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Plmr

Physiol.

Biochcm,

1998, 36 (3). 257-262

Two potential Ca2+-dependent transduction closing in response to abscisic acid


Alain Cowson*, Alain Vavasseur

pathways in stomata1

Laboratoirede BioCnergCtique Cellulaire, CEA/Cadarache-DSV

DEVM, (CNRS-CEA, UMR 163). 13 108 Saint-Paul-lez-Durance cedex, France. * Author to whom correspondence should be addressed (fax 33 (0) 42 25 46 56; e-mail vavasseur@dsvcad.cea.fr)

(Received April 21, 1997; accepted June 7, 1997) Abstract - Withering of abscisic acid (ABA)-insensitive mutants in standard watering conditions as well as under drought suggests that ABA would regulate stomata1 closing not only under water stress but also under other hydric conditions. Depending on its applied concentration, the ABA closing signal might be transduced through different pathways. This possibility was investigated in epidermal peels of Commelina communis by comparing the effects of different Ca + buffers, protein kinase inhibitors, calmodulin (CaM) antagonists and guanosine-triphosphate-binding protein (G protein) modulators on the stomata1 closing responses to 10 nM ABA (ABA,,,) and 100 nM ABA (ABA,,,,). EGTA specifically suppressed the response to ABA,,,, while the Ca*+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N,N X-tetraacetic acid (BAPTA) similarly inhibited the responses to ABA,,, and ABA,,,,. The response to ABA,, was specifically affected by the protein kinase inhibitors KT5926 and I-(5.iodonaphthalene- 1-sulphonyl)- 1H-hexahydro- 1,4-diazepine (ML-7), the CaM antagonist N-(6.aminohexyl)-5-chloro1-naphthalenesulfonamide (W-7), and the phospholipase C inhibitor 1-[6-[[ 17B-3-methoxyestra-1,3,5(10)-trien17-yl]amino]hexyl]-1 Hpyrrole-2,5-dione (LJ73122), whereas the response to ABA,tx, was specifically affected by the G protein antagonist pGlu-GlnD-Trp-Phe-D-Trp-D-Trp-Met-NH? (GP Ant-2) and mas17, an inactive mastoparan analog. These data are consistent with two possible ABA routes, each of them exhibiting specific Cal+-requirements and protein phosphorylations. and being initiated by its own receptor. Furthermore, analogies with animal features suggest that the ABA,,, and ABA,,,,, closing signals might be extracellularly perceived. 0 Elsevier, Paris. Abscisic acid I Ca+ buffers I guanosine-triphosphate-binding stomata1 closing I Commelina communis protein modulators / protein phosphorylation / receptors /

acid I CaM, ABA,,, 10 nM ABA/ ABA,, , 100 nM ABA / BAPTA, 1,2-bis(o-aminophenoxy)ethane-N,N,N,N-tetraacetic complex / DAG, l&diacylglycerol / GP Ant-2, pGlu-Gln-D-Trp-Phe-D-Trp-Dcalmodulin / Ca+-CaM, Ca 1+-calmodulin Trp-Met-NH, / GP Ant-ZA, Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH~ / H-7, 1-(5iso-quinolinesulphonyl)-2-methylpiperazine / InsP3, inositol 1,4,5triphosphate / ML-7, I-(5iodonaphthalene-l-sulphonyl)-lH-hexahydro-1,4-diazepine / MLCK, myosin light chain kinase / U73122, 1-[6-[[17~-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-lH-pyrrole-2,5-dione / U73343, 1-[6-[[17~-3-methoxyestra-1,3,5(lO)-trien-l7-yi]amino]hexyl]-2~-pyrrolidinedione / W-5, N-(6-aminohexyl)-1-naphthalenesulphonamide/ W-7, N-(6-aminohexyl)-5-chloro-l-naphthalenesulphonamide / 7TMS, 7 trans-membrane span

1. INTRODUCTION
Plants respond to water stressby reducing transpiration through abscisic acid (ABA) which induces closing of the stomata1pores in the leaves [4]. It is known that micromolar concentrations of external ABA induce the stomata to close through increasesin cytosolic free Ca+ [14] and protein phosphorylation [ 181.Moreover, recent studies [2] suggestthat, within the ABA signal transduction, G protein-linked seventransmembrane-span (7TMS) receptors regulate plasmamembraneK+ channelsof stomata1 guard cells.
Plant Physiol. Biochem., 0981-9428/98/03/O Elsevier, Paris

Withering of ABA-insensitive mutants [lo] in standard hydric conditions suggests that endogenousABA would regulate stomata1 closing under conditions other than water stress.Also, it is important to question whether or not endogenousABA would respond to different watering conditions by the same regulatory mechanisms of stomata1closing. In the leaves. the concentration of endogenous ABA might depend on the applied watering conditions. In this context, one can suppose that, according to its concentration, endogenous ABA might modulate stomata1 closing via different possible transduction pathways.

