A role for Ca 2+ -conducting ion channels in mechanically-induced signal transduction of airway epithelial cells

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1 Journal of Cell Siene 17, (1994) Printed in Great Britain The Company of Biologists Limited A role for Ca 2+ -onduting ion hannels in mehanially-indued signal transdution of airway epithelial ells Sott Boitano*, Mihael J. Sanderson and Ellen R. Dirksen Department of Anatomy and Cell Biology, UCLA Shool of Mediine, University of California, Los Angeles, CA , USA *Author for orrespondene SUMMARY Mehanial stimulation of a single ell in a ultured monolayer of airway epithelial ells initiates an interellularly ommuniated inrease in intraellular Ca 2+ onentration ([Ca 2+ ] i ) that propagates radially through adjaent ells via gap juntions, forming an interellular Ca 2+ wave. Mehanially-indued interellular Ca 2+ waves also our in the absene of extraellular Ca 2+. However, in Ca 2+ -free medium an inrease in [Ca 2+ ] i of the stimulated ell does not our. Thus, mehanially-indued [Ca 2+ ] i hanges in the stimulated ell are influened by the extraellular Ca 2+ onentration. To investigate if a hannel-mediated Ca 2+ flux aross the plasma membrane ontributes to the elevation of [Ca 2+ ] i in the stimulated ell we used digital image mirosopy to measure mehanially-indued [Ca 2+ ] i hanges in the presene of Ca 2+ hannel blokers. In Ca 2+ -free medium ontaining Gd 3+ (2 µm) mehanial stimulation resulted in an [Ca 2+ ] i inrease in the stimulated ell. The delay time between mehanial stimulation and inrease in [Ca 2+ ] i of the stimulated ell was dependent on extraellular [Gd 3+ ], with a half-maximal effetive onentration of approximately 4 µm. Mehanial stimulation in Ca 2+ -free medium ontaining La 3+ (1 µm) or Ni 2+ (1 µm) gave similar results. Mehanial stimulation in Ca 2+ - free medium ontaining the dihydropyridine Ca 2+ hannel blokers nifedipine (1 µm) and nimodipine (1 µm) also resulted in an inrease of [Ca 2+ ] i of the stimulated ell. Mehanial stimulation of ells treated with thapsigargin to deplete intraellular Ca 2+ stores, in the presene of 1.3 mm extraellular Ca 2+, results in an inrease in [Ca 2+ ] i of the stimulated ell without the propagation of an interellular Ca 2+ wave. Mehanial stimulation of thapsigargintreated ells in an extraellular medium buffered to µm free Ca 2+ still results in an inrease in [Ca 2+ ] i in the stimulated ell. However, the mehanially-indued Ca 2+ inrease in the presene of µm extraellular [Ca 2+ ] an be reversibly bloked by 1 µm Ni 2+. From these results we onlude that a flux of Ca 2+ aross the plasma membrane and through Ca 2+ -onduting hannels ontributes to the mehanially-indued [Ca 2+ ] i hanges in the stimulated ell. Key words: mehanial stimulation, interellular ommuniation, alium wave, alium, Gd 3+, ion hannel, dihydropyridine INTRODUCTION Cells ultured from the airway epithelium respond to loalized mehanial stimulation with an immediate inrease of [Ca 2+ ] i in the stimulated ell that spreads aross the ell from the point of stimulation. After a brief delay at the ell boundary of the stimulated ell this inrease in [Ca 2+ ] i is ommuniated to adjaent ells. Propagation of the inrease in [Ca 2+ ] i ontinues to our through -7 ells in all diretions to form an interellular Ca 2+ wave. In airway epithelial ells interellular Ca 2+ waves are propagated in the absene of extraellular Ca 2+, are initiated by the miroinjetion of IP 3 (Sanderson et al., 199), are bloked by agents that limit IP 3 -dependent Ca 2+ -release (Boitano et al., 1992), are bloked by agents that inhibit gap juntional ommuniation (Sanderson et al., 199), and do not require an extraellular messenger (Hansen et al., 1993). Mehanially-indued interellular Ca 2+ waves also our in glial ells (Charles et al., 1991, 1992, 1993) as well as in a variety of other ell types (Sanderson et al., 1994). Experimental results from a number of studies with epithelial and glial ells support the following model for the mehanism underlying mehanially-indued interellular