Loss of apical monocilia on collecting duct principal cells impairs ATP secretion across the apical

16 Pages·2008·0.54 MB·English

PurinergicSignalling(2008)4:155–170
DOI10.1007/s11302-007-9072-0
ORIGINAL PAPER
Loss of apical monocilia on collecting duct principal cells
impairs ATP secretion across the apical cell surface
and ATP-dependent and flow-induced calcium signals
Michael B. Hovater&Dragos Olteanu&
Elizabeth L. Hanson&Nai-Lin Cheng&Brian Siroky&
Attila Fintha&Peter Komlosi&Wen Liu&
Lisa M. Satlin&P. Darwin Bell&Bradley K. Yoder&
Erik M. Schwiebert
Received:24July2006/Accepted:1November2006/Publishedonline:13November2007
#SpringerScience+BusinessMediaB.V.2007
Abstract Renal epithelial cells release ATP constitutively purinenucleotide inresponsetostimuli.ATPfiltered atthe
under basal conditions and release higher quantities of glomerulus, secreted by epithelial cells along the nephron,
and released serosally by macula densa cells for feedback
: : : :
M.B.Hov:ater D.Olteanu E.L.Hanson N.-L.Cheng signaling to afferent arterioles within the glomerulus has
B.Siroky E.M.Schwiebert important physiological signaling roles within kidneys. In
DepartmentofPhysiologyandBiophysics,
autosomal recessive polycystic kidney disease (ARPKD)
UniversityofAlabamaatBirmingham,
mice and humans, collecting duct epithelial cells lack an
1918UniversityBlvd.,
Birmingham,AL35294–0005,USA apical central cilium or express dysfunctional proteins
: within that monocilium. Collecting duct principal cells
B.K.Yoder E.M.Schwiebert derived from an Oak Ridge polycystic kidney (orpkTg737)
DepartmentofCellBiology,
mouse model of ARPKD lack a well-formed apical central
UniversityofAlabamaatBirmingham,
1918UniversityBlvd., cilium, thought to be a sensory organelle. We compared
Birmingham,AL35294–0005,USA these cells grown as polarized cell monolayers on perme-
: : ablesupportstothesamecellswheretheapicalmonocilium
A.Fintha P.Komlosi P.D.Bell
wasgeneticallyrescuedwiththewild-typeTg737genethat
DepartmentofMedicine,UniversityofAlabamaatBirmingham,
1918UniversityBlvd., encodes Polaris, a protein essential to cilia formation.
Birmingham,AL35294–0005,USA Constitutive ATP release under basal conditions was low
: : : : and not different in mutant versus rescued monolayers.
B.Siroky P.Komlosi P.D.Bell B.K.Yoder
However, genetically rescued principal cell monolayers
E.M.Schwiebert
TheRecessivePKDTranslationalandResearchCoreCenters, releasedATPthree-tofivefoldmorerobustlyinresponseto
UniversityofAlabamaatBirmingham, ionomycin. Principal cell monolayers with fully formed
1918UniversityBlvd.,
apical monocilia responded three- to fivefold greater to
Birmingham,AL35294–0005,USA
hypotonicity than mutant monolayers lacking monocilia. In
:
W.Liu L.M.Satlin support of the idea that monocilia are sensory organelles,
DepartmentsofPediatricsandMedicine, intentionally harsh pipetting of medium directly onto the
MountSinaiSchoolofMedicine,
center of the monolayer induced ATP release in genetically
NewYork,NY10029–6574,USA
rescued monolayers that possessed apical monocilia. Me-
E.M.Schwiebert(*) chanical stimulation was much less effective, however, on
DepartmentsofPhysiologyandBiophysicsandofCellBiology, mutant orpk collecting duct principal cell monolayers that
UniversityofAlabamaatBirmingham,
lacked apical central monocilia. Our data also show that an
MCLM740,1918UniversityBlvd.,
Birmingham,AL35294–0005,USA increase in cytosolic free Ca2+ primes the ATP pool that is
e-mail:[email protected] released in response to mechanical stimuli. It also appears
156 PurinergicSignalling(2008)4:155–170
that hypotonic cell swelling and mechanical pipetting sensory physiology in response to flow, touch, chemical
stimuli trigger release of a common ATP pool. Cilium- and osmotic stimuli [1–5, 15, 16]. MDCK cells and other
competent monolayers responded to flow with an increase cell and tissue models of the renal collecting duct have
incellCa2+derivedfrombothextracellularandintracellular been essential in characterizing cilium-derived cell calcium
stores. This flow-induced Ca2+ signal was less robust in (Ca2+) signals [17–30]. It appears likely that this cilium-
cilium-deficient monolayers. Flow-induced Ca2+ signals in affected Ca2+signalisderivedfromCa2+ entry from extra-
both preparations were attenuated by extracellular gadolin- cellularstoresandCa2+releasefromintracellularstores;the
ium and by extracellular apyrase, an ATPase/ADPase. latter, perhaps, an ER cisternae near the basal body
Taken together, these data suggest that apical monocilia immediately beneath the monocilium [17–30]. Previously
are sensory organelles and that their presence in the apical thought to be a vestigial organelle [1–5] or morphological
membrane facilitates the formation of a mature ATP marker for the cortical collecting duct (CCD) principal cell
secretion apparatus responsive to chemical, osmotic, and (PC) [31], the apical central monocilium is likely a sensor
mechanicalstimuli.TheciliumandautocrineATPsignaling for ductal epithelia [5].
