Differential Coupling of Muscarinic m2 and m3 Receptors to Adenylyl Cyclases V/VI in Smooth Muscle
CONCURRENT m2-MEDIATED INHIBITION VIA Galpha i3 AND m3-MEDIATED STIMULATION VIA Gbeta gamma q*

(Received for publication, February 27, 1997, and in revised form, June 18, 1997)

Karnam S. Murthy and Gabriel M. Makhlouf Dagger

From the Departments of Physiology and Medicine, Medical College of Virginia, Richmond, Virginia 23298-0711

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES


ABSTRACT

Muscarinic m2 and m4 receptors couple preferentially to inhibition of adenylyl cyclase, whereas m1, m3, and m5 receptors couple preferentially to activation of phospholipase C-beta and in some cells to stimulation of cAMP. Smooth muscle cells were shown to express adenylyl cyclases types V and/or VI. Acetylcholine (ACh) stimulated the binding of [35S]GTPgamma S·Galpha complexes in smooth muscle membranes to Galpha q/11 and Galpha i3 antibody. Binding to Galpha q/11 antibody was inhibited by the m3 receptor antagonist, 4-DAMP, and binding to Galpha i3 antibody was inhibited by the m2 receptor antagonist, N,N'-bis[6[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride (methoctramine). The decrease in basal cAMP (35 ± 5%) induced by ACh in dispersed muscle cells was accentuated by 4-DAMP or Gbeta antibody (55 ± 8 to 63 ± 6%). In contrast, methoctramine, pertussis toxin (PTx), or Galpha i3 antibody converted the decrease in cAMP to increase above basal level (+28 ± 5 to +32 ± 6%); the increase in cAMP was abolished by 4-DAMP or Gbeta antibody. In muscle cells where only m3 receptors were preserved by selective receptor protection, ACh caused only an increase in cAMP that was abolished by 4-DAMP. Conversely, in muscle cells where only m2 receptors were preserved, ACh caused an accentuated decrease in cAMP that was abolished by methoctramine or PTx. In conclusion, m2 receptors in smooth muscle couple to inhibition of adenylyl cyclases V/VI via Galpha i3, and m3 receptors couple to activation of the enzymes via Gbeta gamma q/11.


INTRODUCTION

Complementary DNA clones encoding the full sequences of eight isoforms of mammalian adenylyl cyclase (types I-VIII) and the partial sequences of two additional isoforms (types IX and X) have been isolated (1-10). The amino acid sequences (range 1064-1248 residues) are arranged in two cassettes of six transmembrane-spanning domains (9-11). Overall homology is 60% with some cytoplasmic regions exhibiting up to 93% amino acid identity. The presumed catalytic domains (C1a and/or C2a) are homologous to the corresponding domains of membrane-bound homodimeric and soluble heterodimeric guanylyl cyclases (11, 12). Structural homology among the various isoforms is most evident between types II and IV and types V and VI. All the isoforms are expressed in the brain, and types V and VI are the predominant isoforms in the periphery (8-11).

Although functional regulation of adenylyl cyclases is diverse, three broad categories can be distinguished comprising types I, III, and VIII, types II, IV, and probably VII, and types V and VI (10, 11). All adenylyl cyclases are activated by the diterpene, forskolin, and the alpha  subunit of Gs. Types I and VIII, which are expressed exclusively in neurons, are stimulated by submicromolar concentrations of Ca2+ and calmodulin, whereas type III, which is more widely expressed, is stimulated by low micromolar concentrations of Ca2+ (13-16); type I is effectively inhibited by Gbeta gamma but only moderately inhibited by Gi and Go (17-21). Types II and IV are not stimulated by Ca2+/calmodulin or inhibited by Gi but are stimulated by Gbeta gamma (19, 21-24). Stimulation by Gbeta gamma , initially thought to be conditional on concurrent stimulation by Galpha s, is now viewed as highly synergistic, with only modest stimulation by Gbeta gamma alone (21). Types V and VI are inhibited by Gi and by submicromolar concentrations of cytosolic Ca2+ elicited by capacitative Ca2+ influx but not by Ca2+ release from sarcoplasmic stores (24, 25-28); inhibition by Go or stimulation by Gbeta gamma remains uncertain. Both types V and VI contain consensus sequences for phosphorylation and exhibit feedback inhibition, by cAMP-dependent protein kinase (29, 30).

The expression of adenylyl cyclase isoforms in smooth muscle has not been determined. The regulatory pattern suggested by our previous studies in gastrointestinal smooth muscle is consistent with the presence of types V and/or VI. Agonist-induced cAMP formation is mediated by Galpha s (31, 32), and forskolin-stimulated cAMP formation is inhibited, depending on the agonist, by Gi1, Gi2, Gi3, and Go (33-35). Thus, inhibition induced by somatostatin (acting via sstr3) is mediated by Gi1 and Go (33), and inhibition induced by opioid agonists (acting via µ, delta , and kappa  receptors) is mediated by Gi2 and Go (34); inhibition induced by adenosine (acting via A1 receptors) is mediated by Gi3 but not by Go (35). Forskolin-stimulated cAMP formation is inhibited in feedback fashion by cAMP-dependent protein kinase (36). Phorbol esters have no effect on basal or forskolin-stimulated cAMP formation (37). However, inhibition of cAMP formation induced by agonists acting via Gi1 or Gi2 (but not Gi3) is partly reversed by concomitant activation of PKC1; the effect reflects selective PKC-dependent phosphorylation of Galpha i1 and Galpha i2 but not Galpha i3 or Galpha o (37).