Ca 2+ waves: mehanial stimulation initially results in an inrease of inositol 1,4,-trisphosphate (IP 3 ) in the stimulated ell. This IP 3 then releases Ca 2+ from intraellular IP 3 -sensitive stores, resulting in an inrease of [Ca 2+ ] i of the stimulated ell. Subsequently, IP 3 diffuses through gap juntions to initiate the release of intraellular Ca 2+ in adjaent ells. Communiation of IP 3 ontinues until many ells in the ulture partiipate in an interellular Ca 2+ wave (Sanderson et al., 199; Charles et al., 1991, 1992, 1993; Boitano et al., 1992; Sneyd et al., 1994a,b). It is important to emphasize that in Ca 2+ -free onditions an inrease in [Ca 2+ ] i is not deteted in the mehanially-stimulated epithelial ell despite the propagation of an interellular Ca 2+ wave through adjaent ells (Sanderson et al., 199). In fat, in Ca 2+ -free onditions a derease in the [Ca 2+ ] i of the stimulated ell is sometimes deteted, suggesting a mehani-

2 338 S. Boitano, M. J. Sanderson and E. R. Dirksen ally-indued Ca 2+ efflux from the stimulated ell. Similar results have been seen in glial (Charles et al., 1991) and endothelial (Demer et al., 1993) ells. The stimulated ell also displays a distintive response in the presene of extraellular Ca 2+ if IP 3 -dependent Ca 2+ -release is bloked with intraellular heparin (Boitano et al., 1992). Under these onditions mehanial stimulation results in an inrease in [Ca 2+ ] i of the stimulated ell without the propagation of a Ca 2+ wave. Colletively these data suggest that in addition to an IP 3 -dependent release of Ca 2+ from intraellular stores, there is a mehanially-indued Ca 2+ flux aross the plasma membrane of the stimulated ell, and the diretion of this Ca 2+ flux is influened by the extraellular Ca 2+ onentration. The objetive of this study is to determine the role of plasma membrane hannels in the observed mehanially-indued [Ca 2+ ] i hanges of the stimulated ell. We have used digital fluoresene mirosopy and the Ca 2+ -sensitive indiator fura- 2 to observe the hanges in [Ca 2+ ] i. In the presene of a variety of Ca 2+ hannel blokers an inrease in [Ca 2+ ] i ourred in the stimulated ell even in the absene of extraellular Ca 2+. In addition, in Ca 2+ -ontaining medium and when intraellular pools of Ca 2+ were emptied by treatment with thapsigargin, an inrease of [Ca 2+ ] i in response to mehanial stimulation was reversibly bloked by the Ca 2+ hannel bloker Ni 2+. These results support the hypothesis that hanges of the [Ca 2+ ] i in a mehanially stimulated ell our through at least two mehanisms: (1) by Ca 2+ flux through Ca 2+ -onduting hannels in the plasma membrane; and (2) by IP 3 -dependent release from intraellular stores. MATERIALS AND METHODS Cell ulture Primary ell ultures of airway epithelial ells were prepared as desribed elsewhere (Sanderson et al., 199). Briefly, traheas were removed from New Zealand White rabbits sarified by Nembutal injetion. The muosal layer was disseted from the traheas, ut into small squares, plaed onto ollagen-oated overslips and inubated in Dulbeo s Modified Eagle s Medium (DMEM, Gibo BRL, at. no ) supplemented with peniillin, streptomyin, amphoteriin B, and 1% fetal bovine serum. The ells were inubated at 37 C with 1% CO 2 for 7 to days. Mehanial stimulation Mehanial stimulation was performed as desribed by Sanderson and Dirksen (1986). A glass miropipette (approximately 1 µm tip diameter) was positioned near the apial membrane of a single ell by a miromanipulator. The pipette was defleted downward (for approximately milliseonds by a omputer-ontrolled piezoeletri devie) to transiently distort the membrane. Cultures were initially tested for mehanosensitivity and only those ultures that displayed mehanially-indued interellular Ca 2+ waves were used for experiments. Measurement of [Ca 2+ ] i Image analysis of fura-2 fluoresene was performed as desribed elsewhere (Sanderson et al., 199; Charles et al., 1991), with the minor hanges listed below. Cells were inubated in µm fura-2- pentaaetoxymethyl ester (fura-2-am, Calbiohem, La Jolla, CA) in