appear to work in concert to control cell Ca2+. Loss of a Recently, our laboratory has shown that epithelial
cilium-dedicatedautocrinepurinergicsignalingsystemmay sodiumchannel (ENaC)-mediated sodium(Na+)absorption
beacriticalunderlyingetiologyforARPKDandmayleadto is upregulated in mutant cilium-deficient orpk CCD PC
disinhibition and/or upregulation of multiple sodium (Na+) monolayers versus genetically rescued cilium-competent
absorptive mechanisms and a resultant severe hypertensive controls [32]. ENaC activity is present under open-circuit
phenotype in ARPKD and, possibly, other diseases. voltage and short-circuit current measurements in rescued
cellmonolayerswithawell-formedcilium,buttheelectrical
Keywords ATPsecretion.Nucleotidesecretion. signals were three- to sixfold less than mutant monolayers
Purinergicsignaling.Kidney.Renalcollectingduct.Cilia [32]. One of our working hypotheses is that an inhibitory
signalislost(whenthemonociliumisnotwellformed)that
Abbreviations is responsible for tonic attenuation of ENaC function [32].
ATP adenosine 5’-triphosphate Indeed, flow-induced Ca2+ signals have been shown
Ca2+ calcium recently by Praetorius and Leipziger not to be due to the
orpk Oak Ridge polycystic kidney ciliumofMDCKcellspersebuttoautocrineATPsignaling
ARPKD autosomal recessive polycystic that is stimulated by pressure pulses and responsible for
kidney disease Ca2+ spark and wave signal formation [33]. Immature
Na+ sodium MDCK cells without discernible cilia and mature MDCK
Tg737 gene Polaris gene cells with cilia responded similarly [33]. Alternatively,
MDCK Madin-Darby canine kidney Satlin and coworkers have compelling data that monocilia
CCD cortical collecting duct do confer flow-based Ca2+ signals in isolated perfused
PC principal cell CCDs from control mice versus mutant Tg737orpk mice
ER endoplasmic reticulum [20]. In multiple different preparations from tissue to renal
ALU arbitrary light units epithelia to heterologous cells, flow-induced Ca2+ signals
TRP transient receptor potential have been observed [17–30]. However, the concept that an
RVD regulatory volume decrease autacoidmightmediatethesecilium-specificeffectshasnot
K+ potassium been addressed.
−
Cl chloride Herein, we show that the fully formed monocilium does
DIDS 4,4’-diisothiocyanato-stilbene-2, conferamorerobustCa2+signalinrescuedcellmonolayers
2’-disulfonic acid versus mutant cell monolayers that are deficient in well-
NO nitric oxide formed cilia. This finding agrees with the majority of the
literature examining this phenotype [17–30]. However and
more importantly, we show that stimulated or regulated
ATP release is impaired when the monocilium is mal-
Introduction formed. Ionomycin-, hypotonicity-, and mechanically in-
duced ATP release are more robust in cilium-competent
Monociliated ductal epithelial cells are receiving much monolayers versus cilium-deficient monolayers. Varying
attention due to their remodeling in polycystic kidney the order of stimuli also revealed that cell Ca2+ influences
diseases, role in other cystic diseases of the kidney and the mechanically induced secreted ATP pool and that
other tissues, and in sensory physiology [1–12]. Cilia and hypotoniccellswellingandothermechanicalstimulitrigger
flagella from lower organisms have important roles in thereleaseofthesameATPpool.Finally,theflow-induced
PurinergicSignalling(2008)4:155–170 157
Ca2+ signal in this cell model requires autocrine ATP Bioluminescence detection of ATP released from epithelial
release and signaling, as it was blocked by the ATPase/ monolayers In every assay performed, there was an initial
ADPase scavenger, apyrase. These data may reconcile the basal ATP measurement followed by subsequent ATP
different conclusions within the Praetorius and Leipziger measurements in response to different stimuli added in
study [33] and the study of Satlin and coworkers [20]. To different sequences. ATP release assays were performed
our knowledge, this is the first report linking the sensory mainly on well-polarized cell monolayers grown in clear
apical central and nonmotile cilium to ATP secretion. polyester 6.5-mm diameter filter supports. Mutant versus
rescued cell monolayers were studied side by side in each
ATP release protocol. The use of luciferase-luciferin to
Materials and methods indirectly measure the ATP concentration has been pub-
lished previously in detail [34]. Every drug prior to
Cell culture The collecting duct principal cells derived experimentation was tested to ensure that they did not
from an Oak Ridge polycystic kidney (orpk) mouse model interferewiththeluciferaseenzymeactivity[34–37].There
ofautosomalrecessivepolycystickidneydisease(ARPKD) was also no change in cell viability with any of these
and the genetically rescued cells with the wild-type maneuvers as has also been reported previously [34–37].
orpkTg737 gene were a generous gift from Dr. Bradley Each experiment began with a basal ATP measurement or
Yoder (University of Alabama at Birmingham). The the addition of Opti-MEM I medium (GIBCO-BRL) with
“mutant 1” cell clone (94D pcDNA 3.1 cells), the “rescued 1mg/mlluciferase-luciferinreagent(Sigma)beingaddedto
1” cell clone (94D BAP737-1 cells), and the “rescued 2” the apical or basolateral side of the filter’s monolayer.