It is well established that the muscarinic receptors, m2 and m4, are preferentially coupled to inhibition of adenylyl cyclase; the odd-numbered receptors, m1, m3, and m5, are preferentially coupled to phosphoinositide hydrolysis but, in some cells, can also increase the levels of cAMP (38-44). The mechanisms responsible for the increase in cAMP are likely to reflect the type of adenylyl cyclase expressed in various cells. In cells expressing predominantly Ca2+/calmodulin-sensitive adenylyl cyclases types I, III, and VIII, activation could result from Ca2+ mobilization and activation of PKC (43). This, however, is unlikely in cells expressing predominantly Ca2+/calmodulin-insensitive types II and IV or in cells expressing types V and VI that are inhibited by physiological levels of Ca2+ (10, 28). In the present study, we show that types V and/or VI, the isoforms of adenylyl cyclase expressed in smooth muscle cells, are inhibited by m2 receptors via the alpha  subunit of Gi3 and concurrently activated by m3 receptors via the beta gamma subunits of Gq/11.


EXPERIMENTAL PROCEDURES

Dispersion of Smooth Muscle Cells

Muscle cells were isolated from the circular muscle layer of the rabbit stomach by successive enzymatic digestion, filtration, and centrifugation as described previously (31, 33). Briefly, muscle strips were incubated for 30 min at 31 °C in 15 ml of HEPES medium containing 0.1% collagenase (type II) and 0.1% soybean trypsin inhibitor. The composition of the medium was 120 mM NaCl, 4 mM KCl, 2.6 mM KH2PO4, 2 mM CaCl2, 0.6 mM MgCl2, 25 mM HEPES, 14 mM glucose, and 2.1% Eagle's essential amino acid mixture. After washing, the tissues were re-incubated in the same medium for 30 min. The digested tissue was washed with enzyme-free medium, and the cells were allowed to disperse spontaneously for 30 min. Suspensions of single muscle cells were harvested by filtration through 500-µm Nitex mesh. The suspensions were centrifuged twice for 10 min at 350 × g.

In experiments with G protein antibodies, the cells were permeabilized as described previously (31, 33) by incubation for 10 min with 35 µg/ml saponin in a medium containing 20 mM NaCl, 100 mM KCl, 5 mM MgSO4, 1 mM NaH2PO4, 25 mM NaHCO3, 0.34 mM CaCl2, 1 mM EGTA, and 1% bovine serum albumin. The cells were centrifuged at 350 × g for 5 min, washed free of saponin, and resuspended in the same medium.

Radioligand Binding to Muscarinic Receptors in Dispersed Muscle Cells

Radioligand binding to dispersed muscle cells was done as described previously (33). Muscle cells were suspended in HEPES medium containing 1% bovine serum albumin. Triplicate aliquots (0.5 ml) of cell suspension (106 cells/ml) were incubated for 15 min with 1 nM [3H]scopolamine alone or in the presence of acetylcholine, methoctramine, or 4-DAMP. Bound and free radioligand were separated by rapid filtration under reduced pressure through 5-µm polycarbonate Nucleopore filters followed by repeated washing (4 times) with 3 ml of ice-cold HEPES medium containing 0.2% bovine serum albumin. Nonspecific binding was measured as the amount of radioactivity associated with the muscle cells in the presence of 10 µM unlabeled ligand. Specific binding was calculated as the difference between total and nonspecific binding (mean ± S.E. 33 ± 6%). IC50 values were calculated from competition curves using the P.fit program (Biosoft; Elsevier Publishing, Cambridge, UK).

Measurement of cAMP in Dispersed Muscle Cells by Radioimmunoassay

cAMP was measured in dispersed cells by radioimmunoassay as described previously (33, 34). Aliquots (0.5 ml) containing 106 cells/ml were incubated with 0.1 µM acetylcholine, and the reaction was terminated after 60 s with 6% cold trichloroacetic acid (v/v). The mixture was centrifuged at 2,000 × g for 15 min at 4 °C. The supernatant was extracted three times with 2 ml of diethyl ether and lyophilized. The samples were reconstituted for radioimmunoassay in 500 µl of 50 mM sodium acetate (pH 6.2) and acetylated with triethylamine/acetic anhydride (3:1 v/v) for 30 min. cAMP was measured in duplicate using 100-µl aliquots and expressed as pmol/106 cells.

Selective Protection of Muscarinic Receptors

A technique of selective receptor protection was used to determine the presence and function of m2 and m3 receptors. The technique was previously used to determine the co-existence and function of opioid µ, delta , and kappa  receptors (45), 5-hydroxytryptamine 5-HT2 and 5-HT4 receptors (32), histamine H1 and H2 receptors (46), and tachykinin NK1, NK2, and NK3 receptors (47). The technique involves protection of one receptor type with a selective agonist or antagonist followed by inactivation of all unprotected receptors by brief treatment with a low concentration of N-ethylmaleimide. In the present study, the selective m2 receptor antagonist, methoctramine (10 nM), and m3 receptor antagonist, 4-DAMP (10 nM), were used in separate experiments to protect m2 and m3 receptors, respectively. Freshly dispersed muscle cells were incubated with one antagonist at 31 °C for 2 min followed by addition of 5 µM N-ethylmaleimide for 20 min. The cells were centrifuged twice at 150 × g for 10 min to eliminate the protective antagonist and N-ethylmaleimide and resuspended in fresh HEPES medium. The cAMP response of cells treated in this fashion was compared with the response of untreated (naive) cells. Previous studies have shown that the coupling of protected receptors to signaling pathways remains intact (32, 46, 47). Smooth muscle cells incubated with N-ethylmaleimide without protective ligand lost their ability to respond to receptor-linked agonists but retained their ability to respond to agents that bypass receptors (e.g. ionomycin, KCl, forskolin), implying that post-receptor mechanisms were intact (46, 47).