modified Phenol Red-free Hanks Buffered Saline Solution (HBSS) at 37 C for approximately one hour. HBSS onsisted of 1.3 mm CaCl 2,. mm KCl,.3 mm KH 2PO 4,. mm MgCl 2,.4 mm MgSO 4, mm NaCl,.3 mm Na 2PO 4, 1% gluose (made from Gibo BRL, at. no. 146) additionally buffered with 2 mm HEPES, ph 7.2. Cultures were washed in HBSS and allowed to inubate for 3 minutes before use. Fluoresene was observed at room temperature with an inverted mirosope (Nikon Diaphot) equipped with a 4, 1.3 numerial aperture objetive and quartz optial elements. Exitation light was emitted from a 1 W merury lamp through a 34 or 38 nm filter (Omega Optial, Brattleboro, VT) seleted by a omputer-ontrolled stepper wheel. Neutral density filters were used to appropriately balane the exitation intensities. A 4 nm dihroi mirror separated exitation and emission light, and fura-2 fluoresene was viewed through a 1 nm filter with a silione intensified target amera (Cohu, San Diego, CA). Images were reorded with an optial memory dis reorder (Panasoni, TQ226F) and proessed with the aid of a frame grabber and image proessor board (Data Translation, Marlboro, MA, model nos DT2861 and DT2868) in an AT-486 omputer (Gateway, North Sioux City, SD). [Ca 2+ ] i was alulated with ratiometri methods (Grynkiewiz et al., 198) or, when inreased time resolution was neessary (1/3 seond), from single wavelength reordings with an initial referene to a [Ca 2+ ] i map alulated ratiometrially (Charles et al., 1991). Both methods yielded similar values for [Ca 2+ ] i. The resultant [Ca 2+ ] i maps were displayed in a red, green and blue format on a video monitor (Sony PVM-1271Q) and photographed with an Imagereorder (IC 42 Fous Graphis, Foster City, CA). Plots of [Ca 2+ ] i as a funtion of time were obtained from an area of the ell overing 6 6 pixels (approx 2.1 µm by 2.6 µm). The individual points plotted in eah graph were averages of data from either four onseutive video frames (taken at 3 frames per seond) or single frames reorded at intervals of one seond. Plots of hanging [Ca 2+ ] i ontained one point per seond. Exposure to thapsigargin Cells were washed with five to ten times the hamber volume (2 ml) of 1 µm thapsigargin (Calbiohem) in HBSS, whih aused an immediate inrease in [Ca 2+ ] i. To allow suffiient time for the ells to equilibrate [Ca 2+ ] i to resting levels, mehanial stimulation was not performed for at least twenty minutes after appliation of thapsigargin. Cells retained fura-2 fluoresene and displayed iliary ativity for at least one hour after the addition of thapsigargin. When required, solutions were exhanged with Ca 2+ -free HBSS onsisting of. mm KCl,.3 mm KH 2PO 4, 2.8 mm MgCl 2,.4 mm MgSO 4, 138 mm NaCl,.3 mm Na 2HPO 4, 4. mm NaHCO 3, 1% gluose, 1. mm EGTA, and 2 mm HEPES (ph 7.2) or an HBSS buffered to µm free alium (HB), onsisting of.13 mm CaCl 2,. mm KCl,.3 mm KH 2PO 4, 1.27 mm MgCl 2,.4 mm MgSO 4, 14 mm NaCl,.3 mm Na 2HPO 4, 4. mm NaHCO 3,.1 mm EGTA, 1% gluose and 2 mm HEPES (ph 7.2). Exposure to hannel blokers In order to prevent preipitation, Gd 3+ (Aldrih, Milwaukee, WI) was prepared in a PO 4 3 -free HBSS (where KH 2PO 4 and Na 2HPO 4 were replaed with KH 2SO 4 and Na 2SO 4, respetively). La 3+ and Ni 2+ (Sigma) were dissolved in HBSS. Nifedipine (Sigma) and nimodipine (Miles) were initially dissolved in ethanol and then diluted into HBSS. Cultures were washed with five to ten times the hamber volume (2 ml) of HBSS ontaining the respetive hannel bloker. For the Gd 3+ experiments, ultures were first washed for minutes with the PO 4 3 -free HBSS prior to inubation in varying onentrations of Gd 3+. Cells were exposed to the hannel blokers for at least five minutes and up to two hours before mehanial stimulation. Cultures were then washed with the appropriate redued Ca 2+ onentration solutions (HB, Ca 2+ -free HBSS; PO 4-free and Ca 2+ -free HBSS for Gd 3+ experiments) that also ontained the appropriate hannel bloker. Addition of hannel blokers to ells in HBSS or PO 4 3 -free HBSS was not suffiient to ause a hange in [Ca 2+ ] i.

