ophthalmology, Univ of New York, NY; 2Oftalmología, Universidad Nacional Del Nordeste, Corrientes,
Nebraska Medical Ctr, Omaha, NE. Argentina.
Purpose: The purpose of this longitudinal study was to compare aqueous humor Purpose: To determine the effects of vasodilators on intraocular pressure (IOP) and
dynamics and intraocular pressure (IOP) during and after puberty in monkeys with protein content of sheep aqueous humor (AH), because 1) the vasodilators might
unilateral laser-induced glaucoma. increase fluid leakage from the fenestrated capillaries of the ciliary body to the
Methods: Fourteen female cynomolgus monkeys had laser treatments to the trabecular extracellular tissue and directly to the anterior chamber (AC) via the iris, and 2)
meshwork of the left eye to induce a stable and chronic elevation in IOP. Lasering some patients (older than 70) treated with sildenafil have exhibited elevated IOP.
had been done at least 13 months before the first day of measurements. In ketamine- Methods: Experiments were done on domestic sheep residing on a ranch in Argentina.
sedated animals, seated IOPs were measured by pneumatonometry at three different These are docile, easy to handle and compliant animals. They readily swallowed
times throughout the day. Aqueous flow (Fa) was assessed by fluorophotometry, and tablets of sildenafil (50 and 100 mg) and tadalafil (20 mg). IOP was monitored by
outflow facility was evaluated by fluorophotometry (C) and by 2-minute tonography Perkins applanation tonometry in 21 normal sheep orally administered these drugs.
fl
(C ). Uveoscleral outflow was mathematically calculated using either C or C in the In addition, paracentesis was performed on 6 sheep to quantify changes in AH protein
ton fl ton
Goldmann equation to yield Fu or Fu , respectively. Measurements taken on Study levels with the Pierce protein assay.
fl ton
Day I (3.8±0.8 years old) was repeated nearly 3 years later on Study Day II (6.6±2.0 Results: Ingestion of both sildenafil and tadalafil increased sheep IOP from normal
years old). Comparisons between ages and between eyes were made by two-tailed levels of ≈ 9-11 mmHg within 1 hr. The IOP elevation was ≈ 1.6 fold with both doses
paired t-tests (p). of sildenafil. IOP returned to control values within 4 hrs. With the longer-lasting
Results: Comparing the normal eye on Study Day II with Study Day I, the mean IOP vasodilator tadalafil, IOP remained 1.6 to 1.9-fold higher than normal for at least 48
(at noon) decreased by 5.5 mmHg (p=0.001), C increased by 0.13 μl/min/mmHg hrs and returned to control levels within 4 days. The AH protein content was 60%
fl
(p=0.07) and Fu increased 1 to 1.5-fold (p=0.03 for Fu and p=0.07 for Fu). Similar higher after administering 100 mg sildenafil. No other drugs or doses were tested
ton fl
comparisons in the lasered eye found no change in IOP but an increase in C (p=0.03). on this parameter.
ton
Comparing the lasered eye with the normal eye on Study Day I showed a decrease in Conclusions: These data are consistent with a vasodilator-evoked increase in plasma-
mean C of 0.20 μl/min/mmHg (p= 0.02). On Study Day II, compared to the normal like fluid into the AC, which likely accounts for the IOP elevation. As such, the results
ton
eye, the lasered eye had an IOP at noon that was higher by 8.3 mmHg (p=0.003), lower 1) support the existence of a parallel pathway directly from the ciliary stroma to the
mean C by 0.18 μl/min/mmHg (p=0.0001) and higher mean Fu by 1.5 fold (p=0.02). AC, and 2) may be relevant to the management of IOP in senior patients treated with
fl ton
All other comparisons showed no significant differences. these compounds for vascular diseases, especially those patients with glaucoma.
Conclusions: As the young monkey matured, the IOP in the normotensive eye decreased Sheep may be an ideal model for further studies.
because outflow facility and uveoscleral outflow increased. In the contralateral lasered CR: O.A. Candia, None; R. Gerometta, None.
eye, the IOP increased over time because outflow facility decreased. Concurrently, Support: NIH Grants EY000160 and EY001867, and RPB, Inc.
the lasered eye had an increase in the uveoscleral outflow. Apparently, the fluid was
redirected into the healthy uveoscleral outflow pathway from the laser damaged
trabecular outflow pathway.
CR: M.A. Maslonka, None; S. Fan, None; L.S. Stapp, None; C.B. Toris, None.
Support: Research to Prevent Blindness
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
156-159
Monday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1382 - 1406 / B121 - B145
219. Functional Retinal Imaging Organizing Section: MOI
Sunday, May 2, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 140 - 162 / A313 - A335
108. Aqueous Humor Dynamics Organizing Section: PH
160 - A333 161 - A334
Effect of Verapamil on Ciliary Blood Flow and Aqueous Production in Rabbits Cyclic 3′,5′-Adenosine Monophosphate Transiently Stimulates the in vitro Fluid
Transport Across the Isolated Porcine Ciliary Body Epithelium via Protein
J.W. Kiel, A. Maldonado, D.O. Zamora. Ophthalmology, Univ of Texas Hlth Sci Ctr SA,
Kinase A
San Antonio, TX.
C.S. Law1, O.A. Candia2, C.-H. To1. 1School of Optometry, The Hong Kong Polytechnic
Purpose:We have previously found that aqueous production is stable when ciliary
University, Kowloon, Hong Kong; 2Ophthalmology, Mount Sinai School of
blood flow is increased by raising arterial pressure. Under those conditions, neural
Medicine, New York, NY.
or local vascular control mechanisms may have protected the ciliary epithelium
from over-perfusion. Verapamil is a vasodilator that works by blocking calcium Purpose: To study the effects of cyclic 3′,5′-adenosine monophosphate (cAMP)
influx thereby overriding neural and local control mechanisms. This study sought signalling pathway on the fluid flow (FF) and potential difference (PD) across the
to determine whether verapamil-induced vasodilation would increase ciliary blood porcine ciliary body epithelium (CBE) measured by the modified Ussing-type fluid
flow and cause an increase in aqueous production. chamber.
Methods:In anesthetized rabbits (n=10), we measured arterial pressure, IOP, and Methods: A complete annulus of the CBE preparation was excised and mounted in
orbital venous pressure by direct cannulation; carotid blood flow by transit time a modified Ussing-type fluid chamber. The two hemi-chambers were connected to
ultrasound, heart rate by a digital cardiotachometer, ciliary blood flow by laser Doppler different external vessels. On one side, a bubbling reservoir fitted with a pair of Ag/
flowmetry, and aqueous flow by fluorophotometry. The protocol entailed 60 min of AgCl electrodes containing Ringer solution was connected. On the other side, the hemi-
baseline measurements, followed by topical application of verapamil (10 mg/ml, chamber was a close compartment fitted with another pair of Ag/AgCl electrodes and
100 μl). Measurements were then continued for another 135 min. Aqueous flow was a graduated capillary. The capillary level reflected the change of fluid volume inside
calculated with Brubaker’s equation at hourly intervals, as were the mean values for the chamber and the rate of FF was calculated accordingly. The PD was monitored via
the other measured variables. The data were analyzed by repeated measures ANOVA one pair of Ag/AgCl electrodes by a voltage-current clamp unit, and tissue resistance
and Dunnett’s multiple comparisons test (Graphpad Prism). was determined by passing a fixed (10 μA) current across the CBE preparation. The
Results:Blood pressure, carotid blood flow, heart rate and orbital venous pressure effects of several cAMP-regulating agents on FF and PD were examined.