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This hypothesis hasbeen investigated by examining whether different concentrations of exogenously applied ABA trigger different signal transduction pathways leading to stomata] closing in epidermal peels of Commelina communis. For such a purpose, two concentrations of exogenously applied ABA have been retained, e.g. 10 nM ABA (ABA,,,) which significantly limits stomata1 closure and 100 nM ABA . (ABA,,,) which induces almost maximum stomata1 closure [ 141.Indeed, these ABA concentrations would mimic the ABA status encountered in the leaves during either standard watering or water stress. In a comparative study of the stomata1closing responses to various compounds have been ABA,, and ABA,,, assayed such as different Ca+ buffers, calmodulin (CaM) antagonists and selective inhibitors which block protein phosphorylations or prevent different classesof heterotrimeric guanosine-triphosphate-binding regulatory proteins (G proteins) from functioning.

1 EGTA conch

0 BAPTA

1 cont. (mM)

(mM)

Figure 1. Modulation of ABA-induced stomata1 closing by Ca+ buffers. Differential inhibitory effect of the Ca buffers (A) EGTA and (B) BAPTA on the induction of stomata1 closing by IO nM ABA (0) or 100 nM ABA (0). Extent in stomata1 closing is taken as the difterewe between the stomata1 apertures measured just before and 2 h after the addition of ABA. Each data point represents the mean -t SE calculated from 4 independent experiments.

2. RESULTS

AND DISCUSSION

2 .1 . Effect of Ca2+ buffers


In agreement with McAinsh et al. [14], ABA,,, and ABA,, induced the meanstomata1 aperture to decrease by 6.5 and 11.0 urn respectively (figure f). Figure I shows that Ca*+-dependencyof the responseto ABA,,, differs from that exhibited by the responseto ABA,,,,. Indeed, the Ca2+ buffer EGTA largely inhibited stomatal closing to ABA,Y but not to ABA,,, cfigure I A). By contrast, the Ca-+ buffer 1,2-bis(o-aminophenoxy)ethane-N,N, N *, -tetraacetic acid (BAPTA) N almost completely suppressed stomata] closing to ABA,, as well as to ABAr,, @gure I B). Since BAPTA, but not EGTA, could buffer efficiently rapid increases in cytosolic free Cal+ [2], our data suggest that, among the possible Ca2+ transients, rapid ones could be predominantly involved in the response to ABA,. These transients might mediate particular steps within the ABA transduction pathway, including protein phosphorylation. The possibility that the responsesto ABA,,, and ABA,,, might be discriminated by Ca2+-dependent phosphorylations was investigated by using protein kinase inhibitors and CaM antagonistswhich selectively prevent different types of Ca+-dependent processes.

and I-(5-iodonaphthalene-l-sulphonyl)-lH-hexahydro- 1,4-diazepine (ML-7) were tested: ML-7 selectively inhibits myosin light chain kinase (MLCK) [ 171 which is also half inhibited by 97 PM H-7, 17 nM K252a and 18 nM KT5926. Stomata1closing to ABA,,, was specifically suppressed by 1 yM KT5926 and 20 @I ML-7, whereas 1 @I K-252a inhibited the responses both ABA,, and ABA,,,, (figure 2). Given to the inhibitory properties to MLCK exhibited by ML-7 and KT5926, our data suggestthat protein phosphorylations catalysed by kinases of the MLCK type could mediate the responseto ABA,,. This is further supported by the finding that, at the concentration of 20 @I which is not inhibitory to MLCK, H-7 did not affect the responseto ABA ,() (figure 2 A). Although K-252a and KT5926 similarly inhibit MLCK, they differentially affected stomata1closing to ABA,,,,, cfigure 2 B). Therefore, contrasting with the responseto ABA,,, stomata] closing to ABA ,o0might not involve any kinases of the MLCK type. Since MLCK activity depends on the Ca-CaM complex (Ca2+-CaM) [21], our data suggest that Ca2+-CaM dependent protein phosphorylations or protein kinases regulated by a CaM-like domain [16] might be involved in the responseto ABA,, but not to ABA,,,,,. This was investigated by studying the effect of the CaM antagonists N-(6-aminohexyl)-l -naphthalenesulphonamide (W-5) and N-(6aminohexyl)-5-chloro-lnaphthalenesulphonamide(W-7) on the stomata] responsesto ABA,, and ABA,,,,,.