cell clone (94D 737-2 cells) were handled identically and Basolateral ATP release was negligible; therefore, all data
were grown under G418 selection on 6.5-mm diameter reportedareapicalorluminalATPsecretion.Basallevelsof
(Corning Costar) or 12-mm diameter filter supports (Milli- ATPreleaseweremeasuredfor6minin15-s,nonintegrated
cell CM) and bathed in Dulbecco’s modified Eagle’s photon collection periods in a TD-20/20 Luminometer
medium nutrient mixture F-12 HAM with L-glutamine and (Turner Designs) [34–37]. In different orders of addition,
15 mM hydroxyethylpiperazine ethanesulfonic acid we used the following stimuli: (1) ionomycin (2 μM) (to
(HEPES) (Sigma) [32]. This medium was supplemented increase the intracellular calcium concentration), (2) dis-
with 5% fetal bovine serum (FBS) and contained (per tilled water with 1 mg/ml luciferase-luciferin (to dilute the
1,000 ml) dexamethasone (100 μl/l of 2 mg/ml stock), osmotic strength of the Opti-MEM I medium), and (3)
interferon-γ (25 μl of 800 U/μl stock), T3 (10 μl/l of intentionally harsh pipetting in the center of the apical
13 mg/ml stock), G418 (500 μl of 400 μg/ul stock), surface of the cell monolayer (to induce a mechanical
penicillin-streptomycin (10 ml of 100X stock), and ITS stimulus on the apical membrane). Normally, addition of
(10 ml of 0.5 mg/ml insulin transferrin selenium concen- drugordistilledwater(orthesamevolumeofmediumasa
trated stocks). The cells continued to be bathed on both volume addition control) is performed or dispensed very
sides of the filter until a monolayer tight to fluid formed. slowly along the wall of the plastic support that holds the
Measurement of the resistance of the filters was used as an permeable filter so as not to disrupt the tight, confluent
indicatorof theformationofa mature monolayer.Once the monolayer. Therefore, by quickly pipetting the medium up
monolayers were formed for at least 12 h, the cells were anddownontothemonolayers,amechanicalstimulationis
then acceptable for the experimental assay. induced. Luminescence was measured for 6 min after each
stimulus. All experiments ended with the addition of
In addition to these original clones developed in the hexokinase to eliminate any ATP left in the medium. All
Yoder laboratory, we also generated two new clonal lines assays were performed at room temperature.
from the original 94D mixed mutant CCD cell cultures,
namely “mutant 2” and “mutant 3.” The only difference Fura-2/AM imaging of cytosolic free calcium in a cell
between these two subsequent clones and the original monolayer-based perfusion system Fura-2/AM imaging
“mutant 1” clone is the fact that the “mutant 1” clone is was performed as described previously [38–40]. However,
stably transfected with an “empty” pcDNA 3.1 vector that toremainfaithfultothestudyofpolarizedcellmonolayers,
confers G418 resistance. “Mutant 2,” “mutant 3,” and the we designed a cell monolayer-based perfusion chamber
mixed mutant cultures are grown in a medium without system where 12-mm diameter Snapwell Transwell filter
G418.Wealsostudieda“rescuedB2”clonethatwasstably supports are inserted into a homemade perfusion chamber
transfected and rescued with the wild-type Tg737 gene but designed to accommodate this special filter support. Apical
we derived from a different mutant CCD originally, 94E. and basolateral perfusion are then performed separately
Thegenerationoftheseadditionalcloneswasdescribedina through independent injection ports and separate ejection
previous publication [32]. ports are subjected to vacuum. Response to changes in
158 PurinergicSignalling(2008)4:155–170
apical flow from 1 ml/min (a “slow” flow) to 5 ml/min Ionomycin-stimulated ATP release is more robust in
(“high” flow) were performed to induce a flow-induced rescued versus mutant cell monolayers We first assessed
Ca2+ transient akin to that observed by many other labo- the effect of an increase in intracellular free calcium (Ca2+)
ratories.Weassessedmutantandrescuedcellmonolayersin onATPrelease.Thisfirststudywasdoneinpartbecauseof
parallel during all protocols. We also assessed the flow- the emerging role of the primary central monocilium in
induced Ca2+ transient signal in the absence of apical mediating Ca2+ transients, sparks, and waves in cell
extracellular Ca2+ and in the presence of gadolinium monolayers derived from extracellular and intracellular
chloride and apyrase. It was not possible to calibrate the stores [17–30]. This cilium-derived Ca2+ signalis triggered
Fura-2 fluorescence signal to real free cytosolic Ca2+ by touch or flow across this organelle and is mediated, at
values because of ionomycin contamination of the flow least in part, by the polycystin proteins, PC-1 and PC-2
chamber.As such, fluorescence ratiovalues are shown. We [17–30]. Polycystin-2 is a part of the TRPP subfamily of
give estimates of what the free Ca2+ may be based on TRP genes and isa distant relative of the transient receptor
previous calibrations of the same cells grown as nonpo- potentialorTRPCgenefamilyofCa2+entrychannels[17–
larized cells. 30]. Ionomycin (2 μM) applied to the apical surface of
well-polarized cell monolayers triggered a slow, mono-
Materials All reagents and drugs were purchased from phasic rise in ATP release over 3–5 min (see typical time
Sigma. Larger 12-mm diameter filter supports were coursesbelowinFigs.6and8).Thisresponsewasasimilar
obtained from Millipore. Smaller 6.5-mm diameter filter phenotype to that observed in previous studies by our
supports were obtained from Corning Costar. It should be laboratory in human vascular endothelial cell monolayers
noted that we are using less luciferase:luciferin detection [37]. In mutant monolayers deficient in monocilia, an
reagent than in past studies [34–37]. increase in cell Ca2+ increased extracellular ATP from
below 50 nM to 0.4 μM. Figure 2 shows data as biolu-
minescence in arbitrary light units (ALU) with correspond-
ingestimatedATPforeachstepoftheprotocolbasedupon
Results parallel standard curves with known amounts of ATP. In
sharpcontrast,rescuedmonolayersrespondedtoionomycin
Basal ATP release or secretion is not different between stimulation with a rise insecreted ATPfrom approximately
mutant and rescued CCD PC monolayers Basolateral ATP 50nMto2μM,three-tofivefoldhighersustainedamounts
release was near background and was negligible in both on average than in mutant monolayers. The data in Fig. 2
mutant and rescued cell monolayers. This result does not also group the data from the panels of mutant and rescued
mean that ATP is not released across the basolateral cell cloneswhichwereinagreement.Takentogether,thesedata
surface. ATP may, in fact, be released but “trapped” show that cell Ca2+-stimulated ATP release is more robust
between the cell monolayer and the filter support and/or when a well-formed central monocilium is present. In fact,
the detection reagent may not gain sufficient access to this the “releasable” pool of ATP that is sensitive to Ca2+
“trapped” space. Therefore, only apical-directed ATP appears intact in cilium-competent monolayers and defi-
releasewasstudiedexclusivelyinthedetectionexperiments cient in cilium-deficient monolayers.
