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Possible involvement of M5 muscarinic receptor in the enhancing actions of the novel gastroprokinetic agent Z-338 on nifedipine-sensitive voltage-dependent Ca2+ currents in guinea pig stomach.

We investigated the effects of the novel gastroprokinetic agent Z-338 (N-(N-N'-diisopropylaminoethyl)-[2-(2-hydroxy-4,5-dimethoxybenzoylamino)-1,3-thiazole-4-yl] carboxyamide monohydrochloride trihydrate) on L-type voltage-dependent Ca2+ currents (ICa) in guinea pig gastric myocytes by using the whole-cell patch clamp technique. Bath-applied acetylcholine (ACh) produced biphasic effects on ICa, i.e., enhancement (1-100 nM) and inhibition (1-100 microM), both of which were abolished by pretreatment with atropine (10 microM) or intracellular perfusion of GDPbetaS (500 microM). Z-338 (> or = 1 nM, ED50: 120 nM) mimicked the enhancing effects of ACh, but did not inhibit ICa. The effects of Z-338 and ACh were non-additive and blocked by atropine and GDPbetaS, but not by pertussis toxin (PTX) pretreatment (500 ng/ml). ACh (> or = 1 microM) induced slow inward currents via activation of the muscarinic receptor/PTX-sensitive G-protein pathway, but Z-338 was devoid of these effects. Neither pirenzepine (1 microM), AF-DX116 (1 microM), nor oxybutynin (100 nM) could prevent Z-338 (1 microM) and ACh (10 nM) from enhancing ICa, whilst 4-DAMP (100 nM) blocked the effects of Z-338 and ACh. Bath-application of protein kinase C (PKC) activator PDBu (phorbol-12,13-dibutyrate) (250 nM) enhanced ICa, and conversely, pipette inclusion of PKC inhibitor peptide (150 microM) abolished the effects of ACh and Z-338 on ICa. These results collectively suggest that although contribution of the M3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of ICa through activation of M5-like receptor.

Z-338, a novel carboxyamide derivative, has recently been synthesized, in the hope to obtain more potent and clinically safer gastroprokinetic agents having a different mechanism from those of existing gastroprokinetic agents such as cisapride and domperidone, which have been reported to show unfavorable adverse effects on the central nervous and cardiovascular systems (1). It has been shown that Z-338 enhances gastric motor activity in conscious dogs with chronically implanted force transducers and restores gastric emptying suppressed by clonidine treatment in both dog and rat gastroparesis models (1). The mechanisms involved in theses actions of Z-338 have been investigated in more detail by a number of different techniques. In guinea pig stomach strips preincubated with [ 3 H]-labelled choline, Z-338 enhances the electrically stimulated release of [ 3 H]-ACh. This is likely to occur through inhibition of M1 and M2 receptors, since Z-338 can bind to M1 and M2 (but not M3) receptors prepared from rat tissues and effectively block membrane currents associated with activation of recombinantly expressed rat M1 and M2 receptors in Xenopus oocytes (2). Furthermore, it was also found that Z-338 enhances spontaneous contractions as well as electrically evoked excitatory junction potentials and concomitant contractions in guinea pig stomach with no significant changes in inhibitory neuro- transmission, resting membrane potential or basal muscle tone (3). All these findings are consistent with the view that Z-338 facilitates the excitatory neurotransmission in the stomach by increasing ACh release from cholinergic nerve varicosities via inhibition of pre-junctional M1 and M2 receptors. However, it is still unclear what postjunctional mechanisms are involved in the observed increase in spontaneous motor activities of gastric smooth muscle upon application of Z-338. It is also puzzling why Z-388 is able to enhance spontaneous contractions without changing the basal tension level, while ACh affects both. Thus, in the present study, in order to clarify these points, we have investigated the ionic mechanisms underlying the excitatory actions of Z-338 via muscarinic receptors in comparison with those of ACh, by applying the patch clamp technique to single myocytes freshly dissociated from the stomach. Our results show that this compound potentiates the activity of the L-type Ca 2+ channel almost exclusively through stimulation of a muscarinic receptor pharmacologically similar to the M 5 receptor.

