Muscarinic agonist potencies at three different effector systems linked to the M₂ or M₃ receptor in longitudinal smooth muscle of guinea-pig small intestine

Abstract

1. The abilities of muscarinic agonists (arecoline, bethanechol, carbachol, McN-A343, methacholine, pilocarpine) to inhibit isoprenaline-induced cyclic AMP production in chopped fragments (via M₂ receptors), and to evoke cationic current (I_cat) (via M₂ receptors) or calcium store release (via M3 receptors) in enzyme-dispersed, single voltage-clamped cells from longitudinal smooth muscle of the guinea-pig small intestine were examined.

2. All muscarinic agonists (1 – 300 μM) examined inhibited isoprenaline (1 μM)-induced accumulation of cyclic AMP, the IC₅₀ varying from 52 to 248 μM. However, their relative potencies to evoke this M₂ effect were not significantly correlated with their ability to evoke I_cat, also a M₂ effect, whether or not calcium stores were depleted; pilocarpine and McN-A343 inhibited the I_cat response to carbachol.

3. Muscarinic agonists (concentration 300 or 1000 μM), except pilocarpine and McN-A343 which were ineffective, evoked Ca²⁺-activated K⁺ current (I_K-Ca) resulting from Ca²⁺ store release (M₃ effect). Their effectiveness was tested by estimating residual stored calcium by subsequent application of caffeine (10 mM). The relative potencies to evoke Ca²⁺ store release (M₃) and for I_cat activation (M₂) were closely correlated (P<0.001).

4. These data might be explained if M₂-mediated adenylyl cyclase inhibition and I_cat activation involve different G proteins, or involve different populations of M₂ receptors. The observed correlation of agonist potency between I_cat activation and Ca²⁺ store release supports the proposal (Zholos & Bolton, 1997) that M₃ activation can potentiate M₂-cationic channel coupling through Ca²⁺-independent mechanisms.

Introduction

In various gastrointestinal smooth muscles, two different muscarinic receptor types are present as the target for the neurotransmitter acetylcholine. They are defined pharmacologically as M₂ and M₃ corresponding to the genetically defined m₂ and m₃ subtypes (Hulme et al., 1990; Eglen et al., 1996). Evidence for this was obtained initially by radioligand binding studies (Doods et al., 1987; Giraldo et al., 1987; Gomez et al., 1992; Zhang et al., 1991; Michel & Whiting, 1988; 1990) and confirmed by mRNA hybridization (Maeda et al., 1988; Ford et al., 1991) and immunoprecipitation studies (Wall et al., 1991). The number of M₂ receptors is greater than that of M₃ receptors (4 : 1 to 5 : 1). The co-existence of M₂ and M₃ subtypes suggests that both would be activated by acetylcholine or other muscarinic stimulants, and would contribute to the contractile response, but the intracellular signal transduction pathways (G proteins and effectors) involved are likely to be different.

The M₂ receptor subtype in intestinal smooth muscle is well documented to couple via pertussis toxin (PTX)-sensitive G proteins to inhibition of adenylyl cyclase activity. Activation of M₂ receptors, therefore, results in inhibition of the increase in cyclic AMP levels elicited by forskolin, isoprenaline and other compounds that stimulate adenylyl cyclase activity (Peralta et al., 1988; Candell et al., 1990; Griffin & Ehlert, 1992; Reddy et al., 1995). M₂ receptor activation has also been shown to evoke a cationic current (I_cat) (Benham et al., 1985; Bolton & Zholos, 1997; Zholos & Bolton, 1997; Komori et al., 1998). The current response is blocked by PTX (Inoue & Isenberg, 1990a; Komori et al., 1992), but it is not secondary to changes in cytosolic cyclic AMP levels (Komori et al., 1998). Therefore, the M₂ receptor is believed to link to adenylyl cyclase and to cationic channels via separate signalling pathways that involve PTX-sensitive family G proteins. However, the exact nature of the M₂-linked signal transduction mechanisms is still elusive; for example it is unclear whether the G proteins involved in these systems are identical or not.

The M₃ receptors in intestinal smooth muscle are well known to couple via PTX-insensitive G proteins to stimulation of phospholipase C (PLC) leading to formation of inositol-1,4,5-trisphosphate (InsP₃) and in turn to the release of Ca²⁺ from intracellular stores (Prestwich & Bolton, 1995a; Komori & Bolton, 1991). There is abundant evidence to show that Ca²⁺ mobilized from internal stores is an intermediate link between M₂ and M₃ subtypes, whereby M₃ activation can potentiate M₂-mediated I_cat because of the high Ca²⁺ sensitivity of cationic channel opening (Pacaud & Bolton, 1991; Komori et al., 1993, 1996; Kohda et al., 1998). Recently, the existence of another link between both the subtypes was proposed, in which M₃ activation generates a Ca²⁺-independent message potentiating I_cat at the level of receptor or G protein (Bolton & Zholos, 1997; Zholos & Bolton, 1997). The underlying mechanism is still unknown, but such functional linkage implies that without the Ca²⁺-induced facilitation of I_cat, the ability of muscarinic agonist to evoke I_cat via M₂ receptors depends also on its ability to stimulate the M₃ receptor (perhaps via a conformational change in the receptor or activation of associated G proteins). The hypothesis deserves to be tested, in order to validate the proposed functional link between M₂ and M₃ subtypes.