below. Regardless of the filter support used to form cell
monolayersandtheamountofdetectionreagentincludedin Hypotonicity-inducedATPreleaseismorerobustinrescued
the real-time experiments, basal or unstimulated or consti- versus mutant cell monolayers Hypotonic challenge and
tutive ATP release was low and not different between the resultant cell swelling and regulatory volume decrease
panels of mutant and rescued cell monolayers studied. (RVD), a shared mechanism of cell volume regulation by
There was agreement between all mutant clones and all all cells [41–44], is a robust stimulus for ATP release [41–
genetically rescued clones in all protocols below. As such, 46]. It is a shared sentiment by most that ATP release
the data are pooled for mutant cell monolayers and rescued precedes and triggers, at least in part, RVD mechanisms
cell monolayers. Basal ATPrelease data are summarized in suchasopeningofparallelK+andCl−channelstomediate
thebargraphinFig.1as bioluminescence in arbitrary light rapid KCl efflux and to lower cell volume back to basal
units (ALU). In total, the amounts of ATP released were levels [41–46]. As such, we used hypotonic challenge to
below 50 nM. A standard curve was run with each series assess ATP release in mutant cilium-deficient cell mono-
of ATP release experiments. Thus, basal ATP secretion is layers versus rescued cilium-competent cell monolayers.
not different across the apical membrane of mutant and Figure 3 summarizes these data. These stimuli followed
rescued cell monolayers and the amount of ATP released the ionomycin treatment. We show other typical real-time
is barely sufficient to engage the most sensitive P2Y or courses below when hypotonic challenge was performed in
P2X cell surface receptors. the absence of ionomycin (see Fig. 7). In all monolayers,
PurinergicSignalling(2008)4:155–170 159
~~2200--4400 nnMM
2200 11..00 00..5500
SScchheemmaattiicc ooff NNeeww DDeessiiggnn ffoorr
1188 00..99 00..4455 BBeetttteerr LLuummiinneesscceennccee DDeetteeccttiioonn
ooff SSeeccrreetteedd AATTPP ffrroomm PPoollaarriizzeedd
1166 00..88 00..4400
ncencenitsnits 1144 00..77 ~~ 11 nnMM 00..3355 WWaallll ooff CCeellll MMoonnoollaayyeerrss..
eeUU LLuummiinnoommeetteerr
escescght ght U)U) 1122 00..66 00..3300 CChhaammbbeerr 220000 µµll ooff
oluminoluminbitrary Libitrary Lior ALor AL 118800 0000....4455 0000....22225500 ~~00..22 nnMM PSFaPSFannoolluuaaddrrppss ppFFMMttiiooiicclloottrreettnnrroollaayyeerr AMLRAMLR::ppeeLLeeiiaa ddccDDggaaiiuueeeell mmnnttOOeett ccppwwttttiiiiiioottMMhhnn EEMM--11
BiBi(Ar(Ar 66 00..33 00..1155
CCeellll
44 00..22 00..1100 MMoonnoollaayyeerr
LLiidd ooff LL::LL
22 00..11 00..0055 3355 mmmm DDiisshh FFiilltteerr wwiitthh
00..44 µµPPoorreess
00 00 00
ntnt uedued ntnt uedued ntnt uedued 1B1B55aa ssµµooll llooaafftteerraall LLuummiinneesscceennccee
utauta escesc utauta escesc utauta escesc OMMpeettddiiMMiiuuEEmmMM--11 DDDDeeeetttteeeeccccttttooiioorrnn aa WWnnddiinnddooww
MM RR MM RR MM RR
Fig.1 BasalATPreleaseacrosstheapicalcellsurfaceisnotdifferent lyophilized luciferase:luciferin reagent are placed within the cup in
between mutant cilium-deficient and genetically rescued cilium- contactwiththeapicalsurfaceofthecellmonolayer.Detectionisthen
competentorpkkidneycellmonolayers.Inthreeseparatepreparations begun for bioluminescence due to secreted ATP from the cell
of mutant versus rescued cell monolayers on different filter supports monolayerintotheapicalspaceincontinuous15-scollectionperiods
and exposed to different concentrations of luciferase:luciferin detec- delayedonlybythetimetopushthe“start”buttonontheluminometer
tion reagent, ATP release was not different between the cilium- andtorecordtheluminescencetoanExcelspreadsheet.Thestandard
deficient and cilium-competent cell monolayers (n=12 for left set of curve with which approximations were made as to the secreted ATP
bars; n=6 for center and right datasets). In this study, we had a concentrationswasperformedwitheachdataset,wascompiled,andis
breakthrough with regard to what filter support to use for our ATP as follows: 10−10 M ATP, 0.052±0.005 ALU...10−9 M ATP, 0.476±
release experiments studying cell monolayers on filter supports. We 0.042 ALU...10−8 M ATP, 4.95±0.51 ALU...10−7 M ATP, 51.2±4.8
found that the 6.5-mm diameter filter support (the same as used in ALU...10−6 M ATP, 455±23 ALU...10−5 M ATP, 3956±311
open-circuit and Ussing chamber experiments studyingENaC upreg- ALU...10−4 M ATP and 10−3 M ATP, >9999 ALU (exceeded limit
ulation[32])wasidealforthesereal-timeATPreleaseassaysintothe of detection of luminometer). It is important to note that these are
apicalmediumbecausetheapicalcellsurfacewasascloseasitcould estimates of secreted ATP because we do not add inhibitors of ecto-
be and fully exposed to the luminescence detector in the base of the ATPases and it is likely that our ATP release signal is diminished
