The Frequency of Calcium Oscillations Induced by 5-HT, ACH, and KCl Determine the Contraction ...

19 Pages·2005·2.72 MB·English

The Frequency of Calcium Oscillations Induced by 5-HT, ACH,
and KCl Determine the Contraction of Smooth Muscle Cells of
Intrapulmonary Bronchioles
Jose F. Perez and Michael J. Sanderson
Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655
abstract Increased resistance of airways or blood vessels within the lung is associated with asthma or pulmonary
hypertension and results from contraction of smooth muscle cells (SMCs). To study the mechanisms regulating
these contractions, we developed a mouse lung slice preparation containing bronchioles and arterioles and used
phase-contrast and confocal microscopy to correlate the contractile responses with changes in [Ca2(cid:1)] of the
i
SMCs. The airways are the focus of this study. The agonists, 5-hydroxytrypamine (5-HT) and acetylcholine (ACH)
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induced a concentration-dependent contraction of the airways. High concentrations of KCl induced twitching of o
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l agonist concentration and correlated with the extent of sustained airway contraction. In the absence of extracellular fro
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f key words: laser scanning confocal microscopy • asthma • airways • arterioles • mouse • lung slices /5
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n INTRODUCTION Our solution to investigate how cellular physiology 58
ur Gas exchange in the lungs requires an appropriate regulates the contraction of the small airways or arteri- /jgp12
o oles was to examine living lung slices that retain many 56
J matching of ventilation to blood perfusion and this is 53
e influenced by the caliber of the airways and blood structural and functional properties of the lung. Rela- 5.pd
Th vessels. Consequently, an understanding of the mecha- tciovneltyr atchtiilcek rluesnpgo nslsiec eos fh aavirew bayese n( Duasnedd utroa nsdtu deyt tahl.e, f by gu
nisms that control the size of airways and arterioles is e
required to understand lung physiology and the devel- 1993; Martin et al., 1996; Minshall et al., 1997; Adler et st on
al., 1998; Duguet et al., 2001; Martin et al., 2001; 0
opment of obstructive lung disease and pulmonary 9 M
Wohlsen et al., 2001), but thinner lung slices, combined a
hypertension. In general, the mechanisms that control with confocal microscopy, provided us with the ability rch 2
the caliber of intrapulmonary airways or arterioles have 0
to study changes in [Ca2(cid:1)] of SMCs that underlie airway 23
been investigated in either whole lungs or isolated i
contraction (Bergner and Sanderson, 2002a,b, 2003). A
smooth muscle cells (SMCs). While these approaches
subsequent study examined [Ca2(cid:1)] in strips of tracheal
provide valuable data, it is difficult to determine the i
muscle but these do not preserve the lung architecture
site or size of airway or arteriole contraction by mea-
or address the physiology of small airways (Kuo et al.,
surements of air flow in whole lungs or blood pressure
2003). To also examine the equally important role of
in the pulmonary artery. Similarly, it is difficult to relate
blood vessels in lung physiology, we modified the lung
changes in intracellular Ca2(cid:1) concentration ([Ca2(cid:1)])
i slice preparation to preserve the structure and function
of isolated SMCs to the contractile responses of intact
of the arterioles. Although we studied the contraction
airways and arterioles.
Abbreviations used in this paper: ACH, acetylcholine; CPA, cyclopiazonic
The online version of this article contains supplemental material. acid; 5-HT, 5-hydroxytrypamine; IP , inositol 1,4,5-trisphosphate;
3
Correspondence to Michael J. Sanderson: IPR, IP receptor; RyR, ryanodine receptor; SMC, smooth muscle
3 3
[email protected] cell.
535 J. Gen. Physiol. © The Rockefeller University Press • 0022-1295/2005/06/535/19 $8.00
Volume 125 June 2005 535–553
http://www.jgp.org/cgi/doi/10.1085/jgp.200409216
and relaxation of both the airways and arterioles simul- Lung Slices
taneously, we focus here, for clarity, on the responses of
To preserve the normal morphology and study the physiological
the airways. We compare the accompanying responses
response of intrapulmonary airways and blood vessels, we modi-
of the arterioles in a second paper (Perez and Sander- fied the preparation of lung slices (Bergner and Sanderson,
son, 2005). 2002a). Male BALB/C inbred mice (Charles River Breeding Labs,
Although 5-hydroxytryptamine (5-HT) is released by Needham, MA), between 7 and 9 wk old, were killed by intraperi-
toneal injection of 0.3 ml of pentobarbital sodium (Nembutal) as
neuroendocrine cells in the airway mucosa of animals
approved by the IACUC of the University of Massachusetts Medi-
and humans (Lauweryns et al., 1973; Lommel, 2001;
cal School. The trachea was cannulated with an intravenous (IV)
Adriaensen et al., 2003) the role of 5-HT in airway tone catheter tube with two input ports (20G Intima; Becton Dickin-
is not well understood. In mice, rats, and guinea pigs, son) and secured with suture thread (Dexon II, 4–0; Davis and
5-HT induced airway contraction, either in vivo, in iso- Geck) to ensure a good seal. A syringe filled with 3 ml of air was