In this report, we have examined the ability of various muscarinic agonists to elicit a M₃-mediated Ca²⁺-activated K⁺ current (I_K-Ca) that occurs as a result of Ca²⁺ store release, and two M₂-mediated responses, namely the inhibition of isoprenaline-stimulated cyclic AMP accumulation and the activation of I_cat, in longitudinal smooth muscle of guinea-pig small intestine. The I_K-Ca response is used to assess the M₃-stimulating activity. Our data indicated that there is no significant correlation of agonist potency between the two M₂-mediated responses, whereas there was a significant correlation between I_cat activation and M₃ receptor stimulation.

Methods

Male guinea-pigs (300 – 400 g) were killed by either stunning or cervical dislocation, followed by immediate exsanguination. The small intestine was removed and exposed in a physiological medium. The longitudinal muscle layer in the ileal region or the whole length of the small intestine except the terminal 5 cm was carefully peeled from the underlying tissue, and then subjected to procedures for the use in whole-cell current recordings or cyclic AMP measurements.

Whole-cell current recordings

Experiments were performed on single smooth muscle cells from the ileal longitudinal muscle layer obtained after treatment with a combination of collagenase (0.3 – 0.6 mg⁻¹) and papain (0.2 – 0.3 mg⁻¹) at 37°C for 30 min, as described previously (Komori et al., 1998).

Whole cell membrane current was recorded at room temperature with patch pipettes (5 – 7 MΩ in tip resistance) and CEZ-2300 voltage-clamp amplifier (Nihon Kohden, Tokyo, Japan). Current signals were filtered at 1 KHz and then displayed on an oscilloscope (Nihon Kohden, VC9) and a thermal array recorder (Nihon Kohden, RTA-1100). They were also saved on a digital tape using a recorder (TEAC, RD-111T, Tokyo) for later analysis and illustration.

For recording of I_K-Ca, cells were bathed in a conventional physiological salt solution (PSS) that had the following composition (mM): NaCl 126, KCl 6, CaCl₂, 2, MgCl₂ 1.2, glucose 14, HEPES 10.5, pH adjusted to 7.2 with NaOH. Patch pipettes were filled with a solution consisting of (mM): KCl 134, MgCl₂ 1.2, MgATP 1, Na₂GTP 0.1, EGTA 0.05, glucose 14, HEPES 10.5, pH adjusted to 7.4 with KOH. Membrane potential was clamped at a level of 0 mV, close to the reversal potential for I_cat in these ionic conditions, so that the I_K-Ca response was not contaminated by I_cat activated at the same time (Komori et al., 1992). The I_cat response was recorded in an external solution consisting of (mM): CsCl 120, glucose 12, HEPES 10, pH adjusted to 7.4 with CsOH. This solution was introduced to the bath (0.5 ml) 30 – 40 s before the start of I_cat recordings by exchange with PSS. The pipette solution had the following composition (mM): CsCl 80, MgATP 1, Na₂GTP 1, creatine 5, glucose 20, HEPES 10, BAPTA 10, CaCl₂ 4.6 (calculated [Ca²⁺]_i=100 nM), pH adjusted to 7.4 with CsOH. Under these conditions, the I_cat response was neither contaminated by any K⁺ currents including I_K-Ca, nor modulated by intracellular Ca²⁺ or external divalent cations (Pacaud & Bolton, 1991; Zholos & Bolton, 1995, 1997). Unless otherwise stated, I_cat was measured at a holding potential of −40 mV which is suitable for receptor-cationic channel coupling (Zholos & Bolton, 1994). In some experiments, I_cat was recorded where [Ca²⁺]_i was non-clamped, by using PSS as the bath solution and pipettes filled with the following solution (mM): CsCl 134, MgCl₂ 1.2, MgATP 1, NaGTP 0.1, glucose 14, HEPES 10.5, EGTA 0.05, pH adjusted to 7.2 with CsOH (Komori et al., 1996).

Concentration-effect curves of muscarinic agonists for I_cat activation were obtained by their application in an ascending series of six different concentrations (1 – 300 μM), with some exceptional occasions. The curves from individual cells were analysed using computor software (SPSS Inc., DeltaGraph 4.0) that fits the data directly with a logistic function, providing the EC₅₀ value, the maximal current (I_max) and the value (h) of the Hill slope.

Cyclic AMP determination

Smooth muscle fragments were prepared and incubated as described previously (Prestwich & Bolton, 1995b). In brief, the longitudinal muscle layers from the whole length of the small intestine were chopped to 350×350 μm fragments with a McIlwain tissue chopper and placed in Krebs Ringer bicarbonate (KRB) consisting of the following composition (mM): NaCl 120, KCl 5.9, NaHCO₃ 15.4, NaH₂PO₄, 1.2, glucose 11.5, MgCl₂, 1.2; CaCl₂, 2.5, pH adjusted to 7.25 with NaOH. After incubation under aeration with a mixture of 95% O₂ and 5% CO₂ at 37°C for 15 – 20 min, the fragments were transferred to fresh KRB containing IBMX (1 mM), a phosphodiesterase inhibitor, and then divided into 300 μl aliquots in microfuge tubes and placed in a water bath at 37°C for 15 min. If necessary, both thapsigargin (1 μM), a sarcoplasmic reticulum Ca²⁺-ATPase inhibitor and D600 (10 μM), a voltage-dependent Ca²⁺ channel blocker were added to the incubation buffers when IBMX was added.

