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Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro
Life Sciences 74 (2004) 1659 – 1669 www.elsevier.com/locate/lifescie
Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro T. Quinn a, M. Molloy a, A. Smyth a, A.W. Baird a,b,* a
Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b The Conway Institute for Biomolecular and Biomedical Sciences, University College Dublin, Ireland Received 14 February 2003; accepted 27 August 2003
Abstract This study investigates the involvement of capacitative Ca2 + entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM – 1 AM, a sarcoplasmic reticulum Ca2 +-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 AM) after 55 min. Contractile responses to thapsigargin (1 AM) were abolished in a Ca2 +-free medium. Subsequent re-addition of Ca2 + (2.5 mM) produced a sustained tonic contraction (99 F 6% of the carbachol response). The contractile response to Ca2 + re-addition following incubation of tissues in a Ca2 +-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 F 4 % vs 100 F 7 % of carbachol; p < 0.05). Contractile responses to Ca2 + re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2 + channel antagonist, nifedipine (10 AM) and (b) the general inhibitor of Ca2 + entry channels including store-operated channels, SK&F96365 (50 AM and 100 AM). In separate experiments, responses to Ca2 + re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 AM). These results suggest that capacitative Ca2 + entry provides a source of activator Ca2 + for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2 + re-addition following depletion of sarcoplasmic reticulum Ca2 + stores with thapsigargin, are mediated in part by Ca2 + entry through voltage-operated Ca2 + channels and by capacitative Ca2 + entry through store-operated Ca2 + channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2 + entry in this tissue may be modulated by tyrosine kinase. D 2003 Elsevier Inc. All rights reserved. Keywords: Capacitative Ca2+ entry; Contraction; Guinea pig gallbladder smooth muscle; Thapsigargin; Tyrosine kinase
* Corresponding author. Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: +353-1-716-6220; fax: +353-1-716-6219. E-mail address: [email protected] (A.W. Baird). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.08.030
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Introduction Smooth muscle contraction is dependent on an increase in concentration of free cytosolic Ca2 +. This can be caused by an influx of extracellular Ca2 + through ion channels on the plasma membrane or by the release of Ca2 + from the sarcoplasmic reticulum. Extracellular Ca2 + can enter through voltageoperated Ca2 + channels (VOCCs) or alternatively through receptor-activated Ca2 + channels (Barritt, 1991; Sanders, 2001). The relative contribution of intracellular and extracellular sources of Ca2 + depends on the tissue and mode of stimulation. A subgroup of receptor-activated Ca2 + channels that has received much attention is the store-operated Ca2 + channels (SOCCs) (Berridge, 1995). Depletion of the Ca2 + stores in the sarcoplasmic reticulum provides the signal for the opening SOCCs in the plasma membrane through which Ca2 + can enter, thus providing so-called capacitative Ca2 + entry (CCE) (Berridge, 1995; Putney, 1990). Sarcoplasmic Ca2 +-ATPase inhibitors such as thapsigargin have played a crucial role as investigative tools in the development of the concept of CCE. Thapsigargin prevents accumulation of Ca2 + by the sarcoplasmic reticulum, which consequently becomes depleted as Ca2 + passively leaks out into the cytoplasm and is not resequestered (Thastrup et al., 1994). Activation of extracellular Ca2 + dependent responses and Ca2 + influx by thapsigargin is regarded as evidence in favour of the involvement of store-regulated Ca2 + entry in cell signalling (Berridge, 1995; Putney, 1990). Until recently, CCE in smooth muscle was thought to be important only for refilling of the depleted sarcoplasmic reticulum and not as a source of activator Ca2 + for the contractile mechanism. However, evidence indicates that this mechanism may be important for the maintenance of sustained tone in some smooth muscle preparations (Gibson et al., 1998). The proposed regulatory mechanisms of CCE include a diffusible factor and direct coupling of inositol 1,4,5triphosphate receptors with the putative Ca2 + entry channels, but still remain to be elucidated (Parekh and Penner, 1997; Putney et al., 2001). In a number of cell types (Parekh and Penner, 1997) including smooth muscle (Burt et al., 1995; De La Fuente et al., 1995; Low, 1996), it has been reported that the link between store depletion and CCE involves a tyrosine kinase pathway. Recently, we reported that Ca2 + entry through SOCCs may provide a source of activator Ca2 + for bradykinin induced contractions of guinea pig gallbladder smooth muscle (O’Riordan et al., 2001). Since this was not the main focus of that study, this study was undertaken to provide further evidence for involvement of capacitative Ca2 + entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle by pharmacologically characterizing the Ca2 + entry pathways involved in contractions mediated by CCE after depletion of Ca2 + stores by thapsigargin. In addition, the effects of the tyrosine kinase inhibitor, genistein, on the contractile responses to CCE were investigated.