chamber. The left set of bars is data derived from this filter support significantlybyATPdegradationbeforedetection.Theleftsetofbars
and procedure. All other data in subsequent figures derive from this show basal ATP secretion from the 6.5-mm diameter clear polyester
preparation.Adiagramshowshowthefiltersupportwashandledand filter support without feet that is immediately above the light sensor
placed into the luminometer chamber. After washing of serum- andbathedin15μlofserosalmedium.Themiddlesetofbarsshow
containing medium away from the monolayer, a small volume of ATP secretion from a 12-mm polyester clear filter support with feet
Opti-MEM1medium(15μl)isaddedinadroptothelidofa35-mm andbathedin200μlofserosalmedium.Thethirdsetofbarsatright
dishtohydratethepreparationandtopromoteadherencetothelidof showATPsecretionfromapolycarbonatefiltersupportwithfeetand
the dish; 200 μl of Opti-MEM 1 medium containing 1 mg/ml bathedin200μlofserosalmedium
hypotonic challenge of the apical surface (50% dilution of 5–10 μM at the peak of the response and to a lesser
the medium osmolality with distilled water containing sustainedlevelattheendofthehypotonicchallengephase.
1 mg/ml detection reagent) triggered an increase in ATP In volume addition control experiments, ATP release was
release that had transient and sustained components. In triggered that was only transient and increased from 2 to
mutant monolayers, hypotonic challenge increased ATP 4 μM on average (data not shown). Taken together, these
detected in the medium from approximately 0.4 to 3 μM data show that rescued cell monolayers respond more
duringthepeakofstimulationandtoaslightlylesservalue vigorously to hypotonic challenge than mutant cell mono-
at the end of the hypotonicity challenge phase. Addition of layers with regard to ATP secretion, a signal required for
the same volume of isotonic medium (a “volume addition” cell volume regulation. Moreover, increases in cell Ca2+
control)increasedATPsecretionslightlyfrom0.4to0.5μM primeswelling-inducedATPreleaseandallowalargerATP
(datanotshown).Uniformly,rescuedcilium-competentcell secretion signal to visualize.
monolayers responded more vigorously to the hypotonic
stimulus. This stimulus again followed the ionomycin Mechanically-inducedATPreleaseismorerobustinrescued
treatment. ATP release increased from approximately 2 to versus mutant cell monolayers Harsh and fast pipetting of
160 PurinergicSignalling(2008)4:155–170
RReessccuueedd CCeellll MMoonnoollaayyeerrss
†P<0.05 OOvveerr MMuuttaanntt CCeellll MMoonnoollaayyeerrss
11000000 55..00
990000 44..55
~~22 µµMM
880000 44..00
ncencenitsnits 770000 ** cecense)nse) 33..55
eeUU nnoo
escescght ght U)U) 660000 ereereRespResp 33..00
BioluminBiolumin(Arbitrary Li(Arbitrary Lior ALor AL 554433000000000000 ~~4400**00 nnMM Fold-DiffFold-Diff(Ionomycin(Ionomycin 222211......550055
220000 11..00
110000 00..55
00 00
salsal nono salsal nono PPrreeppss:: 11 22 33 44
aa oo aa oo
BB II BB II
MMuuttaanntt RReessccuueedd
Fig.2 Ionomycin-induced ATPrelease acrosstheapicalcellsurface datasetatleftalongwiththreeadditionaldatasets.Althoughthefilter
is markedly attenuated in mutant cilium-deficient versus genetically supports,amountofdetectionreagent,andmagnitudeofluminescence
rescued cilium-competent orpk kidney cell monolayers. Six to nine measureddifferedbetweenpreparationtopreparation(n=6each),the
cell monolayers were assessed at left for the summary data derived fold difference in the ionomycin effect was constant. The asterisk
from the 6.5-mm diameter filter support preparation. The fold reflects P<0.05 by paired Student’s t-test; the cross reflects P<0.05
differenceinionomycin(2μM,addedina2-μlbolusalongtheside significance by analysis of variance (ANOVA) and Tukey’s ad hoc
of the plastic wall into the apical medium)-induced ATP release in test.Thestatisticalanalysisandresultsaregivensimilarlyinallother
rescued cell monolayers over mutant monolayers is graphed for the figures
Fig.3 Hypotoniccellswelling- †P<0.055
inducedATPreleaseacrossthe RReessccuueedd CCeellll MMoonnoollaayyeerrss
apicalcellsurfaceismarkedly OOvveerr MMuuttaanntt CCeellll MMoonnoollaayyeerrss
attenuatedinmutantcilium- ~~55--**1100 µµMM
deficientversusgeneticallyres- 44000000 55..00
cuedcilium-competentorpk
kidneycellmonolayers.Cell 33660000 44..55
monolayerswereassessedatleft
33220000 44..00
fforormthethseu6m.5m-marmyddaitaamdeetreirvfeidlter ncencenitsnits 22880000 cecense)nse) 33..55
supportpreparation.Hypotonic eeUU nnoo
ca(ra2ehnpa0aiegc0lqlaeelμunnactlg)lhwedvamioowtwhlabunsem1ritmenhindoegtuf/owmcdetailhdslelLtibol:alyLfepditachdwaedlaintegr oluminescoluminescbitrary Light bitrary Light or ALU)or ALU) 222211404066000000000000 ~~33** µµMM old-Differeold-Differepotonic Resppotonic Resp 323222......050500
mthreedeiuomth.eTrhperefpoaldradtiiofnfesreisncein BiBi(Ar(Ar 11220000 FF(Hy(Hy 11..55
graphedatrightandwasrela- 880000 11..00
tivelyconstantagain(n=6–9).