Identification of Adenylyl Cyclase Isoforms in Smooth Muscle by Western Blot

Cell homogenates were prepared from dispersed gastric muscle cells and solubilized on ice for 1 h in 20 mM Tris (pH 8.0), 1 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, and 0.5% sodium cholate. For control studies, homogenates were prepared from rat brain. The suspension was centrifuged at 13,000 × g for 5 min. Solubilized membrane proteins (60-70 µg) were resolved by 7.5% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes. After incubation in 5% non-fat dry milk to block nonspecific antibody binding, the blots were incubated for 12 h at 4 °C with antibodies to types II, III, and IV, and a common antibody to types V and VI, and then for 1 h with anti-rabbit IgG conjugated with horseradish peroxidase. The bands were identified by enhanced chemiluminescence.

Identification of Muscarinic Receptor-activated G Proteins

G proteins selectively activated by acetylcholine were identified by the method of Okamoto et al. (48). Ten ml of muscle cell suspension (2 × 106 cells/ml) were homogenized in 20 mM HEPES medium (pH 7.4) containing 2 mM MgCl2, 1 mM EDTA, and 2 mM dithiothreitol. After centrifugation at 27,000 × g for 15 min, the crude membranes were solubilized for 60 min at 4 °C in 20 mM HEPES medium (pH 7.4) containing 2 mM EDTA, 240 mM NaCl, and 1% CHAPS. The membranes were incubated for various periods at 37 °C with 60 nM [35S]GTPgamma S in a solution containing 10 mM HEPES (pH 7.4), 100 µM EDTA, and 10 mM MgCl2. The reaction was stopped with 10 volumes of 100 mM Tris-HCl medium (pH 8.0) containing 10 mM MgCl2, 100 mM NaCl, and 20 µM GTP, and the mixture was placed in wells pre-coated with specific G protein antibodies. After incubation for 2 h on ice, the wells were washed three times with phosphate buffer solution containing 0.05% Tween 20, and the radioactivity from each well was counted. Coating with G protein antibodies (1:1000) was done after the wells were first coated with anti-rabbit IgG (1:1000) for 2 h on ice. The selective m2 receptor antagonist, methoctramine, and m3 receptor antagonist, 4-DAMP, were used to identify the receptor subtype coupled to a given G protein.

Data Analysis

Results were expressed as means ± S.E. of n separate experiments and evaluated statistically using Student's t test for paired or unpaired values.

Materials

125I-cAMP, [3H]scopolamine, and [35S]GTPgamma S were obtained from NEN Life Science Products; HEPES was from Research Organics, Cleveland, OH; soybean trypsin inhibitor and collagenase (type II) were from Worthington; 4-DAMP, methoctramine, p-fluorohexahydro-siladifenidol (p-F-HHSiD) were from Research Biochemicals International, Natick, MA; N-ethylmaleimide and all other chemicals were from Sigma. Antibodies to adenylyl cyclase types II, III, and IV, a common antibody to types V and VI, and antibody to Gbeta were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Pertussis toxin and antibodies to Galpha q/11, Galpha i1-2, Galpha i3, Galpha o, and Galpha s and peptide fragments against which antibodies to Galpha q/11 (QLNLKEYNLV) and Galpha i3 (KNNKECGLY) were raised were obtained from Calbiochem. The ability of these antibodies to block activation or inhibition of specific effector enzymes (phospholipase-beta 1, phospholipase-beta 3, nitric oxide synthase, adenylyl cyclase) has been demonstrated in recent studies (31-35), and a concentration of 10 µg/ml was found to be maximally effective.


RESULTS

Co-expression of Muscarinic m3 and m2 Receptors in Gastric Smooth Muscle Cells

Studies of cloned muscarinic m3 and m2 receptors have established 4-DAMP (Kd 0.5 nM) and methoctramine (Kd 12 nM) as selective antagonists of m3 and m2 receptors, respectively (39, 49-52). Competition binding studies in dispersed gastric smooth muscle cells using [3H]scopolamine as radioligand confirmed the selectivity of the two antagonists. In muscle cells where only m3 receptors were preserved, [3H]scopolamine binding was preferentially inhibited by 4-DAMP (IC50 0.4 ± 0.1 nM) with a potency 104 greater than that of methoctramine (IC50 4 ± 1 µM) (Fig. 1). Conversely, in muscle cells where only m2 receptors were preserved, [3H]scopolamine binding was preferentially inhibited by methoctramine (IC50 8 ± 3 nM) with a potency 2 × 103 greater than that of 4-DAMP (IC50 15 ± 4 µM). The affinities of 4-DAMP and methoctramine in cells where only one native receptor type was preserved matched closely those obtained with cloned m2 and m3 receptors.