Materials and methods Tissue preparation Adult guinea pigs (Dunkin-Hartley) of either sex were killed by cervical dislocation and the gallbladder was removed. The gallbladder was opened and washed several times in Krebs solution
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to remove bile. Three strips of each gallbladder approximately 0.5 cm wide 1.5 cm long were mounted in organ baths containing Krebs solution (15 ml) of the following composition (mM): NaCl, 118; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; glucose, 11.1; NaHCO3, 24.9 and CaCl2, 2.5, maintained at 37jC and gassed with 95% O2 and 5% CO2. A resting pre-load of 0.5 g was applied to each muscle strip which was then allowed to equilibrate for 1 h, during which time the Krebs solution was changed every 20 min. Mechanical activity was recorded using isometric transducers (World Precision Instruments, Stevenage, Herts, U.K.). Tension was continuously monitored and recorded using a MacLab data acquisition system (AD Instruments Ltd., Hastings, U.K.). Experimental protocol Following the equilibration period of 1 h, tissue strips were exposed to carbachol (10 AM) in order to confirm the viability of the tissues and to determine the contractile capacity. Tissues eliciting less than 1 g of tension in response to carbachol were discarded. The carbachol was then washed out and the preparations were left to re-equilibrate for 30 min. To determine the effects of thapsigargin on guinea pig gallbladder smooth muscle, a single dose of thapsigargin (0.1 nM–1AM) was applied to separate tissue strips. The drug was left in contact with the tissue for up to 55 min. Effects of thapsigargin (1 AM) were also determined in a Ca2 +-free Krebs solution containing 1 mM EDTA. To assess the ability of thapsigargin to deplete intracellular Ca2 + stores, we determined the effects of pre-treatment with thapsigargin on the contractile response to carbachol in a Ca2 +-free medium. Control tissues were incubated in a Ca2 +-free Krebs containing 1 mM EDTA with frequent washing over a 5 min period, following which the response to a challenge with carbachol (100 AM) was measured. Other tissues were pre-treated with thapsigargin for 35 min in a normal Krebs solution prior to incubation in the Ca2 +-free Krebs for 5 min and subsequent challenge with carbachol. In another set of experiments, to determine the effects of long term incubation in a Ca2 +-free medium on intracellular Ca2 + stores, tissues were incubated for 35 min in a Ca2 +-free Krebs containing 1 mM EDTA prior to stimulation with carbachol (100 AM). To investigate the presence of store-operated Ca2 + channels we used a Ca2 + add-back procedure following treatment with thapsigargin. Tissue strips were incubated with thapsigargin (1 AM) for 35 min in a Ca2 +-free Krebs containing EDTA (1 mM). The EDTA was removed by washing the tissues over the following 5 min with Ca2 +-free Krebs without EDTA and the tissues left to equilibrate for 20 min. Ca2 + (2.5 mM) was then added to the Krebs solution and the response to this measured. In separate experiments, tissues were incubated in Ca2 +-free Krebs containing EDTA (1 mM) for 35 min in the absence of thapsigargin. Following removal of the EDTA and reequilibration of the tissue, Ca2 + (2.5 mM) was then added to the Krebs solution. To determine the nature of the Ca2 + channels opened in response to depletion of the sarcoplasmic reticulum Ca2 + stores by thapsigargin, the contractile response to Ca2 + re-introduction was determined in the presence of (a) nifedipine (10 AM), an L-type voltage-gated Ca2 + channel antagonist and (b) SK&F96365 (50 and 100 AM), a general inhibitor of Ca2 + entry channels including store-operated channels. Following incubation with thapsigargin in a Ca2 +-free Krebs containing EDTA and removal of the EDTA, tissues were incubated with nifedipine or SK&F96365 for 20 min prior to Ca2 + re-addition. The contractile response to Ca2 + re-addition was also examined following incubation for 20 min with
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the tyrosine kinase inhibitor, genistein (100 AM). In all experiments, separate control and test tissues were studied simultaneously. Statistical analysis Data are presented as mean F S.E.M. The population sample size (n value) refers to the number of animals and not the number of tissue strips. Contractile responses are expressed as a percentage of the carbachol response. Statistical analysis of the data was performed using unpaired Student’s 2-tailed t-test or analysis of variance. P values of less than 0.05 were considered to be significant. Materials Carbachol, nifedipine, thapsigargin, genistein and dimethylsulphoxide were all obtained from Sigma-Aldrich Ltd, Dublin 24, Ireland. SK&F96365 was purchased from CN Biosciences UK, Nottingham, England. Nifedipine, thapsigargin and genestein were dissolved in dimethyl sulphoxide. Carbachol and SK&F 96365 were dissolved in water. Stock solutions of carbachol, thapsigargin and genistein were kept in siliconized plastic tubes, maintained at 18jC. A stock solution of SK&F96365 was prepared and maintained at room temperature. Nifedipine was made up immediately before use.
Results Carbachol (10 AM) produced 2.1 F 0.1 g of tension, (n = 61) in guinea pig gall bladder smooth muscle strips. The carbachol response was used as an indicator of the contractile capacity of the tissue in each individual experiment. All tissue contractile responses were therefore expressed as a percentage of the contraction evoked by carbachol (10 AM). Thapsigargin (0.1 nM–1 AM) caused slowly developing tonic contractions of guinea pig gallbladder smooth muscle (Fig. 1). The peak contractile response to thapsigargin (1 AM) occurred approximately 35 min after its addition (55 F 12% of the carbachol response). The contractile responses to thapsigargin (0.1 AM–0.1 nM) approached but did not appear to reach a maximum over the 55 min experimental period. Analysis of variance showed that the timed contractile responses to thapsigargin (0.1 nM and 10 nM) were significantly different from the timed contractile response to thapsigargin (1 AM) (p < 0.0005). However, there was no significant difference between the final maximal contractile responses obtained with the varying concentrations of thapsigargin used at the final sampling point (55 F 12%, thapsigargin 1 AM at 40 min; 53 F 4%, thapsigargin 0.1 AM at 50 min; 50 F 6%, thapsigargin 10 nM at 50 min; 43 F 9%, thapsigargin 0.1 nM at 55 min). Carbachol (100 AM) elicited a transient contractile response in tissues incubated for 5 min in Ca2 +-free Krebs containing EDTA (1 mM). The contraction was 21.3 F 9% (n = 5) of control contraction in Ca2 + containing Krebs. The contraction returned to basal tension in less than 8 min. After washout with fresh Ca2 +-free solution a second application of carbachol failed to evoke any response. The contractile response to carbachol was abolished in tissues pre-treated with thapsigargin (1 AM) for 35 min prior to incubation in Ca2 +-free Krebs (Fig. 2). Tissues incubated for 35 min in Ca2 +-free Krebs containing EDTA
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Fig. 1. Time course of the contractile responses to varying concentrations of thapsigargin in guinea pig gallbladder smooth muscle strips. Each value represents the mean F SEM of n = 5 – 6 separate experiments. *** P < 0.0005, compared with the response to thapsigargin (1 AM).
Fig. 2. Typical recordings of the effect of pre-treatment with thapsigargin on the contractile response to carbachol in guinea pig gallbladder smooth muscle strips incubated in a Ca2 +-free medium. (A) Control response. Tissues were incubated in a Ca2 +free Krebs containing 1 mM EDTA for 5 min prior to challenge with carbachol (100 AM). A second application of carbachol was made after washout of the first dose. (B) Effect of pre-treatment with thapsigargin. Tissues were pre-treated with thapsigargin for 35 min in a normal Krebs solution prior to incubation in a Ca2 +-free medium for 5 min and subsequent challenge with carbachol.
