Volume 121, Issue 3 p. 451-458
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Specific Gq protein involvement in muscarinic M3 receptor-induced phosphatidylinositol hydrolysis and Ca2+ release in mouse duodenal myocytes

J. L. Morel

J. L. Morel

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France

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N. Macrez

N. Macrez

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France

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J. Mironneau

Corresponding Author

J. Mironneau

Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France

1Laboratoire de Physiologie Cellulaire et Pharmacologie Moléculaire, CNRS ESA 5017, Université de Bordeaux II, 146 rue Léo Saignat, 33076 Bordeaux, France.Search for more papers by this author
First published: 10 February 2009
Citations: 26

Abstract

  • Cytosolic Ca2+ concentration ([Ca2+]i) during exposure to acetylcholine or caffeine was measured in mouse duodenal myocytes loaded with fura-2. Acetylcholine evoked a transient increase in [Ca2+]i followed by a sustained rise which was rapidly terminated after drug removal. Although L-type Ca2+ currents participated in the global Ca2+ response induced by acetylcholine, the initial peak in [Ca2+]i was mainly due to release of Ca2+ from intracellular stores.

  • Atropine, 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, a muscarinic M3 antagonist), pirenzepine (a muscarinic M1 antagonist), methoctramine and gallamine (muscarinic M2 antagonists) inhibited the acetylcholine-induced Ca2+ release, with a high affinity for 4-DAMP and atropine and a low affinity for the other antagonists. Selective protection of muscarinic M2 receptors with methoctramine during 4-DAMP mustard alkylation of muscarinic M3 receptors provided no evidence for muscarinic M2 receptor-activated [Ca2+]i increase.

  • Acetylcholine-induced Ca2+ release was blocked by intracellular dialysis with a patch pipette containing either heparin or an anti-phosphatidylinositol antibody and by external application of U73122 (a phospholipase C inhibitor).

  • Acetylcholine-induced Ca2+ release was insensitive to external pretreatment with pertussis toxin, but concentration-dependently inhibited by intracellular dialysis with a patch pipette solution containing an anti-αq11 antibody. An antisense oligonucleotide approach revealed that only the Gq protein was involved in acetylcholine-induced Ca2+ release.

  • Intracellular applications of either an anti-βcom antibody or a peptide corresponding to the Gβγ binding domain of the β-adrenoceptor kinase 1 had no effect on acetylcholine-induced Ca2+ release.

  • Our results show that, in mouse duodenal myocytes, acetylcholine-induced release of Ca2+ from intracellular stores is mediated through activation of muscarinic M3 receptors which couple with a Gq protein to activate a phosphatidylinositol-specific phospholipase C.

Introduction

Pharmacological identification of muscarinic receptors in intestinal smooth muscles has revealed the existence of several receptor subtypes. Although it appears that muscarinic M3 receptors primarily mediate smooth muscle contraction, the muscarinic M3 receptor subtype accounts for only 30% of the total muscarinic receptors, whereas the remaining majority of receptors (70%) would be of the muscarinic M2 receptor subtype (Michel & Whiting, 1990). Both muscarinic M2 and M3 receptor subtypes have been shown to mediate phosphatidylinositol hydrolysis, resulting in the generation of inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol (Thomas & Ehlert, 1994; Prestwich & Bolton, 1995). InsP3 accumulation has been demonstrated to couple to Ca2+ release from intracellular stores, triggering acetylcholine-induced contraction in ileal smooth muscle (Thomas & Ehlert, 1994).

The muscarinic m1, m3 and m5 receptors have been shown to stimulate phospholipase C-β (PLC-β) via pertussis toxin (PTX)-insensitive G proteins of the Gq family, whereas muscarinic m2 and m4 receptors couple to PTX-sensitive Gi/0 proteins (Ashkenazi et al., 1989). Both PTX-sensitive and PTX-insensitive G proteins can transduce Ca2+ release since the α subunits of the Gq family activate various PLC isoforms, i.e. PLC-β1, -β3 and -β4 (Jhon et al., 1993), whereas the βγ dimers activate PLC-β2 and -β3 (Camps et al., 1992).

The purpose of the present study was two fold: (1) to investigate the changes in [Ca2+]i during exposure to acetylcholine and to assess the contribution of Ca2+ from various sources in single duodenal myocytes, and (2) to identify the receptors, phospholipids and G proteins involved in the acetylcholine-activated Ca2+ release. The results show that the changes in [Ca2+]i are mediated by muscarinic M3 receptor activation and correspond to a transient release of Ca2+ from the intracellular store, as well as to an entry of Ca2+ from outside the cells, partly due to dihydropyridine-sensitive Ca2+ channels. Experiments performed with antibodies directed against phosphatidylinositols and different subtypes of G proteins, with antisense oligonucleotides designed to block synthesis of G protein subunits and with synthetic peptides corresponding to the Gβγ binding domain of the β-adrenoceptor kinase 1 (βARK1) revealed that the muscarinic M3 receptor-activated Ca2+ release from the intracellular store is mediated through activation of a Gq protein and hydrolysis of phosphatidylinositols by a PLC.