Cell dispersion
Hartley guinea pigs of either sex (400 to 700 g) were stunned and killed by decapitation. The whole stomach of each animal was excised out and cut open along the major curvature and divided into two parts. Each part was pinned stretched on the rubber bottom of a dissecting dish filled with 1 mM Ca 2+ containing Krebs solution (for ionic composition see below). The gastric smooth muscle layer was cleaned of attached mucosa, vessels, and connective tissues, and minced into small pieces using fine scissors and forceps. These pieces were incubated in nominally Ca 2+ -free Krebs solution at 36°C for 30 min and transferred consecutively into the Ca 2+ free Krebs solution supplemented with 2 mg /ml collagenase (type I; Sigma, St. Louis, MO, USA) at 36°C for 60 min. Single cells were dispersed by triturating the digested pieces with a large bored Pasteur pipette in Ca 2+ -free Krebs solution and stored in a refrigerator at 4 -10°C until use. Experiments were carried out within 3 h from the time of cell dispersion. For PTX treatment, strips made from adjacent regions of the same gastric muscle were incubated in Krebs solution supplemented with or without 500 ng /ml PTX at 4 -10°C for 12 -18 h (the latter was taken as the control), from which single myocytes were dispersed following the protocol mentioned above. All procedures described above were performed according to the guidelines approved by a local animal ethics committee of Kyushu University.

Electrophysiology
The details of electrophysiological recordings in this study were essentially the same as described previously (4). Briefly, a low-noise, high-impedance patch clamp amplifier (Axopatch 1D; Axon Instruments, Union City, CA, USA) driven by an IBM-compatible computer (Aptiva) was used to generate voltage and acquire current signals, in conjunction with an A /D, D /A converter (TL-1, Axon Instruments). Obtained current signals were low-pass filtered (1 kHz), digitized (2 kHz), and stored on a computer hard disk. The leak subtraction procedure was routinely employed using the P /2 protocol where voltage pulses of half amplitude with an opposite polarity were applied prior to the test pulses. The series resistance of cells examined stayed almost constant throughout the experiments (5 -7 MW), 50 -70% of which was electronically compensated. Data were analyzed offline and an illustration made by the commercial software Clampfit version 6.04 (Axon Instruments) and Kaleida Graph version 3.04 (Synergy Software, Reading, PA, USA). In some cases in which long term recordings were needed, current signals were digitized every 10 ms using the MacLab (AD Instruments, Castel Hill, Australia). In some experiments such as the shown in Fig. 1C and 2B, the nystatin-perforated rather than conventional whole-cell recording was employed to examine the possible role of intracellular Ca 2+ concentration. We found, however, little differences between the results obtained by the two modes of recording ( Fig.1C and Fig.2B). We therefore employed the latter mode throughout the present study, in order to record ICa under better and more stable voltageclamped conditions. The temperature of the bathing solution was adjusted between 25 ± 1°C with an automatic temperature control unit (TC-344B; Warner Instruments, Hamden, CT, USA).
The effects of agonists such as ACh, Z-338 and PDBu on ICa were evaluated after their equilibration, which normally took a few minutes, when I Ca was repetitively evoked by 400-ms pulses at an interval of 20 s. When multiple concentrations were tested, cumulative application was employed. Inhibitors and antagonists were administered 10 min prior to the application of agonists. Considerable variations occurred in the magnitude of responses to ACh or Z-338, due probably to individual variations of experimental animals and a varying efficacy of enzymatic digestion. Particularly, overnight treatment of gastric muscle strips with or without PTX resulted in a significantly attenuated response of dissociated myocytes to Ach or Z-338 as well as a decrease in the current density of ICa, as can be seen in Fig. 4C. Thus, to minimize the influence of these variations, we compared the effects of given drugs between cells dissociated under identical enzymatic conditions from the same batches or adjacent regions of the same muscle strip (Figs. 3 -5).

Statistics
All data were expressed as the mean ± S.E.M. For statistical evaluation, two-tailed paired and unpaired t-tests or one way ANOVA with the pooled variance t-test were used where appropriate.