440000 00..55
StatisticsareshownlikeinFig.2
00 00
alal oo alal oo
ss pp ss pp PPrreeppss:: 11 22 33 44
aa yy aa yy
BB HH BB HH
MMuuttaanntt RReessccuueedd
PurinergicSignalling(2008)4:155–170 161
an 50 μl volume of isotonic medium directly down on the prerequisite for robust ATP secretion to provide a mean-
center of the cilium-competent monolayers (the “mechan- ingful autocrine ATP signal on the luminal surface of
ical stimulus”) in three repetitions triggered an immediate cortical collecting duct principal cells.
ATP secretion that had transient and sustained phases like
the swelling-induced ATP release from 2 to 3–4 μM in Figures 5, 6, 7, and 8 provide typical real-time courses
rescued monolayers. This response was greatly attenuated showing the luminescence data (in arbitrary light units) for
inthemutantmonolayers whereATPreleasewasaugment- mutant and rescued monolayers illustrating the summary
ed only slightly from 0.4 to 0.5 μM. These experiments datapresentedinFigs.1,2,3,and4.With the exception of
were performed after ionomycin challenge and the sum- basal ATP release, all phases of ATP secretion are
mary data are presented in Fig. 4b. In the absence of markedly more robust in cilium-competent models versus
ionomycinpretreatment(Fig.4a),mutantmonolayersfailed cilium-deficientmodelsacrosstheapicalcellsurface.These
to respond to this stimulus altogether, whereas rescued data also revealed that there may be shared and different
monolayers responded significantly with an ATP release pools of “releasable” ATP within the cell. These data also
transient. Again, it is noted that increases in cell Ca2+ also began to reveal that increases in cell Ca2+ influence ATP
appear to prime this mechanically sensitive ATP release secretion markedly.
mechanism. Taken together, these data suggest that the An original physiological role for the apical central
presence of a well-formed apical central monocilium is a monocilium was that of a “mechanosensor.” Figure 5
a No Ionomycin †P<00..0055 b Ionomycciinn PPrreettrreeaattmmeenntt
~~5500 nnMM
**
22224444
†P<<00..0055
22222222
22220000 11550000
~~33 µµMM
**
111888 11335500
111666 11220000
eee ee
ncncncnitsnitsnits 111444 ncncnitsnits 11005500
eeeUUU eeUU
scscscht ht ht ))) 111222 scscht ht )) 990000
mineminemineary Ligary Ligary Ligor ALUor ALUor ALU 111000 ~~1155 nnMM minemineary Ligary Ligor ALUor ALU 775500
oluoluolubitrbitrbitr 888 oluolubitrbitr 660000 ~~550000 nnMM
BiBiBiArArAr 666 BiBiArAr 445500 **
((( ((
444 330000
222 115500
000 00
alal etet alal etet alal etet alal etet
ss pp ss pp ss pp ss pp
BaBa PiPi BaBa PiPi BaBa PiPi BaBa PiPi
MMuuttaanntt RReessccuueedd MMuuttaanntt RReessccuueedd
Fig. 4 Mechanically induced ATP release across the apical cell wasbarelysignificant.Incontrast,rescuedcellmonolayersresponded
surface is markedly attenuated in mutant cilium-deficient versus robustly to the addition of 50 μl of Opti-MEM 1 medium with
genetically rescued cilium-competent orpk kidney cell monolayers. detectionreagentpipettedvigorouslyontothecenterofthemonolayer.