3 Mehanially-indued alium hanges 339 RESULTS Mehanial stimulation in the absene of extraellular Ca 2+ In the absene of extraellular Ca 2+ (Ca 2+ -free HBSS), mehanial stimulation of a single airway epithelial ell resulted in an interellular Ca 2+ wave that was propagated through adjaent ells (Fig. 1A-C). However, the stimulated ell did not display an inrease in [Ca 2+ ] i despite the ommuniated inreases in [Ca 2+ ] i in the adjaent ells. The propagation of a Ca 2+ wave through adjaent ells in Ca 2+ -free HBSS indiated that there was a oordinated release of intraellular Ca 2+ in these ells. When the ultures were returned to Ca 2+ - ontaining media (HBSS), the stimulated ell responded to mehanial stimulation with an inrease in [Ca 2+ ] i (not shown). This demonstrated that the stimulated ell was apable of undergoing an [Ca 2+ ] i hange. Consequently, the failure of an [Ca 2+ ] i inrease in a mehanially-stimulated ell in Ca 2+ -free HBSS suggested that intraellularly released Ca 2+ was lost from the stimulated ell. Ca 2+ -free Thapsigargin Ca 2+ -free + 1 µm Gd µm Calium Conentration (nm) Fig. 1. Mehanially-indued hanges of [Ca 2+ ] i in epithelial ultures. (A-C) The spatiotemporal pattern of inreases in [Ca 2+ ] i indued by mehanial stimulation of a single ell in Ca 2+ -free HBSS. The stimulated ell (arrow) does not display an inrease in [Ca 2+ ] i despite the propagation of a oordinated inrease in [Ca 2+ ] i in adjaent ells (Ca 2+ wave). (D-F) The hanges in [Ca 2+ ] i following mehanial stimulation of thapsigargin-treated ells in the presene of extraellular Ca 2+. Despite an inrease in [Ca 2+ ] i of the stimulated ell (arrow) there is no Ca 2+ wave propagated through adjaent ells. (G-I) The hanges in [Ca 2+ ] i following mehanial stimulation of a single ell in Ca 2+ -free medium ontaining 1 µm Gd 3+. The stimulated ell (arrow) displays an immediate inrease of [Ca 2+ ] i and a Ca 2+ wave is ommuniated to adjaent ells, images are taken at 1 seond (A,D,G), seonds (B,E,H) and 9 seonds (C,F,I) following stimulation The olor bar indiates the sale representing the [Ca 2+ ] i. White lines approximate ell borders. Image sequenes are from single experiments but are representative of >2 (A- C), 12 (D-F) and 14 (G-I) experiments.

4 Delay Time (seonds) [Ca 2+ ] i (nm) 34 S. Boitano, M. J. Sanderson and E. R. Dirksen Mehanial stimulation in the presene of thapsigargin Beause there is an apparent efflux of Ca 2+ from the stimulated ell in Ca 2+ -free HBSS, it followed that a Ca 2+ influx may our in the stimulated ell in Ca 2+ -ontaining HBSS. To test this hypothesis it was neessary to disriminate between an influx of extraellular Ca 2+ and the release of Ca 2+ from intraellular stores. Consequently, the Ca 2+ stores were depleted by transient exposure ( minutes) to thapsigargin, an endoplasmi and saroplasmi retiulum Ca 2+ -ATPase bloker (Thastrup et al., 199; Lytton et al., 1991). Addition of thapsigargin resulted in an immediate inrease in [Ca 2+ ] i, followed by a return of [Ca 2+ ] i to near resting levels within 2 minutes (not shown). Mehanial stimulation of a thapsigargin-treated ell in Ca 2+ - ontaining HBSS resulted in an inrease in the [Ca 2+ ] i of the stimulated ell (Fig. 1D-F; n=12) but did not initiate an interellular Ca 2+ wave, even when the mehanial stimulus was applied 1 hour after exposure to 1 µm thapsigargin. In order to verify that the soure of Ca 2+ ontributing to the inrease of [Ca 2+ ] i in the stimulated ell was extraellular, mehanial stimulation was performed on thapsigargin-treated ells (different ells within the same ulture) after removal of extraellular Ca 2+ by washing with Ca 2+ -free HBSS. Under these onditions no inrease of [Ca 2+ ] i ould be deteted following mehanial stimulation (not shown; n=7). Thus, mehanially-indued inreases in the [Ca 2+ ] i of thapsigargintreated ells were dependent on an influx of extraellular Ca 2+. Mehanial stimulation in the presene of Ca 2+ hannel blokers In the presene of PO 4 3 -free HBSS ontaining Ca 2+ (1.3 mm) and Gd 3+ (2 to 1 µm), mehanial stimulation indued an inrease in the [Ca 2+ ] i of the stimulated ell and an interellular Ca 2+ wave similar to that normally seen in HBSS (n=). Similar results were seen when HBSS solutions were supplemented with Ni 2+ (1 µm; n=), or with the dihydropyridines nifedipine (1 µm, n=4), or nimodipine (1 µm; n=3). Thus, in Ca 2+ -ontaining HBSS, the Ca 2+ hannel blokers did not adversely affet the initiation nor the propagation of an interellular Ca 2+ wave. Experiments were repeated in Ca 2+ -free medium to determine the effets of Ca 2+ hannels blokers on the mehanially-indued hanges in [Ca 2+ ] i of the