Methods

Cell preparation

Swiss mice (20–25 g) were killed by cervical dislocation. The longitudinal layer of the duodenal smooth muscle was cut into several pieces and incubated for 10 min in low Ca2+ (40 μm) physiological solution, then 0.8 mg ml−1 collagenase, 0.2 mg ml−1 pronase E and 1 mg ml−1 bovine serum albumin were added at 37°C for 20 min. After this time, the solution was removed and the pieces of duodenum were incubated again in a fresh enzyme solution at 37°C for 20 min. Tissues were then placed in enzyme-free solution and triturated with a fire-polished Pasteur pipette to release cells. Cells were maintained in short-term primary culture in medium M199 containing 2% foetal calf serum, 2 mm glutamine, 1 mm pyruvate, 20 u ml−1 penicillin and 20 μg ml−1 streptomycin and kept in an incubator gassed with 95% air, 5% CO2 at 37°C and used within 72 h.

Fluorescence measurements

Cells were loaded by incubation in physiological solution containing 1 μm fura-2-acetoxymethylester (fura-2AM) for 30 min at room temperature. These cells were washed and allowed to cleave the dye to the active fura-2 compound for at least 1 h. Fura-2 loading was usually uniform over the cytoplasm and compartmentalization of the dye was never observed.

Measurement of intracellular Ca2+ concentration was carried out by the dual-wavelength fluorescence method, as previously described (Leprêtre et al., 1994a). Briefly, fura-2-loaded cells were mounted in a perfusion chamber and placed on the stage of an inverted microscope (Nikon Diaphot, Tokyo, Japan). A single cell was alternately excited with u.v. light of 340 nm and 380 nm through a 100 × oil-immersion objective (Nikon, 1.3 NA), and emitted fluorescent light from the Ca2+-sensitive dye was collected through a 510 nm long-pass filter with a Charge-Coupled Device camera (Hamamatsu Photonics, Hamamatsu City, Japan). The signal was processed (Hamamatsu Photonics DVS 3000) by correcting each fluorescence image for background fluorescence and calculating 340/380 nm fluorescence ratios on a pixel-by-pixel basis. Averaged frames were usually collected at each wavelength from a single cell every 0.5 s. [Ca2+]i was calculated from mean ratios by use of a calibration curve for fura-2 determined in loaded cells. Some experiments were carried out in the presence of 1 μm oxodipine (a light-stable dihydropyridine derivative) in order to inhibit voltage-dependent Ca2+ channels. All measurements were made at 25 ± 1°C.

Patch-clamp and fluorescence measurements

Voltage-clamp and membrane current recordings were made with a standard patch-clamp technique by use of a List EPC-7 patch-clamp amplifier (Darmstadt-Eberstadt, Germany). The whole-cell recording mode was performed with patch pipettes of 1–3 MΩ resistance. Membrane potential and current records were stored and analysed with an IBM-PC computer (P-clamp system, Axon, Foster City, CA). Simultaneous measurements of intracellular calcium concentration were carried out, as previously described (Leprêtre et al., 1994b). Briefly, 100 μm fura-2 was added to the pipette solution and so entered into the cells following establishment of the whole-cell recording mode. A steady fluorescence was obtained within 3 min. [Ca2+]i was estimated from the 340/380 nm fluorescence ratio by use of a calibration determined within cells. All experiments were carried out at 25 ± 1°C.

The results are expressed as means ± s.e.mean. Significance was tested by means of Student's t test. P values of <0.05 were considered as significant.

Antibodies

Antibodies were added to the pipette solution to allow dialysis of the cell after a break through in whole-cell recording mode for at least 5–8 min, a time longer than that expected theoretically for diffusion of substance in solutions. Purification and specificity of the autoantibody directed against phosphatidylinositol (anti-PtdIns) has been shown previously (Leprêtre et al., 1994a,b). The anti-αq/11 antibody was raised to the C-terminal amino acids (LQLNLKEYNLV) of Gαq/11 subunit. The anti-βcom antibody was raised to the C-terminal amino acids (TDDGMAVATGSWDSFLKIWN) of Gβ1 subunit.