were unchanged. During the first hour after verapamil, IOP, aqueous flow and ciliary Results: Forskolin (10 μM, n = 5), an activator of adenylate cyclase, hyperpolarized the
blood flow were also unchanged. However, during the second hour after verapamil, PD by 100% (P < 0.005) and increased the FF by 42% (P < 0.005). The cAMP analogue
IOP decreased (p<0.01) from 13.4 ±0.9 mmHg to 10.7 ± 0.7 mmHg, aqueous flow 8-Br-cAMP (100 μM, n = 5) hyperpolarized the PD by 190% (P < 0.005) and stimulated
decreased (p<0.05) from 2.66 ± 0.37 μl/mmin to 1.87 ± 0.3 μl/min and ciliary blood the FF by 56% (P < 0.005). The non-specific inhibitor of phosphodiesterases, 3-isobutyl-
flow increased (p<0.05) from 52 ± 5 P.U. to 62 ± 8 P.U. 1-methylxanthine (IBMX, 1 mM, n = 6) only slightly hyperpolarised the PD by 22%
Conclusions:Topical verapamil decreases aqueous production and increases ciliary (P < 0.005) but caused no significant change in the FF (P = 0.74). The pre-treatment of
blood flow. This uncoupling of perfusion from metabolic activity indicates a direct protein kinase A (PKA) inhibitor, H-89 (50 μM, n = 5) partially blocked the cAMP-
inhibitory effect of calcium channel blockade on ciliary ionic transport. stimulated PD and FF by 65% (P < 0.001) and 79% (P < 0.01) respectively.
CR: J.W. Kiel, None; A. Maldonado, None; D.O. Zamora, None. Conclusions: The fluid formation by the CBE can be stimulated by cAMP/PKA
Support: NIH EY09702, the van Heuven endowment, and Research to Prevent Blindness signalling pathway. This pathway likely modulates the transport of ion across the CBE.
CR: C.S. Law, None; O.A. Candia, None; C.-H. To, None.
Support: The Hong Kong Polytechnic University Niche Glaucoma Funding J-BB76,
Research Studentship RP92, and Research Grants Council General Research Fund
(GRF) grant B-Q04F
162 - A335
Melatonin and 5-MCA-NAT Reduce Chloride Efflux in Rabbit Cilliary Non-
Pigmented Epithelial Cells
F. Huete Toral1, A. Crooke2, J. Pintor2. 1Bioquimica y Biologia Molecular IV,
UNIVERSIDAD COMPLUTENSE DE MADRID, MADRID, Spain; 2Bioquimica y
Biologia Molecular IV, Universidad Complutense de Madrid, Madrid, Spain.
Purpose: To investigate if the hypotensive effect of melatonin and 5-MCA-NAT is
due to a reduction in the efflux of chloride in cilliary non-pigmented epithelial cells.
Methods: Immortalized NPE cells, kindly provided by Dr. Coca-Prados, were
seeded in high glucose DMEM until they were confluent. Chloride measurements
were performed with MQAE incubated at 1 mM during 20 h before the experiments
performed. Measurements were carried out by incubating at high Cl- (130 mM, 10
min), then washed and changed by a non-chloride buffer (Cl- is changed by nitrate).
Stern-Volmer constant for chloride efflux in NPE cells was calculated by means of the
double ionophore technique. After that cells were challenged with single doses (100
uM) of melatonin, 5-MCA-NAT and IIK7. Dose-response analysis was performed by
assaying the agonists at concentrations ranging from 0.1 uM to 150 uM.
Results: NPE cells presented changes in the intracellular chloride concentrations
when they were treated with fixed concentrations of melatonin agonists at 100 uM.
Melatonin reduced the Cl- efflux 75±5 % and 5-MCA-NAT (MT3 agonist) 70±7 % of
control (n=8). IIK7 (MT2 agonist), presented a bi-phasic effect first increasing a chloride
influx followed by a chloride efflux. Concentration-response curves for melatonin and
5-MCA-NAT presented pD2 values of 5.8 ± 0.5 and 5.5 ± 0.8, respectively (n=8). Stern-
Volmer constant showed a value of 1.86 M-1, which helped to calculate an intracellular
chloride concentration of 129.43 mM.
Conclusions: The application of melatonins reduced IOP as we have previously
described. Here we have demonstrated that at least in part, the reduction of IOP can
be explained as a reduction in the chloride efflux from the NPE cells, since this ion is
one of the most important in governing the net flow of water in the ciliary processes.
CR: F. Huete Toral, None; A. Crooke, None; J. Pintor, None.
Support: SAF2007/60835
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
160-162
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 682 - 702 / D785 - D805
128. Molecular and Cellular Physiology/Pharmacology Organizing Section: PH
682 - D785 683 - D786
Kir7.1 Channel Mutation (R162W), Associated With Snowflake Vitreoretinal Evaluation of a Major Intracellular Phosphates Regulator I2PP2A in Retina
Degeneration (SVD), Results in a Non-Functional Channel
P. Senanayake1, J. Drazba2A, R. Desnoyer3, S. Karnik2B, J.G. Hollyfield1. 1Ophthalmology,
B.R. Pattnaik1A, A. Sharma2, S. Tokarz1B, T. Schroeder1B, M. Assuma1B, A.O. Edwards3, D.- Cole Eye Institute/Cleveland Clinic Lerner College of Medicine, Cleveland, OH;
A.M. Pillers1C. APediatrics, Ophthalmology and Visual Sci, Eye Research Institute, AImaging Core/Molecular Medicine, BMolecular Cardiology/Molecular Medicine,
BPediatrics, CPediatrics, Eye Research Institute, 1Univ of Wisconsin, Madison, WI; 2Lerner Research Institute/Cleveland Clinic Lerner College of Medicine, Cleveland,
2Experimental Pathology, Mayo Clinic, Rochester, MN; 3Institute for Molecular OH; 3Molecular Cardiology, Lerner Research Institute, Cleveland, OH.
Biology, Univ of Oregon, Eugene, OR.