Fig. 1. Inhibition of [3H]scopolamine binding by muscarinic antagonists in dispersed gastric smooth muscle cells. Muscle cells (0.5 × 106 cells in 0.5 ml) were incubated for 15 min with 1 nM [3H]scopolamine. The cells were first treated with either methoctramine or 4-DAMP so as to protect m2 or m3 receptors, respectively, and unprotected receptors were inactivated by a 20-min incubation with 5 µM N-ethylmaleimide as described under "Experimental Procedures." Nonspecific binding was measured in the presence of 10 µM acetylcholine. A, high affinity binding of methoctramine (IC50 8 ± 3 nM) in cells where only m2 receptors were preserved. B, high affinity binding of 4-DAMP (0.4 ± 0.1 nM) in cells where only m3 receptors were preserved. Values are means ± S.E. of three experiments each done in triplicate.
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Schild analysis of the relative potencies of the two antagonists in inhibiting acetylcholine-induced contraction confirmed that 4-DAMP was about 6 × 103-fold more potent than methoctramine as an antagonist of m3 receptors that mediate contraction. Contraction in dispersed muscle cells was measured by scanning micrometry as described previously (33, 34). Muscle cells in which only m2 receptors were preserved lost the ability to contract in response to acetylcholine, whereas muscle cells in which only m3 receptors were preserved retained fully their ability to contract (EC50 0.4 nM in muscle cells expressing one or both receptor types). The concentration of acetylcholine that elicited maximal contraction (0.1 µM) abolished [3H]scopolamine binding. The radioligand binding and pharmacological studies in dispersed gastric smooth muscle cells confirmed the selectivity of the antagonists and the validity of the receptor protection technique.

Expression of Adenylyl Cyclase Isoforms in Gastric Smooth Muscle

Western blot analysis of homogenates derived from dispersed gastric smooth muscle cells using antibodies to adenylyl cyclase type II, type III, type IV, and a common antibody to types V and VI disclosed the presence of type V and/or type VI only (Fig. 2). In contrast, homogenates from rat brain disclosed the presence of types II, III, IV, and V/VI (Fig. 2). The selective expression of types V and/or type VI in smooth muscle conforms to the predominant expression of these two types in peripheral tissues (8-11). The properties of adenylyl cyclase in gastric and intestinal smooth muscle are consistent with the properties of types V and VI; both isozymes are inhibited by Galpha i and Galpha o (unlike types II and IV) (33, 34) and are not stimulated by Ca2+ or calmodulin (unlike types I, III, and VIII) (10, 28).


Fig. 2. Expression of adenylyl cyclase types V and/or VI in gastric smooth muscle. Western blot analysis was performed on homogenates prepared from dispersed gastric smooth muscle cells and rat brain. The homogenates were solubilized with sodium cholate in Tris buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis, electrophoretically transferred to nitrocellulose membranes, and probed with specific antibodies to the adenylyl cyclase (AC) types II, III, IV, and a common antibody to types V and VI. Immunoreactive bands for types II, III, IV, and V/VI were detected in rat brain; a band corresponding to type V/VI only was detected in smooth muscle.
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Identification of G Proteins Coupled to Muscarinic m2 and m3 Receptors

Incubation of solubilized muscle cell membranes with acetylcholine (0.1 µM) and [35S]GTPgamma S (60 nM) for 20 min caused a significant, time-dependent increase in the binding of [35S]GTPgamma S·Galpha complexes to wells pre-coated with specific antibody to Galpha q/11 and Galpha i3 but not to wells pre-coated with antibodies to Galpha s, Galpha i1-2, or Galpha o (Fig. 3 and Table I). The increase in bound radioactivity reflected acetylcholine-dependent activation of the dissociated alpha  subunits of Gq/11 and Gi3 by [35S]GTPgamma S. The increase in the binding of [35S]GTPgamma S·Galpha complexes to wells pre-coated with Galpha q/11 antibody was abolished by the m3 receptor antagonists, 4-DAMP (0.1 µM), and p-fluorohexahydro-siladifenidol (0.1 µM) but not by the m2 receptor antagonist, methoctramine (0.1 µM) (Fig. 3 and Table I); control binding in the absence of acetylcholine was not affected by 4-DAMP or methoctramine (control binding at 20 min 3414 ± 377 versus 3620 ± 467 and 3547 ± 587 with 4-DAMP and methoctramine, respectively). Conversely, the increase in the binding of [35S]GTPgamma S·Galpha complexes to wells pre-coated with Galpha i3 antibody was abolished by methoctramine but not by 4-DAMP or p-fluorohexahydro-siladifenidol (Fig. 3 and Table I); control binding in the absence of acetylcholine was not affected by 4-DAMP or methoctramine (control binding at 20 min 3287 ± 419 versus 3711 ± 653 and 3428 ± 592 with 4-DAMP and methoctramine, respectively). The pattern implied that m3 receptors were coupled to Gq/11 while m2 receptors were coupled to Gi3.