Microinjection of oligonucleotides

Myocytes were seeded at a density of about 103 cells per mm2 on glass slides imprinted with squares for localization of injected cells and maintained in short-term primary culture in medium M199 containing 2% foetal calf serum, 2 mm glutamine, 1 mm pyruvate, 20 u ml−1 penicillin, and 20 μg ml−1 streptomycin; they were kept in an incubator gassed with 95% air, 5% CO2 at 37°C. The sequences of the oligonucleotides used in this study were determined by sequence comparison and multiple alignment by use of Mac Molly Tetra software (Soft Gene, Berlin, Germany). Injection of oligonucleotides was performed into the nucleus of myocytes by a manual injection system (Eppendorf, Hamburg, Germany). The injection solution contained 10 μm oligonucleotides in water; approximately 10 fl were injected with commercially available micro-capillaries (Femtotips, Eppendorf) with an outlet diameter of 0.5 μm. In some control experiments, myocytes were injected only with water and tested in comparison with non-injected cells and cells injected with antisense oligonucleotides. The myocytes were cultured for 3 days in culture medium and the glass slides were transferred into a perfusion chamber for intracellular Ca2+ measurements. The sequences of the anti-αq and anti-α11 oligonucleotides have been published previously (Dippel et al., 1996). The sequence of anti-αq/11com is ATGGACTCCAGAGT, of sense αq/11com is ACTCTGGAGTCCAT corresponding to nt 4–17 of αq cDNA (Strathmann & Simon, 1990) and of scrambled anti-αq/11com is TACGGTCCAGAGTA corresponding to a scrambled sequence of nt 4–17 of αq cDNA (Strathmann & Simon, 1991).

Solutions

The normal physiological solution contained (in mm): NaCl 130, KCl 5.6, MgCl2 1, CaCl2 2, glucose 11 and HEPES 10, pH 7.4 with NaOH. The basic pipette solution contained (in mm): KCl 130, HEPES 10, pH 7.3 with NaOH. Ca2+-free solution was prepared by omitting CaCl2 and by adding 0.5 mm EGTA. Muscarinic agonists and caffeine were applied to the recorded cell by pressure ejection from a glass pipette for the period indicated on the records. Before each experiment a fast application of physiological solution was tested and cells with movement artefacts were excluded.

Chemicals and drugs

Collagenase was obtained from Worthington (Freehold, NJ); pronase (type E), bovine serum albumin, acetylcholine, carbachol, oxotremorine, pilocarpine, pirenzepine, gallamine, thapsigargin, pertussis toxin (PTX), heparin and chondroitin sulphate were from Sigma (St Louis, MO). M199 medium was from Flow Laboratories (Puteaux, France). Foetal calf serum was from Flobio (Courbevoie, France). Streptomycin, penicillin, glutamine and pyruvate were from Gibco (Paisley, U.K.). 4-Diphenylacetoxy-N-(2-chloroethyl)-piperidine hydrochloride (4-DAMP), atropine and methoctramine were from RBI (Natick, MA, U.S.A.). Oxodipine was a gift from Dr Galiano (IQB, Madrid, Spain). Caffeine was from Merck (Nogent sur Marne, France). Fura-2AM, fura-2 and ryanodine were from Calbiochem (Meudon, France). Synthetic peptides corresponding to the Gβγ binding domain of βARK1 (peptide G) or to a region outside the Gβγ binding site (peptide A) were from Genosys (Cambridge, U.K.). 1-(6-((17β-3methoxystra-1,3,5 (10) - trien - 17 - yl) amino) hexyl) - 1H - pyrrole - 2,5 - dione (U73122) and 1-(6-((17β-3methoxystra-1,3,5(10)-trien-17-yl) amino)hexyl)-1H-pyrrolipine-dione (U73343) were from Biomol (Plymouth Meeting, PA, U.S.A.). Anti-βcom antibody (SC378) was from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Anti-αq/11 antibody was a gift from G. Guillon (Montpellier, France) and oligonucleotides were a gift from F. Kalkbrenner (Berlin, Germany).

Results

Effects of acetylcholine and caffeine on [Ca2+]i in duodenal myocytes

In single myocytes isolated from mouse duodenum, ejection of 10 μm acetylcholine for 10 s caused a clear biphasic Ca2+ response (Figure 1Aa). On average, [Ca2+]i increased rapidly from a resting level of 48 ± 2 nm to a peak of 215 ± 26 nm, which was followed by a sustained [Ca2+]i elevation of 79 ± 4 nm (n = 14). The rapid, initial increase in [Ca2+]i was still present in Ca2+-free external solution for 1 min but was decreased by 40 ± 6% (n = 5), whereas the sustained phase was removed (Figure 1Ab). This indicates that the transient increase in [Ca2+]i is largely due to release of Ca2+ from intracellular stores whereas the later sustained Ca2+ response is maintained by Ca2+ entry into the cell from the extracellular space. In the presence of 1 μm oxodipine (a light-resistant di-hydropyridine) for 5 min, the transient Ca2+ response induced by 10 μm acetylcholine was reduced by 25 ± 5% (Figure 1B; n = 9) and the sustained Ca2+ response (measured at the end of acetylcholine ejections) was decreased by 49 ± 6% (Figure 1B; n = 9) whereas the resting Ca2+ level was unchanged. These results suggest that part of the transient and sustained Ca2+ response is due to activation of voltage-dependent L-type Ca2+ channels.