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(1 mM) failed to elicit a contractile response to carbachol. Each of these conditions was reversible in that upon restoration of Ca2 +, carbachol responsiveness was restored. The contractile response to thapsigargin (1 AM) was abolished in a Ca2 +-free Krebs containing EDTA (1 mM). Subsequent re-addition of Ca2 + (2.5 mM) caused a tonic contraction which was 99 F 5.8% of the contractile response to carbachol (10 AM). The contractile response to Ca2 + re-addition following incubation of tissues in a Ca2 +-free Krebs in the absence of thapsigargin was significantly less than in its presence (79 F 4% vs 100 F 7% of the contractile response to carbachol; Fig. 3A). The rate of onset of response following Ca2 + re-addition was greater in the tissues pre-treated with thapsigargin (Fig. 3B). The contractile response to Ca2 + re-addition following treatment of the tissues with thapsigargin (1 AM) in a Ca2 +-free medium was attenuated by nifedipine (10 AM; 54 F 7% of the carbachol response) and SK&F96365 (50 and 100 AM; 19 F 3% and 9 F 3% of the carbachol response, respectively) and
Fig. 3. A: Contractile responses to Ca2 + (2.5 mM) re-addition following incubation of guinea pig gallbladder smooth muscle strips in Ca2 +-free Krebs containing EDTA (1 mM) in the presence and absence of thapsigargin (1 AM). Values represent contractile responses measured 14 min after addition of Ca2 +. B: Time-course of the contractile response to Ca2 + re-addition. Each value represents the mean F SEM of n = 5 – 8 separate experiments. * P < 0.05.
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Fig. 4. The contractile responses to Ca2 + re-addition following treatment of guinea pig gallbladder smooth muscle strips with thapsigargin (1 AM) in Ca2 +-free Krebs containing EDTA (1 mM) are markedly attenuated by nifedipine and SK&F96365 and essentially abolished by genistein. Each value represents the mean F SEM of n = 5 – 8 separate experiments. Differs significantly from control as follows: ** P < 0.005; *** P < 0.0005. Bracketed * indicates P < 0.05 between identified groups.
essentially abolished by genistein (100 AM; 0.8 F 0.7% of the carbachol response) as shown in Fig. 4. Nifedipine 50 AM had no further inhibitory effect (data not shown).
Discussion Sarcoplasmic reticulum Ca2 +-ATPase inhibitors have been widely used in the study of CCE since they cause receptor-independent depletion of intracellular Ca2 + stores (Berridge, 1995). They have been shown to increase tone in a variety of smooth muscle preparations, suggesting that CCE provides a source of activator Ca2 + for the contractile machinery (Gibson et al., 1998). Previously, we reported that thapsigargin (1 AM) produces extracellular Ca2 + dependent sustained contractions of guinea pig gallbladder smooth muscle (O’Riordan et al., 2001), suggesting that CCE plays a role in excitationcontraction coupling in this tissue. In this study, we confirmed these findings and investigated the effects of varying concentrations of thapsigargin on the contractile response. Interestingly, we found that the contractile responses to thapsigargin (0.1 nM–1 AM) in guinea pig gallbladder smooth muscle were graded with respect to concentration, only in respect of rates of action rather than in terms of efficacy. This suggests that higher concentrations of thapsigargin inhibit the sarcoplasmic Ca2 +-ATPase pumps more rapidly than lower concentrations, resulting in a more rapid depletion of intracellular Ca2 + stores and a faster generated stimulus for Ca2 + influx. Therefore, there would be greater accumulation of cytosolic Ca2 + to signal contraction per unit time. The lack of contractile response to thapsigargin in the absence of extracellular Ca2 + suggests that the leak of Ca2 + from the sarcoplasmic reticulum is slow and insufficient to reach a level to initiate contraction.
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CCE has been shown to be activated by a variety of stimuli, which can release stored Ca2 +. These include normal agonists, a range of pharmacological agents or simply incubation of cells in Ca2 +-free conditions (Berridge, 1995). It has been reported that intracellular Ca2 + stores in gallbladder muscle are quickly depleted in the absence of extracellular Ca2 + (Lee et al., 1989). In this study we functionally assessed the ability of thapsigargin and incubation in a Ca2 +-free medium to deplete intracellular Ca2 + stores in guinea pig gallbladder smooth muscle. Consistent with previous reports, in the absence of extracellular Ca2 + carbachol elicited a contractile response reflecting release of Ca2 + from intracellular stores (Parkman et al., 1996; Akici et al., 2000). A second application of carbachol after washout of the first dose did not elicit any contractile response, indicating that carbachol sensitive Ca2 + internal stores were depleted. Pre-treatment with thapsigargin abolished the contractile response to carbachol in a Ca2 +free medium suggesting that it mobilizes Ca2 + from the same intracellular store as carbachol. Furthermore, the contractile response to carbachol was abolished following incubation for 35 min in a Ca2 +-free medium containing 1 mM EDTA indicating depletion of intracellular Ca2 + stores. Classically, CCE is studied by first depleting the sarcoplasmic Ca2 + stores with thapsigargin or another sarcoplasmic Ca2 +-ATPase inhibitor, in a Ca2 +-free medium. Ca2 + re-addition then reveals the unique inward Ca2 + influx (Takemura et al., 1989). Having established that intracellular Ca2 + stores in gallbladder muscle are depleted by thapsigargin and by incubation in a Ca2 +-free medium, we compared the contractile response to Ca2 + re-addition following incubation of tissue strips in a Ca2 +-free medium in the presence and absence of thapsigargin. In both cases a large tonic contraction occurred in response to Ca2 + re-addition suggesting that depletion of intracellular Ca2 + stores provides the stimulus for Ca2 + influx. However, the maximum contractile response to Ca2 + re-addition was less than that following treatment with thapsigargin as was the rate of development of contractile force. These findings can be explained by the ’superficial buffer barrier’ hypothesis, in which part of the Ca2 + entering smooth muscle through the plasma membrane is actively taken up by the sarcoplasmic reticulum Ca2 +-ATPase into Ca2 + stores before it can reach the contractile myofilaments and activate contraction (Van Breemen et al., 1995). Inhibition of the sarcoplasmic reticulum Ca2 +-ATPase by thapsigargin would interrupt this process thereby leading to an increased cytosolic Ca2 + and an enhanced contractile response as observed. Taken together these results provide additional evidence for the contribution of a CCE pathway to guinea