To investigate the effects, if any, of muscarinic agonists on isoprenaline-induced increases in cyclic AMP levels, the fragments were incubated for 10 min in the presence or absence of each muscarinic agonist (1 – 300 μM). After this incubation, isoprenaline was added at desired concentrations (0.1 – 100 μM), followed by further 5 min incubation. The reaction was then stopped with 10% (final concentration) trichloroacetic acid (TCA). The fragments were incubated for 15 min on ice, centrifuged for 20 min at 13,000×g, and the supernatant removed. The pellet was stored ready for protein determination (the average protein concentration was 1.10±0.02 mg/tube, n=20). TCA in the supernatant was removed by diethyl ether extraction, and then TCA-free samples were neutralized using 1 mM NaOH, and aliquots removed for cyclic AMP determination by a competition binding assay using a [³H]-cyclic AMP-labelled kit (Amersham International Ltd), as described previously (Prestwich & Bolton, 1995b).

Chemicals

Ten muscarinic agonists were used; acetylcholine chloride, McN-A343 (4-(N-[3-chlorophenyl]-carbamoyloxy)-2-butynyl-trimethylammonium chloride), oxotremorine, methacholine chloride, pilocarpine hydrochloride (purchased from Sigma, St Louis, MO, U.S.A.), carbachol chloride (from WAKO, Tokyo, Japan), arecoline HBr, bethanechol chloride, OXA-22 (cis-2-methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide), and oxotremorine-M (oxotremorine methiodide) (from Research Biochemicals, Natick, MA, U.S.A.). All the muscarinic agonists were dissolved in distilled water and stocked at −20°C in concentrations 100 times or more higher than those used in experiments.

Guanosine 5′ triphosphate (GTP, sodium salt), isobutylmethylxanthine (IBMX), thapsigargin, methoxyverapamil (D600), and 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid (BAPTA) were obtained from Sigma, and caffeine from WAKO. All other chemicals were obtained from Sigma or WAKO.

Data analysis

Values in the text are given as means±s.e.mean of the number of experiments with smooth muscle fragments or cells (n). Student's paired or unpaired t-test was used for statistical comparison and when P<0.05, differences were considered significant. Correlationships between agonist potencies at different responses were analysed with a Pearson's correlation coefficient test.

Results

Inhibition of cyclic AMP accumulation

The ability of muscarinic agonists to inhibit isoprenaline-stimulated cyclic AMP accumulation was examined in chopped fragments of longitudinal intestinal smooth muscle. These experiments were carried out to ascertain a suitable concentration of isoprenaline such that application of muscarinic agonist might decrease or increase the cyclic AMP response. Basal cyclic AMP levels were estimated to be 22.0±4.9 pmol mg⁻¹ protein (n=5). Incubation with isoprenaline (0.1 to 100 μM) increased cyclic AMP levels above the basal level in a concentration-dependent manner (Figure 1). The level of cyclic AMP reached in 100 μM isoprenaline was 108.0±12.6 pmol mg⁻¹ protein (n=4), and by 1 μM, 54.8±6.3 pmol mg⁻¹ protein (n=4), about half of the former mean value.

Figure 1.

The effect of muscarinic agonists on the concentration-effect curve for isoprenaline-induced increases in cyclic AMP levels. Smooth muscle fragments were incubated with isoprenaline (0.1 – 100 μM) for 5 min in the absence or presence of each indicated muscarinic agonist (100 μM) applied 10 min before. Changes in cyclic AMP were measured in the continuous presence of 1 mM IBMX, a phosphodiesterase inhibitor, using a ³H-cyclic AMP labelled kit. Results represent the mean±s.e.mean of four experiments performed on separate occasions and are expressed as absolute pmol mg⁻¹ protein.

Six muscarinic agonists (carbachol, arecoline, bethanechol, methacholine, pilocarpine and McN-A343) were tested; none of these up to 100 μM affected basal cyclic AMP levels. However, they all (at 100 μM) inhibited the increase of cyclic AMP elicited by isoprenaline (1 to 100 μM), although there were variations in the extent of inhibition (Figure 1). For example, the level of cyclic AMP reached by 100 μM isoprenaline (108.0±12.6 pmol mg⁻¹ protein) was decreased to 44.5±11.7 and 73.0±12.8 pmol mg⁻¹ protein (each n=4) by carbachol and bethanechol, respectively, and to intermediate values by other agonists. The maximum concentration used (300 μM) may not have achieved a maximum response but 1 mM or more may have had non-specific effects.

The ability of muscarinic agonists to inhibit the rise in cyclic AMP levels produced by 1 μM isoprenaline was studied. Carbachol inhibited the accumulation of cyclic AMP in a concentration-dependent manner, with an average 72.2±2.7% (n=4) inhibition at 300 μM; 52 μM carbachol (by interpolation) reduced the response to 1 μM isoprenaline to half (see open circles in Figure 2a). The other five agonists also behaved similarly (Figure 2b – f); the per cent inhibitions at 300 μM and the concentrations producing 50% inhibition of the 1 μM isoprenaline response were 64.8±7.1% and 103 μM for methacholine, 62.7±4.4% and 117 μM for arecoline, 58.5±2.0% and 127 μM for bethanechol, 66.4±5.7% and 65 μM for pilocarpine and 53.1±3.3% and 248 μM for McN-A343 (each, n=4). The potencies at 300 μM relative to carbachol ranged between 0.73 (McN-A343)and 0.92 (pilocarpine) and the rank order was carbachol> pilocarpine>methacholine>arecoline>bethanechol>McN-A343 (see Figure 7). The rank order for 50% inhibition was the same.

Figure 2.

Concentration-effect curves of six muscarinic agonists (a – f) for inhibition of 1 μM isoprenaline-stimulated cyclic AMP accumulation in the absence or presence of 1 μM thapsigargin and 10 μM D600. Cyclic AMP levels were determined as described in Figure 1. Results represent the mean±s.e.mean of four experiments performed on separate occasions and are expressed as the per cent inhibition of the increase in cyclic AMP compared to control.