attached to one port while the other port was closed. The chest
lated lungs, or in isolated trachea rings (Levitt and
cavity was opened by cutting along the sternum and the ribs adja-
Mitzner, 1989; Eum et al., 1999; Fernandez et al., 1999;
cent to the diaphragm. To reduce the intrapulmonary blood ves-
Held et al., 1999; Moffatt et al., 2004). However, this sel resistance and facilitate vessel perfusion with gelatin, the col-
contraction seemed sensitive to atropine and 5-HT lapsed lungs were gently reinflated to approximate their total
was thought to act via a muscarinic pathway. In hu- lung capacity by injecting (cid:1)1.5 ml of air. A warm (37(cid:2)C) solution D
o
mans, inhaled 5-HT did not produce bronchoconstric- opfe rgfeulsaetdin t h(tryopueg hA ,t hpeo ricnitnrea psuklimn,o 3n0a0r yb blolooomd, v6e%ss eilns, svHiaB tShSe) pwuals- wnloa
tion (Raffestin et al., 1985; Cushley et al., 1986) but monary artery, by inserting the hypodermic needle of an infusion ded
could facilitate cholinergic-induced bronchoconstric- set (SV x S25BL; Terumo Corporation) into the right ventricle of from
tion (Dupont et al., 1999). In asthmatics, plasma levels the heart and slowly injecting (cid:1)1 ml of gelatin solution. A small http
of 5-HT are elevated and correlate with clinical severity cotton–wool swab soaked in ice-cold sHBSS was placed only on ://ru
the heart to solidify the gelatin before the perfusion needle was p
(Lechin et al., 1996), but its role in asthma remains re
unknown. rpermesosuvered.. AT hsyer inlugneg fis lwleedr ew idthe flaa wteadr mb y( 3r7e(cid:2)leCa)s isnoglu tthioen poof s2it%ive a gaair- ss.org
Another unresolved aspect of airway SMC contractil- rose (type VII or VII-A: low gelling temperature) in sHBSS was at- /jgp
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ifltyu xis o tfh eex rtroalece ollfu mlare mCab2r(cid:1)a n(Jea ndsespeonl,a 2r0iz0a2ti)o. nB eacnadu steh ec oinn-- tcalacmhepde dt op rtohxei msealc oton dth ep otrrat chofe at hane dc patuhregteedr. oTf haier wIVit ht uthbee awgaas- rticle-p
traction induced by either KCl or agonists appears to rose solution by allowing the trapped air to escape via a 27G nee- df/1
require extracellular Ca2(cid:1) (Li et al., 2003), it is believed dwlaes irnesmerotevded i natnod t hteh eIV lu tunbgse wpreorex irmeainl fltoa ttehde cblya minpje. cTtihneg I(cid:1)V 1c.l3am mpl 25/6/5
that a sustained Ca2(cid:1) influx via voltage-, store-, or re- of agarose-sHBSS. Subsequently, (cid:1)0.2 ml of air was injected into 35/1
2
ceptor-operated channels mediates contraction. How- the airways to flush the agarose-sHBSS out of the airways and into 21
4
ever, sustained elevations of Ca2(cid:1) are often toxic to the distal alveolar space. Immediately after agarose inflation, the 58
many cell types including SMCs. Consequently, we re- lpulnacgesd waet r4e(cid:2) Cw afoshr e1d5 mwiitnh. Tichee-c loulndg saHnBdS hSe, aartn dw etrhe er eamnoimveadl awnads /jgp125
examined the mechanisms of agonist-induced (5-HT or placed in sHBSS (4(cid:2)C) and cooled for an additional 30 min to en- 653
acetylcholine [ACH]) contraction and voltage-depen- sure the complete gelling of the gelatin and agarose. 5.p
d
dent (KCl) contraction. We found that 5-HT and ACH To cut thin lung slices, a single lung lobe was removed from the f b
induced Ca2(cid:1) oscillations in airway SMCs with a fre- respiratory tree by cutting the main bronchus. The lung lobe was y gu
e
quency that determines the size of the airway contrac- trimmed near the bronchus to produce a flat surface that was ad- st o
tion. By contrast, KCl induced low frequency Ca2(cid:1) oscil- hered to the mounting block of a vibratome (model EMS-4000; n 09
Electron Microscope Sciences) using cyanoacrylate glue (Krazy M
ltahtoioungsh tbhoatth imndeuchceadn istmwist crheiqnugi reind aanir winafly uSxM oCf sC. aA2(cid:1)l,- gwlause s,u Abrmoner (cid:3)ge; dE lienc tar obnat hM oicfr soHscBoSpSe mScaiienntaciense).d T aht e(cid:1) m4(cid:2)oCu. nTthede lluonbeg arch 2
0
this was found to be primarily required to refill internal lobe was sectioned into slices (cid:1)130 (cid:4)m thick starting at the lung 23
stores to maintain the Ca2(cid:1) oscillations that mediated periphery. The initial slices consisted mainly of agarose-filled alve-
oli and respiratory bronchioles, but as slicing advanced deeper
contraction.
into the lobe, small arterioles and airways start to appear. Airways
were readily identified by their lining of epithelial cells with beat-
MATERIALS AND METHODS ing cilia. Serial sections with these features were collected and
transferred individually to wells of a 24-well plate containing
Materials DMEM supplemented with antibiotics and anti-mycotics and
NaHCO. Fetal serum was not added because it can contain 5-HT
3
Cell culture reagents were obtained from Invitrogen and GIBCO (3–5 (cid:4)M when used at 10%) that can stimulate contraction of
BRL. Other reagents were obtained from Sigma-Aldrich. Hanks’ SMCs (Abdullah et al., 1994). Slices were kept in culture media at
balanced salt solution (GIBCO BRL) was supplemented with 20 37(cid:2)C and 10% CO for up to 3 d. At 37(cid:2)C, the gelatin in the blood
mM HEPES buffer (sHBSS) and adjusted to pH 7.4. K(cid:1)-sHBSS vessel lumen dissol2ved, leaving the blood vessel lumen empty.