Complex actions of ACh on membrane currents
When voltage step pulses more positive than -40 mV were applied to single gastric myocytes from a holding potential of -60 mV with Cs + (140 mM) in the pipette, rapidly activating and inactivating inward currents were elicited ( Fig. 1: Aa, b; for I-V relationship, see Fig. 2C). These currents were not significantly affected by substitution of external Na + by N-methyl, D-glucamine, but completely abolished in the presence of nifedipine (³1 mM), suggesting that high voltage-activated L-type Ca 2+ channels are responsible for its generation (ICa). The properties of I Ca were in agreement with those previously reported in the same preparation (5, 6), but in some cells, transient inward currents showing much faster kinetics compared with I Ca were recorded prior to the activation of I Ca . These currents are likely to result from activation of voltagedependent Na + channels, since they were completely abolished in the presence of 1 m M TTX or upon total elimination of external Na + . Thus, in the rest of the present study, to avoid the contamination of voltage-dependent Na + currents, 1 mM TTX was routinely added in the bath. Figure 1A demonstrates typical actions of bath-applied ACh on the holding current and ICa evoked by depolarizing pulses to 0 mV at two extreme concentrations. At a relatively low concentration of ACh (10 nM), there was almost no change in the basal current level, but the magnitude of I Ca was significantly enhanced. In contrast, at a high concentration (100 mM), a slow sustaining inward current developed after addition of ACh, and in parallel with this, the magnitude of I Ca decreased progressively ( Fig. 1: Aa, b; for summary, see Fig. 1B). The reduction of ICa at high ACh concentrations was due unlikely to superimposition of slow inward current, since its reversal potential was close to 0 mV (see the inset in Fig. 1C), at which ICa was evaluated. Both enhancing and inhibiting effects of ACh occurred in a concentration-dependent fashion and did not seem to be strongly affected under weak Ca 2+ -buffering conditions (open circles in Fig. 1C: data from nystatinperforated recording). As summarized in Fig. 1C, the enhancing effects were already evident in the nanomolar range of ACh, whereas the effective concentrations of ACh to inhibit ICa mostly spans the micromolar range (Fig. 1C). The magnitude of the slow sustaining inward current also depended on ACh concentration, the range of which almost coincided with that for ICa inhibition (data not shown). The slow inward current was cationic (Na + and Ca 2+ removal abolished it) with a reversal potential of about 0 mV (inset in Fig. 1C), and its activation was strongly suppressed by pretreatment with AF-DX116 (1 mM), a relatively specific blocker for M2 muscarinic receptor subtype, or by overnight incubation of gastric muscle strips with PTX (500 ng /ml; see Materials and Methods) (data not shown). These results strongly suggest that the channels underlying the slow inward current are cation channels activated through stimulation of the M 2 receptor /G i/ o protein pathway that have ubiquitously been observed in the whole gastrointestinal tract (7,8).

Z-338 solely enhances ICa
Z-338 also enhanced ICa in a concentration-dependent fashion (ED 50 = 0.12 mM) (Fig. 2: A and B). However, different from ACh, Z-338 did not exhibit any discernible inhibition of ICa even at a concentration as high as 100 mM (Fig. 2B), and it failed to evoke slow inward currents. The enhancing actions of Z-338 on I Ca are voltage-independent, as indicated by almost symmetrical current voltage relationships before and after application of ACh (Fig. 3C). Over a wide range of membrane potentials, the extent of I Ca potentiation by Z-338 remained nearly the same. Furthermore, the enhancing effects on ICa (open circle in Fig. 2B) or inability to induce inward cationic currents of Z-338 were not changed, when the currents were recorded with nystatin-perforated recording, suggesting a less significant role of intracellular Ca 2+ concentration in these effects. The effects of Z-338 and ACh to enhance ICa, appear to be mutually occlusive, since after ICa had already been enhanced by 10 nM ACh, further application of Z-338 (1 mM) was ineffective, and vice versa (Fig. 3A). These results strongly suggest that common signaling pathways are involved in the enhancing actions of Z-338 and ACh.
The effects of ACh and Z-338 on I Ca are likely to occur through activation of muscarinic recptor /Gq/11 G-protein /PKC pathways. Firstly, these effects were completely eliminated by bath application of atropine (10 mM) or intra- cellular perfusion of GDPbS (500 mM) via the pipette (Fig. 3B). Secondly, overnight incubation with PTX (500 ng /ml, see Materials and Methods) did not significantly affect the efficacy of ACh or Z-338 to cause potentiation and inhibition of ICa (Fig. 4C), thus precluding an active role of Gi/o G-protein in these signal transduction pathways. Thirdly, intracellular perfusion of PKC inhibitory peptide (150 mM) resulted in almost complete abolition of both enhancing and inhibitory actions of ACh and Z-338 on I Ca (Fig. 4A), and conversely, a potent PKC activator phorbol butyrate (PDBu, 250 nM) caused the potenti-ation of I Ca , which occurred in a non-additive fashion to the effects of 1 m M Z-338 (Fig. 4B).