Figure 7.

Summary of muscarinic agonist potency for cyclic AMP and I_cat responses. The former potency was defined by the per cent inhibition (at 300 μM) of the isoprenaline-stimulated cyclic AMP accumulation (Figure 2) and the latter by the maximum response of I_cat (the I_max in Table 1). These respective potencies were normalized by the value for carbachol. The relative potencies are shown for the cyclic AMP responses in the absence and presence of both 1 μM thapsigargin and 10 μM D600 and for the I_cat response of the indicated individual agonists.

Similar experiments were carried out under conditions where internal Ca²⁺ stores were depleted by pretreatment with thapsigargin (1 μM) and voltage-dependent Ca²⁺ channel activity blocked by D600 (10 μM). Under these conditions, all six agonists were also effective in inhibiting the accumulation of cyclic AMP (see closed circles in Figure 2). For carbachol, methacholine and arecoline, the per cent inhibitions at 300 μM and concentrations reducing the 1 μM isoprenaline response to 50% were 71.0±1.6% and 36 μM, 66.0±1.9% and 58 μM and 61.5±2.8% and 91 μM, respectively (all, n=4), values which were similar to the corresponding values obtained in the absence of thapsigargin and D600. For pilocarpine, bethanechol and McN-A343, however, the per cent inhibition at 300 μM was not as great as in the absence of thapsigargin and D600, being 51.8±3.1, 36.8±7.0 and 29.1±7.7% (all, n=4), respectively. Consequently, 253 μM pilocarpine inhibited the response to isoprenaline by 50%, while neither bethanechol nor McN-A343 achieved 50% inhibition. The relative potencies of the five agonists at 300 μM relative to carbachol fell between 0.41 (McN-A343) and 0.93 (methacholine) and the apparent rank order was carbachol>methacholine> arecoline>pilocarpine>bethanechol>McN-A343 (see Figure 7). It was noted that the position of pilocarpine in the rank order shifted from the second to the fourth when thapsigargin and D600 were present.

Activation of nonselective cationic current, I_cat

The above six muscarinic agonists and four others (acetylcholine, oxotremorine-M, oxotremorine and OXA-22) were studied for their ability to evoke I_cat. Intracellular calcium concentration [Ca²⁺]_i was clamped at 100 nM by the use of a BAPTA/CaCl₂ combination in order to prevent I_cat being modulated by changes in [Ca²⁺]_i during muscarinic stimulation (Pacaud & Bolton, 1991; Komori et al., 1993). Five to 6 min after establishing whole cell recording mode, the solution bathing the cells was changed from PSS to Cs⁺-based solution in order to block K⁺ currents (see closed triangles in Figure 3), which resulted in the generation of an inward current, probably carried by Cs⁺ ion (Zholos & Bolton, 1995). The Cs⁺ current gradually developed to reach a steady level (up to 50 pA in size) in 20 s or so, and then each muscarinic agonist was applied in ascending concentrations of 1, 3, 10, 30, 100 and 300 μM, unless otherwise stated.

Figure 3.

Cationic current (I_cat) induced by muscarinic agonists (a – g) in single ileal smooth muscle cells. The current response was recorded at a holding potential of −40 mV and under conditions where [Ca²⁺]_i was clampled at 100 nM with a BAPTA/CaCl₂ combination. Five to 6 min after establishment of whole-cell recording mode, a physiological salt solution (PSS) bathing the cells was replaced with a Cs⁺-rich solution (at closed triangle) and then each indicated agonist was applied at ascending concentrations (1 to 300 μM) as indicated by the arrows. The agonists applied were washed away with PSS (at open triangle). The dotted lines indicate the levels of holding current in the Cs⁺-rich solution, which were used as base lines to determine the amplitude of I_cat (see Figure 4). (a – g) are from different cells.

Figure 3a shows a typical I_cat response to application of the ascending series of carbachol concentrations. The current response developed in a graded manner and it reached a new sustained level in 10 – 20 s after the increase in agonist concentration; maximum current was usually achieved at 100 μM. The averaged relationship between the amplitude of I_cat and carbachol concentration (n=14) is shown in Figure 4a. I_cat amplitudes were determined by taking the initial sustained level of current in Cs⁺ solution as a base line. Mean values for the I_max, EC₅₀ and slope of the curve (h) estimated by curve fitting of a series of data from the individual cells were 1028.6±123.1 pA, 7.5±1.6 μM and 1.2±0.1 (n=14, Table 1), respectively. The EC₅₀ value was close to that (7.6 μM) reported by Zholos & Bolton (1997).

Figure 4.

Averaged concentration-effect curves of muscarinic agonists for I_cat activation. The curves were obtained from experiments as described in Figure 3. The data points for each agonist were fitted with a logistic function. The curves, depending on whether significantly different from carbachol's curve in the maximum response, are classified into the two groups; (a), not significant and (b), significant (P<0.05). The dotted curve in (b) is for carbachol. Each point indicates the mean±s.e.mean of measurements in 5 – 14 cells.

Table 1.