2.2. Effect of protein antagonists

kinase inhibitors

and CaM

The protein kinase inhibitors I-(5-isoquinolinesulphonyl)-2-methylpiperazine (H-7), K-252a, KT.5926


Plant Physiol. B&hem.

Ca*+-dependent

stomata1 closing responses to abscisic acid

259

Figure 2. Influence of protein kinaae inhibitors on ABA-induced stomatal closing. Differential eflect of the protein kinase inhibitors H-7 (20 @I). K-252a (I pM), KT.5926 (I ~.IM) and ML-7 (20 FM) on the induction of stomata1 closing by (A) 10 nM ABA (ABA,,,) and (B) lOOnM ABA (ABA,,,,). Stomata] apertures were measured just before (base line) and 2 h after (columns) the application of ABA. Each experiment was at least duplicated.

Figure 3. Influence of calmodulin antagonists on ABA-induced stomatal closing. Differential effect of the calmodulin antagonists W-S (20 ~.IM) and W-7 (20 FM) on the induction of stomata1 closing by (A) IO nM ABA (ABA,,) and (B) 100 nM ABA (ABA,,,). Stomata1 apertures were measured just before (base line) and 2 h after (columns) the application of ABA. Each experiment wab at lcast duplicated.

The CaM antagonist W-7 (20 @VI) almost completely inhibited stomata1 closing to ABA,, (figure 3 A) but did not affect the responseto ABA,,,,, (figure 3 B), whereas 20 pill W-5 did not significantly change any of the two stomata1responses(&uue 3 A and B). The differential effect of W-5 and W-7 on ABA,,,-induced stomat.al closing would be explained by the less inhibitory action of W-5 on Ca+-CaM compared to W-7 [9]. Therefore, these data are consistent with a possible implication of Ca-CaM dependent processesin the stomata1responseto ABA,,,. In contrast, the fact that 20 pM W-7 did not inhibit stomatal closing to ABA,,,,, suggestthat this last response would not involve any Ca+-CaM dependent process. This viewpoint is supported further by the fact that other CaM antagonists, such as trifluoperazine and fluphenazine gave similar results to those obtained with W-7 (data not shown). From all these data emergesthe possibility that each of the two ABA closing signals could be transduced through specific protein phosphorylations. In an attempt to characterize the ABA,,, and ABAloo closing signals. the concentration of the protonated form of ABA, ABA(H), was modified by varying the external pH at a constant concentration of exogenously applied ABA. Figure 4 shows that acidification of the bathing solution induced a loss of sensitivity to ML-7 and W-7 for the stomata1 closing responses to ABA,,, and

ABAloo. This change in sensitivity was observed at a higher pH for the response to ABA,, (figure 4 C and D) compared to the responseto ABA,,, (figure 4 A and B), which suggeststhat the responsesto ABA,,, and ABA,,,, would be determined by the ABA(H) concentration. It is not possible from these results to conclude whether the protonation is required for ABA uptake or for interacting with a receptor.

2.3. Effect of inhibitors

of G protein functioning

As suggested by figure I, the stomata1 closing responsesto ABA,,, and ABA,, would differentially depend on Ca2+, which might reflect differences in their Ca7+ mobilization. To investigate whether particular steps in Ca2+ mobilization would characterize the type of ABA response, we searchedfor a possible differential effect of the aminosteroid 1-[6-[ [ 17p-3methoxyestra-I ,3,5( lO)-trien-l7-yl]amino]hexyl]-lHpyrrole-2,5-dione (U73 122) on the ABA,,, and ABA,,,, responses.Indeed, U73 122 is a selective inhibitor of receptor- coupled phospholipase C-dependent processesin mammalian cells, such as the production of inositol I ,4.5-triphosphate (InsP,) and I ,2-diacylglycerol (DAG) and Cal+ mobilization [ 191. The aminosteroid U73122 (3 nM) half inhibited the response to ABA,, @gure 5B), whereas it did not affect stomata1closing to ABA,(,,, @gur-e5 C) even at concentrations increasing up to the micromolar range.
vol. 36, no 3 - 1998

260

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stomata1 closing as changed by the external pH. Influence of the external pH on the modulation of ABA- induced stomata1 closing by ML-7 (20 FM) and W-7 (20 yM). Stomata1 apertures were measured just before (base line) and 2 h after (columns) the addition of 10 nM ABA (ABA,,,) at (A) pH 6 or (B) pH 5, and of 100 nM ABA (ABA,,,) at (C) pH 7 or (D) pH 6. Each experiment was at least duplicated.