Purpose: Aim of this study was to evaluate I2PP2A and the AT2 receptor in human,
Purpose: Hereditary snowflake vitreoretinal degeneration (SVD) causes degeneration rat and mouse retina. High glucose increases Angiotensin II (Ang II) in Müller cells by
of multiple ocular tissues due to a mutation in Kir7.1 channel. Kir7.1 channel is ten fold. In experimental diabetes Ang II and Ang II, AT2 receptors are significantly
predominantly present in the apical membrane of retinal pigment epithelium (RPE) increased. Ang II is also increased in the human diabetic retina. AT2 receptor activation
that faces photoreceptor outer segment. This channel is highly conserved amongst leads to stimulation of tyrosine and threonine phosphatases. I2PP2A is a major
mammals and rat Kir7.1 mutant channel produced non-selective cation current. As a first regulator of intracellular phosphates activity.
step to understand the underlying biology, we characterized the human (Kir7.1) mutant Methods: Donor human eyes were obtained from the Cleveland Eye Bank within
expressed in mammalian cells. 12 hours postmortem and fixed in PBS-paraformaldehyde. C57BL/6 12 week old
Methods: Wildtype (hKir7.1WT) and 484 C>T (R162W) mutant (hKir7.1M) constructs mice were purchased from Jackson Laboratory. WKY 11 week old male rats were
in pCMV6-XL5 were co-transfected into CHO-M1 cells along with pcDNA3.1/NTGFP purchased from Charles River Lab. Mice and rats were sacrificed by carbon dioxide
using Fugene-6. Kir channel currents were studied by whole-cell configuration of patch asphyxiation. The eyes were enucleated and fixed in PBS- paraformaldehyde. The
clamp electrophysiology. Bath solution: (in mM) 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, eyes were then frozen in OCT and stored at -80º C. AT2 receptor and I2PP2A was
1.8 CaCl2 and 1 MgCl2 pH 7.4 using NaOH. We used 20 mM Cs+ in place of equimolar localized by immunohistochemistry and fluorescence imaging. Retinal sections were
Na+ to block Kir7.1 current. For selectivity bath Na+ was replaced with either K+ or Rb+.
labeled with polyclonal antibodies against AT2 receptor (1:100, Alpha Diagnostics,
Recording electrodes contained (in mM): 30 KCl, 83 K-gluconate, 10 HEPES, 5.5 EGTA,
San Antonio, TX) and I2PP2A (B13008, 1:400, Stratagene, La Jolla , CA) and resolved
0.5 CaCl, 4 Mg-ATP, and 0.5 GTP adjusted to pH 7.2 using KOH.
2 with goat anti-rabbit Alexa 488 or 594 conjugated labeled secondary antibodies. Slides
Results: GFP positive cells expressing hKir7.1WT channels had a resting membrane
were mounted in anti-fade medium containing DAPI for labeling nuclei (Vectorshield
potential of -45 ± 4.6 mV (n=10) compared to -3.2 ± 1.8 mV (n=15) for the hKir7.1M channel
Laboratories, Inc., Burlingame, CA). Images were taken with a Leica confocal laser-
transfected cells (p < 0.005). Current-voltage curves for the hKir7.1WT channel showed
scanning microscope (Heidelberg, Germany).
typical inward rectification. This current was inhibited by 20 mM Cs+. The inward current
of hKir7.1WT was at least 6-fold larger in the presence of Rb+ and showed higher preference Results: I2PP2A was detected in the ganglion cell layer (GCL), inner nuclear layer
for Rb+ over K+. The hKir7.1M channel on the otherhand exhibited no current over the (INL) and outer nuclear layer (ONL). The I2PP2A has a nuclear and perinuclear
whole voltage range in which hKir7.1WT is activated. Further application of 20 mM Cs+ distribution. The AT2 was also detected in the GCL, INL and ONL and has nuclear
or 135 mM Rb+ solution had no effect on the hKir7.1M channel current. and perinuclear localization. In addition, AT2 IR was detected in the inner limiting
Conclusions: We provide evidence that the hKir7.1M subunit does not form functional membrane, Müller cell endfeet and in the Müller cell processes.
channels. Kir7.1 is a mildly inwardly rectifying potassium channel that facilitates Conclusions: AT2 receptor and I2PP2A may be colocalized. The link between I2PP2A
potassium homeostasis around the photoreceptor outer segment and also facilitates and the AT2 receptor in retina remains to be established.
directional fluid flow by RPE. This non-functional Kir7.1 channel therefore by altering CR: P. Senanayake, None; J. Drazba, None; R. Desnoyer, None; S. Karnik, None; J.G.
RPE physiology perhaps recapitulates SVD etiology like the minute crystalline deposits Hollyfield, None.
and retinal detachments. Support: Supported by NIH ( JGH015638), & an Unrestricted Grant from Research
CR: B.R. Pattnaik, None; A. Sharma, None; S. Tokarz, None; T. Schroeder, None; M. to Prevent Blindness.
Assuma, None; A.O. Edwards, None; D.-A.M. Pillers, None.
Support: NIH Grant EY014467 (AOE), FFB (AOE), RRF (BRP), UW Pediatrics, College of
Medicine and Public Health, and Graduate School (DMP)
684 - D787 685 - D788
ERK 1/2 is an Important Mediator of Oxidative Stress and Iron Metabolism in Unique and Analogous Functions of AQP0 for Lens Fiber Cell Architecture and
Lens and Retinal Pigmented Epithelial Cells Transparency
M.C. McGahan, J. Harned, M. Lall. Dept of Molec Biomed Sci, North Carolina State K. Varadaraj, S.S. Kumari, R.T. Mathias. Physiology and Biophysics, State University
University, Raleigh, NC. of NY, Stony Brook, NY.
Purpose: Activation of the ERK 1/2 pathway has been associated with protection Purpose: To investigate whether aquaporin 0 (AQP0) performs only water channel
against oxidative stress in a number of cell types. Studies indicate that oxidative function or has additional function/s to contribute towards lens transparency.
stress induced by hydrogen peroxide is dependent upon iron and is mediated by the Mutations as well as knockout of AQP0 result in dominant lens cataract. Among
ERK 1/2 pathway. The purpose of this study was to determine how oxidative stress the multiple functions speculated for AQP0, only water channel function has been
affects iron metabolism and to investigate if ERK 1/2 is involved in these responses. authentically proven. Lens switches protein expression from AQP1 in the equatorial
Methods: Cultured canine lens epithelial cells (LEC) and retinal pigmented epithelial epithelial cells to 40 times less efficient AQP0 in the differentiating secondary fiber
cells (RPE) were used in these studies. Hydrogen peroxide was added either as a bolus cells. If AQP0 has only water permeability function, can the more efficient water
(250 µM) or generated at a steady state of 2.5 µM by glucose oxidase (GO) added to channel, AQP1, transgenically expressed in the fiber cells compensate and restore lens
the medium. Ferritin was measured by ELISA, ERK 1/2 activation was determined by transparency in the AQP0 knockout (AQP0 -/-) mouse lens? If not, it can be inferred
Western analysis of ERK 1/2 phosphorylation, de novo ferritin synthesis was measured that AQP0 performs distinctive role/s in the lens besides the water channel function.
by metabolic labeling with 35S-methionine. Methods: We first generated a transgenic wild type mouse line (TgAQP1+/+) expressing
Results: ERK 1/2 was activated in cultured LEC and RPE treated with GO over a 24h human AQP1 in the lens fiber cells using the fiber cell-specific alphaA-crystallin
period (constant 2.5 µM hydrogen peroxide). U-0126, a specific inhibitor of ERK 1/2 promoter. TgAQP1+/+ mice were crossed with AQP0 knockout (AQP0-/-) mice to
activation decreased both baseline and hydrogen peroxide induced ERK 1/2 activation. generate AQP0-/- mice expressing AQP1 in the lens fiber cells (TgAQP1+/+/AQP0-/-) to
Both hydrogen peroxide (bolus and GO generated) increased ferritin concentration see whether lens transparency can be restored. RT-PCR, immunostaining, western
in LEC 1.3- and 3-fold, respectively. Interestingly, U-0126 did not inhibit the effects blotting, confocal microscopy, electron microscopy, membrane water permeability
of hydrogen peroxide on ferritin levels. On the contrary, it was additive to hydrogen assay and lens transparency evaluation techniques were used in this study.