Maximum amplitude (I_max), half-effective concentration (EC₅₀), and slope factor (h) in the concentration-effect curves for muscarinic agonist-evoked cationic current (I_cat)

Other agonists except pilocarpine and McN-A343 were also effective in eliciting I_cat. Figure 3b – e shows typical examples of I_cat responses to methacholine, OXA-22, bethanechol and arecoline. The I_cat response to OXA-22 often decreased when the agonist concentration was increased to 100 or 300 μM (Figure 3c). As seen from the averaged concentration-effect curves in Figure 4, all the effective agonists, except bethanechol, produced a maximal response at 30 to 300 μM. Bethanechol often required a higher concentration of 1000 μM to do so. The mean values of I_max for the seven effective agonists were all smaller than the corresponding value for carbachol, and this was statistically significant (P<0.05) for oxotremorine, bethanechol and arecoline (Table 1). The mean EC₅₀ values for acetylcholine, oxotremorine-M and oxotremorine were 3.5 to 4.5 times lower (P<0.05) and bethanechol nine times higher (P<0.05), than carbachol. The mean h values for all agonists were not significantly differed from carbachol.

Pilocarpine had little or no effect in eliciting I_cat (Figures 3f and 4b); however, when applied at 300 μM, it produced a small, noisy inward current (up to 120 pA) in five of 11 cells. The current amplitude averaged 23.5±15.1 pA (n=11), only 2% of the I_max value for carbachol (Table 1). McN-A343 was completely ineffective in all seven cells (Figures 3g and 4b). Further characterization of pilocarpine and McN-A343 was performed in different experimental conditions. Their effects on the carbachol concentration-effect curve for I_cat activation were investigated. Both agonists (100 μM) shifted the curve to the right with depression of the maximum response (Figure 5); the EC₅₀ value for carbachol was increased from 7.5±1.6 μM (Table 1) to 178.6±65.4 μM (n=5) by pilocarpine and to 40.8±6.5 μM (n=5) by McN-A343 (both P<0.05) and the I_max value decreased from 1028.6±123.1 pA (Table 1) to 635.5±101.1 pA (n=5) and 487.2±76.1 pA (n=5) by the respective agonists (both P<0.05). Their ability to induce I_cat was examined over a broader range of membrane potential using a negative going ramp pulse from 40 to −80 mV over 0.5 s (Figure 6a). Current-voltage (I-V) relationships in the presence of either agonist were obtained after leakage subtraction. In two of six cells, the I-V curve for pilocarpine (300 μM) lay on the voltage axis, indicating no activation of I_cat in these cells. In the remaining four cells, a detectable, but slight, current activation occurred in the voltage range between −20 and −60 mV with a maximum at some −40 mV. The I-V curve with the most clear activation is shown in Figure 6b. In this curve, current amplitude at any voltage was less than 5% of that at the corresponding voltage in a typical I-V curve for 300 μM carbachol (n=5; Figure 6a,b). The I-V curve for 300 μM McN-A343 overlapped the voltage axis in all five cells tested (Figure 6b). Finally, their I_cat-eliciting activity was examined in cells immersed in PSS and internally dialyzed with a pipette solution without the BAPTA/CaCl₂-buffer (Komori et al., 1996). At a holding potential of −40 or −50 mV, neither pilocarpine nor McN-A343 (100 – 1000 μM) produced an appreciable I_cat (n=12 and 9, respectively), whereas in cells which had been insensitive to these agonists, carbachol (3 – 100 μM) invariably elicited a sustained or oscillatory pattern of I_cat (n=8; data not shown). These results show that pilocarpine and McN-A343 have binding affinity but little or no potency at receptors mediating I_cat.

Figure 5.

The effect of pilocarpine and McN-A343 on the carbachol concentration-effect curve for I_cat activation. The current response to carbachol was recorded as described in Figure 1, except for the presence of 100 μM pilocarpine (a) or McN-A343 (b) in the bath solution as indicated by the lines. (c) The averaged curves for carbachol in the presence of pilcarpine or McN-A343 and the control curve repeated from Figure 4a. Each point for the former two curves indicates the mean±s.e.mean of measurements in five cells.

Figure 6.

The ability of pilocarpine, McN-A343 and carbachol to activate I_cat over a broad range of membrane potential. (a) Current traces from three different cells held at −40 mV to which a negative going ramp pulse from 40 to −80 mV over 0.5 s was applied before (open sqaure) and during application (closed square) of 300 μM carbachol (CCh), pilocarpine (Pilo) or McN-A343. (b) Current-voltage (I-V) curves for CCh, Pilo and McN-A343 from the respective cells in (a), which were constructed by subtracting the I-V curve before application of the agonists from that obtained during their application. See text for details.

Relationship between agonist potencies at the twoM₂-mediated responses

Using the data from the group of six agonists whose activity was examined for both cyclic AMP inhibition and I_cat activation, we investigated mutual relation of potencies for both responses. Potencies of the individual agonists for the cyclic AMP response and I_cat activation, expressed by taking potency of carbachol as unity, are summarized in Figure 7. The relative potencies for the cyclic AMP response were estimated from the inhibition attained at 300 μM of cyclic AMP accumulation in the absence and presence of thapsigargin and D600 (Figure 2) and those for I_cat activation from the maximum I_cat response (I_max value in Table 1). As seen from Figure 7, the relative potency in evoking the two responses varied considerably among the different agonists, regardless of the use of thapsigargin and D600 in cyclic AMP assays; for example, arecoline and pilocarpine were as potent as methacholine for the cyclic AMP response, but obviously less potent for the I_cat response. The relation between the relative potencies for both responses was statistically tested. The calculated correlation coefficient had a value of 0.582 when cyclic AMP was assayed without thapsigargin and D600, and a value of 0.679 when assayed with these drugs present. These values were smaller than the value (0.729, when n=6) for a significant correlation (P<0.05), suggesting a lack of a parallelism of agonist potency between the two effector systems linked to the M₂ subtype receptor.