was prepared by replacing the sodium salts of sHBSS with potas-
sium counterparts. High KCl isotonic solutions were prepared by Measurement of the Contractile Response of Airways and
mixing K(cid:1)-sHBSS with normal sHBSS to obtain the desired con- Arterioles Induced by Agonists
centration in K(cid:1). Hanks’ “0” Ca2(cid:1) solution was prepared by sup-
plementing sHBSS without Ca2(cid:1) and Mg2(cid:1) with 0.9 mM MgSO Lung slices containing at least one airway and an accompanying
4
and 1 mM of NaH-EGTA. arteriole that were cut to reveal a transverse section were se-
2 2
536 Ca2(cid:1) Signaling and Contraction of Bronchiole SMCs
lected. Care was taken to ensure that the airways were lined with cence ((cid:6)510 nm) was detected by a photomultiplier tube
epithelial cells showing ciliary activity and that the blood vessel (PMT). A video frame capture board (Raven; Bit Flow, Inc.) digi-
wall was intact and not collapsed. It was not uncommon to find tizes the PMT signal (1 PMT per channel) to form an image (480
blood vessels with elements of their walls separated from the lung pixels (cid:5) 400 lines) that was recorded using the software Video
parenchyma; these vessels were not used. Slices were transferred Savant to a hard disk (a stripped SCSI volume). For time lapse, a
to a custom-made perfusion chamber consisting of a Plexiglas recording rate of 2 Hz was used, otherwise video rate (30 Hz) was
support for a 45 (cid:5) 50 mm coverglass. A lung slice was placed in used. For the higher speed recordings of Ca2(cid:1) waves and elemen-
the center of the coverglass and held in place with a small sheet tal Ca2(cid:1) events, images were acquired at 60 Hz (480 pixels (cid:5) 170
of nylon mesh (210 (cid:4)m opening; Small Parts Inc.). To ensure the lines). A line-scan analysis of these images was performed by ex-
nylon mesh did not influence the contractile responses of the air- tracting a row of pixels from each image and placing them se-
ways and arterioles or interfere with the phase-contrast optics, a quentially, as a time sequence, in a single image. Alternatively,
small hole was cut in the mesh, and this was centered over the se- changes in fluorescence intensity within the images were ana-
lected airway and arteriole. A second custom-cut (11 (cid:5) 40 mm) lyzed by selecting regions of interest (ROI) of (cid:1)5 (cid:5) 5 pixels in a
coverglass edged with silicone grease (Valve Sealant; Dow Corn- single SMC. Average fluorescence intensities of an ROI were ob-
ing Co.) was placed over the slice and nylon mesh. tained, frame-by-frame, using the Scion Image software with cus-
Perfusion of the lung slice in the chamber was performed by tom written macros that allow tracking of the ROI within an SMC
applying a gravity-fed flow of solution at one end of the glass as it moved with contraction. When necessary, a bleach correc-
chamber and suction at the other end of the chamber. The vol- tion was calculated from the bleaching rate observed during a
ume of the chamber was (cid:1)100 (cid:4)l with a perfusion rate of 800 (cid:4)l/ period of 30 s before the perfusion of drugs. Final fluorescence D
o
min. The application of different solutions was controlled by us- values were expressed as a fluorescence ratio (Ft/F0) normalized wnlo
ing a custom-built perfusion system consisting of eight solution to the initial fluorescence (F0). ade
rtuesbeer v(oWirasr (n3e0r mInls)t rcuomnnenectst eIdn cto.) .a Tmhaen iflfoolwd fwriotmh ae saicnhg lree oseurtvpouirt Immunocytochemistry d from
wttarinoaoF snli on rgsrevoe egnfprutehwtleraaadatsret eeemd-.dc iob cbnyry ota rsa acvnsoat p levmelee ic(c(DtLrrooiFasnVpciAohcp; o iyLtn, et3ele0ur 0nCf;ag ocN mesi lkipaconaennds) y tw)wh euiert nheimd aoe abr2g s0Tee(cid:5) rTav Lceoq dbcu joeiosncni--- L(BT(cid:7)uShAne2 0g (s(cid:2) lssCiHlci)ceB esfS osw Srw -eB2er0rSee A m iw)n.iac nAsu,h nbaetanditbde iod nwd safieHosshrB ew1Sde S rh,ie n fi a dxstiH elrudBot oewSdmSit h1 ct o:ce2nom5tl0adp i ien1nri0 ans0tHgu% rB1 e aS mcSwe-giBtt/ohSmnA ael. http://rupress.org
FITC-conjugated mouse monoclonal antibody against (cid:3)-smooth /jg
toPfirfuvo elmn0, .ia 6x t–z)ho 1edo. 0emC pC (cid:4)eaDnmdd a/cipnaptmgoix reeo, rlna an otudhfs eaint hrzgeoe do aum CccC oisnDmegt p tlicuenantgmes.r et Ioimrna gta eigv(reemfs aa ocw deme era(le Rg nToreiaMficdco- 6arR7tdiu1oen0dn;- mscHruoBssccSolSep- BaeSc Autisn, ian (ngSd i4g fl8mu8a o-nrAemlsdc reeixncchcie)ta. wtSiaolisnc re elsic gwohretdr eea dnw dwa siathh eb dtah ntehd crwoeiend fttohimc a(cid:6)el s5m 1ini0- p/article-pdf/1
nIOer ;i nBdituFsltorwie,s I, nIcn.c). )a.n Dd iigmitaagl ei macaqgueiss i(t6io4n8 s(cid:5)of t4w8a4r ep (ixVeidlse)o w Searvea nret;- nnmeo ufoslry ewmithis stihoen .fl Puhoarsees-cceonnctera ismt aimgeasg besy wuseirneg rae csoercdoendd sPimMuTl ttao- 25/6/53
corded in time lapse (0.5 Hz) and stored directly on a hard drive. detect the laser light transmitted by the specimen. 5/1
2
Images were converted to stacks of TIFF images and analyzed 21
Statistics 4
with the PC software NIH Image/Scion image (Scion Corpora- 58
tion; download: www.scioncorp.com). After image calibration A paired Student’s t test was used to test for significant dif- /jgp1
and the selection of an appropriate grayscale threshold to distin- ferences between means. All statistical values are expressed as 256
guish the lumen from the surrounding tissue, the area of the air- mean (cid:8) SEM. 535
way and arteriole lumen was calculated, with respect to time, by .pd
pixel summing. Values were normalized to the prestimulation lu- Online Supplemental Material f by g
minal area (initial area). All measurements were performed at u
e
room temperature. Videos consisting of sequences of phase-contrast or fluorescence st o
images were produced in Video Savant by exporting the images n 0
Measurements of Intracellular Ca2(cid:1) as “mpeg” files. To show the time of addition of agonists, labels 9 M
a
were added to each frame using a custom written script file. Vid- rch
Aul astro Pckro sboelus)t iownas o pf r2e pmaMre dO wreitgho n2 0g r(cid:4)ele nan 4h8y8d BroAuPsT DAM-ASMO (aMnodl e5c0- ecaotse dar feo rp reeascehn tveidde ion. Vtiimdeeo lsa p1–se4 aanred atvhaeil apblaley baat chkt tspp:e/e/dw wisw i.njgdpi-. 2023
(cid:4)g Oregon green-AM. An equal volume (20 (cid:4)l) of 20% Pluronic org/cgi/content/full/jgp.200409216/DC1.