peroxide in both delivery systems, increasing ferritin concentration as much as 4 fold. Results: Transgenic mice (TgAQP1+/+) showed increase in membrane water permeability
Both U-0126 and hydrogen peroxide increased de novo ferritin synthesis, but their without causing defects in lens morphology, ultrastructure or transparency compared
effects were not additive. Addition of the iron chelator, Dp44mT inhibited the effects to the wild type. AQP0-/- compromised homeostasis of the lenses as demonstrated by the
of both bolus and continuous exposure to hydrogen peroxide as well as U-0126 on development of cataract. Water permeability of TgAQP1+/+/AQP0-/- mice was 2.6 times
ferritin concentration. more than that of the wild type. Transgene AQP1 reduced the severity of lens cataract
Conclusions: Exposure of LEC and RPE to hydrogen peroxide causes an increase and lessened the pace of acceleration of cataractogenesis; however, lens transparency
in ERK 1/2 activation, which was inhibited by U-0126. Both bolus and continuous was not completely restored. Lens fiber cells showed less-compact arrangement.
addition of hydrogen peroxide caused an increase in ferritin concentration, which Conclusions: Partial restoration of lens transparency in AQP0 null mice expressing
was not mediated by the ERK 1/2 signaling pathway as U-0126 did not inhibit this transgenic AQP1 in the fiber cells suggests that AQP0 and AQP1 have at least one
response. On the contrary U-0126 was additive to the peroxide induced increase in analogous function, of being water channels. Using an animal model, this study
ferritin levels. Ferritin synthesis is increased acutely at the translational level when also reveals that AQP1 cannot fully replace AQP0 functionally, implying AQP0 may
iron content of cells is increased. Therefore, the ERK 1/2 pathway may function to have unique function/s. Absence of AQP0 could be the cause for the less-compact
keep iron levels in the cells low since inhibiting it caused a large increase in ferritin architecture of the fiber cells, indicating a possible cell-to-cell adhesion structural
synthesis and concentration. In addition, the effects of U-0126 and hydrogen peroxide role for AQP0.
are inhibited by the iron chelator Dp44mT. All of this evidence points to an important CR: K. Varadaraj, None; S.S. Kumari, None; R.T. Mathias, None.
role of the ERK 1/2 pathway in iron metabolism. Support: NIH Grant EY06391 and Alcon Research Ltd.
CR: M.C. McGahan, None; J. Harned, None; M. Lall, None.
Support: NIH Grant EY-04900 and Funds from the State of North Carolina
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
682-685
Monday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1382 - 1406 / B121 - B145
219. Functional Retinal Imaging Organizing Section: MOI
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 682 - 702 / D785 - D805
128. Molecular and Cellular Physiology/Pharmacology Organizing Section: PH
686 - D789 687 - D790
Knockdown of Pannexin-1 Hemichannel Inhibits ATP Release by Human Cytoskeletal Remodeling Regulates ATP Release by Human Trabecular
Trabecular Meshwork Cells Meshwork Cells
A. Li1A, C.T. Leung1A, K. Peterson-Yantorno1A, C.H. Mitchell1A,1B, M.M. Civan1A,1C. M.M. Civan1A,1B, C.T. Leung1A, K. Peterson-Yantorno1A, A. Li1A. APhysiology, BMedicine,
APhysiology, School of Medicine, BAnatomy and Cell Biology, School of 1Univ of Pennsylvania School of Medicine, Philadelphia, PA.
Dental Medicine, CMedicine, School of Medicine, 1University of Pennsylvania,
Purpose: To test whether remodeling the cytoskeleton and shape of human trabecular
Philadelphia, PA.
meshwork (TM) cells modulates ATP release, the first step in purinergic regulation
Purpose: To test whether down-regulation of pannexin-1 (Px1) hemichannels in human of aqueous humor dynamics.
trabecular meshwork (TM) cells reduces ATP release, the enabling step in purinergic Methods: Human TM (hTM5) cells were subcultivated and plated into 96-well
regulation of aqueous humor dynamics. We previously identified Px1, connexin (Cx) microplates or stretching chambers 48hrs before experiments. ATP release was
hemichannels and ionotropic P2Xreceptor in TM cells by RT-PCR with DNA sequencing. measured by the luciferin-luciferase reaction using a luminometer, and ATP
7
Methods: Cultured human TM (hTM5) cells were transduced with shRNA lentiviral concentrations were calibrated in isotonic and hypotonic solutions with/without drugs.
particles and selected with puromycin (0.5µg/mL) for stable Px1 knockdown. Efficiency After pre-incubation isotonically with/without drugs for 1hr, cells were stimulated
was determined by real-time PCR and semi-quantitative immunoblotting. ATP release was either by 50% hypotonicity or graded mechanical stretch. Cytoskeletal-remodeling
measured with a luminometer by the luciferin-luciferase reaction 24-48hrs after plating agents, including dexamethasone (DEX, 1µM for 14 days), H-7 (5µM and 50µM for 1hr)
cells into 96-well plates. Cells were pre-incubated isotonically with/without drugs for 1hr, and cytochalasin D (CCD, 25µM for 1hr), were added and continuously maintained
and stimulated by 50% hypotonicity or by raising cytosolic Ca2+ with 10µM ionomycin. in corresponding cell baths at indicated time points prior to hypotonic exposure.
ATP concentrations were monitored in isotonic and hypotonic solutions with/without Cell viability was estimated by the MTT method after hypotonicity or by propidium
drugs. Intracellular Ca2+ levels were similarly measured with fluorescent dye Fluo-4. iodide staining after stretch.
Results: Lentivirus-mediated shRNA produced stable knockdown of Px1 mRNA by 67% Results: ATP concentration in control isotonic solution was 10.4+0.2nM (mean+SE,
(n=21) and protein by 39% (n=8) for >5 weeks. ATP concentration for the non-targeted
n=200 wells), rising to 47.4+0.8nM (n=171, P<0.001) after 50% hypotonicity. Promoting
control (NC) was 9.6+0.1nM (mean+SE, n=543 wells), increasing 3.6-fold to 31.3+0.5nM
actin-cytoskeleton polymerization with 1µM DEX reduced hypotonically-induced ATP
(n=746, P<0.01) after hypotonic exposure. Px1 knockdown (KD) did not change the isotonic
release by 78.9+1.3% (n=28, P<0.01), whereas facilitating depolymerization with 25µM
baseline (n=147, P=0.71), but reduced the hypotonically-elicited ATP release by 27+2%
CCD, 5µM H-7 and 50µM H-7 increased swelling-activated release by 39.7+9.2% (n=54,
(n=287, P<0.001). Relatively selective blockers of Px1 (100µM Probenecid), Cxs (1mM
P<0.01), 41.1+6.6% (n=36, P<0.01), and 50.4+6.1% (n=54, P<0.01), respectively. Altering
Heptanol) and P2X receptor (1µM KN-62) inhibited hypotonicity-induced release by
7 cell shape by stretch also triggered ATP release. Compared with ATP levels before
42+2% (n=246), 39+3% (n=61), and 33+3% (n=120) in NC, and by 12+2% (n=377), 63+2%
(n=160), and 44+3% (n=120) in KD, respectively. KN-62 (1µM) abolished ionomycin- stretch, 10% and 30% extensions of the chamber surface area elicited 1.8±0.2 fold (N=17,
stimulated ATP release in both groups (NC: 100+1%, n=80; KD: 98+1%, n=120) without P<0.01) and 6.5±1.0 fold (N=18, P<0.001) increments, respectively. Cell viability was
affecting Ca2+ influx. unaffected by stretch or hypotonicity with/without drug applications.