stimulated ell. In a Ca 2+ -free solution ontaining 1 µm Gd 3+, mehanial stimulation of a single ell resulted in an inrease in [Ca 2+ ] i of the stimulated ell and an interellular Ca 2+ wave similar to that seen in HBSS (n=14) (Fig. 1G-I). This result was in ontrast to the lak of an inrease of [Ca 2+ ] i of the stimulated ell in Ca 2+ -free HBSS without Gd 3+ (Fig. 1A-C), and suggested that Gd 3+ had bloked the efflux of Ca 2+ from the stimulated ell. In the presene of 1 µm Gd 3+ and Ca 2+ -free onditions the inrease in [Ca 2+ ] i of the stimulated ell frequently ourred immediately, although delays of one seond or more were sometimes observed (Fig. 2A). A similar inrease in [Ca 2+ ] i of the stimulated ell in Ca 2+ -free solutions ould also A B 1 2 C 3 D Time (seonds) Fig. 2. Mehanially-indued hanges in [Ca 2+ ] i of the stimulated ell in Ca 2+ - and PO 4 3 -free solutions supplemented with Gd 3+. Plots represent the [Ca 2+ ] i versus time in Ca 2+ -free solutions ontaining (A) 1 µm Gd 3+ ; (B) µm Gd 3+ ; and (C) 3 µm Gd 3+. [Gd 3+ ] are indiated at the top right. The time of stimulation is indiated by the arrow. In all ases a Ca 2+ wave was observed to propagate through adjaent ells. (D) Dose-response relationship between the extraellular Gd 3+ onentration and the time taken for [Ca 2+ ] i to reah the 1/2 maximal [Ca 2+ ] i (delay time) following stimulation in Ca 2+ -free solution ontaining Gd 3+. As the [Gd 3+ ] is inreased, the delay time in the stimulated ell dereases. Bars represent standard deviation [Gd 3+ ] (µm)

5 Mehanially-indued alium hanges 341 [Ca 2+ ] i (nm) A Ni 2+ B C be observed at lower Gd 3+ onentrations following a longer delay (Fig. 2B). At the lowest effetive Gd 3+ onentrations (2-3 µm) delay times were typially between 4 and 7 seonds (Fig. 2C), but 2 seond delay times were sometimes observed. The delay time between mehanial stimulation and a 1/2 maximal [Ca 2+ ] i hange was omposed of delays in both the onset of the initial [Ca 2+ ] i hange and in the time needed from the initial hange in [Ca 2+ ] i to a 1/2 maximal value. The half-maximal effetive onentration for the onset of the Ca 2+ response in the stimulated ell was approximately 4 µm Gd 3+ (Fig. 2D). Similar results were obtained using another lanthanide series Ca 2+ hannel bloker, La 3+ (not shown; n=2). Similarly to Ca 2+ -free HBSS ontaining 1 µm Gd 3+, Time (seonds) 2 Nif 2 Nim Fig. 3. Mehanially-indued hanges in Ca 2+ -free HBSS supplemented with Ca 2+ hannel blokers. The time of stimulation is indiated by an arrow. (A) 1 µm Ni 2+ ; (B) 1 µm nifedipine; and (C) 1 µm nimodipine. In all ases a mehanially-indued inrease in the [Ca 2+ ] i of the stimulated ell was followed by a Ca 2+ wave propagated through adjaent ells. Traes are from single experiments representative of 6 (A), 1 (B) and 4 (C) experiments. 2 mehanial stimulation of a single ell in a Ni 2+ (1 µm, n=6; or 1 mm, n=3) supplemented Ca 2+ -free solution resulted in an inrease in [Ca 2+ ] i of the stimulated ell within 1 seond of mehanial stimulation (Fig. 3A) in addition to a propagated interellular Ca 2+ wave. When Ni 2+ was removed by washing with Ca 2+ -free HBSS and mehanial stimulation repeated on the same ell, the stimulated ell did not inrease its [Ca 2+ ] i despite the propagation of a Ca 2+ wave to adjaent ells (not shown; n=3). In Ca 2+ -free HBSS supplemented with the dihydropyridine hannel blokers nifedipine (1 µm) or nimodipine (1 µm) there was a mehanially-indued inrease of [Ca 2+ ] i of the stimulated ell following a slight delay (Fig. 3B,C; nifedipine, n=1; nimodipine, n=4). The inrease of [Ca 2+ ] i in the stimulated ell was followed by the propagation of an interellular Ca 2+ wave (not shown). Upon removal of the dihydropyridines by washing with Ca 2+ -free HBSS, mehanial stimulation of the same ell resulted in an interellular Ca 2+ wave without an inrease of [Ca 2+ ] i in the stimulated ell (nifedipine, n=2; nimodipine, n=1; not shown). Mehanial stimulation in the presene of Ni 2+ and thapsigargin In HB, a solution with the extraellular Ca 2+ onentration adjusted to µm, mehanial stimulation of a single ell resulted in an inrease in the [Ca 2+ ] i of the stimulated ell and a normal interellular Ca 2+ wave (Fig. 4A-D; n=8). Similar to mehanial stimulation of thapsigargin-treated ultures in HBSS, mehanial stimulation of thapsigargin-treated ells in HB displayed an inrease of [Ca 2+ ] i of the stimulated ell without a propagation of a Ca 2+ wave (not shown). However, after bathing the thapsigargin-treated ells in HB supplemented with 1 µm Ni 2+, mehanial stimulation did not inrease the [Ca 2+ ] i of the stimulated ell, even after repeated