Activation of Ca²⁺-activated K⁺ current, I_K-Ca

M₃ receptor activation in this type of cell induces I_K-Ca via Ca²⁺ release from the stores (Komori & Bolton, 1991; Komori et al., 1998). To assess potency for Ca²⁺ store release, we examined the ability to elicit I_K-Ca of the same group of 10 agonists as tested for I_cat activation. The individual agonists were applied at a maximally effective concentrations of 300 or 1000 μM and 30 – 40 s later, 10 mM caffeine, a potent Ca²⁺-store releaser, was routinely applied to check if releasable Ca²⁺ remained in the stores (Komori et al., 1998).

Application of carbachol evoked I_K-Ca lasting 3 – 10 s (Figure 8a), as previously reported (Komori et al., 1998), the peak amplitude of which amounted to 3.2±0.7 nA on average (n=6). Subsequent application of caffeine in the presence of carbachol was usually without effect (Figure 8a; Table 2). All other agonists, except bethanechol, pilocarpine and McN-A343, also evoked a sizable I_K-Ca (Figure 8b – d). The mean peak amplitude varied from 2.0 to 3.1 nA among the six agonists; acetylcholine, oxotremorine-M, arecoline, OXA-22, methacholine and oxotremorine (Table 2). Subsequent caffeine was without effect or evoked I_K-Ca with varied amplitude, depending on the previously applied agonist and different cells stimulated with the same agonist (Table 2).

Figure 8.

Examples of Ca²⁺-activated K⁺ current (I_K-Ca) evoked by muscarinic agonists. (a – g) Current responses of different cells to (a) carbachol, (b) acetylcholine (ACh), (c) oxotremoline-M (Oxo-M), (d) arecoline (Are), (e) bethanechol (BCh), (f) pilocarpine (Pilo) and (g) McN-A343 applied at a maximally effective concentration (300 or 1000 μM) and subsequent application of 10 mM caffeine as indicated by the lines. The cells were held at 0 mV, a level close to the reversal potential for I_cat and exposed to the drugs 5 min after establishment of whole-cell recording mode. Mean values for the amplitudes of agonist-evoked I_K-Ca, subsequent caffeine-evoked I_K-Ca and the sum of both currents are given in Table 2.

Table 2.

Ca²⁺-activated K⁺ current (I_K-Ca) evoked by muscarinic agonists at maximally effective concentrations and by subsequent application of caffeine

The ability of bethanechol (300 μM) varied too extensively among different cells to be reliably assessed. However, when used at a higher concentration of 1000 μM, the agonist invariably evoked I_K-Ca with a peak amplitude of 1.6±0.4 nA (n=7) but subsequent caffeine also invariably evoked I_K-Ca which averaged 1.5±0.6 nA (n=7) (Figure 8e). Neither pilocarpine nor McN-A343 was able to evoke an appreciable I_K-Ca, but caffeine applied following the respective agonists evoked I_K-Ca of 3.7±1.2 nA (n=15) and 2.2±0.5 nA (n=11). Even at 1000 μM, both agonists were without effect (n=3, respectively). When pilocarpine (100 μM) was previously added in the bath solution, carbachol evoked only a small I_K-Ca of 0.1 – 0.2 nA followed by subsequent caffeine-evoked I_K-Ca of some 2.0 – 3.0 nA (n=3). Substantially similar results were obtained when carbachol was applied in the presence of McN-A343 (100 μM). Therefore, pilocarpine and McN-A343 seemed to behave as antagonists rather than agonists at M₃ receptors.

When the amplitude of I_K-Ca evoked by a given agonist was compared with the summed amplitude of this and the subsequent caffeine current, there was a significant (P<0.05) difference for bethanechol, arecoline and oxotremoline, as well as pilocarpine and McN-A343, but not for carbachol, acetylcholine, OXA-22, methacholine and oxotremoline-M (Table 2). The results indicated that these agonists have different potencies for Ca²⁺ store release.

Relationship between agonist potency for Ca²⁺ store release and for I_cat activation

We compared the relationship of agonist potencies for Ca²⁺ store release and for I_cat activation, using the data obtained so far. The potency of the former was estimated from the proportional size of agonist-evoked I_K-Ca to the sum of this and subsequent caffeine current and normalized by the value of 0.97 for carbachol-evoked I_K-Ca (see Agonist/Sum. in Table 2). The potency for the latter determined and normalized on the basis the I_max value for carbachol. Figure 9 illustrates plots of the relative potency at I_cat activation against that at Ca²⁺ store release for the 10 muscarinic agonists. The data points are scattered along or near the solid line drawn to indicate a positive correlation of potency between both of them. The calculated correlation coefficient of 0.923 was greater than the value (0.872, when n=10) expected for a significant correlation (P<0.001).

Figure 9.

Correlation between muscarinic agonist poptency for I_cat activation and for Ca²⁺ store release. The former potency was defined by the I_max values in Table 1 and the latter, by the proportional size of agonist-evoked I_K-Ca to the sum of this and subsequent caffeine current in Table 2 (see column Sum/Agonist), and these respective potencies were normalized by the value for carbachol and plotted for Ca²⁺ store release on the X axis and for I_cat activation on the Y axis. The solid line indicates a regression line calculated using the data points from the 10 agonists, as expressed by Y=0.865X. The obtained Pearson's correlation-coefficient of 0.923 was greater than the expected value for a significant correlation (0.872, when n=10 and P<0.001).