Details are in the caption following the image

Effects of applications of 10 μm acetylcholine (ACh) in single mouse duodenal myocytes. The cells were loaded with fura-2AM and not patch-clamped. (A) In normal physiological solution with 2 mm Ca2+ (a), a long ejection of ACh (10 s) induced a transient increase in [Ca2+]i followed by a sustained phase which decreased rapidly to baseline resting [Ca2+]i upon removal of ACh. In Ca2+-free, 0.5 mm EGTA-containing solution (b), a transient increase in [Ca2+]i was still observed whereas the sustained phase was abolished. (B) Both transient and sustained Ca2+ responses induced by ACh in control conditions (a) were decreased by application of 1 μm oxodipine for 5 min (b).

In order to demonstrate the involvement of the intracellular Ca2+ store in the transient Ca2+ response induced by acetylcholine, complete depletion of the store was evoked by application of ryanodine and caffeine. Ryanodine has been shown to lock the Ca2+ release channels of the intracellular Ca2+ store in an open state so that the stored Ca2+ is progressively reduced (Meissner, 1986). Short (3 s) applications of 10 mm caffeine caused transient increases in [Ca2+]i (Figure 2Aa), the amplitudes of which (190 ± 22 nm, n = 17) were not significantly different from those induced by acetylcholine applications (215 ± 26 nm, n = 14, P>0.05). When the time interval between two successive applications was 3 min, the second transient increase in [Ca2+]i induced by caffeine or acetylcholine was similar to the first Ca2+ response. When the cells were preincubated in the presence of 10 μm ryanodine for 60 min (Figure 2b), the first caffeine application induced a reduced Ca2+ response (166 ± 37 nm, n = 10) whereas the basal [Ca2+]i level was progressively increased to 110 ± 5 nm (n = 10). A second application of caffeine, 3 min later, was ineffective (n = 10). Similarly, the acetylcholine-induced transient increase in [Ca2+]i was not observed after a first application of caffeine (n = 15; Figure 2c), suggesting that acetylcholine released Ca2+ from the intracellular store which possesses ryanodine- and caffeine-sensitive channels. Finally, we used thapsigargin to induce Ca2+ store discharge (Thastrup et al., 1990) by inhibiting the Ca2+-ATPases. Under these conditions, both acetylcholine- and caffeine-induced Ca2+ responses were abolished (n = 8; data not shown), indicating that thapsigargin prevented refilling of an internal Ca2+ store which can be mobilized by acetylcholine and caffeine. In the following experiments, acetylcholine-induced Ca2+ release was measured in Ca2+-free, 0.5 mm EGTA-containing solution for 30 s.

Details are in the caption following the image

Effects of caffeine and ryanodine on the intracellular Ca2+ store. The cells were loaded with fura-2AM and not patch-clamped. (a) A short (3 s) application of 10 mm caffeine (Caf) caused only a transient increase in [Ca2+]i, the amplitude of which was similar to that induced by a second application of caffeine, separated by a 3 min interval. (b) When the cells were preincubated in the presence of 10 μm ryanodine for 60 min, the first 10 mm caffeine application induced a Ca2+ response, but the second one was ineffective. (c) Similarly, no response was evoked with a second application of 10 μm ACh.

Effects of muscarinic agonists and antagonists

The pharmacological profile of the acetylcholine-induced Ca2+ release was examined with compounds displaying selective affinity sequences for muscarinic receptor subtypes. In Figure 3a, the concentration-response curves for acetylcholine and carbachol indicate that maximal Ca2+ release was obtained at 100–300 μm for each compound. The concentrations producing half-maximal response (EC50) were estimated to be 1.6 ± 0.4 μm for acetylcholine and 22.3 ± 1.5 μm for carbachol (n = 4). The Hill coefficients obtained from Hill plots were close to unity. In contrast, oxotremorine produced a limited Ca2+ response which reached about 20–25% of the maximal response and pilocarpine was completely ineffective. A series of compounds antagonized the acetylcholine-induced Ca2+ release (Figure 3b) with the following concentrations producing half-inhibition of the control response (IC50; n = 4–9): 0.6 ± 0.2 nm for 4-DAMP, 0.8 ± 0.2 nm for atropine, 447 ± 78 nm for pirenzepine, 629 ± 67 nm for methoctramine and 50 ± 5 μm for gallamine. The rank order of potency was, therefore, 4-DAMP atropine > pirenzepine methoctramine > gallamine. α-Bungarotoxin (300 nm) had no effect on the acetylcholine-induced Ca2+ release (n = 9).