pig gallbladder smooth muscle contraction. A number of studies have indicated that the contractile response to sarcoplasmic reticulum Ca2 +ATPase inhibitors in smooth muscle is caused by Ca2 + entry through both VOCCs and SOCCs; the relative contribution of each depending on the smooth muscle type (Gibson et al., 1998). In this study, the L-type VOCC blocker, nifedipine partially inhibited the contractile response to Ca2 + re-addition following depletion of the sarcoplamic reticulum Ca2 + stores with thapsigargin. Approximately 45% of the contractile response to Ca2 + re-addition in the guinea pig gallbladder may be attributed to Ca2 + influx through VOCCs. Likewise, the contractile response to sarcoplasmic reticulum Ca2 +-ATPase inhibitors is partially reduced by nifedipine in mouse anococcygeus (Wallace et al., 1999), cat gastric fundus (Petkov and Boev, 1996) and guinea pig trachea (Takemoto et al., 1998). In contrast, in the rat aorta the contractile response is abolished by nifedipine (Low et al., 1996), whereas in rat pulmonary artery (Ng and Gurney, 2001) and rat spleen (Burt et al., 1995) the response is largely nifedipine resistant. It is well documented that CCE is insensitive to classical inhibitors of VOCCs but is blocked by the non-selective Ca2 + channel blocker, SK&F96365 (Putney, 1986: Hoth and Penner, 1992; Fasolato et al., 1994). SK&F96365, originally introduced as a blocker of receptor-operated Ca2 + channels has been
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shown to block VOCCs (Merritt et al., 1990). It has also been reported to block CCE after depletion of Ca2 + stores in a variety of cell types (Schilling et al., 1992; Jan et al., 1999; Wallace et al., 1999; Ohta et al., 2000) by direct inhibition of SOCCs. In this study, the contractile response to Ca2 + re-addition was markedly attenuated by SK&F96365. Since this study was carried out in the absence of receptor activation, it is reasonable to postulate that the inhibitory effect of SK&F96365 is due to inhibition of both VOCCs (the nifedipine sensitive component of the contraction) and SOCCs (the nifedipine resistant component). The mechanisms linking store depletion to the opening of SOCCs still remain elusive. The most consistent information appears to concern tyrosine phosphorylation which has been reported to modulate CCE in a variety of cells (Montero et al., 1994; Sargeant et al., 1994; Tepel et al., 1994; Yule et al., 1994; Sharma and Davis, 1996). Genistein, one of the most commonly used tyrosine kinase inhibitors, has been shown to inhibit contractions induced by CCE in pulmonary (De La Fuente et al., 1995) and mesenteric arteries (Low, 1996), in mouse anococcygeus (Wallace et al., 1999) and in rat spleen (Burt et al., 1995). It has been reported that genistein can inhibit CCE without inhibition of protein tyrosine phosphorylation, raising some doubts about the regulatory role of tyrosine kinase in CCE (Krause et al., 1996; Vostal and Shafer, 1996). Genistein has also been reported to have an inhibitory action on L-type channels (Liu and Sperelakis, 1997). In the present study, genistein abolished the contractions induced by Ca2 + re-addition following depletion of intracellular Ca2 + stores with thapsigargin. Furthermore, it abolished the contractile response to high K+ (60 mM, data not shown). These findings are consistent with its known inhibitory actions on CCE and VOCCs. However, whether the inhibitory effect of genistein on the contractile response to CCE in guinea pig gallbladder smooth muscle involves inhibition of tyrosine kinase remains to be elucidated. Conclusion The present study has shown that thapsigargin induces sustained tonic contractions of guinea pig gallbladder smooth muscle. The contractile response to Ca2 + re-addition following depletion of sarcoplasmic reticulum Ca2 + stores with thapsigargin is mediated in part by Ca2 + entry through VOCCs and CCE through SOCCs. These results suggest that CCE plays a role in excitation-contraction coupling in guinea pig gallbladder smooth muscle. In addition, a tyrosine kinase may be involved in the signal transduction pathway for CCE in this tissue. References Akici, A., Karaalp, A., Iskender, E., Christopoulos, A., El-Fakahany, E.E., Oktay, S., 2000. Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder. European Journal of Pharmacology 388 (1), 115 – 123. Barritt, G.J., 1991. Receptor-activated Ca2 + inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2 + signalling requirements. The Biochemical Journal 337 (Pt 2), 153 – 169. Berridge, M.J., 1995. Capacitative calcium entry. The Biochemical Journal 312, 1 – 11. Burt, R.P., Chapple, C.R., Marshall, I., 1995. The role of capacitative Ca2 + influx in the a1h-adrenoreceptor-mediated contraction to phenylephrine of the rat spleen. British Journal of Pharmacology 116 (4), 2327 – 2333. De La Fuente, P.G., Savineau, J.P., Marthan, R., 1995. Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2 + pump blockers; thapsigargin and cyclopiazonic acid. Pflugers Archiv: European Journal of Physiology 429 (5), 617 – 624.
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Takemura, H., Hughes, A.R., Thastrup, O., Putney Jr., J.W. 1989. Activation of calcium entry by the tumor promoter thapsigargin in parotid acinar cells: Evidence that an intracellular calcium pool and not an inositol phosphate regulates calcium fluxes at the plasma membrane. Journal of Biological Chemistry 264 (21), 12266 – 12271. Tepel, M., Kuhnapfel, S., Theilmeier, G., Teupe, C., Schlotmann, R., Zidek, W., 1994. Filling state of intracellular Ca2 + pools triggers trans plasma membrane Na+ and Ca2 + influx by a tyrosine kinase-dependent pathway. Journal of Biological Chemistry 269 (42), 26239 – 26242. Thastrup, O., Dawson, A.P., Scharff, O., Foder, B., Cullen, P.J., Drobak, B.K., Bjerrum, P.J., Christensen, S.B., Hanley, M.R., 1994. Thapsigargin, a novel molecular probe for studying intracellular calcium release and storage. Agents and Actions 43 (3 – 4), 187 – 193. Van Breemen, C., Chen, Q., Laher, I., 1995. Superficial buffer barrier function of smooth muscle sarcoplasmic reticulum. Trends in Pharmacological Sciences 16 (3), 98 – 105. Vostal, J.G., Shafer, B., 1996. Thapsigargin-induced calcium influx in the absence of detectable tyrosine phosphorylation in human platelets. Journal of Biological Chemistry 271 (32), 19524 – 19529. Wallace, P., Aymam, S., McFadzean, I., Gibson, A., 1999. Thapsigargin-induced tone and capacitative calcium influx in mouse anyococcygeus smooth muscle cells. Nauyn-Schmiedeberg’s Archives of Pharmacology 360 (4), 368 – 375. Yule, D.I., Kim, E.T., Williams, J.A., 1994. Tyrosine kinase inhibitors attenuate ‘‘capacitative’’ Ca2 + influx in rat pancreatic acinar cells. Biochemical and Biophysical Research Communications 202 (3), 1697 – 1704.