F-127 (Molecular Probes) in anhydrous DMSO was mixed with
the stock solution and diluted in 2 ml of sHBSS containing 100
(cid:4)M sulfobromophthalein (an inhibitor used to prevent dye ex- RESULTS
trusion through anion exchangers) to produce a final loading so- Characteristics and Morphology of Lung Slices
lution of 20 (cid:4)M Oregon green-AM, 0.2% pluronic, and 100 (cid:4)M
sulfobromophthalein. Slices were incubated for 30 min at 30(cid:2)C Throughout the lungs, the intrapulmonary airways and
followed by 30 min at room temperature in the loading solution, arteries have a close anatomical association that follows
followed by an additional hour at room temperature in sHBSS
containing 100 (cid:4)M sulfobromophthalein to allow for the deester- a parallel course. As a result, an airway and an accom-
panying arteriole (a bronchiole–arteriole pair) was eas-
ification of the dye. Lung slices were mounted in the perfusion
chamber as described above. ily identified and visualized in a single microscopic
Fluorescence imaging was performed using a custom-built field of view (Fig. 1, A and B). This also makes the iden-
video-rate confocal microscope (Sanderson and Parker, 2003) tification of pulmonary veins (not shown) easier be-
modified to capture four channels simultaneously (Sanderson,
cause they are found as individual structures at some
2004). In brief, the 488-nm line of an argon or diode laser was
distance away from the bronchiole–arteriole pair. This
scanned across the specimen with two oscillating mirrors (for X-
and Y-scan) in an inverted microscope. The resultant fluores- anatomical separation precludes a direct comparison
537 Perez and Sanderson
of the arteriole and vein responses in the same experi-
ment. Each bronchiole–arteriole pair, when observed
in transverse section, usually consists of a larger airway
and a smaller arteriole. The airway is characterized
by a lining of cuboidal epithelial cells with actively
beating cilia. The arteriole lumen is lined with a low
profile, squamous endothelium. Both structures are
surrounded by a dense layer of tissue that often has a fi-
brous appearance. More distally, the airway and arteri-
ole are surrounded by the alveolar parenchyma consist-
ing of thin-walled sacs (Fig. 1, A and B). Specific
antibody staining for SMC (cid:3)-actin (Fig. 1 A) reveals
that the SMCs are located in the surrounding fibrous
layer, directly below the epithelium or endothelium. It
is important to note that in the lung slices used, only
the alveoli remain filled with agarose. Before gelling, D
o
w
the agarose is flushed out of the airways with air. The nlo
a
gelatin is absent from the arterioles because it dissolves de
d
during incubation at 37(cid:2)C. Consequently, the luminal fro
m
compartments do not offer resistance to contraction. h
Agarose does not dissolve at 37(cid:2)C but remains in the al- ttp://ru
p
veoli to keep the alveoli inflated and airways open. re
ss.o
The Contractile Response of Bronchiole–Arteriole Pairs to rg/jg
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ACH, 5-HT, and KCl /a
rticle
To establish the contractile sensitivity of bronchiole– -p
d
arteriole pairs to ACH, 5-HT, and high KCl, we mea- f/12
5
sured changes in the luminal area with respect to time. /6/5
In response to 1 (cid:4)M ACH, the airway contracted and 35/1
2
reduced its lumen area by (cid:1)60% within 2 min (Fig. 1, 21
4
5
B and C, blue line; Video 1, available at http://www. 8/jg
p
jgp.org/cgi/content/full/jgp.200409216/DC1) and af- 1
2
5
ter 5 min, the average airway luminal area had de- 65
3
creased to 57 (cid:8) 7% (n (cid:9) 8 slices from 3 mice). Upon 5.p
d
washout of ACH, the airway quickly relaxed, attaining f by g
80–90% of its initial size within 3 min, but continued to ue
slowly relax over the next 5–7 min. In contrast to the st on
0
airways, the arterioles had no contractile response to 9 M
a
ACH (Fig. 1 C, red line). rch
The exposure of the same bronchiole–arteriole pair 20
2
to 1 (cid:4)M 5-HT induced the contraction of both the air- 3
way and arteriole (Fig. 1, B and C). In the airway, 5-HT
Figure 1. The localization of SMCs and the contractile responses induced a contraction at a similar rate compared with
of an airway and arteriole in a lung slice induced by ACH, 5-HT,
that induced by ACH and reduced the luminal area by
and KCl. (A) A lung slice was fixed and stained with FITC-conju-
gated antibodies against smooth muscle (cid:3)-actin. Fluorescence and
phase-contrast images were recorded with a confocal microscope.
The fluorescence image was assigned a green pseudo-color and
superimposed on the phase-contrast image to show colocalization. of the lumen of an airway (blue line) and arteriole (red line) with
The final image consists of a montage of six different images. The respect to time in response to ACH, 5-HT, and KCl (top bars).
bronchiole airway (A) and the accompanying arteriole (a) are ACH induced a contraction of the airway but not of the arteriole.
surrounded by alveolar parenchyma. A prominent layer of SMCs 5-HT induced a greater contraction in the arteriole than in the
(green) is located below the epithelial (EPC) and endothelial airway. KCl induced twitching in both the airway and arteriole and
(ENC) cells of the bronchiole and arteriole, respectively. (B) A a sustained contraction of the arteriole. Upon washout of agonists
series of phase-contrast images, recorded at different times (indi- or KCl, the airway and arteriole relaxed. A movie of these data is
cated by arrows in C) showing the appearance of an airway and shown in Video 1 (available at http://www.jgp.org/cgi/content/
arteriole before (1) and after stimulation with 1 (cid:4)M ACH (2), 1 full/jgp.200409216/DC1). Representative experiment of six
(cid:4)M 5-HT (3), and 100 mM KCl (4). (C) The cross-sectional area different slices from three mice.
538 Ca2(cid:1) Signaling and Contraction of Bronchiole SMCs
Figure 2. The effect of concentration
on 5-HT–, ACH-, and KCl-induced
airway contraction. Lung slices with
airways of similar sizes were stimulated
with increasing concentrations of 5-HT,
ACH, or KCl (perfusion time of 8 min).
Relaxation of the airways between expo-
sure to agonists or KCl was achieved by
washing with sHBSS for 10 min. An
image was recorded every 2 s and the
airway lumen cross-sectional area was
calculated and plotted with respect to
time. (A) 5-HT induced a concentration-
dependent increase in the contraction
of airways in the range of 0.01–0.5 (cid:4)M.