Conclusions: Px1 knockdown decreases swelling-activated ATP release and largely Conclusions: The results indicate that ATP is released by physiologically-relevant
nullifies the inhibitory effect of the Px1 blocker probenecid. Antagonists of other stretch of TM cells. The data also provide the first evidence that actin polymerization
ATP conduits become correspondingly more efficacious. In contrast, the abolition of inhibits, whereas depolymerization enhances hypotonically-triggered ATP release.
intracellular Ca2+-evoked ATP release by KN-62 suggests that a Px1-independent P2X Changes in ATP release will alter ecto-enzymatic formation of adenosine, thereby
pathway predominates after ionomycin stimulation. The results demonstrate that the7 modulating release of metalloproteinase-2, mediated by A1 adenosine receptors in TM
conduits recruited by TM cells are stimulus-specific, and that Px1 contributes to swelling- cells. This observation may be relevant to the dependence of aqueous humor outflow
initiated ATP release. on the actin cytoskeleton, whose mechanism is not yet established.
CR: A. Li, None; C.T. Leung, None; K. Peterson-Yantorno, None; C.H. Mitchell, CR: M.M. Civan, None; C.T. Leung, None; K. Peterson-Yantorno, None; A. Li, None.
None; M.M. Civan, None. Support: NIH Grant EY13624 (M.M.C.) and Core Grant EY 01583
Support: NIH Grants EY 13624 (M.M.C.), EY 015537 and EY 013434 (C.H.M.) and Core
Grant EY 01583
688 - D791 689 - D792
Prostaglandins and NO Mediate the Urocortin2/ CRF2 Induced Relaxation of Autonomic Innervation to the Intraocular Muscles of the Anterior Segment in
the Iris Sphincter Muscle Chicks
A.A. Rocha-Sousa1, M. Tavares-Silva1, S. Fonseca2, S. Cardoso1, S. Azevedo- Pinto1, A. L.A. Ostrin1, Y. Liu1, J. Mok Yee1, V. Choh2, C.F. Wildsoet3. 1School of Optometry,
Leite-Moreira1. 1Physiology, Faculty of Medicine University of Porto, Porto, Portugal; University of California Berkeley, Berkeley, CA; 2School Optometry, University of
2Ophthalmology, Hospital de São João, Porto, Portugal. Waterloo, Waterloo, ON, Canada; 3Center for Ocul Disease & Dvlpmt, University of
California, Berkeley, Berkeley, CA.
Purpose: Urocortin 2 (Ucn2) is an endogenous peptide member of the CRF family that
is expressed in the retinal pigmented epithelium and has a vasodilator effect in the Purpose: Studies show that the embryological chick iris has only muscarinic receptors,
retinal circulation. Our aim is to characterize the effect of Ucn2 in the iris sphincter while the mature iris muscles have both nicotinic and muscarinic receptors. Mature
muscle and its subcellular pathways. chick ciliary muscle is reported to have nicotinic receptors; whether they also have
Methods: The effect of increasing Ucn2 concentrations (10e-10-10e-6M) on carbacol- muscarinic receptors during early development has not been fully resolved. The
precontracted (10e-6M) rabbit iris sphincter muscles were tested alone and (n=9) in the possibility of dual cholinergic receptors has implications for chick emmetropization
presence of: (i)Nω-nitro-L-Arginine (L-NA;10e-5M;n=9), (ii)indomethacin (10e-5M;n=8) studies. An in vitro pharmacological study was undertaken to characterize receptor
and (iii)antisauvagine-30 (10e-6M;n=8). subtypes on intraocular muscles of chick anterior segment at various ages.
Results: Ucn2 promoted a concentration-dependent decrease on active tension, Methods: Eyes from 42 normal White-Leghorn chickens, aged embryonic day 18 to
maximal at 10-7M (18.6±6.1%). This effect was blunted with indomethacin (1.7±3.2%), postnatal 6 weeks, were used. Eyes were enucleated and anterior segments mounted
L-NA (8.5±6.6%) and antisauvagine-30 (a CRF2 selective antagonist; .0±5.1%). and scanned in a lens scanning instrument (ScanTox). As indices of muscle contraction,
Conclusions: Ucn2 is a new neurohumoral factor that modulates the relaxation of the lens back vertex distance (BVD), a measure of accommodation, and pupil size were
iris sphincter muscle. This effect is mediated by prostaglandins and is also dependent determined. Individual preprations were tested with increasing concentrations of
on nitric oxide production. CRF2 is the receptor involved in this relaxation. either pilocarpine (muscarinic agonist), carbachol (muscarinic/nicotinic agonist) or
CR: A.A. Rocha-Sousa, None; M. Tavares-Silva, None; S. Fonseca, None; S. Cardoso, nicotine (nicotinic agonist). The response to a maximum effective concentration of
None; S. Azevedo- Pinto, None; A. Leite-Moreira, None. nicotine was also measured from both eyes in 6 previously monocularly iridectomized
Support: Supported by the “Concurso Investigação na Pré-Graduação da U.Porto / chicks, aged 6 wks.
Santander Totta” Results: Nicotine had the greatest effect on accommodation and pupil size, with a 100
µM concentration producing the maximum decrease in BVD of 35.5±11%, and pupil size
of 54.0±16%, averaged across all ages. With carbachol, a higher concentration (5000 µM)
was required to achieve maximal decreases in BVD and pupil size, 29.6±8 and 15.4±16%
respectively, which were less than that of nicotine. Up to 1000 µM pilocarpine had
little effect on either BVD or pupil size, even for the youngest chicks (4.4±7%, 4.1±3%
respectively, E18 to D7;). Iridectomized eyes showed minimal decrease in BVD to 100
µM nicotine (6.2±5% compared to 41.5±20% for fellow eyes).
Conclusions:Nicotinic receptors primarily mediate the response of the ciliary muscle
and iris in chicks, from age E18 onwards. A possible role of muscarinic receptors in the
youngest eyes is suggested by subtle decreases in BVD and pupil size with pilocarpine,
and the iris was more sensitive to carbachol than the older eyes. The lack of response
to nicotine in iridectomized eyes confirmed results of a previous study and implies
that the iris is necessary for accommodation in vitro, although a parallel in vivo study
of the same eyes showed that accommodation was unaffected.
CR: L.A. Ostrin, None; Y. Liu, None; J. Mok Yee, None; V. Choh, None; C.F. Wildsoet,
None.