Fig. 3. Time course of binding of acetylcholine-stimulated GTPgamma S·Galpha complexes to Galpha q/11 and Galpha i3 antibodies. Membranes, isolated from dispersed gastric muscle cells, were solubilized in CHAPS and incubated with [35S]GTPgamma S in the presence or absence of 0.1 µM acetylcholine for various periods. Aliquots were added to wells pre-coated with Galpha i3 antibody (A) or Galpha q/11 antibody (B) for 2 h, and bound radioactivity was measured (cpm/mg protein). Acetylcholine caused a significant (p < 0.001) increase in binding of [35S]GTPgamma S·Galpha complexes to wells pre-coated with Galpha i3 antibody (A) or Galpha q/11 antibody (B) but not to wells pre-coated with Galpha s, Galpha i1-2, or Galpha o antibody (see Table I). Binding of complexes to Galpha q/11 antibody was blocked by 4-DAMP (0.1 µM) (B, open circles), and binding of complexes to Galpha i3 antibody was blocked by methoctramine (0.1 µM) (A, open circles). Peptide I against which Galpha i3 antibody was raised abolished binding to Galpha i3 antibody but not Galpha q/11 antibody (see Table I). Peptide II against which Galpha q/11 antibody was raised abolished binding to Galpha q/11 antibody but not Galpha i3 antibody (see Table I). Values are means ± S.E. of four experiments.
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Table I. The binding of acetylcholine-stimulated GTPgamma -S-Galpha complexes in smooth muscle membranes to specific G protein antibodies (Ab)

CHAPS-solubilized membranes from dispersed gastric muscle cells were incubated for 20 min with [35S]GTPgamma S in the absence (GTPgamma S alone) or presence of 0.1 µM acetylcholine and then added to wells precoated with specific G protein antibodies. p-F-HHSiD, p-fluorohexahydro-siladifenidol; peptide I, Galpha i3 sequence against which Galpha i3 antibody was raised; peptide II, Galpha q/11 sequence against which Galpha q/11 antibody was raised. The m2 and m3 receptor antagonists inhibited the increase in binding induced by acetylcholine. Peptides I and II inhibited the binding of GTPgamma S-Galpha complexes as well as the increase in binding induced by acetylcholine. Values are means ± S.E. of four experiments.

Binding of [35S]GTPgamma S-Galpha complex to antibody-coated wells
Galpha q/11 Ab Galpha i3 Ab Galpha i1-2 Ab Galpha s Ab Galpha 0 Ab

cpm/mg protein
GTPgamma S alone 3414  ± 377 3287  ± 419 3525  ± 604 2477  ± 273 1067  ± 214
Acetylcholine (0.1 µM) 7594  ± 895 7948  ± 801 3572  ± 435 2512  ± 377 1100  ± 278
  + Methoctramine (0.1 µM) 7268  ± 768 3375  ± 345a NTb NT NT
  + 4-DAMP (0.1 µM) 3715  ± 498a 7562  ± 580 NT NT NT
  + p-F-HHSiDc (0.1 µM) 3428  ± 342a 7618  ± 624 NT NT NT
  + Peptide I (1 nM) NT 3607  ± 482a NT NT NT
  + Peptide I (1 µM) 7433  ± 624 156  ± 85a NT NT NT
  + Peptide II (1 nM) 3628  ± 568a NT NT NT NT
  + Peptide II (1 µM) 232  ± 178a 7638  ± 745 NT NT NT

a Significant inhibition, p < 0.01 to 0.001.
b NT, not tested.
c p-F-HHSiD, p- fluorohexahydro-siladifenidol.

Peptides I and II, comprising the G protein sequences against which the Galpha i3 and Galpha q/11 antibodies, respectively, were raised, were used to block the binding of GTPgamma S·Galpha complexes to the corresponding antibody. Peptide I inhibited the binding of GTPgamma S·Galpha complexes to Galpha i3 antibody in a concentration-dependent fashion, whereas peptide II had no effect (Fig. 3 and Table I). Conversely, peptide II inhibited the binding of GTPgamma S·Galpha complexes to Galpha q/11 antibody in a concentration-dependent fashion, whereas peptide I had no effect (Fig. 3 and Table I). It is noteworthy that the peptides inhibited the control binding of GTPgamma S·Galpha complexes to the corresponding antibody as well as the increase in binding induced by acetylcholine.

Dual cAMP Response of Dispersed Smooth Muscle Cells to Acetylcholine

Acetylcholine (0.1 µM) caused a significant 35 ± 5% decrease in basal cAMP levels of dispersed gastric muscle cells (basal level, 3.9 ± 0.6 pmol/106 cells) (Fig. 4). Neither 4-DAMP nor methoctramine alone had any effect on basal cAMP (4.5 ± 0.4 pmol/106 cells). Pretreatment of the cells with 0.1 µM methoctramine converted the decrease induced by acetylcholine to a significant 32 ± 6% increase above basal level (Fig. 4); the increase in the presence of methoctramine was inhibited in a concentration-dependent fashion by 4-DAMP (EC50 0.5 nM) and abolished by 0.1 µM 4-DAMP (Figs. 4 and 5). Pretreatment of the cells for 1 h with 200 ng/ml PTx so as to uncouple m2 receptors from Gi3 also converted the acetylcholine-induced decrease in cAMP to a significant 36 ± 5% increase above basal level; the increase in cAMP was abolished by 0.1 µM 4-DAMP (Fig. 4).