Capacitative calcium entry in guinea pig gallbladder smooth muscle in vitro T. Quinn a, M. Molloy a, A. Smyth a, A.W. Baird a,b,* a
Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland b The Conway Institute for Biomolecular and Biomedical Sciences, University College Dublin, Ireland Received 14 February 2003; accepted 27 August 2003
Abstract This study investigates the involvement of capacitative Ca2 + entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle. Thapsigargin (0.1 nM – 1 AM, a sarcoplasmic reticulum Ca2 +-ATPase inhibitor) produced slowly developing sustained tonic contractions in guinea pig isolated gallbladder strips. All contractions approached 50% of the response to carbachol (10 AM) after 55 min. Contractile responses to thapsigargin (1 AM) were abolished in a Ca2 +-free medium. Subsequent re-addition of Ca2 + (2.5 mM) produced a sustained tonic contraction (99 F 6% of the carbachol response). The contractile response to Ca2 + re-addition following incubation of tissues in a Ca2 +-free bathing solution in the absence of thapsigargin was significantly less than in its presence (79 F 4 % vs 100 F 7 % of carbachol; p < 0.05). Contractile responses to Ca2 + re-addition following treatment with thapsigargin were attenuated by (a) the L-type voltage-operated Ca2 + channel antagonist, nifedipine (10 AM) and (b) the general inhibitor of Ca2 + entry channels including store-operated channels, SK&F96365 (50 AM and 100 AM). In separate experiments, responses to Ca2 + re-addition were essentially abolished by the tyrosine kinase inhibitor, genistein (100 AM). These results suggest that capacitative Ca2 + entry provides a source of activator Ca2 + for guinea pig gallbladder smooth muscle contraction. Contractile responses to Ca2 + re-addition following depletion of sarcoplasmic reticulum Ca2 + stores with thapsigargin, are mediated in part by Ca2 + entry through voltage-operated Ca2 + channels and by capacitative Ca2 + entry through store-operated Ca2 + channels which can be blocked by SK&F96365. Furthermore, capacitative Ca2 + entry in this tissue may be modulated by tyrosine kinase. D 2003 Elsevier Inc. All rights reserved. Keywords: Capacitative Ca2+ entry; Contraction; Guinea pig gallbladder smooth muscle; Thapsigargin; Tyrosine kinase
* Corresponding author. Department of Veterinary Physiology and Biochemistry, Faculty of Veterinary Medicine, University College Dublin, Belfield, Dublin 4, Ireland. Tel.: +353-1-716-6220; fax: +353-1-716-6219. E-mail address: [email protected] (A.W. Baird). 0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.08.030
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Introduction Smooth muscle contraction is dependent on an increase in concentration of free cytosolic Ca2 +. This can be caused by an influx of extracellular Ca2 + through ion channels on the plasma membrane or by the release of Ca2 + from the sarcoplasmic reticulum. Extracellular Ca2 + can enter through voltageoperated Ca2 + channels (VOCCs) or alternatively through receptor-activated Ca2 + channels (Barritt, 1991; Sanders, 2001). The relative contribution of intracellular and extracellular sources of Ca2 + depends on the tissue and mode of stimulation. A subgroup of receptor-activated Ca2 + channels that has received much attention is the store-operated Ca2 + channels (SOCCs) (Berridge, 1995). Depletion of the Ca2 + stores in the sarcoplasmic reticulum provides the signal for the opening SOCCs in the plasma membrane through which Ca2 + can enter, thus providing so-called capacitative Ca2 + entry (CCE) (Berridge, 1995; Putney, 1990). Sarcoplasmic Ca2 +-ATPase inhibitors such as thapsigargin have played a crucial role as investigative tools in the development of the concept of CCE. Thapsigargin prevents accumulation of Ca2 + by the sarcoplasmic reticulum, which consequently becomes depleted as Ca2 + passively leaks out into the cytoplasm and is not resequestered (Thastrup et al., 1994). Activation of extracellular Ca2 + dependent responses and Ca2 + influx by thapsigargin is regarded as evidence in favour of the involvement of store-regulated Ca2 + entry in cell signalling (Berridge, 1995; Putney, 1990). Until recently, CCE in smooth muscle was thought to be important only for refilling of the depleted sarcoplasmic reticulum and not as a source of activator Ca2 + for the contractile mechanism. However, evidence indicates that this mechanism may be important for the maintenance of sustained tone in some smooth muscle preparations (Gibson et al., 1998). The proposed regulatory mechanisms of CCE include a diffusible factor and direct coupling of inositol 1,4,5triphosphate receptors with the putative Ca2 + entry channels, but still remain to be elucidated (Parekh and Penner, 1997; Putney et al., 2001). In a number of cell types (Parekh and Penner, 1997) including smooth muscle (Burt et al., 1995; De La Fuente et al., 1995; Low, 1996), it has been reported that the link between store depletion and CCE involves a tyrosine kinase pathway. Recently, we reported that Ca2 + entry through SOCCs may provide a source of activator Ca2 + for bradykinin induced contractions of guinea pig gallbladder smooth muscle (O’Riordan et al., 2001). Since this was not the main focus of that study, this study was undertaken to provide further evidence for involvement of capacitative Ca2 + entry in excitation-contraction coupling in guinea pig gallbladder smooth muscle by pharmacologically characterizing the Ca2 + entry pathways involved in contractions mediated by CCE after depletion of Ca2 + stores by thapsigargin. In addition, the effects of the tyrosine kinase inhibitor, genistein, on the contractile responses to CCE were investigated.