Support: NIH grant K12 to UCB
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
686-689
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 682 - 702 / D785 - D805
128. Molecular and Cellular Physiology/Pharmacology Organizing Section: PH
690 - D793 691 - D794
Activation of Src Family Kinases Causes Inhibition of Na,K-ATPase Activity in P2Y2 Nucleotide Receptors Increase the Presence of Aquaporin-1 in Rabbit Non
Porcine Nonpigmented Ciliary Epithelium Pigmented Ciliary Epithelial Cells
M. Shahidullah, N.A. Delamere. Department of Physiology, University of Arizona, A. Martin-Gil1, J. Pintor2. 1Bioquimica y Biologia Molecular IV, UNIVERSIDAD
Tucson, AZ. COMPLUTENSE DE MADRID, Madrid, Spain; 2Bioquimica y Biologia Molecular
IV, Universidad Complutense de Madrid, Madrid, Spain.
Purpose:Nonpigmented ciliary epithelium (NPE) is the tissue responsible for aqueous
humor secretion in the eye. Na,K-ATPase is pivotal in the secretion of this fluid. Src Purpose:To investigate the role of P2Y2 purinergic receptors in the raise of aquaporin-1
family kinases (SFK) have been shown to influence Na,K-ATPase activity in porcine levels in NPE cell membranes and cytoplasm.
ocular lens epithelium (Bozulic, Dean et al. 2005). Here we show that activation of Methods:Immortalized NPE cells, kindly provided by Dr. Coca-Prados, were seeded
SFK by DIDS, SNP or by lowering cytoplasmic pH inhibits Na,K-ATPase activity. in high glucose DMEM until they were subconfluent. Immunocytochemical studies
Methods:Src activation in porcine NPE lysate was detected by western blot followed were performed by means of an antibody raised against aquaporin-1 (AQP1) from
by densiometric analysis. Na,K-ATPase activity was studied in porcine cultured NPE Santa Cruz. Immunolabeling of the cells was performed by incubating them after the
by measuring ouabain-sensitive potassium (86Rb) uptake. corresponding treatment (see below) with a dilution of 1:100 of the primary antibody
Results:Sodium nitroprusside (100 µM), DIDS (100 µM) and low pH (6.5) bathing followed by a dilution 1:50 of a secondary antibody labelled with FITC. Previous to
medium caused significant phosphorylation of Src and ERK1/2. The Src phosphorylation this immunostaining cells were challenged with 100 uM of the dinucleotide Ap4A,
was abolished by pre-treating the cells with a selective Src family kinases inhibitor, at 15, 30, 60, 90 and 120 min. To avoid Ap4A degradation, the medium was changed
PP2. Interestingly, PP2 also blocked DIDS-induced ERK1/2 phophorylation, indicating every 15 min. Antagonists of the P2Y receptors, suramin, reactive blue 2 and PPADS
that Src phosphorylation was the precedeing event. Sodium nitroprusside, DIDS (all at 100 uM), were pre-incubated 30 min before Ap4A (100 uM) was added and
and low pH bathing medium caused marked inhibition of Na,K-ATPase activity as antagonists were present during the dinucleotide application.
measured by the reduction of ouabain-sensitive potassium (86Rb) uptake in cultured Results:The application of Ap4A to the NPE cells demonstrated a gradual increase in
NPE. DIDS-induced inhibition of Na,K-ATPase activity was blocked by PP2. DIDS or the presence of AQP1 which was time-dependent from the beginning to 60 min, when
low pH medium caused significant reduction of cytoplasmic pH. the maximal expression of the protein is reached (200 % of increase when compared to
Conclusions:Activation of Src family kinases causes inhibition of Na,K- control) (n=8). Times longer than 60 min, gradually returned the fluorescent values to
ATPase activity in NPE. Lowering cytoplasmic pH may have the potential to the initial control values (at 120 min). Concerning the distribution of AQP1, although at
activate Src family kinases. Acid activation of Src family kinases can be a novel the beginning its presence was both in the cytoplasm and in the membrane, at 60 min,
way of controlling fluid secretion in the NPE and possibly in other epithelia. there was a significant increase of AQP1 in the plasma membrane. The experiments
Reference Bozulic, L. D., W. L. Dean, et al. (2005). “The influence of SRC-family tyrosine perfomed with the antagonist of the P2Y receptors demonstrated the involvement of
kinases on Na,K-ATPase activity in lens epithelium.” Investigative Ophthalmology this receptor in the action of Ap4A. In particular reactive blue 2 was the best reversing
& Visual Science 46(2): 618-622. the effect of Ap4A. In the case of this antagonist, it was able to inhibit up to 80 % of
CR: M. Shahidullah, None; N.A. Delamere, None. the effect depicted by Ap4A.
Support: NIH EY00691519 Conclusions:Diadenosine tetraphosphate, a good P2Y2 agonist, was able to mobilize
AQP1 which was more visible after 60 min incubation with this substance. This increase
in AQP1 expression can be the explanation to what was found in the whole rabbit in
which P2Y2 agonists clearly increase intraocular pressure.
CR: A. Martin-Gil, None; J. Pintor, None.
Support: BSCH-UCM GR58/08
692 - D795 693 - D796
Functional Expression of Human Aquaporin Water Channels AQP1, AQP4 and Expression of Aquaporin 4 in Corneal Endothelial Cells
AQP9
S.P. Patel1, A.N. Van Hoek2, N.C. Joyce3, K. Colby4. 1Ophthalmology, University at
A.N. Van Hoek, D. Brown, S. Breton. Center for Systems Biology, Massachusetts Buffalo, Buffalo, NY; 2Center for Systems Biology and Program in Membrane
General Hospital, Boston, MA. Biology, Massachusetts General Hospital, Boston, MA; 3Ophthalmology, Schepens
Eye Research Institute, Harvard Med School, Boston, MA; 4Ophthalmology,
Purpose: Aquaporin (AQP) water channels are present in cornea, ciliary body, lens
Massachusetts Eye & Ear Infirmary, Boston, MA.
and retina but their functional contributions to trans epithelial and endothelial water
transport is not understood other then providing facilitated pathways for water Purpose: Aquaporin (AQP) water channels are present in corneal endothelium, but their
movement. We have created CHO cell lines stably expressing human aquaporins with role in corneal water transport is unidentified. Prior functional studies of AQPs in corneal
the goal to compare and contrast functional responses to anisosmotic solutions in endothelium have focused on aquaporin 1 (AQP1), a major mammalian water channel.
intact CHO cells. Using intact cells it will permit measurement of direction-dependent The purpose of this study was to identify and localize aquaporin 4 (AQP4) in corneal
osmotic water permeability coefficients of AQP1, AQP4 and AQP9. The dissection of endothelial cells in several species. This may provide additional insight into mechanisms
properties specific to an aquaporin may provide novel concepts to how aquaporins of corneal water homeostasis.
contribute to water homeostasis in health and disease. Methods: Normal human donor corneas were obtained from the National Disease Research
Methods: Large amount of cells were obtained by culturing CHO cells in suspension. Interchange (Philadelphia, PA). Donor confidentiality was maintained according to the
The water transport function of CHO cells stably expressing human water channels Declaration of Helsinki. Fuchs endothelial dystrophy corneas were obtained at the time of
AQP1, AQP4 and AQP9 was measured at 10 °C following rapid mixing in the stopped penetrating keratoplasty under an institutional review board approved protocol. Human
flow apparatus with an equal amount of a hyper- or hypo-osmotic solution. Cells in corneal specimens were stored in Optisol GS for transport. Bovine eyes were transported
275 mOsm HEPES buffered saline (HBS) were usually mixed with 3xHBS to produce on ice from a local slaughterhouse. Whole rat eyes were fixed in paraformaldehyde.