Stimulation with 10 (cid:4)M ACH induced a
larger contraction as compared with
10 (cid:4)M 5-HT. (B) A representative exper-
iment showing the effect of increasing Do
w
isotonic concentrations of KCl on the n
lo
contraction of airways. (C and D) Sum- ad
e
mcoanrtyr aoctfi litthye o fc othnec eanirtrwaatiyos nt-od e(pCe)n AdCenHt d from
(open squares) and 5-HT (closed cir- http
cles) or (D) KCl, calculated after 5 min ://ru
of agonist or KCl exposure. Each point pre
represents the mean (cid:8) SEM from at ss.o
least three different experiments on rg/jg
different slices from at least two mice. p/a
For each agonist, the contractility data rticle
were fitted with a logistic function -p
d
curve. f/1
2
5
/6
/5
3
41 (cid:8) 5% after 5 min (n (cid:9) 9 slices from 3 mice). In the Consequently, in this paper, we have focused on charac- 5/1
2
2
arteriole, 5-HT induced an initial fast contraction that terizing and correlating the contractile responses and 14
5
reduced the lumen by up to 80% within 2 min. This was the underlining Ca2(cid:1) signaling of the airway SMCs to 8/jg
p
1
followed by a second phase of slower contraction. The 5-HT, ACH, and KCl. The responses of the arteriole 2
5
average arteriole luminal area was reduced by 75 (cid:8) 6% SMCs to these agonists are addressed in a separate 653
5
after 5 min (n (cid:9) 6 slices from 3 mice). Upon 5-HT study (Perez and Sanderson, 2005). .pd
f b
washout, both the airway and arteriole relaxed; how- Concentration Dependence of Airway Contraction to 5-HT, y gu
ever, it is important to note that the airway relaxed ACH, and KCl est o
quickly, whereas the arteriole relaxation had a delayed n 0
9
onset and occurred more slowly (Fig. 1 C). To determine the relative sensitivity of the contraction M
a
The same bronchiole–arteriole pair responded to iso- of the airways to 5-HT, ACH, and KCl, we measured the rch
2
tonic sHBSS containing 100 mM KCl with a large reduc- changes in luminal area in response to sequentially in- 02
3
tion of the arterial lumen but only a small reduction of creasing concentrations of agonist (Fig. 2). Increasing
the airway lumen (Fig. 1, B and C). However, unlike the concentrations of 5-HT or ACH (from 0.01 to 0.5 (cid:4)M)
sustained contraction induced by ACH and 5-HT, the induced an increasing reduction in luminal size (Fig. 2,
contraction induced by KCl was irregular or spasmodic A and C). At low concentrations of 5-HT or ACH (0.01
(Video 1). This was especially evident in the airway (cid:4)M), the airway contraction was small and consisted of
where uncoordinated transient contractions (or twitch- uncoordinated twitching of the airway wall. At higher
ing) of individual airway SMCs only resulted in a small agonist concentrations, the airway contraction pro-
decrease in luminal area. After 5 min, KCl had induced ceeded quickly and smoothly without twitching. While
a luminal reduction in airways of 14 (cid:8) 3% (n (cid:9) 7 slices a maximal luminal reduction was induced by concen-
from 3 mice) and arterioles of 64 (cid:8) 8% (n (cid:9) 6 slices trations of (cid:10)1 (cid:4)M for each agonist, the maximal re-
from 3 mice). Removal of the KCl resulted in arteriole sponse to 5-HT was less than that induced by the maxi-
and airway relaxation although some airways continued mal concentration of ACH (Fig. 2 C). This relative sen-
to display transient contractions (four out of seven). sitivity was verified in each lung slice by the addition of
These results indicate that the SMCs of airways and the complementary agonist at the end of each experi-
arterioles respond very differently to different agonists. ment (Fig. 2 A). The addition of 10 (cid:4)M ACH, after 10
539 Perez and Sanderson
(cid:4)M 5-HT, resulted in a larger contraction (69% and
59%, respectively; Fig. 2 A). Conversely, the addition of
10 (cid:4)M 5-HT, after the addition of 10 (cid:4)M ACH, resulted
in a smaller airway contraction (38% and 49%, respec-
tively). 5-HT induced a smaller airway contraction than
ACH at all concentrations (Fig. 2 C). An average maxi-
mal contraction of 48 (cid:8) 4% (n (cid:9) 5 from 3 mice) and
59 (cid:8) 2% (n (cid:9) 6 from 3 mice) was induced by 10 (cid:4)M
5-HT and ACH, respectively. The half maximal concen-
tration for airway contraction (EC ) was 31 and 30 nM
50
for 5-HT and ACH, respectively.
Exposure of the airway to a range of isotonic KCl
concentrations between 25 and 150 mM commonly in-
duced a transient contraction that subsided into a
smaller sustained contraction of the lumen that varied
little over the range of KCl concentrations tested (Fig. D
o
w
2, B and D). It is worth noting that, at all KCl concen- nlo
a
trations, the small overall contraction was very similar de
d
to that induced by a very low concentration of agonist fro
m
and was achieved by uncoordinated twitching. How- h
ttp
ever, the maximum response to KCl was small, consist- ://ru
ing of a contraction of only 11 (cid:8) 1% in response to 100 pre
mM KCl (n (cid:9) 4 from 3 mice). The order of the sensitiv- ss.o
rg
ity of the contractile response of airways was ACH (cid:10) /jg
p
5-HT (cid:6)(cid:6)(cid:6) KCl. /article
-p
Ca2(cid:1) Oscillations in Airway SMCs Induced by 5-HT and ACH df/1
2
5
In response to 1 (cid:4)M 5-HT, the SMCs of the airway re- /6/5
3
sponded with an initial increase in [Ca2(cid:1)]i that was Figure 3. Ca2(cid:1) signaling induced by 5-HT in airway SMCs in 5/12
quickly followed by the occurrence of oscillations in lung slices. (A) Selected images recorded at times indicated in 214
[cChrao2(cid:1)n]oi u(sFlyig, .w 3it)h. eTahcehs ec eClla d2(cid:1)is polsacyiilnlagti roenpse oticticvuer irnecdr eaasysens- e5a-HchT .p Baanr,e 2l 0( a(cid:4)–mf). (Tahned r desapshonedse lsi nofe tsh irne eB )d idffuerrienngt SthMeC esx (pinodsuicraet etod 58/jgp1
in [Ca2(cid:1)] at different times with respect to neighboring by arrows in a) are highlighted. After stimulation with 1 (cid:4)M 5-HT, 2565
i the fluorescence in each SMC increases and begins to oscillate 35
SMCs (Fig. 3; Video 2, available at http://www.jgp.org/ .p
asynchronously (thick arrows, b–f) above the basal level (thin d
cgi/content/full/jgp.200409216/DC1). Individual Ca2(cid:1) arrows). Due to airway contraction, the cells are displaced toward f by g
oscillations occurred as Ca2(cid:1) waves propagating through- the bottom right. (B) In all three airway SMCs, the Ca2(cid:1) signaling ue
out the length of the cell (Video 3; also shown in Fig. induced by 5-HT was characterized by an initial increase in [Ca2(cid:1)]i st on
o13n)e, SbMutC ntoo aCna a2(cid:1)d jwacaevnest SwMerCe. Tobhsee irnveitdia ttioo nsp orfe tahde fCroa2m(cid:1) pafoti lxl2oe lwHs)ez,d .a sbT ydh eCefi anfl2(cid:1)eu doo irsncei stlclhaeetni oScMnes C.c hFs laausno ignreedssi ccfearnotecmde iisnmm Aaag,l ela s,R wwOeerIrese p(rle(cid:1)oct5ot er(cid:5)dde ad5s 09 March
oscillations in the SMCs was accompanied by a sustained a ratio (Ft/F0) with respect to time. A representative experiment of 2023
contraction of the airway; the SMCs shortened and were at least five trials from different slices from three mice is shown. A
displaced toward the airway lumen (Video 2). movie showing the effect of 5-HT on Ca2(cid:1) signaling in airway
SMCs is shown in Video 2 (available at http://www.jgp.org/cgi/
With the exception of differences in frequency, the
content/full/jgp.200409216/DC1).