Details are in the caption following the image

Effects of muscarinic agonists and antagonists on [Ca2+]i in single cells loaded with fura-2 AM and not patch-clamped. (a) Concentration-response curves to (▪) ACh, (▴) carbachol, (▾) oxotremorine and (♦) pilocarpine. [Ca2+]i values were expressed as a percentage of the maximal response induced by ACh. (b) Inhibition of the ACh-stimulated [Ca2+]i changes by (•) 4-DAMP, (▪) atropine, (Δ) pirenzepine, (○) methoctramine and (□) gallamine. [Ca2+]i values are expressed as a percentage of the response obtained with 5 μm ACh. Each point represents the mean for 5–9 cells; vertical lines show s.e.mean.

Moreover, the Ca2+ responses induced by 100 μm oxotremorine were inhibited in a dose-dependent manner by low concentrations of 4-DAMP (IC50 = 0.6 ± 0.2 nm, n = 6) and high concentrations of methoctramine (IC50=315 ± 37 nm, n = 6), suggesting that the oxotremorine-induced Ca2+ response occurred through activation of muscarinic M3 receptors.

In order to ensure the absence of muscarinic M2 receptor-induced Ca2+ response, we initially used 100 nm methoctramine (a concentration that completely but reversibly blocks muscarinic M2 receptors, Doods et al., 1993). Under these conditions, the acetylcholine-induced Ca2+ release reached 148 ± 23 nm (n = 8) and was not significantly different from control values (155 ± 16 nm, n = 14). Second, we pretreated myocytes with 10 nm 4-DAMP mustard and 100 nm methoctramine (in order to protect muscarinic M2 receptors from 4-DAMP mustard alkylation) for 60 min at 37°C (Eglen & Harris, 1993). After washing (90 min at 37°C), Ca2+ release in response to applications of 5 or 10 μm acetylcholine was not detectable (data not shown; n = 38), indicating that muscarinic M2 receptor activation was not involved in intracellular Ca2+ mobilization in duodenal myocytes.

Effects of heparin, anti-PdtIns antibody and phospholipase C inhibitor

We used heparin to block inositol trisphosphate receptors and any further Ca2+ release via these receptors (Guillemette et al., 1989). The effects of acetylcholine and caffeine were studied with heparin in the pipette solution (Figure 4a). In cells held at –70 mV, the basal [Ca2+]i (119 ± 8.5 nm, n = 20) was larger than that measured in non patch-clamped cells loaded with fura-2AM. In the presence of 5 mg ml−1 heparin for 5 min, the basal [Ca2+]i was not different from that observed under control conditions (120 ± 20 nm, n = 7). Application of 10 μm acetylcholine was unable to evoke a noticeable transient Ca2+ release (Figure 4a, n = 5) whereas application of 10 mm caffeine induced a Ca2+ release (135 ± 25 nm, n = 5) similar to control values (130 ± 21 nm, n = 9). This effect was specific to heparin, as, when chondroitin sulphate (5 mg ml−1) was added to the pipette solution instead of heparin, the amplitude of the acetylcholine-induced Ca2+ release was not significantly affected (control: 145 ± 12 nm; in the presence of chondroitin sulphate: 147 ± 18 nm; n = 12). To determine whether phosphatidylinositol hydrolysis is involved in the generation of second messengers in response to muscarinic receptor activation, we tested the effects of an anti-PtdIns antibody on the acetylcholine-induced Ca2+ release. As shown in Figure 4b, intracellular application of 15 μg ml−1 anti-PtdIns antibody for 7 min inhibited the acetylcholine-induced Ca2+ release (n = 8). When anti-PtdIns antibody (15 μg ml−1) was inactivated by heating at 95°C for 30 min, the acetylcholine-induced Ca2+ release was unaffected (control: 150 ± 21 nm, n = 12; in the presence of inactivated anti-PtdIns antibody: 141 ± 26 nm, n = 12). We also tested the effects of U73122 which has been proposed as a phospholipase C inhibitor (Sohn et al., 1993; Macrez-Leprêtre et al., 1996). Pretreatment of the cells with 100 μm U73122 for 5 min largely inhibited the acetylcholine-induced Ca2+ release (Figure 4b; n = 10) while it did not significantly affect the caffeine-induced Ca2+ response (n = 14; data not shown). In contrast, pretreatment of the cells with 100 μm U73343 (the inactive analogue of U73122) did not significantly affect acetylcholine-induced Ca2+ release (Figure 4b).