Fig. 4. Effect of the selective m2 receptor antagonist, methoctramine, and pertussis toxin on the cAMP response to acetylcholine in dispersed smooth muscle cells. cAMP levels were expressed as percent change from basal levels (3.9 ± 0.6 pmol/106 cells). The response to ACh (0.1 µM) was measured before and after a 10-min treatment with methoctramine (0.1 µM) or a 60-min treatment with PTx (200 ng/ml). Methoctramine or PTx converted the decrease in cAMP to increase above basal level; the increase was abolished by 4-DAMP (0.1 µM). Values are means ± S.E. of four experiments. ** p < 0.01.
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Fig. 5. Concentration-dependent effects of 4-DAMP and methoctramine on the cAMP response to ACh. A, muscle cells were pretreated with 0.1 µM methoctramine to elicit ACh-induced increase in cAMP, and the ability of 4-DAMP to block the increase was then determined. B, muscle cells were pretreated with 4-DAMP to accentuate the decrease of cAMP induced by ACh, and the ability of methoctramine to block the decrease was then determined. Results are expressed as percent change from basal cAMP levels. Values are means ± S.E. of three experiments.
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Conversely, pretreatment of the cells with 0.1 µM 4-DAMP accentuated the decrease in cAMP induced by acetylcholine (-63 ± 6% versus control response -35 ± 5% with acetylcholine alone; p < 0.01); the decrease was reversed in a concentration-dependent fashion by methoctramine (EC50 1 nM) and abolished by 0.1 µM methoctramine (Figs. 5 and 6). The accentuated decrease in cAMP in the presence of 0.1 µM 4-DAMP was abolished by pretreatment of the cells with PTx (Fig. 6). The pattern of response to acetylcholine reflected concurrent inhibition of cAMP mediated by m2 receptors and stimulation mediated by m3 receptors.


Fig. 6. Effect of the selective m3 receptor antagonist, 4-DAMP, on the cAMP response to ACh in dispersed smooth muscle cells. cAMP levels were expressed as percent change from basal levels (3.9 ± 0.6 pmol/106 cells). The response to ACh (0.1 µM) was measured before and after treatment with 0.1 µM 4-DAMP. ACh decreased basal cAMP (p < 0.01); 4-DAMP accentuated the decrease in cAMP induced by ACh (**, p < 0.01 from decrease with ACh alone); the decrease was blocked by a 10-min treatment with methoctramine (0.1 µM, Methoc) or a 60-min treatment with PTx (200 ng/ml). Values are means ± S.E. of four experiments.
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The protein kinase C inhibitor, calphostin C (1 µM), had no effect on the increase in cAMP induced by acetylcholine in the presence of methoctramine or PTx (+37 ± 4 and +39 ± 5%, respectively, versus +35 ± 5%) or the accentuated decrease in cAMP induced by acetylcholine in the presence of 4-DAMP (-59 ± 5% versus -63 ± 6%).

Identification of G Proteins Coupled to m2 and m3 Receptors by Functional Blockade with Antibodies

Permeabilized muscle cells were used to identify the G proteins coupled to m2 and m3 receptors by functional blockade with G protein antibodies. Basal cAMP and the decrease in cAMP induced by acetylcholine were not affected by permeabilization (basal level, 4.2 ± 0.5 pmol/106 cells; acetylcholine-induced decrease of cAMP, -33 ± 3%). Pretreatment of permeabilized muscle cells with methoctramine, PTx, or Galpha i3 antibody (10 µg/ml) converted the decrease in cAMP to an increase above basal level (+27 ± 5, +35 ± 5, and +28 ± 5%, respectively) (Fig. 7). Preincubation of the cells for 1 h with Gbeta antibody (10 µg/ml) abolished the increase in cAMP induced by all three agents (Fig. 7), and preincubation with antibodies to Galpha q/11 or Galpha i1-2 (each 10 µg/ml) had no effect (range of response +27 ± 7 to +30 ± 6%). The increase in cAMP induced by Galpha i3 antibody was also abolished by 4-DAMP (2 ± 5%).


Fig. 7. Effect of G protein antibodies on the cAMP response to ACh in permeabilized smooth muscle cells. cAMP was measured in permeabilized smooth muscle cells and expressed as percent change from basal levels. Permeabilization had no effect on basal levels (4.2 ± 0.5 pmol/106 cells). Cells were treated for 10 min with methoctramine (0.1 µM), 60 min with PTx (200 ng/ml), or 60 min with Galpha i3 antibody (10 µg/ml). Treatment with methoctramine, PTx, or Galpha i3 antibody converted the decrease in cAMP induced by ACh (0.1 µM) to increase above basal level; the increase was abolished by preincubation of the cells for 60 min with Gbeta antibody (10 µg/ml). Values are means ± S.E. of four experiments. **, p < 0.01.
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Preincubation of permeabilized muscle cells for 1 h with Gbeta antibody (10 µg/ml) accentuated the decrease in cAMP induced by acetylcholine (-51 ± 6% versus control response -35 ± 5%; p < 0.05) (Fig. 8); preincubation with antibodies to Galpha q/11 or Galpha i1-2 had no effect (-33 ± 6% and -29 ± 3%). The accentuated decrease in cAMP induced by Gbeta antibody was abolished by methoctramine, PTx, or Galpha i3 antibody (Fig. 8). 4-DAMP also accentuated the decrease in cAMP induced by acetylcholine (-56 ± 4% versus control response -33 ± 5%; p < 0.01) (Fig. 8). The accentuated decrease induced by 4-DAMP was abolished by preincubation of the cells with Galpha i3 antibody (10 µg/ml) (Fig. 8) but not with antibodies to Galpha q/11 or Galpha i1-2 (-53 ± 6% and -57 ± 3%).