Involvement of M 5 receptor in Z-338 induced I Ca potentiation
To determine the muscarinic receptor subtypes involved in the enhancing and inhibitory actions of Z-338 and ACh, we examined the effects on ICa of several widely used muscarinic antagonists having different subtype specificities (9, 10). These include pirenzepine (1 mM; relatively M1 subtype-selective; pA2 = 8.0), AF-DX116 (1 mM; rela- tively M2 subtype selective; pA2 = 8.2 -9.0), 4-DAMP (100 nM; almost equally effective for M1, M3 and M5 subtypes; pA 2 = 9.2, 9.2 and 8.9, respectively), and oxybutynin (100 nM; shows higher inhibitory efficacies for M1, M3 and M4 subtypes than for M2 and M5 subtypes; pA 2 = 8.5, 7.8, 8.7, 8.2 and 7.6 for M 1 -M 5 receptors, respectively). As summarized in Fig. 5, neither pirenzepine, AF-DX116, nor oxybutynin significantly altered, whereas 4-DAMP almost completely eliminated the enhancing effects of Z-338. Similarly, both potentiation (not shown) and inhibition (Fig. 5B) of ICa by ACh were greatly reduced by pretreatment with 0.1 mM 4-DAMP (pirenzepine, AF-DX116; ineffective), but interestingly, in the presence of 0.1 m M oxybutynin, inhibition by high concentrations of ACh (100 m M) was reverted to potentiation, which was probably unmasked by elimination of concurrent inhibition. The extent of this potentiation did not become greater by further addition of Z-338 (Fig. 5B). However, when the concentration of oxybutynin was raised to greater than 1 mM, the potentiation was also significantly inhibited (data not shown). These results collectively indi-  cate that ICa potentiation by Z-338 or low concentrations of ACh is mediated by a muscarinic receptor subtype most sensitive to 4-DAMP, while ICa inhibition by high concentrations of ACh is via one preferring both 4-DAMP and oxybutynin, thus suggesting the involvement of muscarinic receptors showing similar pharmacological profiles to M3 and M 5 receptors, respectively (see Discussion).