basic characteristics of the Ca2(cid:1) signals induced by
ACH appeared similar to those induced by 5-HT (Fig.
4). In general, the pattern of Ca2(cid:1) oscillations consisted cillation consisted of an initial rapid Ca2(cid:1) transient fol-
of two phases. The initial phase consisted of low ampli- lowed by a slower phase of declining [Ca2(cid:1)]. The mean
i
tude and high frequency Ca2(cid:1) oscillations superim- duration of the Ca2(cid:1) oscillation in 1 (cid:4)M 5-HT or ACH
posed on an elevated [Ca2(cid:1)] that lasted (cid:1)1 min (Fig. 4, was 0.7 (cid:8) 0.2 s or 0.8 (cid:8) 0.2 s, respectively. Although the
i
A and C). During the second phase, the Ca2(cid:1) oscilla- magnitude of the increase of the fluorescence (F/F )
t 0
tions occurred with a stable frequency and similar am- during the first and subsequent Ca2(cid:1) oscillations fluctu-
plitude superimposed on a decreasing baseline of ated between values of 1.5 and 3.0, no major differ-
[Ca2(cid:1)] (Fig. 4, B and D, and Fig. 8). This second phase ences between agonists were identified.
i
persisted for the remaining time while the agonist was In the range from 10(cid:7)8 to 10(cid:7)6 M, both 5-HT and
present (see Fig. 8, C and D). Each individual Ca2(cid:1) os- ACH induced a concentration-dependent increase in
540 Ca2(cid:1) Signaling and Contraction of Bronchiole SMCs
the frequency of the Ca2(cid:1) oscillations calculated during
the second phase (Fig. 4 E). At all concentrations, 5-HT
induced a Ca2(cid:1) oscillation frequency that was slower
than that induced by ACH. For example, 1 (cid:4)M 5-HT in-
duced a frequency of 18 (cid:8) 2 cycles min(cid:7)1 (n (cid:9) 5 slices
of 3 animals) while 1 (cid:4)M ACH induced a frequency of
26 (cid:8) 2 cycles min(cid:7)1 (n (cid:9) 6 slices of 3 animals, P (cid:9)
0.018, t test.). The half maximum concentration was 88
and 45 nM for 5-HT and ACH, respectively.
Ca2(cid:1) Signaling in Airway SMCs Induced by KCl
High concentrations of KCl also induced increases in
[Ca2(cid:1)] and Ca2(cid:1) oscillations (Fig. 5; Video 4). How-
i
ever, the KCl-induced Ca2(cid:1) oscillations occurred more
slowly and an initial phase of high frequency Ca2(cid:1) os-
cillations was absent. The frequency of the Ca2(cid:1) oscilla- Do
w
tions induced by KCl was low (one to three per nlo
a
d
minute) at all concentrations tested (25–100 mM; Fig. e
d
5 D). An average frequency of 2.8 (cid:8) 1.4 cycles min(cid:7)1; fro
m
(n (cid:9) 4 slices of 3 animals) occurred at 50 mM KCl but http
this was not significantly different from the frequen- ://ru
p
cEiaecsh r eCcao2r(cid:1)d terda nasti ehnitg ohre ro socri lllaotwioenr hKaCdl ac olonncegn dtruartaiotinosn. ress.org
(11.8 (cid:8) 2.6 s) (Fig. 5 B). In some cases, the Ca2(cid:1) oscil- /jgp
/a
lations of neighboring cells had similar frequencies rticle
and appeared to be synchronized (four airways, Fig. -p
d
5). However, in other cases, neighboring cells showed f/12
5
asynchronous Ca2(cid:1) oscillations with different frequen- /6/5
3
cies (four airways). In each case, the Ca2(cid:1) oscillations 5/1
2
were accompanied by local transient contractions of ei- 21
4
5
ther multiple or individual cells (Fig. 5 C; Video 4, 8/jg
p
available at http://www.jgp.org/cgi/content/full/jgp. 1
2
5
200409216/DC1). 65
3
5
.p
Correlation of Airway Contraction with Ca2(cid:1) df b
Oscillation Frequency y gu
e
st o
The correlation between the increasing frequency of n
0
the Ca2(cid:1) oscillations and the increasing airway contrac- 9 M
a
tion with increasing concentrations of 5-HT and ACH rch
suggests that frequency of the Ca2(cid:1) oscillations deter- 202
3
mines the extent of airway contraction (Fig. 6). By con-
trast, an increasing correlation between airway contrac-
Figure 4. Comparison of Ca2(cid:1) oscillations induced by 5-HT and
tion and the frequency of Ca2(cid:1) oscillations induced by
ACH in airway SMCs. Airway SMCs of lung slices stimulated with
(A) 1 (cid:4)M 5-HT or (C) 1 (cid:4)M ACH. The Ca2(cid:1) responses to these KCl did not exist because the frequencies of the Ca2(cid:1)
agonists were characterized by an increase in [Ca2(cid:1)]i followed oscillations induced by a range of KCl concentrations
by Ca2(cid:1) oscillations that persisted until the removal of the agonist. were all very low and similar (Fig. 6). However, the con-
(B and D) An expanded region of 1 min, indicated by the lower traction induced by these low frequency Ca2(cid:1) oscilla-
bar in A and C, to show the details of the Ca2(cid:1) oscillations induced
tions in response to high KCl matched the contraction
by each agonist. Representative traces of at least three experiments
from different slices from two mice. A movie of the first 1 min after induced by low frequency Ca2(cid:1) oscillations induced by
stimulation with 5-HT is shown in Video 3 (available at http:// low doses of agonists.