Figure 4. ABA-induced

0 L

Moreover. in the absence of external ABA, U73122 did not modify the mean stomata1 aperture (figure 5 A). Therefore, since InsP, [6] and DAG [ 111 respectively induce stomata1closing and opening, our finding could be explained by supposing that, within the ABA,, transduction pathway, U73122 could inhibit phospholipaseC activity, resulting in the concomitant decreasesin InsP, and DAG concentrations: the drop in cellular DAG level would close partially the stomata1 pore, thereby preventing full inhibition of stomata1 closing by U73 122. Since InsP, mobilizes Ca2+reserves in vivo [6] and releasesCa*+ from vacuoles in vitro [I], our data suggestthat putative inhibition of phos holipase C by U73122 might block at least one Caf mobilizing step specific to the ABA,,, response. Hypothesis that U73122 could inhibit phospholipase C in C. communis guard cells is supported by the finding that the aminosteroid I-[6-[[ 17p-3methoxyestra- 1,3,5( 10)-trien- 17-yl]amino]hexyl l-2,5pyrrolidine-dione (U73343), a close analogue of U73 122 which does not affect the activity of phospholipase C in mammalian cells [19], did not inhibit the response to ABA,,, at concentrations ranging from nanomolar to micromolar values (figure 5 B, data not shown). Within the surface receptor- G protein- phospholipase C transduction unit of mammalian cells, the action site of U73122 could be at the level of G, proteins coupled to phospholipase CD 120, 22, 231 and linked to specific 7TMS receptors [3]. These features,

of inhibitors of G protein functioning on ABAinduced stomata1 closing. Differential effect of the G protein modulators U73122 (3 nM), U73343 (3 nM), GP Ant-2A (10 FM), GP Ant-2 (10 FM) and masl7 (5 FM) on (A) the maintenance of stomata1 opening in the light under COz-free air (-CO? + Light) and on the induction of stomata1 closing by (B) 10 nM ABA (ABA,,) or(C) 100 nM ABA (ABA,,,,). Stomata1 aperture is measured just before (base line) and 2 h after (columns) the addition of ABA. Each experiment was replicated at least 4 times.

Figure 5. Influence

together with the presenceof G protein-linked 7TMS receptors in the plasma membrane of stomata1guard cells [2], suggestthat the ABA,, signal might be extracellularly perceived by a 7TMS-like receptor and specifically transduced through G, protein mediated activation of phospholipaseCp. Reinforcing this hypothetical scheme is the inhibition of the response to ABA,, by the G protein antagonist Arg-Pro-Lys-ProGln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH? (GP Ant 2A) C&w-e 5 B) which selectively prevents activation of G, proteins in a competitive manner with 7TMS receptors for G, protein binding [15]. However, by itself @gut-e 5 A) or in the presence of 100 nM ABA (figure 5 C), GP Ant-2A increased the mean stomata1 aperture, indicating that inhibition of the response to ABA,,, by GP Ant-2A might also result from a side effect. Furthermore, the responseto ABA,,, was specifically affected by the inactive mastoparan analog mas17 and the G protein antagonist pGlu-Gln-D-TrpPhe-D-Trp-D-Trp-Met-NH2 (GP Ant-2) @gure 5). In animal systems, these compounds selectively inhibit activation of G proteins other than G, proteins, such as Gi proteins [7, 151 which have also been identified in plant systems [ 131.In animal cells, the G, and Gi proteins are linked to different types of receptor [7, 15, 31.