Fig. 8. Effect of 4-DAMP and Gbeta antibody on the cAMP response to ACh in permeabilized smooth muscle cells. cAMP levels in permeabilized smooth muscle cells were expressed as percent change from basal levels (4.2 ± 0.5 pmol/106 cells). Muscle cells were treated for 10 min with 4-DAMP (0.1 µM) or preincubated for 60 min with Gbeta antibody (10 µg/ml). Treatment with 4-DAMP or Gbeta antibody accentuated the decrease in cAMP induced by ACh (0.1 µM). The accentuated decrease in cAMP induced by 4-DAMP was abolished by preincubation of the cells for 60 min with Galpha i3 antibody. The accentuated decrease induced by Gbeta antibody was abolished by a 10-min treatment with methoctramine (0.1 µM, Methoc), or a 60-min preincubation with either PTx (200 ng/ml) or Galpha i3 antibody (10 µg/ml). Values are means ± S.E. of four experiments. **, p < 0.01 from control response with ACh alone.
[View Larger Version of this Image (12K GIF file)]

The pattern of response elicited by treatment of permeabilized muscle cells with specific G protein antibodies implied that the acetylcholine-induced decrease in cAMP was mediated by m2 receptors via Galpha i3, whereas the increase in cAMP was mediated by m3 receptors via Gbeta gamma q/11.

cAMP Response in Muscle Cells with One Muscarinic Receptor Type

The results obtained in naive muscle cells expressing both m2 and m3 receptors were corroborated in cells where only one receptor type was preserved. In cells where only m3 receptors were preserved, acetylcholine caused only an increase in cAMP (+27 ± 3%), similar to that elicited in naive cells when m2 receptors were blocked with methoctramine or uncoupled with PTx, or when Galpha i3 was blocked with Galpha i3 antibody (Fig. 9). The increase in cAMP induced by acetylcholine was abolished by 4-DAMP but was not affected by methoctramine or PTx.


Fig. 9. Cyclic AMP response to ACh in dispersed muscle cells where either m2 or m3 receptors were preserved. Dispersed smooth muscle cells in which either m3 or m2 receptors were preserved were prepared as described under "'Experimental Procedures." Inactivation of either receptor type had no effect on basal cAMP levels (3.6 ± 0.4 pmol/106 cells). A, in muscle cells where only m3 receptors were preserved; ACh (0.1 µM) caused only an increase in cAMP that was abolished by 4-DAMP. B, in cells where only m2 receptors were preserved, ACh (0.1 µM) induced an accentuated decrease in cAMP levels that was abolished by PTx or methoctramine (Methoc). Values are means ± S.E. of four experiments. *, p < 0.02; **, p < 0.01.
[View Larger Version of this Image (11K GIF file)]

In cells where only m2 receptors were preserved, acetylcholine elicited an accentuated decrease in cAMP (-52 ± 4%) (Fig. 9), similar to that elicited in naive cells in the presence of 4-DAMP or after treatment with Gbeta antibody (Figs. 6 and 9). The accentuated decrease in cAMP was abolished by methoctramine or PTx but was not affected by 4-DAMP (Fig. 9).


DISCUSSION

The present study confirmed the co-existence of muscarinic m2 and m3 receptors on gastric smooth muscle cells and demonstrated the differential coupling of the two receptor types to adenylyl cyclase. Muscarinic m2 receptors, the predominant receptor type expressed in gastrointestinal smooth muscle (40, 53), are known to be coupled to inhibition of adenylyl cyclase via a PTx-sensitive G protein (40, 54-56). The present study identified this G protein as Gi3. In addition, the present study demonstrated a direct coupling of m3 receptors to activation of adenylyl cyclase via the beta gamma subunits of Gq/11. Although coupling of m3 receptors to activation of adenylyl cyclase was known to occur in some cell types (38, 41), the mechanism(s) underlying this effect had not been determined.

The evidence for the differential coupling of m2 and m3 receptors was based on a combination of experimental strategies. (a) GTPgamma S·Galpha complexes activated by m3 receptors bound selectively to Galpha q/11 antibodies, whereas GTPgamma S·Galpha complexes activated by m2 receptors bound selectively to Galpha i3 antibodies. No acetylcholine-induced increase in binding to Galpha i1-2 or Galpha o antibodies could be detected. The binding of GTPgamma S·Galpha complexes to Galpha i3 or Galpha q/11 antibodies was selectively blocked by peptide fragments against which these antibodies were raised. (b) Blockade of m2 receptors or their uncoupling from G proteins by PTx converted the decrease in cAMP induced by acetylcholine to increase above basal level; the increase was blocked by a selective m3 receptor antagonist and by a common antibody to Gbeta implying that activation of adenylyl cyclase was mediated by Gbeta gamma derived from m3-dependent activation of Gq/11. (c) Concurrent activation of adenylyl cyclase mediated by m3 receptors attenuated the predominant inhibition mediated by m2 receptors; blockade of the stimulatory effect with an m3 receptor antagonist or Gbeta antibody accentuated the decrease in cAMP; the accentuated decrease was abolished by methoctramine, PTx, and Galpha i3 antibody. (d) The results obtained in naive muscle cells expressing both receptor types were corroborated using muscle cells in which only one receptor type was preserved. Thus, cells where only m2 receptors were preserved responded to acetylcholine by an accentuated decrease in cAMP which was abolished by an m2 receptor antagonist or PTx; conversely, cells where only m3 receptors were preserved responded to acetylcholine with only an increase in cAMP that was abolished by an m3 receptor antagonist.