Upon arrival in the lab, tissues were mounted in OCT embedding medium for frozen
a 2xHBS mixture, creating an outwardly directed 275 mOsm gradient across the cell
sectioning. Immunolocalization for AQP4 was performed with an AQP4-specific antibody
plasma membrane, which shrink cells to 50% of their original size, as the result of
(Millipore, Billerica, MA). Images were acquired with epifluorescence and confocal
volume flow of water. To produce an inwardly directed osmotic gradient of the same
microscopy. Immunoblot analysis was performed on normal human corneal tissue
strength, cells in 1xHBS were premixed with 3xHBS, and the equilibrated cells in
to verify the presence of AQP4. The presence of AQP1 by immunolocalization (AQP1
2xHBS were then mixed in the stopped flow apparatus with an equal amount of plain
antibody, Millipore) in the corneal endothelium was also evaluated.
water to produce the inwardly directed 275 mOsm gradient.
Results: AQP4 expression was found in human corneas from both normal and Fuchs’
Results: In shrinkage assays we found facilitated transport of water through AQP1 and
dystrophy patients, and was also noted in bovine corneal endothelium. Confocal
AQP9 but not through AQP4. In re-swelling assays we found facilitated transport of microscopy showed a speckled cytoplasmic localization in endothelial cells for all samples.
water through all three water channels. In addition we observed overshoots (shrink- However, AQP4 was not detected in rat corneal endothelium, but, as has been previously
swell-shrink, respectively, swell-shrink-swell) in the osmotic responses and were able noted, was robustly expressed in non-pigmented ciliary epithelium and retinal Mueller
to fit traces with a damped sinusoidal function with a period of ~1 second superimposed cells. The cytoplasmic localization of AQP4 in human and bovine cells was compared
on an exponential function. Revisiting our earlier stopped-flow experiments from to AQP1. AQP1 was similarly present in a cytoplasmic distribution in human samples.
almost 20 years ago multiple oscillations (period ~0.2 sec) of water transfer can be Apical localization was noted in bovine endothelial cells.
appreciated in kidney brush border membrane vesicles expressing AQP1. Conclusions: This is the first demonstration, to our knowledge, of species-specific
Conclusions: This is the first demonstration of direction-dependent water transport expression of AQPs in ocular tissue. Future functional studies of AQPs in corneal
through AQP4, signifying a rectifying property for this water channel. Further, endothelium may address the potential contribution of AQP4 in water balance as well
rhythmic water permeation through aquaporins suggests inertial mass-movement as the potential consequences of cytoplasmic localization of AQPs.
(body of water), setting up a harmonic oscillation around the dissipating osmotic CR: S.P. Patel, None; A.N. Van Hoek, None; N.C. Joyce, None; K. Colby, None.
pressure, damped by a resisting force, possibly caused by the cytoskeleton. Support: Unrestricted departmental Challenge Grant from Research to Prevent Blindness,
CR: A.N. Van Hoek, None; D. Brown, None; S. Breton, None. New York, NY (SPP); NEI R01 EY12700 (NCJ); New England Corneal Transplant Research
Support: NIH Grants HD045821, DK38452 Fund (KC)
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
690-693
Monday, May 4, 8:30 AM - 10:15 AM Hall B/C Poster Session Program Number/Board # Range: 1382 - 1406 / B121 - B145
219. Functional Retinal Imaging Organizing Section: MOI
Sunday, May 2, 11:15 AM - 1:00 PM Hall B/C Poster Session Program Number/Board # Range: 682 - 702 / D785 - D805
128. Molecular and Cellular Physiology/Pharmacology Organizing Section: PH
694 - D797 695 - D798
DUSP6/MKP3 Overexpression in Corneal Epithelial Cells Inhibits EGF-Induced ERK-DUSP5 Interactions Specifically Control the EGF Proliferative Effect in
Erk1/2, NKCC1 Phosphorylation and Cell Proliferation Primary and SV40-Immortalized Corneal Epithelial Cells
H. Yang1, Z. Wang1, J.E. Capó-Aponte2, J.M. Wolosin3, P.S. Reinach1. 1Biological P.S. Reinach1, Z. Wang1, H. Yang1, J.E. Capó-Aponte2, J.M. Wolosin3. 1Biological
Sciences, SUNY College of Optometry, New York, NY; 2U.S. Army Aeromedical Sciences, SUNY College of Optometry, New York, NY; 2U.S. Army Aeromedical
Research Laboratory, Fort Rucker, AL; 3Ophthalmology, Mt. Sinai School of Research Laboratory, Fort Rucker, AL; 3Ophthalmology, Mt. Sinai School of
Medicine, New York, NY. Medicine, New York, NY.
Purpose: Mitogen activated protein kinase (MAPK) signaling depends on the Purpose: Membrane receptor activation induces phosphorylation of terminal kinases
magnitude and duration of kinase phosphorylation whereas signaling termination is (tKs) of the Erk1/2, p38 and JNK cascades. The tKs transpose into the nucleus to
elicited by dual-specificity phosphatases (DUSPs).DUSP6 selectively dephosphorylates activate their targets. Dual specificity phosphatases (DUSP), a 26 plus member family,
cytosolic Erk1/2. In human corneal epithelial cells (HCEC), mitogenic responses to modulate the duration and magnitude of tKs activation. This study identified the
epidermal growth factor (EGF) are elicited through ERK pathway signaling and DUSPs in human corneal epithelial cells that modulate mitogenic responses to EGF
Na:K:2Cl cotransporter (NKCC1) activation. To determine if EGF-induced increases through changes in ERK pathway activation.
in HCEC proliferation are modulated by DUSP6, the time dependent effects of DUSP6 Methods: DUSPs gene expression was profiled in both human corneal epithelium
overexpression were evaluated on changes in the phosphorylation patterns of Erk1/2 (HCE) and SV40-immortalized HCE cells (imHCEC) using microarrays. An expanded
and NKCC1. population of primary cells (prHCEC) was obtained by sequential passage in EpiLifetm.
Methods: A HCEC subline strongly overexpressing DUSP6 (DUSP6+) was created HCEC sublines expressing shRNAs for inhibition of DUSP1, DUSP5 and JNK1
using the pLEX lentivector (Open Biosystems). Lentiviral particles were produced in gene expression (DUSP1i, DUSP5i and JNKi), were used pGIPz lentivectors (Open
293 cells, concentrated and used to transduce SV40-HCEC with the aid of polybrene. Biosystems). Transduced cells were selected based on strong puromycin resistance
Transduced cells were selected based on the strong puromycin resistance conferred and GFP fluorescence conferred to the cells by the pGIPz expression. Western blots
by the pLEX transduction. Western blots were used to monitor Erk1/2 and NKCC1 assessed the effects of shRNA expression on, phosphatase expression and target
phosphorylation status. [3H] thymidine incorporation evaluated cell proliferation. phosphorylation. Relative proliferation responses to EGF after serum starvation were
Results: In unmodified cells EGF (5 ng/ml) induces transient increases in Erk1/2 determined by measuring [3H] thymidine uptake.