www.jgp.org/cgi/content/full/jgp.200409216/DC1). (E) Concen-
tration–response curves for the frequency of Ca2(cid:1) oscillations
induced by 5-HT (solid circles) and ACH (open squares).
Changes in [Ca2(cid:1)] were determined in single SMCs for each
i
agonist concentration in separate experiments. Each point different slices from at least two mice. Data points of 5-HT and
represents the mean (cid:8) SEM from at least five different cells from ACH were each fitted with a logistic function curve.
541 Perez and Sanderson
Figure 6. Relationship between Ca2(cid:1) oscillation frequency
and airway contraction induced by 5-HT, ACH, and KCl. Data D
from concentration–response curves of the frequency of the Ca2(cid:1) ow
n
oscillations and the contractility for each agonist were replotted. loa
d
Data were fitted with a sigmoidal curve (Boltzmann). For ACH ed
(open squares) and 5-HT (filled circles), the airway contraction fro
m
increases as a saturating function of the frequency of the Ca2(cid:1) h
(ofisclliellda ttiroinans.g Tlehs)e inlodwu cfreedq ounelnyc ay sCmaa2l(cid:1)l coosncitlrlaactitoionns .induced by KCl ttp://rup
re
ss.o
rg
/jg
p
Type of 5-HT Receptor Involved in Airway Contraction /a
rticle
Because 5-HT and 5-HT receptors are known to pro- -p
duce increase2s in [Ca2(cid:1)3]i, 5-HT–specific antagonists df/125
and agonists were used to determine which receptor /6/5
3
type mediates airway contraction. Ketanserin, a 5-HT2– 5/12
specific antagonist, at 10 nM blocked the contraction 21
4
induced by 1 (cid:4)M 5-HT (Fig. 7 A). In addition, sequen- 58/jg
tial stimulation with 10(cid:7)8, 10(cid:7)7, and 10(cid:7)6 M DOI (2, p1
2
5
5-dimethoxy-4-iodoamphetamine hydrochloride), a 65
3
5
5-HT –specific agonist, induced reversible airway con- .p
tractio2n (three airways from three mice). On the other df by g
hand, SR 57227 (4-amino-1-(6-chloro-2-pyridyl)-piperi- ue
dsuinbest ahnytdiraol ccholnotrriadcet)io, na a5t-H 1T0(cid:7)3 7a ogro n1i0s(cid:7)t,6 dMid ( nthoet dinedcurecaes ea st on 09 M
in airway lumen area was (cid:11)10%, three airways from arch
Figure 5. Ca2(cid:1) signaling induced by KCl in airway SMCs. (A) three mice). These results suggest that the 5-HT2 recep- 202
3
Selected images taken at different times (indicated in B by arrows tor is responsible for mediating 5-HT responses.
and dashed lines) during the exposure to 100 mM KCl (bar)
showing two cells responding with transient increases in [Ca2(cid:1)] Alternative Mechanisms of Action for 5-HT and KCl
i
(thick arrows) over a basal level (thin arrows). (B) Fluorescence
changes acquired from a 5 (cid:5) 5 pixel ROI within each SMC indi- Because a cholinergic pathway has been proposed as a
cated in A showing the Ca2(cid:1) oscillations induced by KCl. (C) A mechanism by which 5-HT induced airway contraction
line-scan plot, from the dotted line indicated in A, showing the in trachea and isolated lungs (Levitt and Mitzner, 1989;
Ca2(cid:1) oscillations induced by KCl in SMC 1 (white lines) and
Eum et al., 1999; Fernandez et al., 1999; Held et al.,
the accompanying transient contraction (downward deflections,
1999; Moffatt et al., 2004), we examined the effect of at-
arrows) toward the lumen (lower black area). (D) Concentration–
response curve for the frequency of Ca2(cid:1) oscillations induced by ropine on the 5-HT responses of lung slices to deter-
KCl. Each point represents the mean (cid:8) SEM from six different mine if any 5-HT effects occurred indirectly. Atropine
cells from three different slices from three mice. Data points were (1 (cid:4)M) had no effect on the airway contractile re-
joined with a straight line. A movie of the effect of KCl is shown
sponse (Fig. 7 B) when added, either before or after ex-
in Video 4 (available at http://www.jgp.org/cgi/content/full/ posure to 1 (cid:4)M 5-HT. By contrast, 1 (cid:4)M atropine totally
jgp.200409216/DC1).
abolished the airway contractile response induced by 1
(cid:4)M ACH. Similarly, 1 (cid:4)M atropine induced a full relax-
542 Ca2(cid:1) Signaling and Contraction of Bronchiole SMCs
exposure, the lung slices were under constant perfu-
sion, a condition that would be expected to quickly
wash away any small concentrations of agonists released
from nerves. However, the twitching response induced
by KCl persisted with an approximately constant fre-
quency during several minutes of perfusion with KCl.
Furthermore, this response was elicited by several se-
quential stimulations with KCl (Fig. 2 B, Fig. 5 B, and
Fig. 12), which would have been expected to run-down
the release of agonists from isolated nerve terminals.