Materials and methods Tissue preparation Adult guinea pigs (Dunkin-Hartley) of either sex were killed by cervical dislocation and the gallbladder was removed. The gallbladder was opened and washed several times in Krebs solution
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to remove bile. Three strips of each gallbladder approximately 0.5 cm wide 1.5 cm long were mounted in organ baths containing Krebs solution (15 ml) of the following composition (mM): NaCl, 118; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.2; glucose, 11.1; NaHCO3, 24.9 and CaCl2, 2.5, maintained at 37jC and gassed with 95% O2 and 5% CO2. A resting pre-load of 0.5 g was applied to each muscle strip which was then allowed to equilibrate for 1 h, during which time the Krebs solution was changed every 20 min. Mechanical activity was recorded using isometric transducers (World Precision Instruments, Stevenage, Herts, U.K.). Tension was continuously monitored and recorded using a MacLab data acquisition system (AD Instruments Ltd., Hastings, U.K.). Experimental protocol Following the equilibration period of 1 h, tissue strips were exposed to carbachol (10 AM) in order to confirm the viability of the tissues and to determine the contractile capacity. Tissues eliciting less than 1 g of tension in response to carbachol were discarded. The carbachol was then washed out and the preparations were left to re-equilibrate for 30 min. To determine the effects of thapsigargin on guinea pig gallbladder smooth muscle, a single dose of thapsigargin (0.1 nM–1AM) was applied to separate tissue strips. The drug was left in contact with the tissue for up to 55 min. Effects of thapsigargin (1 AM) were also determined in a Ca2 +-free Krebs solution containing 1 mM EDTA. To assess the ability of thapsigargin to deplete intracellular Ca2 + stores, we determined the effects of pre-treatment with thapsigargin on the contractile response to carbachol in a Ca2 +-free medium. Control tissues were incubated in a Ca2 +-free Krebs containing 1 mM EDTA with frequent washing over a 5 min period, following which the response to a challenge with carbachol (100 AM) was measured. Other tissues were pre-treated with thapsigargin for 35 min in a normal Krebs solution prior to incubation in the Ca2 +-free Krebs for 5 min and subsequent challenge with carbachol. In another set of experiments, to determine the effects of long term incubation in a Ca2 +-free medium on intracellular Ca2 + stores, tissues were incubated for 35 min in a Ca2 +-free Krebs containing 1 mM EDTA prior to stimulation with carbachol (100 AM). To investigate the presence of store-operated Ca2 + channels we used a Ca2 + add-back procedure following treatment with thapsigargin. Tissue strips were incubated with thapsigargin (1 AM) for 35 min in a Ca2 +-free Krebs containing EDTA (1 mM). The EDTA was removed by washing the tissues over the following 5 min with Ca2 +-free Krebs without EDTA and the tissues left to equilibrate for 20 min. Ca2 + (2.5 mM) was then added to the Krebs solution and the response to this measured. In separate experiments, tissues were incubated in Ca2 +-free Krebs containing EDTA (1 mM) for 35 min in the absence of thapsigargin. Following removal of the EDTA and reequilibration of the tissue, Ca2 + (2.5 mM) was then added to the Krebs solution. To determine the nature of the Ca2 + channels opened in response to depletion of the sarcoplasmic reticulum Ca2 + stores by thapsigargin, the contractile response to Ca2 + re-introduction was determined in the presence of (a) nifedipine (10 AM), an L-type voltage-gated Ca2 + channel antagonist and (b) SK&F96365 (50 and 100 AM), a general inhibitor of Ca2 + entry channels including store-operated channels. Following incubation with thapsigargin in a Ca2 +-free Krebs containing EDTA and removal of the EDTA, tissues were incubated with nifedipine or SK&F96365 for 20 min prior to Ca2 + re-addition. The contractile response to Ca2 + re-addition was also examined following incubation for 20 min with
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the tyrosine kinase inhibitor, genistein (100 AM). In all experiments, separate control and test tissues were studied simultaneously. Statistical analysis Data are presented as mean F S.E.M. The population sample size (n value) refers to the number of animals and not the number of tissue strips. Contractile responses are expressed as a percentage of the carbachol response. Statistical analysis of the data was performed using unpaired Student’s 2-tailed t-test or analysis of variance. P values of less than 0.05 were considered to be significant. Materials Carbachol, nifedipine, thapsigargin, genistein and dimethylsulphoxide were all obtained from Sigma-Aldrich Ltd, Dublin 24, Ireland. SK&F96365 was purchased from CN Biosciences UK, Nottingham, England. Nifedipine, thapsigargin and genestein were dissolved in dimethyl sulphoxide. Carbachol and SK&F 96365 were dissolved in water. Stock solutions of carbachol, thapsigargin and genistein were kept in siliconized plastic tubes, maintained at 18jC. A stock solution of SK&F96365 was prepared and maintained at room temperature. Nifedipine was made up immediately before use.
Results Carbachol (10 AM) produced 2.1 F 0.1 g of tension, (n = 61) in guinea pig gall bladder smooth muscle strips. The carbachol response was used as an indicator of the contractile capacity of the tissue in each individual experiment. All tissue contractile responses were therefore expressed as a percentage of the contraction evoked by carbachol (10 AM). Thapsigargin (0.1 nM–1 AM) caused slowly developing tonic contractions of guinea pig gallbladder smooth muscle (Fig. 1). The peak contractile response to thapsigargin (1 AM) occurred approximately 35 min after its addition (55 F 12% of the carbachol response). The contractile responses to thapsigargin (0.1 AM–0.1 nM) approached but did not appear to reach a maximum over the 55 min experimental period. Analysis of variance showed that the timed contractile responses to thapsigargin (0.1 nM and 10 nM) were significantly different from the timed contractile response to thapsigargin (1 AM) (p < 0.0005). However, there was no significant difference between the final maximal contractile responses obtained with the varying concentrations of thapsigargin used at the final sampling point (55 F 12%, thapsigargin 1 AM at 40 min; 53 F 4%, thapsigargin 0.1 AM at 50 min; 50 F 6%, thapsigargin 10 nM at 50 min; 43 F 9%, thapsigargin 0.1 nM at 55 min). Carbachol (100 AM) elicited a transient contractile response in tissues incubated for 5 min in Ca2 +-free Krebs containing EDTA (1 mM). The contraction was 21.3 F 9% (n = 5) of control contraction in Ca2 + containing Krebs. The contraction returned to basal tension in less than 8 min. After washout with fresh Ca2 +-free solution a second application of carbachol failed to evoke any response. The contractile response to carbachol was abolished in tissues pre-treated with thapsigargin (1 AM) for 35 min prior to incubation in Ca2 +-free Krebs (Fig. 2). Tissues incubated for 35 min in Ca2 +-free Krebs containing EDTA
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Fig. 1. Time course of the contractile responses to varying concentrations of thapsigargin in guinea pig gallbladder smooth muscle strips. Each value represents the mean F SEM of n = 5 – 6 separate experiments. *** P < 0.0005, compared with the response to thapsigargin (1 AM).