stimuli (Fig. 4E; n=9). After washout of Ni 2+, mehanial stimulation indued an inrease of the [Ca 2+ ] i in the stimulated ell (Fig. 4F; n=6). DISCUSSION In this study we have shown that mehanial stimulation of a single ell auses a flux of Ca 2+ aross its plasma membrane that an be reversibly bloked by Gd 3+, La 3+, Ni 2+, nifedipine and nimodipine. These results suggest that Ca 2+ -onduting hannels play a role in mehanial signal transdution and are relevant to mehanially-indued interellular Ca 2+ waves in airway epithelia (Sanderson et al., 199; Boitano et al., 1992; Hansen et al., 1993), glia (Charles et al., 199, 1991, 1992) and in a variety of other ell types (Sanderson et al., 1994). Interellular Ca 2+ waves may allow for the oordination of several ells to respond as a unit. This is most learly demonstrated by the oordinated inrease in iliary beat frequeny of multiple airway epithelial ells following mehanial stimulation of a single ell (Sanderson et al., 1988). The urrent hypothesis for the ommuniation of mehanially-indued interellular Ca 2+ waves has been formulated from a number of studies (Sanderson et al., 199; Charles et al., 1991, 1992, 1993; Boitano et al., 1992; Sneyd et al., 1994a,b). In this hypothesis, mehanial stimulation is believed to ativate phospholipase C, whih auses an inrease in the IP 3 onentration of the stimulated ell. IP 3 then ats at

6 342 S. Boitano, M. J. Sanderson and E. R. Dirksen Fig. 4. Mehanial stimulation in HB with external [Ca 2+ ] buffered to µm. (A-D) hanges in [Ca 2+ ] i versus time in a series of adjoining ells (ell positions shown at right) following mehanial stimulation of a single ell (arrow). There was a mehanially-indued inrease of [Ca 2+ ] i in the stimulated ell followed by a propagated Ca 2+ wave through adjaent ells. The time before the initiation of the [Ca 2+ ] i inrease in the adjoining ells was dependent on the ell distane from the stimulated ell. (E) Mehanially-indued hanges in [Ca 2+ ] i versus time of a thapsigargin-treated ell in HB supplemented with 1 µm Ni 2+. An inrease in [Ca 2+ ] i of the stimulated ell is not observed when the intraellular Ca 2+ stores are emptied by thapsigargin and Ca 2+ -onduting hannels are bloked by Ni 2+, even after several stimulations (arrows). (F) [Ca 2+ ] i versus time of the same ell depited in (E) after washout of the Ni 2+ with HB. Mehanially-indued [Ca 2+ ] i inrease (arrow) is restored one the hannel bloker is washed away. Traes are of single experiments representative of 8 (A-D), 9 (E) and 6 (F) experiments. the IP 3 reeptor to indue release of Ca 2+ from intraellular stores. This Ca 2+ inrease is amplified by a Ca 2+ -indued Ca 2+ release mehanism via the IP 3 reeptor to propagate an intraellular Ca 2+ wave. Subsequently, IP 3 diffuses through gap juntions to adjaent ells and releases intraellular Ca 2+ to bring about a ommuniated inrease in [Ca 2+ ] i. This ommuniation ontinues to many ells to reate an interellular Ca 2+ wave. In support of the hypothesis that an interellular Ca 2+ wave onsists of a oordinated release of intraellular Ca 2+ we have shown that mehanial stimulation in the absene of extraellular Ca 2+ initiates an interellular Ca 2+ wave (Fig. 1A-C; Sanderson et al., 199; Charles et al., 1991; Demer et al., 1993). However, it is important to note that in Ca 2+ -free onditions these waves propagate through adjaent ells without an inrease in [Ca 2+ ] i of the stimulated ell. This result appears to be in ontradition to the proposed hypothesis, whih predits that an inrease in [Ca 2+ ] i in the stimulated ell should our due to the prodution of IP 3 and subsequent IP 3 - dependent Ca 2+ release from intraellular stores. We demonstrate here that in the absene of extraellular Ca 2+ the proposed IP 3 -dependent Ca 2+ release in the stimulated ell an be visualized by externally applying a variety of Ca 2+ hannel blokers. It has been shown that influx of extraellular Ca 2+ an alter the frequeny and the veloity of IP 3 -dependent intraellular Ca 2+ waves in Xenopus ooytes (Girard and Clapham, 1993), or the triggering of [Ca 2+ ] i hanges in several ell types (for review see Rooney and Thomas, 1993). Consequently, we suggest that under Ca 2+ -free onditions a Ca 2+ -efflux ontributes to the delayed onset or omplete inhibition of [Ca 2+ ] i hanges in mehanially-stimulated airway epithelial ells. The fat that epithelial, glial (Charles et al., 1991) and endothelial (Demer et al., 1993) ells adjaent to the stimulated ell display [Ca 2+ ] i inreases in Ca 2+ -free onditions supports the idea that extraellular Ca 2+ is not essential for the initiation of intraellular Ca 2+ waves. These onlusions are