Details are in the caption following the image

Effects of heparin anti-PdtIns antibody, U73122 and U73343 on the increase in [Ca2+]i induced by ACh or caffeine. In voltage-clamped cells at a holding potential of –70 mV, fura-2 (100 μm) was added to the pipette solution. (a)(i) Ca2+ responses were induced in control conditions by 10 μm ACh. In cells dialysed with a pipette solution containing 5 mg ml−1 heparin for 5 min, Ca2+ responses were induced by ACh (ii) or caffeine (iii). (b) Peak increases in [Ca2+]i evoked by 10 μm ACh in control conditions, in a cell dialysed with 15 μg ml−1 anti-PdtIns antibody or boiled anti-PdtIns antibody (95°C for 30 min) for 7 min through the patch clamp pipette and in cells superfused with 100 μm U73122 or U73343 for 5 min. Data are given as means ± s.e.mean with number of experiments in parentheses. External solution was a Ca2+-free, 0.5 nm EGTA-containing solution. *Values significantly different from those obtained under control conditions, P<0.01.

Inhibition of G protein function and expression

Pertussis toxin (PTX) ADP ribosylates both Gi and Go proteins, thus preventing their activity. Cells were incubated in a culture medium containing 0.5 μg ml−1 PTX for 20 h. This pretreatment suppressed α2-adrenoceptor-induced stimulation of Ca2+ channels, which has been demonstrated to depend on activation of a Gi protein (Macrez-Leprêtre et al., 1995). Acetylcholine-induced Ca2+ release was not affected by the PTX pretreatment (control: 147 ± 20 nm, n = 12; PTX-pre-treated cells: 144 ± 17 nm, n = 12) while intracellular application of 1 mm GDP-β-S for 5–6 min completely abolished acetylcholine-induced Ca2+ release (n = 7), suggesting the involvement of PTX-insensitive G proteins. Antibodies directed against the carboxyl terminus of the α subunits of G proteins have been shown to be useful tools for identifying transduction pathways. When an anti-αq11 antibody was added to the basic pipette solution for 6 min, the acetylcholine-induced Ca2+ release was concentration-dependently inhibited, as illustrated in Figure 5a. Maximal inhibition was obtained with a concentration of 20 μg ml−1 anti-αq11 antibody. Intracellular application of the anti-αq11 antibody (20 μg ml−1) inactivated by heating at 95°C for 30 min did not significantly affect the acetylcholine-induced Ca2+ release (control: 135 ± 25 nm, n = 9; in the presence of inactivated anti-αq11 antibody: 125 ± 21 nm, n = 15; Figure 5a).

Details are in the caption following the image

Effects of anti αq/11 antibody and antisense oligonucleotides on the increase in [Ca2+]i induced by 10 μm ACh. (a) In other voltage-clamped cells (at a holding potential of –70 mV) fura-2 (100 μm) was added to the pipette solution. Shown are ACh-induced Ca2+ responses in control conditions, in cells dialysed with a pipette solution contained various concentrations of anti-αq/11 antibody or 20 μg ml−1 boiled anti-αq/11 antibody (95°C for 30 min) for 6 min. (b) Peak increases in [Ca2+]i evoked by 10 μm ACh (open columns) or 10 mm caffeine (hatched columns) in non-injected cells and in cells injected with 10 μm anti-αq/11, anti-αq or anti-α11 antisense oligonucleotides. Myocytes were used 3 days after injection. *Values significantly different from those obtained in non-injected cells, P<0.01. External solution was a Ca2+-free, 0.5 mm EGTA-containing solution.

In order to discriminate between Gq and G11 protein, we used antisense oligonucleotides designed to inhibit selectively the expression of Gq or G11 protein. For each experiment, we compared the Ca2+ responses of antisense oligonucleotide-injected cells located within a marked area on the glass slide to sense or scrambled oligonucleotide-injected cells or non-injected cells outside this marked area. This procedure ensures that antisense oligonucleotides-injected cells were always compared to control cells that were otherwise grown, treated and analysed under identical conditions, i.e. culture, incubation, microinjection and loading with fura-2 AM. The increases in [Ca2+]i evoked by 10 μm acetylcholine and 10 mm caffeine, in Ca2+-free, 0.5 mm EGTA-containing solution for 30 s, were measured for each cell, and mean values were calculated from all the cells of each experiment. As illustrated in Figure 5b, the acetylcholine-induced Ca2+ release was similarly inhibited in cells injected with 10 μm anti-αq/11com and anti-αq oligonucleotides while the caffeine response was not significantly affected. In contrast, cells injected with 10 μm anti-α11 oligonucleotide showed no inhibition of either acetylcholine- or caffeine-induced Ca2+ responses. Increasing the concentration of injected anti-α11 oligonucleotide to 20 μm did not induce a further inhibition (n = 12). Furthermore, we used sense αq/11com and scrambled anti-αq/11com oligonucleotides which did not significantly anneal to the target sequence of Gαq/11 subunits. Acetylcholine-induced Ca2+ release was not significantly affected by injection of these oligonucleotides (non-injected cells: 148 ± 14 nm, n = 15; sense αq/11com-injected cells: 155 ± 18 nm, n = 12 and scrambled anti-αq/11com-injected cells: 142 ± 11 nm, n = 12). These results suggest different tasks for Gq and G11 proteins since Gq protein transduces the muscarinic signal for Ca2+ release while G11 protein does not.