Fig. 2. Typical recordings of the effect of pre-treatment with thapsigargin on the contractile response to carbachol in guinea pig gallbladder smooth muscle strips incubated in a Ca2 +-free medium. (A) Control response. Tissues were incubated in a Ca2 +free Krebs containing 1 mM EDTA for 5 min prior to challenge with carbachol (100 AM). A second application of carbachol was made after washout of the first dose. (B) Effect of pre-treatment with thapsigargin. Tissues were pre-treated with thapsigargin for 35 min in a normal Krebs solution prior to incubation in a Ca2 +-free medium for 5 min and subsequent challenge with carbachol.
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(1 mM) failed to elicit a contractile response to carbachol. Each of these conditions was reversible in that upon restoration of Ca2 +, carbachol responsiveness was restored. The contractile response to thapsigargin (1 AM) was abolished in a Ca2 +-free Krebs containing EDTA (1 mM). Subsequent re-addition of Ca2 + (2.5 mM) caused a tonic contraction which was 99 F 5.8% of the contractile response to carbachol (10 AM). The contractile response to Ca2 + re-addition following incubation of tissues in a Ca2 +-free Krebs in the absence of thapsigargin was significantly less than in its presence (79 F 4% vs 100 F 7% of the contractile response to carbachol; Fig. 3A). The rate of onset of response following Ca2 + re-addition was greater in the tissues pre-treated with thapsigargin (Fig. 3B). The contractile response to Ca2 + re-addition following treatment of the tissues with thapsigargin (1 AM) in a Ca2 +-free medium was attenuated by nifedipine (10 AM; 54 F 7% of the carbachol response) and SK&F96365 (50 and 100 AM; 19 F 3% and 9 F 3% of the carbachol response, respectively) and
Fig. 3. A: Contractile responses to Ca2 + (2.5 mM) re-addition following incubation of guinea pig gallbladder smooth muscle strips in Ca2 +-free Krebs containing EDTA (1 mM) in the presence and absence of thapsigargin (1 AM). Values represent contractile responses measured 14 min after addition of Ca2 +. B: Time-course of the contractile response to Ca2 + re-addition. Each value represents the mean F SEM of n = 5 – 8 separate experiments. * P < 0.05.
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Fig. 4. The contractile responses to Ca2 + re-addition following treatment of guinea pig gallbladder smooth muscle strips with thapsigargin (1 AM) in Ca2 +-free Krebs containing EDTA (1 mM) are markedly attenuated by nifedipine and SK&F96365 and essentially abolished by genistein. Each value represents the mean F SEM of n = 5 – 8 separate experiments. Differs significantly from control as follows: ** P < 0.005; *** P < 0.0005. Bracketed * indicates P < 0.05 between identified groups.
essentially abolished by genistein (100 AM; 0.8 F 0.7% of the carbachol response) as shown in Fig. 4. Nifedipine 50 AM had no further inhibitory effect (data not shown).
Discussion Sarcoplasmic reticulum Ca2 +-ATPase inhibitors have been widely used in the study of CCE since they cause receptor-independent depletion of intracellular Ca2 + stores (Berridge, 1995). They have been shown to increase tone in a variety of smooth muscle preparations, suggesting that CCE provides a source of activator Ca2 + for the contractile machinery (Gibson et al., 1998). Previously, we reported that thapsigargin (1 AM) produces extracellular Ca2 + dependent sustained contractions of guinea pig gallbladder smooth muscle (O’Riordan et al., 2001), suggesting that CCE plays a role in excitationcontraction coupling in this tissue. In this study, we confirmed these findings and investigated the effects of varying concentrations of thapsigargin on the contractile response. Interestingly, we found that the contractile responses to thapsigargin (0.1 nM–1 AM) in guinea pig gallbladder smooth muscle were graded with respect to concentration, only in respect of rates of action rather than in terms of efficacy. This suggests that higher concentrations of thapsigargin inhibit the sarcoplasmic Ca2 +-ATPase pumps more rapidly than lower concentrations, resulting in a more rapid depletion of intracellular Ca2 + stores and a faster generated stimulus for Ca2 + influx. Therefore, there would be greater accumulation of cytosolic Ca2 + to signal contraction per unit time. The lack of contractile response to thapsigargin in the absence of extracellular Ca2 + suggests that the leak of Ca2 + from the sarcoplasmic reticulum is slow and insufficient to reach a level to initiate contraction.