In a where no ionomycin pretreatment was performed, four mutant In two additional repetitions, 50 μl of apical medium was drawn up
and four rescued cell monolayers were compared. In b where and ejected onto the center of the monolayer before luminescence
mechanical stimulation was performed after ionomycin challenge, readingscontinued.Figure5showsthenatureoftheresponseversus
six mutant and six rescued cell monolayers were compared. Without thehypotonic challengeandtheionomycinstimulation. Statistics are
ionomycin, there was no response from the mutant cell monolayers. shownlikeinFig.2
With ionomycin, there was a response from mutant monolayers that
162 PurinergicSignalling(2008)4:155–170
Fig. 5 Typical real-time course
TThhiirrdd
SSeeccoonndd
ofmechanicallyinducedATP
PPiippeett
releaseacrosstheapicalcellsur- PPiippeett
faceinmutantversusrescuedcell
monolayers.Inthisandthree 2244
subsequentfigures,thegreen MMMuuutttaaannnttt
(closedorsolid)ballsreflectdata 2222 RRReeessscccuuueeeddd
fromcilium-competentcell 2200 FFiirrsstt
monolayersandthewhite(open) PPiippeett
ballsreflectdatafromcilium- 1188
ddeofincoietnctocnenlelcmtothneolpaoyienrtss.bLei-nes ncencenitsnits 1166
eeUU
cseacuosendtshebreetwareeenderleaaydsinogfsa.Ofenwe escescght ght U)U) 1144
cAaTnPorbesleeravseettrhaantstiheenrtesianrdeuccleedarin minminary Liary Lior ALor AL 1122
rmoebsescceuhreavdneidcceafllrloimmnsoumnltousl.taaTynhetrecsesblilgyntahles BioluBiolu(Arbitr(Arbitr 118800
monolayersaregreatly
66
attenuated
44
22
00
00 11 22 33 44 55 66 77 88 99 1100 11111122 1133 1144 1155 1166 11771188 1199 2200 2211 2222 2233 2244
TTiimmee
((MMiinnuutteess))
validates this idea using ATP secretion as the biological monolayers. The responses in mutant cilium-deficient cell
endpoint.Repeatedpipettingofisotonicmediumcontaining monolayers were greatly attenuated. This comparison was
the same amount of detection reagent revealed repeated performed on the same day with the same preparation of
stimulation of ATP release transients in cilium-competent luciferase:luciferin detection reagent. Figure 6 shows a
Fig. 6 Typical real-time course
PPiippeett
ofionomycinandmechanically
inducedATPreleaseacrossthe
22440000
apicalcellsurfaceinmutant MMMuuutttaaannnttt
versusrescuedcellmonolayers. 22220000
Ionomycininducesaslow IIoonnoo RRReeessscccuuueeeddd
monophasicsustainedincrease 22000000
inATPsecretionthatisthree-to
11880000
fivefoldmorerobustinrescued
coeflliomnoomnoylcaiynersst.imInultahteiopnr,etsheence ncencenitsnits 11660000
eeUU
mmeocrehapnriocnaoluinnscueldtsApTroPdsueccereetvioenn escescght ght U)U) 11400
twhhateniscmelolrCeas2u+stiasineleedvaintedn.ature minminary Liary Lior ALor AL 111220000000000
Nevertheless,thesignalsob- oluolubitrbitr
servedfrommutantcellmono- BiBiArAr 800
layersremaingreatlyattenuated ((
600
440000
200
0
00 11 22 33 44 55 66 77 88 99 1100 11111122 1133 1144 1155 1166 11771188 1199 2200 2211 2222 2233 2244
TTiimmee
((MMiinnuutteess))
PurinergicSignalling(2008)4:155–170 163
similar experiment but with ionomycin pretreatment. Here, transient in the rescued cell monolayers versus the mutant
the classic ionomycin-induced ATP secretion phenotype is cellmonolayers.Ionomycinalsoproducedafourfoldgreater
shown. It is a slow and monophasic increase in secreted sustainedATPreleaseinrescuedversusmutantmonolayers.
ATP that is robust; however, the response of rescued Hypotonic challenge also triggered a more robust response
monolayers is fourfold greater than mutant monolayers. in the mutant cell monolayers. A large degree of the
Subsequent mechanical stimulation via repeated pipetting stimulated ATP release was inhibited by the broad speci-
showed a more vigorous response in the cilium-competent ficity anion transport inhibitor, DIDS, suggesting that an
monolayersversusthecilium-deficientmonolayers.Figure7 ATPtransportprocessmaybemediatingthereleaseofATP
provides multiple compelling illustrations. First, the re- from intracellular pools. In the presence of DIDS inhibi-
sponse to hypotonic challenge is shown and it is much tion, release is inhibited while degradation of released ATP
more robust in rescued versus mutant cell monolayers. In- proceeds unabated, leading to a sharp decline in the signal.
terestingly, however, the repeated harsh pipetting stimulus Incontrast,ionomycin-stimulated ATPreleaseisattenuated
is without effect in both rescued and mutant cells after the by performing the luminescence experiment at 4°C (data
hypotonic challenge. In fact, the luminescence decreases not shown), suggesting a vesicular mechanism of release.
modestly. These data suggest that these two mechanical Hexokinase is added at the end of every protocol to scav-
stimuli may mobilize the same “releasable” pool of ATP. enge the ATP and abolish the luminescence signal.