Discussion

Muscarinic receptors in intestinal smooth muscle mediate multiple responses including adenylyl cyclase inhibition, I_cat activation, Ca²⁺ store release and contraction. The former two responses are mediated by M₂ subtype receptors coupling to PTX-sensitive G proteins (Candell et al., 1990; Griffin & Ehlert, 1992; Zholos & Bolton, 1997; Komori et al., 1998), and the latter two by M₃ subtype receptors coupling to PTX-insensitive G proteins (Komori et al., 1992, 1998; Eglen et al., 1996).

In the present study, the six agonists of carbachol, methacholine, bethanechol, arecoline, pilocarpine and McN-A343, all inhibited the isoprenaline-evoked cyclic AMP response believed to be mediated via M₂ receptors. However pilocarpine and McN-A343 were virtually without ability to evoke I_cat which is also believed to be evoked via M₂ receptors. Moreover, the relationship of relative potencies for the two responses varied considerably among the different agonists (Figure 7) and statistical tests indicated no significant correlation between them. If M₂ receptors utilize one multifunctional G protein to mediate both cyclic AMP and I_cat responses, the individual agonists tested might be expected to display a parallelism in potency for the two responses. Therefore, it is possible that the G proteins involved in the individual responses are different. Based on this, it is postulated that pilocarpine and McN-A343, if they occupy M₂ receptors, cannot or poorly induce the required conformational change in the receptor to activate the G protein linked to the cationic channel system. In agreement with this, both agonists acted as antagonists on the I_cat response to carbachol. However, if a receptor subtype is predisposed to couple preferentially to one G protein/effector system (see Gudermann et al., 1996), then another possibility is that different populations of M₂ receptors exist, one coupling to a G protein that affects adenylyl cyclase and a second to another G protein that affects cationic channels. In this case pilocarpine and McN-A343 might behave as an agonist at the former receptor, and as an antagonist at the latter.

In the present study, the two M₂-mediated responses were recorded under considerably different experimental conditions. The inhibition of the cyclic AMP response was measured in chopped fragments of smooth muscle without control of [Ca²⁺]_i and membrane potential (hence GTP was not added to the bathing solution), whereas the I_cat response was recorded in single isolated cells whose [Ca²⁺]_i and membrane potential were both clamped. These differences would mean some difficulty in comparing agonist potencies for both responses. However, when thapsigargin and D600 were used to block intracellular Ca²⁺ mobilization as well as electrical membrane activity in cyclic AMP assays, in an attempt to mimic the conditions used for I_cat recording, the same conclusion that the agonist potencies for cyclic AMP and I_cat responses did not correlate was reached. Conversely, to mimic the cyclic AMP assay system, we tried I_cat recording conditions both in a physiologically relevant range of membrane potential (−80 to 40 mV) and without clamping [Ca²⁺]_i as well as the presence of extracellular Ca²⁺. However, McN-A343 and pilocarpine still had little or no effect in eliciting I_cat. These results confirmed the comparison of agonist potency, nevertheless measurement of cyclic AMP or adenylyl cyclase activity in conditions similar to those used for I_cat recording would be of interest.

The presence of thapsigargin and D600 in assay systems resulted in reduced abilities to elicit a cyclic AMP response. This suggests the action of internal Ca²⁺ mobilized by Ca²⁺ store release and/or by an increase in voltage-dependent Ca²⁺ channel activity may affect the adenylyl cyclase response. In other words, the M₂/adenylyl cyclase system might be under a Ca²⁺-dependent regulation by the M₃/PLC/InsP₃ system and the M₂/cationic channel system which causes voltage-dependent Ca²⁺ channels to open by depolarizing the cell membrane. These possible interactions would be expected to have important functional implications in vivo. However, the extent to which agonist ability was affected by thapsigargin and D600 varied from one agonist to another; a relatively severe change in the ability was seen for bethanechol, pilocarpine and McN-A343 which are rather weak in their Ca²⁺-mobilizing activity, as judged from their weak ability to evoke I_K-Ca. Therefore, beside the inhibition of intracellular Ca²⁺ mobilization, some nonspecific actions of thapsigargin and/or D600 (Kobayashi et al., 1991) might be involved.

The 10 muscarinic agonists showed a significant correlation for their abilities to evoke I_K-Ca and I_cat indicating a relationship between their ability to release Ca²⁺ stores and to evoke I_cat activation (Figure 9). It is well known that the I_cat response is strongly potentiated by a rise in [Ca²⁺]_i due to Ca²⁺ store release (Pacaud & Bolton, 1991; Inoue & Isenberg, 1990b). However, this mechanism seems unlikely to be involved in the observed correlation of agonist potency, because I_cat was recorded under conditions where [Ca²⁺]_i was clamped at 100 nM with the Ca²⁺ buffer (10 mM BAPTA/4.6 mM CaCl₂). Therefore, some other mechanism, independent of Ca²⁺, seems to underlie it. In this context, an idea has been proposed that M₃ activation, as well as inducing Ca²⁺ store release, can potentiate I_cat through an action at the level of receptor or G protein (Bolton & Zholos, 1997; Zholos & Bolton, 1997). This hypothesis implies that independently of I_cat modulation by [Ca²⁺]_i the ability of an agonist to elicit I_cat depends on its ability to stimulate M₃ receptors leading to a conformational change in receptor or activation of associated G proteins. The present observation, i.e. the close correlation of agonist potencies, substantiates this implication, corroborating the possible existence of a Ca²⁺-independent functional link between M₂ and M₃ receptors (Bolton & Zholos, 1997; Zholos & Bolton, 1997).