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CCE has been shown to be activated by a variety of stimuli, which can release stored Ca2 +. These include normal agonists, a range of pharmacological agents or simply incubation of cells in Ca2 +-free conditions (Berridge, 1995). It has been reported that intracellular Ca2 + stores in gallbladder muscle are quickly depleted in the absence of extracellular Ca2 + (Lee et al., 1989). In this study we functionally assessed the ability of thapsigargin and incubation in a Ca2 +-free medium to deplete intracellular Ca2 + stores in guinea pig gallbladder smooth muscle. Consistent with previous reports, in the absence of extracellular Ca2 + carbachol elicited a contractile response reflecting release of Ca2 + from intracellular stores (Parkman et al., 1996; Akici et al., 2000). A second application of carbachol after washout of the first dose did not elicit any contractile response, indicating that carbachol sensitive Ca2 + internal stores were depleted. Pre-treatment with thapsigargin abolished the contractile response to carbachol in a Ca2 +free medium suggesting that it mobilizes Ca2 + from the same intracellular store as carbachol. Furthermore, the contractile response to carbachol was abolished following incubation for 35 min in a Ca2 +-free medium containing 1 mM EDTA indicating depletion of intracellular Ca2 + stores. Classically, CCE is studied by first depleting the sarcoplasmic Ca2 + stores with thapsigargin or another sarcoplasmic Ca2 +-ATPase inhibitor, in a Ca2 +-free medium. Ca2 + re-addition then reveals the unique inward Ca2 + influx (Takemura et al., 1989). Having established that intracellular Ca2 + stores in gallbladder muscle are depleted by thapsigargin and by incubation in a Ca2 +-free medium, we compared the contractile response to Ca2 + re-addition following incubation of tissue strips in a Ca2 +-free medium in the presence and absence of thapsigargin. In both cases a large tonic contraction occurred in response to Ca2 + re-addition suggesting that depletion of intracellular Ca2 + stores provides the stimulus for Ca2 + influx. However, the maximum contractile response to Ca2 + re-addition was less than that following treatment with thapsigargin as was the rate of development of contractile force. These findings can be explained by the ’superficial buffer barrier’ hypothesis, in which part of the Ca2 + entering smooth muscle through the plasma membrane is actively taken up by the sarcoplasmic reticulum Ca2 +-ATPase into Ca2 + stores before it can reach the contractile myofilaments and activate contraction (Van Breemen et al., 1995). Inhibition of the sarcoplasmic reticulum Ca2 +-ATPase by thapsigargin would interrupt this process thereby leading to an increased cytosolic Ca2 + and an enhanced contractile response as observed. Taken together these results provide additional evidence for the contribution of a CCE pathway to guinea pig gallbladder smooth muscle contraction. A number of studies have indicated that the contractile response to sarcoplasmic reticulum Ca2 +ATPase inhibitors in smooth muscle is caused by Ca2 + entry through both VOCCs and SOCCs; the relative contribution of each depending on the smooth muscle type (Gibson et al., 1998). In this study, the L-type VOCC blocker, nifedipine partially inhibited the contractile response to Ca2 + re-addition following depletion of the sarcoplamic reticulum Ca2 + stores with thapsigargin. Approximately 45% of the contractile response to Ca2 + re-addition in the guinea pig gallbladder may be attributed to Ca2 + influx through VOCCs. Likewise, the contractile response to sarcoplasmic reticulum Ca2 +-ATPase inhibitors is partially reduced by nifedipine in mouse anococcygeus (Wallace et al., 1999), cat gastric fundus (Petkov and Boev, 1996) and guinea pig trachea (Takemoto et al., 1998). In contrast, in the rat aorta the contractile response is abolished by nifedipine (Low et al., 1996), whereas in rat pulmonary artery (Ng and Gurney, 2001) and rat spleen (Burt et al., 1995) the response is largely nifedipine resistant. It is well documented that CCE is insensitive to classical inhibitors of VOCCs but is blocked by the non-selective Ca2 + channel blocker, SK&F96365 (Putney, 1986: Hoth and Penner, 1992; Fasolato et al., 1994). SK&F96365, originally introduced as a blocker of receptor-operated Ca2 + channels has been
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shown to block VOCCs (Merritt et al., 1990). It has also been reported to block CCE after depletion of Ca2 + stores in a variety of cell types (Schilling et al., 1992; Jan et al., 1999; Wallace et al., 1999; Ohta et al., 2000) by direct inhibition of SOCCs. In this study, the contractile response to Ca2 + re-addition was markedly attenuated by SK&F96365. Since this study was carried out in the absence of receptor activation, it is reasonable to postulate that the inhibitory effect of SK&F96365 is due to inhibition of both VOCCs (the nifedipine sensitive component of the contraction) and SOCCs (the nifedipine resistant component). The mechanisms linking store depletion to the opening of SOCCs still remain elusive. The most consistent information appears to concern tyrosine phosphorylation which has been reported to modulate CCE in a variety of cells (Montero et al., 1994; Sargeant et al., 1994; Tepel et al., 1994; Yule et al., 1994; Sharma and Davis, 1996). Genistein, one of the most commonly used tyrosine kinase inhibitors, has been shown to inhibit contractions induced by CCE in pulmonary (De La Fuente et al., 1995) and mesenteric arteries (Low, 1996), in mouse anococcygeus (Wallace et al., 1999) and in rat spleen (Burt et al., 1995). It has been reported that genistein can inhibit CCE without inhibition of protein tyrosine phosphorylation, raising some doubts about the regulatory role of tyrosine kinase in CCE (Krause et al., 1996; Vostal and Shafer, 1996). Genistein has also been reported to have an inhibitory action on L-type channels (Liu and Sperelakis, 1997). In the present study, genistein abolished the contractions induced by Ca2 + re-addition following depletion of intracellular Ca2 + stores with thapsigargin. Furthermore, it abolished the contractile response to high K+ (60 mM, data not shown). These findings are consistent with its known inhibitory actions on CCE and VOCCs. However, whether the inhibitory effect of genistein on the contractile response to CCE in guinea pig gallbladder smooth muscle involves inhibition of tyrosine kinase remains to be elucidated. Conclusion The present study has shown that thapsigargin induces sustained tonic contractions of guinea pig gallbladder smooth muscle. The contractile response to Ca2 + re-addition following depletion of sarcoplasmic reticulum Ca2 + stores with thapsigargin is mediated in part by Ca2 + entry through VOCCs and CCE through SOCCs. These results suggest that CCE plays a role in excitation-contraction coupling in guinea pig gallbladder smooth muscle. In addition, a tyrosine kinase may be involved in the signal transduction pathway for CCE in this tissue. References Akici, A., Karaalp, A., Iskender, E., Christopoulos, A., El-Fakahany, E.E., Oktay, S., 2000. Further evidence for the heterogeneity of functional muscarinic receptors in guinea pig gallbladder. European Journal of Pharmacology 388 (1), 115 – 123. Barritt, G.J., 1991. Receptor-activated Ca2 + inflow in animal cells: a variety of pathways tailored to meet different intracellular Ca2 + signalling requirements. The Biochemical Journal 337 (Pt 2), 153 – 169. Berridge, M.J., 1995. Capacitative calcium entry. The Biochemical Journal 312, 1 – 11. Burt, R.P., Chapple, C.R., Marshall, I., 1995. The role of capacitative Ca2 + influx in the a1h-adrenoreceptor-mediated contraction to phenylephrine of the rat spleen. British Journal of Pharmacology 116 (4), 2327 – 2333. De La Fuente, P.G., Savineau, J.P., Marthan, R., 1995. Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2 + pump blockers; thapsigargin and cyclopiazonic acid. Pflugers Archiv: European Journal of Physiology 429 (5), 617 – 624.
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