phosphorylation that peak at ~ 15 min and are followed by a gradual decline to near Results: DUSP gene expression levels in both HCE and imHCEC follow the order:
baseline levels 60 min later. Similar biphasic phosphorylation changes occurred with DUSP1>>DUSP5>> DUSP4~DUSP6 >>DUSP14>>>all other DUSPs. In control, im and
a slight delay in NKCC1. Cell proliferation increased 1.6-fold. Preincubation with 50 pr HCECs, EGF caused a rapid spike in the phosphorylation of all tKs, followed by
µM bumetanide blocked the EGF-linked NKCC1 phosphorylation without affecting decreases to near baseline levels, pre-EGF levels, which temporally coincided with
the time course and magnitude of Erk1/2 phosphorylation, indicating the critical dramatic increases in the levels of DUSP1 and 5 protein expression. In contrast,
role of NKCC1 activation in cell cycling. In the DUSP6+ cells, EGF failed to induce in DUSP1i, the DUSP1 increase was abolished and the tKs maintained a peak
transient changes in Erk1/2 or NKCC1 phosphorylation and simultaneously abolished phosphorylation level for at least 2 hr; in DUSP5i the same temporal difference occurred
the proliferative response to EGF. The EGF effects on the other two terminal MAPK, exclusively in ERK tKs. In JNK1i, the target kinase was essentially knocked out.
JNK and p38 in the DUSP6+ cells were essentially identical to those in the native cells. Proliferation measurements showed that in DUSP5i cells the EGF response was 3-fold
Conclusions: DUSP6 controls the magnitude and duration of EGF-induced Erk1/2 larger than in control cells, whereas in DUSP1i it barely changed. Surprisingly, JNK1i
phosphorylation. EGF-induced HCEC mitogenesis is dependent on Erk1/2 mediated cells displayed a dramatic larger than 5-fold increase in thymidine uptake.
phosphorylation of NKCC1. Conclusions: DUSP5 selectively controls ERK pathway activity and proliferation. The
CR: H. Yang, None; Z. Wang, None; J.E. Capó-Aponte, None; J.M. Wolosin, None; P.S. lack of an effect of DUSP1 inhibition on proliferation can be attributed to its pan-MAPK
Reinach, None. effect. The expected pro-proliferative effect due to enhanced phosphorylation of ERK
Support: EY04795, EY014878, TRUE Research Foundation W81XWH090163 does not occur because a JNK antiproliferative effect is simultaneously unleashed.
CR: P.S. Reinach, None; Z. Wang, None; H. Yang, None; J.E. Capó-Aponte, None; J.M.
Wolosin, None.
Support: EY04795, EY014878, TRUE Research Foundation W81XWH090163
696 - D799 697 - D800
Kinetic Simulation of Active Drug Transport in the Cornea The Role of Bicarbonate and Carbonic Anhydrase in Lactic Acid Transport
Across Corneal Endothelium
K.-S. Vellonen1A,1B, E. Järvinen1B, A. Urtti1A. ACentre for Drug Research, BDivision
of Biopharmaceutics and Pharmacokinetics, 1University of Helsinki, Helsinki, T.T. Nguyen, C. Liu, J. Bonanno. Optometry, Indiana University, Bloomington, IN.
Finland.
Purpose: The cornea produces a significant amount of lactate, a potent osmolyte, that
Purpose: Topically applied ocular drugs may be absorbed by simple diffusion or active needs to be efficiently removed in order to avoid corneal edema. We asked whether
transport. Corneal epithelium expresses several influx and efflux transporters that bicarbonate and carbonic anhydrase (CA) activity facilitate the transport of lactic acid
influence drug permeation. We simulated the importance of active corneal transport in corneal endothelium (CE).
in different cases. Methods: Physiological experiments were performed in vitro using fresh rabbit
Methods: Compartmental ocular models with tear fluid, corneal epithelium and CE mounted in a double-sided perfusion chamber. CE cells were loaded with the
anterior chamber compartments were built using Stella® 9.0.1 program. Ocular intracellular pH sensitive fluorescent dye BCECF-am. Change in the rate of lactate-
bioavailability and drug concentrations in aqueous humor were simulated. Simulations induced acidification (LIA) was measured in the presence of HCO- free (BF) and
3
were carried out for hydrophilic and lipophilic compounds with different affinities HCO- rich (BR) medium and in the presence and absence of 100uM acetazolamide, a
3
(K ) and maximum velocities (V ) of active influx and efflux. Impact of drug release carbonic anhydrase inhibitor. Endogenous lactate efflux was also determined in BF
m max
rate from formulation was also simulated. and BR ringer using bovine CE cells grown to confluence on tissue culture inserts. To
Results: Fast drug loss from the pre-corneal space (by drainage or conjunctival systemic examine physiological function in vivo, 11 white New Zealand rabbits were injected
absorption) and passive diffusion in the corneal epithelium were the most significant intracamerally in one eye with shRNA directed against NBCe1 cotransporter and the
determinants of ocular bioavailability. Active transport has the most prominent effect other with scrambled shRNA delivered by lentiviral vector. Corneal thickness (CT)
on absorption when the drug has slow passive diffusion and/or small dose. High initial was measured before and after challenge with the topical CAI Azopt. Two weeks post-
drug concentration after instillation and rapid clearance from the tear fluid clearly injection, corneas were harvested and processed for western blotting or RT-PCR and
diminish the role of active transport in ocular drug absorption. However, transporters, lactate analysis. Seven separate rabbits were treated topically with Azopt in one eye
especially influx carriers, may have significant effect on the bioavailability when the and PBS in the other. CT measurements were taken every hr for 4-5 hrs. Subsequently,
drug is administered with topical controlled release formulation. the rabbits were euthanized and corneas were processed for lactate analysis. Lastly, 4
Conclusions: Kinetic simulation model is a useful tool for probing transporter effects rabbits were injected intracamerally with 25µl 1mM ouabain in one eye. Similarly, CT
on ocular drug absorption. measurements were taken and corneas were subjected to lactate analysis.
CR: K.-S. Vellonen, None; E. Järvinen, None; A. Urtti, None. Results: pHi decreased (~.05) following exposure to lactate on the basal and apical
Support: Academy of Finland surface in tissues perfused in BF ringer. In BR ringer, there was a smaller decrease (~.02)
in pHi (BL surface) & no decrease apically. In the presence of HCO-, acetazolamide
3
increased the rate of LIA on the BL surface (from .035 pH units/min to .101 pH
units/min). Endogenous lactate efflux was 30% greater in the presence of HCO-.
3
Six rabbits had moderate to significant (50-98%) NBCe1 knockdown (KD). Lactate
concentration in these 6 rabbits were 38+58% greater in the KD eye, although not
statistically significant. Topical application of Azopt induced a 10+ 2% increase in
CT. Corneal lactate concentration was 23+19 % greater in the eyes treated with Azopt
(n=7, p=.015). Ouabain induced an average of 28+ 6% increase in CT and 43+ 40% in
lactate concentration (n=4, p=0.15).
Conclusion: Transport of lactate from the corneal stroma to the anterior chamber is
facilitated by HCO- /CA and active transport activity of the CE.
3
CR: T.T. Nguyen, None; C. Liu, None; J. Bonanno, None.
Support: NIH Grants EY019537 & EY008834
Copyright 2010 by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved.
For permission to reproduce any abstract, contact the ARVO Office at [email protected]. Commercial Relationships are noted at the end of each abstract by “None” or with codes.
694-697