Ca+-dependent

stomata1 closing responses to abscisic acid

261

Therefore, taken together, our data support that each of the two Ca+-dependent stomata1 closing responses to ABA might be initiated by its own receptor. Furthermore, they do not exclude the possible involvement of InsP, in the Ca2+ mobilizing step of the ABA,, transduction pathway, as recently suggested [121. Up to now, only phosphoinositide-specific phospholipase C isozymes closely related to the 6 subfamily have been shown to be induced in plants by high ABA levels [S]. Since regulation of phospholipase C6 in mammalians would occur through reversible binding to the plasma membrane [5], activity of these plant phospholipase C isozymes might be stimulated by putative surface receptors via the functioning of as yet to be identified G proteins. In this context, one can propose that the signal would be specifically transduced ABA,00 through a putative transmembrane unit composed of a surface receptor, a Gi protein and a phospholipase C6 related isozyme. 3. CONCLUSION Our data are consistent with the hypothesis that, depending on the concentration of its protonated form ABA(H) in the apoplast, ABA might modulate stomatal closing through one of two potential transduction pathways that would differ in their Ca+ dependencies and protein phosphorylation events. Furthermore, they suggest that each of these two putative ABA routes might be initiated by its own receptor. The concentration of endogenous ABA(H) might vary in response to different watering conditions. Thus, according to water ava.ilability, ABA might regulate guard cell turgor via two possible Ca2+-dependent transduction pathways. This scheme can contribute to our understanding of the molecular functioning of ABA in the guard cell and, more generally, to our concepts that the plant may respond differently to a given hormone in a concentration-dependent manner. 4. METHODS
4.1. Plant material. Commelina communis (L.) seeds were germinated on moistened cellulose tissue for 10 d. The seedlings were then transferred to pots of coarse sand in a growth chamber with a 14 h photoperiod (25 C, RH 60 %) followed by IO h of darkness (20 C, RH 80 %). Light (250 pmol . m-* s- PPF) was supplied by 150 W mercury lamps (HQI-TS, Osram, Germany). The pots were watered five times a day with half-strength Hoagland solution.

4.2. Bioassay with epidermal strips. Epidermal strips (IO mm x 5 mm) were peeled from the abaxial surface of the youngest fully expanded leaves of 4 week old plants of C. communis. The leaves were harvested at the end of the dark period. Opened stomata were obtained by incubating the peels for 2 h 30 min at 20 C under a photosynthetic photon flux of 270 pmol . me2 SC in 20 mM KCl, IO mM 2-[Nmorpholinolethane sulphonic acid (Sigma), pH 6, aerated with CO; free air (-CO,) obtained by passing dry air over sodalime (Soda Asbestos, Prolabo. Paris, France). Afterwards, irradiation continued for 2 h under -CO, in the presence of (+/-) cis-trans abscisic acid (ABA; Sigma) at IO or 100 nM. The external pH was varied by buffering the bathing solution at pH 5 (with 10 mM citrate) and pH 7 (with IO mM Hepes) at the beginning of the experiment. It was verified that, in the absence of external ABA, these pH buffers did not modify the mean stomata1 aperture. 4.3. Experiments with Ca + buffers. To buffer cytosolic free Ca+, EGTA (Sigma) or BAPTA (Sigma) was added to the bathing solution at the start of the experiment. The 50 mM stock solutions of EGTA and BAPTA contained K+ at I53 and 220 mM respectively. Control bathing solutions contained the K+ salt of the imino-diacetic acid (Interchim) to ad,just their final Kf concentrations at the same values as those exhibited by the EGTA- and BAPTA-containing solutions. 4.4. Experiments with protein kinase inhibitors and calmodulin antagonists. To block protein phosphorylation and Ca*+-calmodulin (CaM) complex dependent processes. the following inhibitors were separately supplied to the bathing solution 30 min before applying ABA: the protein kinase inhibitors H-7. K-252a, KT5926 and ML-7 at the respective concentrations of 20,1, 1 and 20 PM, and the CaM antagonists W-5 and W-7 at 20 yM. These concentrations were retained from preliminary dose/response studies. It was verified that, at these concentrations, H-7, K-252a. KT5926. ML-7. W-5 and W-7 alone changed the mean stomata1 aperture by less than I.5 pm. These inhibitors were dissolved in dimethyl sulphoxyde (DMSO; Sigma), except H-7 which was dissolved in distilled water. The control bathing solutions contained DMSO at the same concentrations (I 0.1 % v/v) as the inhibitor-containing solutions. All these inhibitors were purchased from Biomol Research Laboratories Inc. (Plymouth Meeting, UK). 4.5. Experiments with inhibitors of G protein functioning. To modulate G protein functioning, the following compounds were separately added to the bathing solution 30 min before applying ABA: the aminosteroid U73 122 and its analogue U73343, the G protein antagonist pGlu-Gln-D-TrpPhe-D-Trp-D-Trp-Met-NH* (GP Ant-2) and its analogue GP Ant-2A, and mas 17, an inactive analogue of mastoparan. All these modulators were obtained from Biomol Research Laboratories Inc, (Plymouth Meeting, UK) and used at optimal concentrations, retained from preliminary dose/response studies. Whilst GP Ant-2A and mas 17 were dissolved in stervol. 36, no 3 - 1998