Figure8 showsthe relative effects ofthe three stimuli used Taken together, these data suggest that cell Ca2+ is
in this study on rescued cilium-competent cell monolayers critically important for priming the ATP release machinery.
and on mutant cilium-deficient cell monolayers. Although These data also suggest that the “releasable” pools of ATP
greatlydiminishedbecauseoftheY-axisscale,themechan- are present in cilium-competent cell monolayers beneath
ical stimulus triggered a fourfold greater ATP secretion the apical cell surface, while they may be impaired or
PPiippeett
120 HHyyppoo
MMMuuutttaaannnttt
110
RRReeessscccuuueeeddd
100
90
ncencenitsnits 80
eeUU 70
minescminescary Light ary Light or ALU)or ALU) 6500
uurr
ololbitbit
BiBiArAr 40
((
30
20
10
0
00 11 22 33 44 55 66 77 88 99 1100 11111122 1133 1144 1155 1166 11771188 1199 2200 2211 2222 2233 2244
TTiimmee
((MMiinnuutteess))
Fig.7 Typicalreal-time courseofhypotonic challenge-induced ATP that both stimuli are indeed mechanical in nature and that the same
release across the apical cell surface in mutant versus rescued cell poolof“releasable”ATPisbeingaffectedbyeachstimulus.Theslight
monolayers. Hypotonicity induced a marked increase in ATP release drop in signal with the pipetting stimulus is the addition of 50 μl of
that was fivefold more robust in rescued cell monolayers versus isotonic medium to the 400 μl of 50% diluted medium and the
mutantcellmonolayers.Ofinterest,however,mechanicalstimulation resultant change in osmotic strength at the apical cell surface. The
via repeated pipetting near the center of the cell monolayers had no hypotonicity-inducedsignalobservedfrommutantcellmonolayersis
effectonATPreleaseafterhypotoniccellswelling.Thesedatasuggest againgreatlyattenuated
164 PurinergicSignalling(2008)4:155–170
HHyyppoo
6000 MMMuuutttaaannnttt
5500 RRReeessscccuuueeeddd DDIIDDSS
5000
4500
eee
ncncncnitsnitsnits 4000
eeeUUU
escescescght ght ght U)U)U) 3500
oluminoluminoluminbitrary Libitrary Libitrary Lior ALor ALor AL 32050000 PPiippeett IIoonnoo HHKK
BiBiBiArArAr 2000
(((
1500
1000
500
0
00 11 22 33 44 55 66 77 88 99 1100 11111122 11331144 11551166 11771188 1199 2200 221122222233 2244225522662277 2288 22993300
TTiimmee
((MMiinnuutteess))
Fig.8 Typicalreal-timecourseofallthreestimuligiveninanorder areobservedthatapproach∼15–20μMintheapicalmediumbathing
whereeachstimulationcanbeobservedinmutantversusrescuedcell cilium-competent cell monolayers. In these plots, the effects of the
monolayers. Although the large scale diminished the mechanically broad specificity anion transport inhibitor, DIDS, are shown. DIDS
induced signal and data, an ATP release transient is observed in diminishes ATP release in both types of monolayers and in other
rescued cell monolayers and not in mutant cell monolayers. The epithelial cell monolayer preparations to different degrees. The
monophasicionomycinresponseisthenobserved,againmorerobust inhibition is partial and DIDS does not affect the detection reagent.
inrescuedcellmonolayers.Then,hypotonicchallengeisperformedin Hexokinase is added at the end of each experiment to eliminate
the presence of ionomycin. Here, the largest values of secreted ATP secretedATPandtodiminishtheluminescencesignaltolowlevels
missing in cilium-deficient cell monolayers. These ideas
will be revisited and discussed below. 11..3355
oo 11..33
Flow-induced calcium signals are attenuated in cilium- atiati
RR11..2255
deficient mutant monolayers versus cilium-competent e e
monolayers An original physiological role for the apical ncnc 11..22
ee
central monocilium was that of a mediator of flow- or cc11..1155 MMuuttaanntt
ss
touch-induced Ca2+ signals in MDCK cells by Praetorius rere 11..11 RReessccuueedd 22
oo
and Spring [17–19]. Subsequent studies in heterologous uu
FlFl11..0055
cells, renal collecting duct cell models, and renal collecting a-2 a-2 11 BBaassaall FFllooww --11 mmll//mmiinn
ducts showed that an intact cilium is required for the flow- rr
induced Ca2+ signal [20–30]. To be faithful to a polarized FuFu00..9955 HHiigghh FFllooww --55 mmll//mmiinn
LLooww NNaa++,, MMgg22++ RRiinnggeerr ((BBaassaall FFllooww ñ–11 mmll//mmiinn))
epithelial cell monolayer system used routinely for this 00..99
study and other studies [32, 34–40], we devised a flow 9898 4848 9494 3939 8484 3030 7575 2121 6767 1313 5959
2.2. 11 22 44 55 77 88 1010 1111 1313 1414
chamber where the apical versus basolateral sides of the
TTiimmee ((sseeccoonnddss)
monolayercouldbeperfusedindependentlyandatdifferent Fig. 9 Typical real-time courses of flow-induced Ca2+ transient
flow rates. The 12-mm diameter Snapwell transwell filter signals across the apical cell surface in mutant versus rescued cell
can then be inserted into this chamber for selective monolayers.FluorescenceratiovalueswereshownforFura-2.Itwas
very difficult to perform ionomycin-based calibrations to derive real
perfusion and fluorescence imaging. With a constant low
cytosolicfreeCa2+valuesinthismonolayerpreparationthatcouldbe
rate of perfusion of the basolateral surface of the mono-
trusted. Moreover, the ionomycin used contaminated the perfusion
layers, low versus high rates of perfusion were performed lines for subsequent experiments. The flow-induced responses are
on the apical surface in rescued versus mutant monolayers. observedinthecenterofeachtraceandthefloweffectwasreversed
by returning to low flow.The use of the low Na+, low Mg2+ Ringer
allowedustoviewthistransientmoreeasily;itwasmorerobustand
Figure 9 shows the response to modifications in saline long-livedundertheseionicconditionswheretheCa2+entrychannel
Na and Mg and to high flow (5 ml/min) versus low flow inhibitors,Na+andMg2+,wereminimized