Effects of anti-βcom antibody and βARK1 peptides

The anti-αq11 antibody and antisense oligonucleotide block of the acetylcholine-induced Ca2+ response cannot distinguish whether α or βγ subunits are transducing the signal that activates Ca2+ release from the intracellular stores. Therefore, we dialysed an anti-βcom antibody into the cell by the patch pipette for 8 min. Anti-βcom antibody (10 μg ml−1) induced a slight increase of acetylcholine-induced Ca2+ release (Figure 6). In a second set of experiments, we dialysed peptides corresponding to fragments of βARK1 (Nair et al., 1995) into the cell by the patch pipette for 5 min. Carboxyl-terminal fragments of βARK1 have been used to bind Gβγ subunits and to block activation of effectors (Stehno-Bittel et al., 1995; Nair et al., 1995). Intracellular applications of 100 μm peptide G (corresponding to the Gβγ binding region of βARK1) or peptide A (corresponding to a domain of βARK1 not involved in Gβγ binding) had no significant effects on acetylcholine-induced Ca2+ release (Figure 6). Taken together, these results suggest that Gβγ subunits are not involved in the muscarinic M3 receptor-activated transduction coupling leading to Ca2+ release from intracellular stores.

Details are in the caption following the image

Effects of anti βcom antibody and βARK1 peptides on the increase in [Ca2+]i induced by 10 μm ACh. In voltage-clamped cells (held at –70 mV), antibodies (10 μg ml−1) and peptides (100 μm) were dialysed intracellularly for 5–8 min with the pipette solution containing 100 μm fura-2. Data are given as means ± s.e.mean with number of experiments in parentheses. External solution was a Ca2+-free, 0.5 nm EGTA-containing solution.

Discussion

Our results show that, in mouse duodenal myocytes, the muscarinic M3 receptor is specifically coupled to the Gq protein and activates a phosphatidylinositol-specific phospholipase C leading to intracellular Ca2+ release. This conclusion is based on experiments with antibodies raised against the carboxyl terminus of Gα subunits to block interactions of G proteins with muscarinic receptors, antisense oligonucleotides to block expression of G protein subunits and synthetic peptides acting as Gβγ subunit inhibitors.

In duodenal myocytes, our results are consistent with the idea that a single intracellular Ca2+ store is mobilized by acetylcholine as well as by caffeine and ryanodine. This conclusion is supported by the following observations: (1) when Ca2+-ATPases were blocked with thapsigargin, which induces Ca2+ leaks from the intracellular Ca2+ store, acetylcholine- and caffeine-induced Ca2+ responses were abolished; (2) intracellular application of heparin (5 mg ml−1), which completely inhibits InsP3 binding at sites responsible for the Ca2+ mobilizing effect of InsP3, suppressed the acetylcholine-induced Ca2+ response; (3) pretreatment with ryanodine and caffeine abolished the intracellular Ca2+ store sensitive to acetylcholine; (4) the basal [Ca2+]i was increased (from 50 to 110 nm) in the presence of ryanodine and caffeine suggesting that depletion of the Ca2+ store leads also to increased Ca2+ entry into the cell (Missiaen et al., 1990). These results suggest that the InsP3- and caffeine-sensitive Ca2+ store may represent, at least functionally, a single releasable Ca2+ pool, in agreement with previous observations in smooth muscle cells (Leprêtre & Mironneau, 1994; Zholos et al., 1994).

Using affinity sequences for agonists and antagonists and the method of irreversible alkylation of muscarinic M3 receptors with 4-DAMP mustard in conjunction with protection of muscarinic M2 receptor by methoctramine (Eglen & Harris, 1993), we showed that only the muscarinic M3 receptor subtype is involved in intracellular Ca2+ release in duodenal myocytes. Evidence supporting this conclusion was obtained from the following results: (1) acetylcholine and carbachol were equally effective at inducing maximal [Ca2+]i increases, while oxotremorine showed partial agonist properties that were antagonized by low concentrations (nm) of 4-DAMP. Pilocarpine (a muscarinic M1 receptor agonist, Baumgold et al., 1995) had no effect at all. The effect of acetylcholine on [Ca2+]i was not affected by α-bungarotoxin indicating that functional nicotinic receptors are not present in duodenal myocytes; (2) 4-DAMP and atropine showed a high affinity (around 1 nm) for inhibiting the acetylcholine-induced Ca2+ release, whereas pirenzepine, methoctramine and gallamine show a low affinity. This affinity sequence is typical of muscarinic M3 receptors (Doods et al., 1994): (3) after alkylation of the muscarinic M3 receptor population by 4-DAMP mustard and protection of muscarinic M2 receptors, application of acetylcholine did not initiate a transient increase in [Ca2+]i. These results are consistent with previous data on the pharmacological profile of muscarinic receptors in smooth muscles (Doods et al., 1993; Eglen & Harris, 1993).