262

A. Cousson, A. Vavasseur rabbit aortic strips, J. Pharmacol. Exp. Ther. 2 17 (198 1) 494-499. [IO] Koomneef M., Reuling G., Karssen C. M., The isolation and characterization of abscisic acid-insensitive mutants of Aruhidopsis thulium, Physiol. Plant. 61 (1984) 377-383. ] Lee Y., Assmann S. M., Diacylglycerols induce both ion pumping in patch-clamped guard-cell protoplasts and opening of intact stomata, Proc. Nat]. Acad. Sci. USA 88 (1991) 2127-2131. ] Lee Y., Choi Y. B., Suh S., Lee J., Assmann S. M., Joe C. O., Kelleher J. F., Crain R. C., Abscisic acid-induced phosphoinositide turnover in guard cell protoplasts of Viciafaba, Plant Physiol. 110 (1996) 987-996. ] Ma H., Yanofsky M. F., Huang H., Isolation and sequence analysis of TGAl cDNAs encoding a tomato G protein o! subunit, Gene 107 (1991) 189-195. [14] McAinsh M. R.. Brownlee C., Hetherington A. M., Abscisic acid-induced elevation of guard cell cytosolic Cazc precedes stomata1 closure, Nature 343 (1990) 186-188. [IS] Mukai H., Munekata E., Higashijima T.. G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding, J. Biol. Chem. 267 (1992) 16237.16243. 1161 Roberts D. M., Harmon A. C., Calcium-modulated proteins: targets of intracellular calcium signals in higher plants, Ann. Rev. Plant Physiol. Plant Mol. Biol. 43 (1992) 375-414. [17] Saitoh M., lshikawa T., Matsushima S., Naka M., Hidaka H., Selective inhibition of catalytic activity of smooth muscle myosin light chain kinase, J. Biol. Chem. 262 ( 1987) 7796-7801. [ 181 Schmidt C., Schelle l., Liao Y.-I., Schroeder J. I., Strong regulation of slow anion channels and abscisic acid signaling in guard cells by phosphorylation and dephosphorylation events, Proc. Natl. Acad. Sci. USA 92 (1995) 9535-9539. [ 191 Smith R. J., Sam L. M., Justen J. M., Bundy G. L., Bala G. A., Bleasdale J. E., Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness, J. Pharmacol. Exp. Ther. 253 (1990) 688-697. [20] Smrcka A. V., Hepler J. R., Brown K. O., Stemweis P. c., Regulation of polyphosphoinositide-specific phospholipase C activity by purified Gq, Science 25 I ( 1991) 804-807. 1211 Somlyo A. I?. Somlyo A. V., 1994. Signal transduction and regulation in smooth muscle, Nature 372 (1994) 23 l-236. 1221 Taylor S. J., Chae H. Z., Rhee S. G., Exton J. H., Activation of the PI isozyme of phospholipase C by a subunits of the G,, class of G proteins, Nature 350 (1991) 516-518. 1231 Yule D. I., Williams J. A., U73122 inhibits Cal+ oscillations in response to cholecystokinin and carbachol but not to JMV-180 in rat pancreatic acinar cells, J. Biol. Chem. 267 (1992) 13830- 13835.

ile double distilled water, U73122, U73343 and GP Ant-2 were dissolved in DMSO (Sigma), and control bathing solutions contained DMSO at the same concentrations (2 0.1 % v/v) as the U73122-, U73343- and GP Ant-2-containing solutions. 4.6. Data analysis. Stomata1 apertures were measured with an optical microscope (Nikon, Optiphot, Tokyo, Japan) fitted with a camera lucida and a digitizing table (Houston Instrument, Austin, TX) linked to a personal computer (Compaq, Houston, TX). For each treatment. five epidermal strips were floated on 10 ml of bathing solution and 10 stomata1 apertures were measured from each epidermal strip. Except when otherwise noted in the figures, each data point represents the mean of 50 stomata1 apertures with the confidence limits to the mean for o! = 0.05. Acknowledgments. The authors thank Prof. Nam Hai Chua (The Rockefeller University, New York, USA) and Dr Andre Vermeglio (CEA, Cadarache, France) for commenting on the manuscript.

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