in ontrast to the suggestion that some extraellular Ca 2+ is required as a mediator of the mehanially-indued [Ca 2+ ] i inrease in endothelial ells (Sigurdson et al., 1993). Beause experiments using Ca 2+ hannel blokers and Ca 2+ - free medium suggest that Ca 2+ -onduting hannels are opened in response to mehanial stimulation, we might expet Ca 2+ to move into the ell following mehanial stimulation in a Ca 2+ -ontaining medium. In order to disriminate between the [Ca 2+ ] i hanges aused by mehanially-indued influx of Ca 2+ and those from IP 3 -dependent intraellular Ca 2+ release, intraellular Ca 2+ stores were emptied by the Ca 2+ -ATPase inhibitor thapsigargin (Thastrup et al., 199; Lytton et al., 1991). In Ca 2+ -ontaining medium, mehanial stimulation of thapsigargin-treated ells resulted in inreases in the [Ca 2+ ] i of the stimulated ell without the propagation of an interellular Ca 2+ wave. Beause the intraellular Ca 2+ stores in thapsigargin-treated ells are devoid of Ca 2+, the observed inrease of the [Ca 2+ ] i in the stimulated ell is due to an influx of Ca 2+ from the extraellular medium. This restrition of the inrease in the [Ca 2+ ] i to the mehanially-stimulated ell is also observed in glial ells treated with thapsigargin (Charles et al., 1993) and airway epithelial ells loaded with heparin, a bloker of IP 3 -dependent Ca 2+ release (Boitano et al., 1992). Sine thapsigargin is a speifi inhibitor of endoplasmi retiulum and saroplasmi retiulum Ca 2+ -ATPases, it is possible that mehanial stimulation is diretly ativating plasma membrane Ca 2+ -ATPases to alter the [Ca 2+ ] i. However, the diversity of Ca 2+ hannel blokers used to inhibit mehanially-indued Ca 2+ influx or efflux (see above) makes non-speifi inhibition of plasma membrane Ca 2+ -ATPases less likely, and supports the idea that these Ca 2+ fluxes our through Ca 2+ -onduting hannels. An alternative pathway for mehanially-indued Ca 2+ -flux is through transient holes in the plasma membrane. However, the ability of hannel blokers to alter the Ca 2+ flux and the fat that fluoresent dyes are not lost from the stimulated ell following mehanial stimulation argues against the mediation of Ca 2+ fluxes aross the plasma membrane through transient holes.

7 Mehanially-indued alium hanges 343 A potential mehanism ontributing to the lak of an observed mehanially-indued inrease in [Ca 2+ ] i of the stimulated ell under Ca 2+ -free onditions is the biphasi response of the IP 3 -reeptor/ca 2+ hannel to the [Ca 2+ ] i (Bezprozvanny et al., 1991; Finh et al., 1991; Iino and Endo, 1992). At both low and high onentrations of Ca 2+, Ca 2+ -indued Ca 2+ release (CICR) via the IP 3 reeptor is inhibited. Thus, the subsequent CICR following mehanial stimulation would be inhibited if the [Ca 2+ ] i of the stimulated ell is kept suffiiently low. This ould be aomplished if the small amount of Ca 2+ that is released by IP 3 immediately leaves the ytosol through mehanially-ativated Ca 2+ -onduting hannels. This would require that the membranes ontaining IP 3 reeptors and intraellular Ca 2+ stores be situated lose to the plasma membrane. The delayed rapid release of Ca 2+ from intraellular stores at relatively low extraellular Gd 3+ onentration is ompatible with a slow aumulation of ytosoli Ca 2+ due to the gradual release from IP 3 -sensitive stores and an inomplete blokage of Ca 2+ -onduting hannels. In this ase the rapid inrease in the [Ca 2+ ] i would our one suffiient Ca 2+ is available for effiient IP 3 -dependent Ca 2+ release and CICR via the IP 3 reeptor. An alternative explanation for the inhibition of the [Ca 2+ ] i inreases in the stimulated ell under Ca 2+ -free onditions is that intraellular Ca 2+ is released in the stimulated ell by an IP 3 -dependent mehanism and subsequent CICR, but in the absene of plasma membrane hannel blokers, the released Ca 2+ leaves the ell before being deteted by digital image mirosopy. Although it is possible that Ca 2+ hanges may go undeteted (see Negulesu and Mahen, 1988), the robust nature of the intraellular Ca 2+ waves in airway epithelial ells suggests that this mehanism is less likely to our. One potential route for the mehanially-indued Ca 2+ flux is through streth-ativated hannels. Streth-ativated hannels have been desribed in numerous ell types (Morris, 199; Sahs, 1992; Hamill and MBride, 1993), inluding epithelial ells (Christensen, 1987; Okada et al., 199; Kim et al., 1993). Although Gd 3+ in the 1-2 µm range has been used to identify streth-regulated hannels in several ell types (Yang and Sahs, 1989; Frano et al., 1991; Sigurdson et al., 1992; Davis et al., 1992; Naruse and Sokabe, 1993), we only