The selectivity of the antagonists used for receptor protection was demonstrated by radioligand binding, and the measured IC50 values closely matched those derived from measurements in cells expressing cloned m2 or m3 receptors (39, 49-52). Pharmacological analysis confirmed the validity of the receptor protection technique that had previously been used to characterize a variety of receptors co-expressed on smooth muscle cells and coupled to the same or distinct signaling pathways (e.g. histamine H1 and H2 receptors (46), 5-HT2 and 5-HT4 receptors (32), adenosine A2b and A1 receptors (35), tachykinin NK1, NK2, and NK3 receptors (47), and opioid µ, delta , and kappa  receptors (34)). Only receptors were inactivated while post-receptor mechanisms were spared; in particular, neither basal nor forskolin-stimulated cAMP formation was affected (32, 35).

Increasing awareness of the diverse regulation of various isoforms of adenylyl cyclases requires that the proposed mechanisms for activation or inhibition of the enzymes be consistent with the properties of the adenylyl cyclase(s) expressed in a given cell type (8-11). In the present study, adenylyl cyclase types II, III, and IV could not be detected in dispersed gastric muscle cells. Type V and/or type VI was detected by Western blot analysis since the common antibody could not distinguish between the two types. The tissue expression and regulatory features of adenylyl cyclase in smooth muscle are consistent with the absence of types I and VIII which are confined to neurons (13-16), and with the absence of types II, IV, and possibly VII which are not susceptible to inhibition by Gi (11, 21-23).

The types of adenylyl cyclase (V and/or VI) expressed in smooth muscle are regulated in similar fashion and known to be inhibited by various isoforms of Gi and by feedback phosphorylation by cAMP-dependent protein kinase (25, 26, 29, 30). Both these properties are evident in smooth muscle. Forskolin-stimulated cAMP formation in gastric smooth muscle, for example, is augmented by selective inhibition of cAMP-dependent protein kinase activity with myristoylated protein kinase A inhibitor (36). Inhibition of adenylyl cyclase in smooth muscle by various isoforms of Gi and Go appears to be receptor-specific. Inhibition by somatostatin sstr3 is mediated additively by Gi1 and Go (33), whereas inhibition by opioid µ, delta , and kappa  receptors is mediated additively by Gi2 and Go (34). Go is not involved in inhibition by adenosine A1 or muscarinic m2 receptors that are coupled to Gi3 (35, 37).

Inhibition by submicromolar concentrations of Ca2+ resulting from capacitative entry of Ca2+ is a distinctive regulatory feature of adenylyl cyclases types V and VI (10, 21); this feature, however, could not be demonstrated under our experimental conditions. The measurement of cAMP in the present study was made during the first 60 s when the concomitant rise in cytosolic Ca2+ induced by acetylcholine is determined by inositol 1,4,5-trisphosphate-dependent release of Ca2+ from sarcoplasmic stores and thus precedes store depletion and capacitative Ca2+ entry (57, 58). Furthermore, suppression of phosphoinositide hydrolysis and inositol 1,4,5-trisphosphate-dependent Ca2+ release by Galpha q/11 antibody had no effect on the increase in cAMP mediated by m3 receptors. The concurrent activation of PKC also had no effect on adenylyl cyclase activity in smooth muscle since inhibition of PKC activity by calphostin C had no effect on cAMP levels. As previously shown (37), however, PKC can influence adenylyl cyclase activity in gastrointestinal smooth muscle indirectly by selective phosphorylation of Galpha i1 and Galpha i2; concurrent activation of PKC attenuated the inhibition of forskolin-stimulated cAMP mediated by somatostatin sstr3 receptors coupled to Gi1 and by opioid µ, delta , and kappa  receptors coupled to Gi2, but not by adenosine A1 or muscarinic m2 receptors coupled to Gi3 (33-35).

A novel aspect of this study was the ability of Gbeta gamma derived from the dissociation of Gq/11 to activate smooth muscle adenylyl cyclases type V and/or VI. Activation was not conditional on concurrent activation of Gs, as is the case for activation of types II and IV (11, 21). The low abundance of Gbeta gamma in peripheral tissues, particularly when derived from the dissociation of Gq/11, raises the possibility that, when expressed in smooth muscle, types V and VI may be unusually sensitive to activation by Gbeta gamma or by specific combinations of beta gamma subunits.

In summary, muscarinic m2 receptors are coupled to inhibition of adenylyl cyclases V and/or VI in smooth muscle via the alpha subunit of Gi3, whereas m3 receptors are coupled to activation of the enzymes via the beta gamma subunits of Gq/11. The cAMP response to muscarinic agonists reflects the predominant inhibitory influence of m2 receptors.


FOOTNOTES

*   This work was supported by Grant DK-28300 from the NIDDKD of the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: P.O. Box 980711, Medical College of Virginia, Richmond, VA 23298-0711. Tel.: 804-828-9601; Fax: 804-828-2500.
1   The abbreviations used are: PKC, protein kinase C; 4-DAMP, 4-diphenylacetoxy-N-methylpiperidine; ACh, acetylcholine; methoctramine, N,N'-bis[6[[(2-methoxyphenyl)methyl]amino]hexyl]-1,8-octanediamine tetrahydrochloride; PTx, pertussis toxin; GTPgamma S, guanosine-5'-O-(3-thio)triphosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid.

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