Role of Extracellular Ca2(cid:1) in SMC Contraction
and [Ca2(cid:1)] Signaling
i
In the presence of extracellular Ca2(cid:1), 5-HT or ACH stim-
ulated a sustained contraction of the airway (Fig. 8, A
and B). In the absence of Ca2(cid:1), 5-HT or ACH induced a Do
w
similar initial contraction but this was followed by relax- nlo
a
ation (Fig. 8, A and B). In Ca2(cid:1)-free conditions, the con- de
d
traction induced by ACH was 83% at 1 min but only 11% fro
m
after 5 min. Similar relative changes were obtained with http
5-HT (Fig. 8 A). These results indicate that the extracel- ://ru
p
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The basis for these two different responses was found /jgp
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no effect on 5-HT–induced airway contraction but completely of the agonist (Fig. 8, G and H). In the absence of ex- 8/jg
blocked or relaxed ACH-induced contraction. Representative tracellular Ca2(cid:1), the initial increase in [Ca2(cid:1)]i and stim- p125
traces of at least three different slices from three mice. ulation of Ca2(cid:1) oscillations was observed, but the fre- 65
3
quency of the Ca2(cid:1) oscillations began to decline after 5.p
d
ation of an airway that was precontracted with ACH (cid:1)1.5 min, and the Ca2(cid:1) oscillations ceased (cid:1)2–3 min f by g
(viFaAi gml.t h7uo sBcu)ag.r hiTn whicee s rbee ecrleeipesuvtoelt rsts h iinantd tiKhcCaet les im st haaalcltt ia5ni-rgHw vTaiya ds mooefe smm nbiocrtea .ancet glqaruteeesrns i(cvFye i ogrf.e 8ltah,x eEa –CtiHoa2n)(cid:1). oTofhs ctihisl elpa ratoiiorgwnrase yscs oiinvre rt ehdlaeet cearbdes awesneit chien t ohthfe e Cp farr2oe(cid:1)-- uest on 09 M
a
depolarization of the SMCs, it is possible that KCl may (Fig. 8, A and B). By contrast, in the absence of extra- rch
also act via membrane depolarization of neural termi- cellular Ca2(cid:1), high KCl did not stimulate a change in 202
nals within the lung slice to locally release neurotrans- the [Ca2(cid:1)]i, a result that correlates with the inability of 3
mitters. However, we found that KCl induced contrac- KCl to induce airway contraction under these condi-
tion of the airways in the presence of atropine and ket- tions (four airways from three mice).
anserin, receptor antagonists that fully blocked the
Effect of Ni2(cid:1) and Nifedipine on Airway Contraction
effect of ACH and 5-HT, respectively. Similarly, the pos-
sibility that KCl acts via the release of an (cid:3) -agonist was The extent of the Ca2(cid:1) influx during agonist- or KCl-
1
ruled out by the fact that the airways were totally un- induced contraction was investigated with Ca2(cid:1) channel
responsive to phenylephrine. Because ATP (but not blockers. Stimulation with 1 (cid:4)M 5-HT in the presence
AMP or adenosine) can stimulate airway contraction of 1 mM Ni2(cid:1) induced an airway contraction that was
(Bergner and Sanderson, 2002b) and may be core- slightly smaller in magnitude than the control (Fig. 9
leased from nerve terminals, we added 10 U/ml apy- A). Similarly, stimulation with 1 (cid:4)M ACH in the pres-
rase, a potent ATPase, during KCl stimulation. Under ence of 1 mM Ni2(cid:1) induced airway contraction, al-
these conditions, a twitching response of the airways though the airway subsequently showed a slow relax-
similar to that induced by KCl alone was observed. In ation (Fig. 9 B). Airway contraction induced by 5-HT or
addition, it should be noted that during agonist or KCl ACH was not affected by 10 (cid:4)M nifedipine. By contrast,
543 Perez and Sanderson
D
o
w
n
lo
a
Figure 8. Role of extracellular Ca2(cid:1) de
d
during the contraction and the Ca2(cid:1) fro
m
signaling of airway SMCs induced by h
5re-HspTo nasneds AofC Hair. w(aAy sa nind Bdi)f fCeroennttr alcutnilge ttp://rup
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presence or absence of extracellular /jg
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wCaay2(cid:1)s . fRreopmr estehnrteaet ivme itcrea.c e(s Co–fH fi)v e Tahire- /article
changes in [Ca2(cid:1)] in small ROIs of -pd
single SMCs were dietermined in lung f/12
5
slices stimulated with (C, E, and G) 1 /6/5
(cid:4)M 5-HT or (D, F, and H) 1 (cid:4)M ACH in 35
/1
the (C and D) presence or (E and F) 22
absence of extracellular Ca2(cid:1) as indi- 145
8
cated by top bars. In the absence or /jg
presence of extracellular Ca2(cid:1), both p12
faoglolonwisetds ibnyd uCcae2d(cid:1) aonsc iinllcartieoanses. iAnl t[hCoau2(cid:1)gh]i 56535.p
Ca2(cid:1) oscillations persisted in the df b
presence of extracellular Ca2(cid:1), they y g
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panrodg srteospsipveedly inr etdhue caeb sethnecier offr eexqtureancecly- est on
lular Ca2(cid:1). (G and H) The frequency of 09 M
laagtoend iste-ivnedruy ce2d0 Cas,2 (cid:1) ions citllhaeti onpsr,e cseanlccue- arch 2
(squares) or absence of Ca2(cid:1) (triangles) 023
with respect to time. Symbols and bars
are the mean (cid:8) SEM of five to eight
SMCs in five paired experiments from
different lung slices from three mice.
both nifedipine and Ni2(cid:1) completely blocked airway of the airway (Fig. 9 C). After the initial increase in
contraction stimulated by high KCl. These results indi- [Ca2(cid:1)] and the onset of Ca2(cid:1) oscillations, the fre-
i
cate that while L-type Ca2(cid:1) channels may have a signifi- quency of Ca2(cid:1) oscillations progressively decreased and
cant role in KCl-induced contraction, they have little this was accompanied by the relaxation of the airway.
influence in agonist-induced contraction and suggest After removal of Ni2(cid:1), but still in the presence of ACH,
that other Ca2(cid:1) channels serve to sustain contraction. the frequency of the Ca2(cid:1) oscillations increased again
To investigate the cause of the slow relaxation of the and the airway recontracted. These results suggest that
airway observed during the stimulation with ACH in the Ni2(cid:1) blocked Ca2(cid:1) channels that contribute to the
the continued presence of Ni2(cid:1), we simultaneously maintenance of the Ca2(cid:1) oscillations and perhaps serve
measured the changes in SMC [Ca2(cid:1)] and contraction to refill internal Ca2(cid:1) stores.
i
544 Ca2(cid:1) Signaling and Contraction of Bronchiole SMCs