observed effiient blokage of the Ca 2+ response of epithelial ells using Gd 3+ at onentrations between and 1 µm. Beause Gd 3+ has also been reported to blok voltage-sensitive Ca 2+ hannels at these higher onentrations (Biagi and Enyeart, 199; Boland et al., 1991; Canzoniero et al., 1993) we examined the effets of a diverse group of Ca 2+ hannel blokers on the mehanially-indued Ca 2+ response. The tested blokers were not assoiated with the bloking of streth-ativated hannels, but were suesful in reversibly bloking the flux of Ca 2+ aross the plasma membrane of the mehanially-stimulated ell. Although we annot rule out that streth-ativated hannels may ontribute to the Ca 2+ fluxes in the stimulated ell, we onlude that other types of Ca 2+ -onduting hannels are also opened indiretly by mehanial stimulation. The indiret ativation of Ca 2+ -onduting hannels by mehanial stimulation may involve seond messengers, G- proteins, or membrane voltage. The possibility of a seond messenger-regulated, streth-ativated hannel has been desribed in airway epithelium (Kim et al., 1993). It is important to note that only the Ca 2+ response of the mehanially-stimulated ell is strongly influened by the extraellular Ca 2+ onentration in omparison to adjaent ells and, that mehanially-indued interellular Ca 2+ waves are ommuniated via gap juntions by an IP 3 -mediated mehanism (Charles et al., 1992; Boitano et al., 1992). In order to aount for these experimental observations any seond messenger proposed to modulate the observed hannel ativity: (1) must be produed in the stimulated ell; and (2) annot preede the propagation of the IP 3 -dependent Ca 2+ wave to the adjaent ell. These riteria make it unlikely that the observed hannel regulation in the stimulated ell is due to IP 3 (or any of its breakdown produts), or any other seond messenger that may traverse gap juntions. It is interesting to note that when IP 3 is made in a ell by phospholipase C, a similar quantity of diaylglyerol (DAG) is also produed (see Berridge, 1993). DAG is a membrane bound seond messenger that would not likely be ommuniated from ell to ell, and thus would be in an ideal position to diretly ativate plasma membrane hannels. In addition, DAG an ativate protein kinase C, another potential hannel modulator that would be restrited from moving to adjaent ells. It is also possible that mehanial stimulation ativates speifi G-proteins of the stimulated ell. In addition to the potential for the ativation of phospholipase C, G-proteins ould also diretly or indiretly modulate hannels within the stimulated ell and would not be expeted to traverse gap juntions to adjaent ells. The sensitivity of the [Ca 2+ ] i hanges to the voltageregulated Ca 2+ hannel bloker Ni 2+, and to the L-type hannel blokers nifedipine and nimodipine suggests that voltagesensitive hannels are also opened by mehanial stimulation. Cultured epithelial ells have been shown to be eletrially oupled (Sanderson et al., 1988); thus, we might surmise that any voltage hange in the stimulated ell would be passed on to adjaent ells. The timing of the voltage hanges (almost instaneous) as opposed to the the timing of the propagation of the interellular Ca 2+ wave (an approximate 1 seond delay at the ell borders) as well as the fat that interellular Ca 2+ waves are propagated under both hyperpolarizing and depolarizing onditions (Sanderson et al., 1994; S. Boitano and A. C. Charles, unpublished data), exludes voltage hanges as being the major signal transduer for interellular Ca 2+ wave propagation. Beause of the different [Ca 2+ ] i responses in the stimulated and adjaent ells, the observed [Ca 2+ ] i hanges are probably not solely the onsequene of voltage-gated hannels. An alternative explanation is that the opening of a seond messenger-mediated hannel in the stimulated ell also displays some sort of voltage sensitivity. The fat that a variety of hannel blokers are effetive in bloking whole ell ondutane of Ca 2+ emphasizes the need to haraterize further the speifi hannels mediated by mehanial stimulation. This haraterization should be possible with single hannel reording tehniques. We thank Dr Andrew Charles for his help in onduting the experiments and extensive disussion in preparing the manusript. We thank Dr Mihael Woodruff for his ritial reading of the manusript and Jennifer Felix for ulturing the epithelial ells. S.B. is a Parker B. Franis Family Fellow. This work was supported by the Smokeless Tobao Researh Counil, In. and the Tobao Related Disease Researh Program of the University of California. M.J.S. is supported by NIH grant no. HL49288.

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