In studies using several muscarinic agonists there was a close correlation between their abilities to evoke contraction of longitudinal muscle from isolated guinea pig ileum and phosphoinositide hydrolysis in CHO cells with expressed m₃ receptor (Ehlert et al., 1999). The rank orders of potencies for the agonists to cause contraction or phosphoinositide hydrolysis were very similar to the results presented in this study supporting a link between M₃ receptor activation, Ca store release and contraction. However Mc-N-343 and pilocarpine were antagonists or ineefective in our studies on single cells whereas they were weakly effective in the experiements of Ehlert et al. (1999).

The present and other results lead us to suggest possible arrangements of the functional expression of muscarinic receptors in intestinal smooth muscle. One possible relationship consists of an M₂ and an M₃ subtype receptor, the former coupling to two different PTX-sensitive G proteins that affect either adenylyl cyclase or the cationic channel (see above), and the latter coupling to PTX-insensitive G proteins, potentially G_q/G₁₁, linked to the PLC/InsP₃ system. In this scheme, it would be predicted that the Ca²⁺-independent potentiation of I_cat by M₃ activation would arise through associated G proteins or some downstream factors other than Ca²⁺. The prediction, however, contradicts our recent observation that anti-G_q/G₁₁ protein antibodies had no significant effect on the I_cat response to carbachol, but blocked the agonist-evoked I_K-Ca (Yan et al., 2000). Thus, the M₃ effect on M₂ activation is likely to be upstream of the G_q/G₁₁ protein or a separate system.

Another possibility is the existence of two different types of M₂ receptors, a population of independent M₂ receptors and a second population of M₂/M₃ receptor complexes (Zholos & Bolton, 1997) which would be consistent with the fact that M₂ binding sites are much more abundant than M₃ binding sites (4 : 1 to 5 : 1; Giraldo et al., 1987). The discrete M₂ receptor couples via a PTX-sensitive G protein to the adenylyl cyclase system, while the M₂ part of the M₂/M₃ complex couples via another PTX-sensitive G protein to a cationic channel system, and the M₃ part, via G_q/G₁₁ proteins, to a PLC/InsP₃ system. When an agonist occupies the M₂/M₃ complex, the resulting conformational change in the M₃ receptor generates a Ca²⁺/G protein-independent message to potentiate M₂-mediated I_cat, and also activates the PLC/InsP₃ system via G_q/G₁₁, leading to Ca²⁺ store release and in turn Ca²⁺-induced potentiation of I_cat. Our recent experiments using antibodies against various G proteins provided evidence favorable for involvement of G_o proteins in the activation of I_cat (Yan, Zholos, Unno, Komori & Bolton; unpublished data), as suggested in guinea-pig gastric myocytes (Kim et al., 1998). Together with the general belief that adenylyl cyclase inhibition involves G_i proteins, it may be speculated that the major G proteins coupling to the discrete and complex-type M₂ receptors are a G_i and a G_o G-protein, respectively. The model discussed here is a working hypothesis amenable to various molecular biological and pharmacological tests such as have been made in the opioid system, where it has been demonstrated that G protein-coupled opioid δ and κ subtypes dimerize to form a functional receptor (Jordan & Devi, 1999).

In the present study, pilocarpine and McN-A343 failed to evoke I_K-Ca, but were able to inhibit the I_K-Ca-evoking activity of carbachol, indicating their antagonist behaviour at M₃ receptors. Such properties are also indicated from other studies in which their effects on phosphoinositide turnover and Ca²⁺ store-dependent changes in [Ca²⁺]_i or tension were examined (Gardner & Mitchelson, 1988; Hishinuma et al., 1997; Morel et al., 1997; Wang et al., 1992). Both agonists also shifted the carbachol concentration-effect curve for I_cat activation to the right with depression of the maximum response (Figure 5). Similar changes in the carbachol curve are seen with certain muscarinic antagonists, and it is suggested that the rightward shift is due to M₂ blockade and depression of the maximum, due to M₃ blockade (Zholos & Bolton, 1997). These properties of pilocarpine and McN-A343 combined with their obvious potency for the cyclic AMP response can be readily explained by the expression pattern we postulate for the M₂ receptor and M₂/M₃ complexes.

In conclusion, the present study shows that in longitudinal smooth muscle of guinea-pig small intestine, there was no significant correlation of muscarinic agonist potency between the M₂/adenylyl cyclase and the M₂/I_cat system, while a significant correlation exists between the potencies of the M₂/I_cat and M₃/PLC/InsP₃ systems. These results combined with others provide a hypothetical model for the functional expression pattern of muscarinic receptors. However, further evaluation of this model will require investigation to clarify the precise muscarinic signalling mechanisms involved.

Abbreviations

BAPTA

1, 2-bis (2-aminophenoxy) ethan-N,N′ N′-tetraacetic acid

[Ca²⁺]_i

intracellular calcium concentration

D600

methoxyverapamil

EGTA

ethyleneglycol-bis (β-aminoethyl ether) N,N,N′,N′-tetraacetic acid

G protein

GTP binding protein

IBMX

isobutylmethylxanthine

I_cat

nonselective cationic current

IC₅₀

concentration required to produce 50% inhibition

I_K-Ca

Ca²⁺-activated K⁺ current

I_max

maximum current

InsP₃

inositol-1,4,5-triphosphate

McN-A343

4-(N-[3-chlorophenyl]-carbamoyloxy)-2-butynyl-trimethylammonium chloride

OXA-22

cis-2-methyl-5-trimethylammoniummethyl-1,3-oxathiolane iodide

PLC

phospholipase C

PTX

petrussis toxin