Finally, we showed that the transduction pathway activated by muscarinic M3 receptors leads to stimulation of a Gq protein and hydrolysis of phosphatidylinositols by a phospholipase C. Hydrolysis of phosphatidylinositols by phospholipase C in response to activation of muscarinic M3 receptors was supported by the observations that intracellular applications of heparin or anti-PtdIns antibody inhibited the acetylcholine-induced Ca2+ release. In addition, external application of U73122 (a phospholipase C inhibitor) also blocked the acetylcholine-induced Ca2+ release. Identification of the Gq protein which couples muscarinic M3 receptor to phospholipase C was obtained by using antibodies directed against different subtypes of G proteins and antisense oligonucleotides designed to block synthesis of G protein subunits. Our results indicate that the acetylcholine-induced Ca2+ release is selectively inhibited by an anti-αq/11 antibody. As this antibody is directed against the carboxyl terminus of the Gα subunit, it cannot discriminate between αq and α11 subunits. Therefore, we performed selective inhibitions of αq or α11 subunit by nuclear injection of antisense oligonucleotides. Only inhibition of the αq subunit expression was able to suppress the acetylcholine-induced Ca2+ release in duodenal myocytes. This is in contrast with a previous study on muscarinic m1 receptor-induced Ca2+ responses by Dippel et al. (1996); they proposed a similar role for αq and α11 subunits in inducing release of stored Ca2+. There are at least two explanations for these discrepancies. First, as the latter experiment was performed in Ca2+-containing solution, acetylcholine-induced increases in [Ca2+]i may depend on both Ca2+ release from intracellular stores and Ca2+ influx from the external medium. It is possible that αq and α11 subunits may have distinct functions and couple to Ca2+ release and Ca2+ entry, respectively. It has been recently shown that a nonselective cation channel, the Drosophila trpl channel, may be stimulated in a membrane confined way by the Gα11 subunit (Obukhov et al., 1996). As our experiments were performed in Ca2+-free EGTA-containing solution, we measured only the acetylcholine-induced Ca2+ release from the intracellular store. Second, the receptor-G protein interaction may depend on both the membrane environment and structural constraints achieved by the receptor/G protein/effector complex (Sato et al., 1995). These possibilities could also explain why, depending on the cell type or in vitro assays used, it has been proposed that phospholipase C-β can be activated by several different Gα proteins including Gα0 (Blitzer et al., 1993), Gαi1, Gαi2, Gαi3 (Kaneko et al., 1992; Dell' Acqua et al., 1993), Gαq (Aragay et al., 1992; Wu et al., 1992), Gα11 (Aragay et al., 1992) and Gαs (De la Pena et al., 1995). Although phospholipase C-β activation through the α subunit of Gq and G11 proteins has been largely described, it has been recently shown that, in Xenopus oocytes, activation of overexpressed muscarinic m3 receptors leads to Ca2+ release via the βγ sub-units of Gq/G11 proteins (Stehno-Bittel et al., 1995). In contrast, in duodenal myocytes, the muscarinic M3 receptor-activated Ca2+ release was not affected by an anti-βcom antibody or by synthetic peptides derived from βARK1, which selectively bind to the Gβγ subunits. It is likely that Gβγ sub-units are bound to βARK1 peptides and anti-βcom antibody as these substances, used at similar concentrations, inhibit the angiotensin II-induced stimulation of Ca2+ channels in vascular myocytes (N. Macrez, J.L. Morel, F. Kalkbrenner, P. Viard, G. Schultz & J. Mironneau, unpublished data). Our results suggest that the stimulation of phospholipase C by activation of muscarinic M3 receptors in duodenal myocytes occurs via the α subunit of the Gq protein. As the PLC-β isoforms mediating Ca2+ release from the intracellular store have not been identified in either cell, it can be postulated that in duodenal myocytes, the Gαq subunit predominantly activates PLC-β1 (Jhon et al., 1993) whereas in Xenopus oocytes, the Gβγ subunit predominantly activates PLC-β2 (Camps et al., 1992).

In conclusion, in mouse duodenal myocytes, the release of Ca2+ from the intracellular store is mediated through activation of M3 muscarinic receptors which couple with the Gq protein and activate a phosphatidylinositol-specific phospholipase C-β.

This work was supported by grants from Centre National de la Recherche Scientifique et Centre National des Etudes Spatiales, France. We are indebted to Dr G. Guillon (INSERM 469, Montpellier) for giving us anti-αq11 antibodies and to Pr G. Schultz and Dr F. Kalkbrenner (Institut für Pharmakologie, Berlin) for giving us oligonucleotides.