DISCUSSION
The major finding of the present study is that Z-338, a novel gastroprokinetic drug, which has been reported to inhibit pre-junctional muscarinic autoreceptors (2), enhances voltage-dependent L-type Ca 2+ current in gastric smooth muscle through stimulation of a muscarinic receptor showing biochemical and pharmacological properties consistent with those of the M5 subtype. This is supported by several different lines of evidence. The effects of Z-338 are: i) resistant to PTX treatment, ii) almost completely eliminated by bath application of atropine or intracellular perfusion of GDPb S or PKC-selective inhibitory peptide, iii) mimicked by PDBu or low concentrations of ACh, both of which occurred in a non-additive fashion with respect to Z-338. These results strongly suggest the involvement of PI turnover-linked, G-protein coupled receptors; i.e., either M1, M3 or M5 muscarinic receptor subtypes. iv) The observed pharmacological profile of the effects of Z-338 as well as ACh is most consistent with the reported profile of M5 subtype; pirenzepine and AF-DX116 were ineffective, and oxybutynin showed a lower antagonistic efficacy than 4-DAMP against I Ca potentiation by Z-338 or ACh ( Fig. 5; refs. 10, 11). Calculation with reported pA2 values of muscarinic antagonists (see above) and an ED50 value of Z-338 obtained in this study (0.12 mM, Fig. 2B) suggests that expected occupancy by Z-338 (1 mM) of M1 and M2 receptors in the presence of pirenzepine (1 mM) and AF-DX116 (1 mM), respectively, and of M3 and M5 receptors in the presence of 0.1 mM 4-DAMP is less than 10%, whereas that for M3 and M5 receptors with 0.1 mM oxybutynin is 14% and 63%, respectively. Notably, when 1 mM oxybutynin is present, the expected occupancy of M5 receptor by Z-338 (1 m M) reduces to 17%, all values calculated being most compatible with the involvement of M 5 -receptor. Thus, the most likely interpretation of these data is, although other nonspecific effects of Z-338 such as on G-protein and downstream effectors cannot entirely be precluded, that a muscarinic receptor subtype similar to M5 subtype is functionally expressed in gastric smooth muscle and coupled specifically to voltage-dependent Ca 2+ channel via the G q /11 /PKC pathway. On the other hand, inhibition of ICa caused by high concentrations of ACh, which is deficient in the actions of Z-338, seems mediated by activation of another PI turnover-linked muscarinic receptor in gastric smooth muscle, most likely the M3 subtype, since the inhibition was not affected by pirenzepine or AF-DX116, and comparably suppressed by 4-DAMP and oxybutynin. Inability of Z-338 to cause I Ca inhibition is compatible with the fact that no detectable binding occurs between tritiated Z-338 and M3 muscarinic receptor (2).
Muscarinic receptors in the GI tract have been characterized mostly by means of radioligand binding techniques, and it has been reported that the predominant subtypes expressed in gut smooth muscles are M2 and M3 (12,13). In contrast, in pre-junctional tissues in the GI tract such as myenteric neurones, M 1 and M 2 receptors are preponderant and thought to play primary roles as negative autoreceptors for ACh release (14,15). It has been suggested that the M 3 subtype is functionally most important for mediating contractions in GI muscle, since potent M3receptor antagonists such as 4-DAMP strongly attenuate peristaltic movements of whole gut and contractions of isolated muscle preparations induced neurally or by exogenously applied cholinomimetic drugs (12,16). In contrast, despite its abundant expression, the extent of contribution of M2 receptor to excitatory responses in the GI tract remains rather elusive; only a limited role in antagonizing the relaxant effects of cAMP-increasing agents via inhibition of adenylate cyclase has been suggested (17). However, recent patch clamp experiments in guinea pig gastric and ileal smooth muscle cells revealed that M 2 receptors also activate nonselective cation channels via a PTX-sensitive Gi/o G-protein, thereby depolarizing the membrane and in turn causing Ca 2+ entry through the nifedipine-sensitive voltage-dependent pathway (18 -20). Since these channels are very susceptible to changes in intracellular Ca 2+ concentration (i.e., its elevation strongly potentiates the channel activities; 21,22) as well as extracellular Ca 2+ concentration (23), this M2 receptor-mediated mechanism may significantly contribute to generating maintained contractions in concert with IP 3 -mediated Ca 2+ release caused by concomitant activation of M3 receptor (7). Significant contribution of M2 receptor mediated pathways to contractile responses of GI muscle has been clearly demonstrated in M3-receptor knock-out mice (13,24).
Very recently, more modern biochemical and molecular strategies such as RT-PCR and immunocytochemical techniques have provided new lines of evidence for the distribution profile of muscarinic receptors in GI muscle. In both freshly dissociated and primary cultured human esophageal smooth muscle cells, mRNA transcripts for and plasma membrane-localized immunoreactivities to all muscarinic receptor subtypes including the M5 subtype were detected using their specific primers and antibodies (25,26). The M 5 receptor was originally found to be enriched in specialized regions of the brain, but later shown to be expressed more ubiquitously throughout the brain and also in nonneuronal tissues such as blood cells and salivary glands (10,27). However, very little is yet known about what molecular targets and functions are associated with M5 receptors and their downstream signaling cascades, except for some indirect suggestions of their possible implications in modulating dopaminergic transmission and salivary gland secretion and activating nitric oxide synthase (10,27). In this respect, our present results have provided the intriguing possibility that a muscarinic receptor pharmacologically indistinguishable from the M5 receptor upregulates L-type Ca 2+ channel activities via activation of a specific PKC pathway and may contribute to enhancing the gastric motility (see below). Recently, modulation of Ltype Ca 2+ channels by muscarnic receptors was investigated by recombinantly expressing five subtypes of muscarinic receptors (m1 -m5) into an established fibroblast cell line, NIH 3T3 cells (28). Somewhat unexpectedly, stimulation of PI turnover-linked receptors, i.e., m1, m3 and m5 receptors by ACh all resulted in inhibition of Ca 2+ currents via activation of PKC, thus seemingly contradicting the results of the present study. Although there is no good explanation for this discrepancy, it is worthwhile noting that in several native tissues, stimulation of PKC often biphasically regulates (29) or solely enhances (30 -32) voltage-dependent Ca 2+ channel activities. A possible molecular background for such differences may be the involvement of splice variants of a 1C subunit containing different PKC phosphorylation sites at the N-terminal (e.g., a 1Ca versus a 1Cb; 29,33) or different PKC isoforms (34,35). Obviously, further studies are required to distinguish between these possibilities.
A previous study employing recombinant expression of muscarinic receptors in Xenopus oocytes has shown that Z-338 is capable of binding M1 and M2 (but not M3) receptors and acts antagonistically on these receptors to inhibit activation of Ca 2+ -dependent Clcurrents by IP 3 -mediated Ca 2+ release via M1 receptor and that of co-expressed GIRK1, an inward-rectifying K + channel via the M2 receptor /PTX-insensitive G-protein pathway (2). These antagonistic effects of Z-338 are consistent with its observed enhancing actions on electrically evoked ACh release and excitatory junction potentials in guinea pig stomach muscle strips (2, 3), which can be accounted for by the inhibition of M1 and M2 autoreceptors residing in prejunctional cholinergic nerve terminals and negatively regulating ACh release (14,36). In contrast, the data of the present study have clearly shown that Z-338 agonistically enhances Ltype voltage-dependent Ca 2+ channel activities probably through stimulation of post-junctional M5 receptor (see above), but cannot activate nonselective cation channels on the smooth muscle membrane even at high micromolar concentrations. The latter observation is consistent with the previous findings of microelectrode experiments that Z-338 (10 mM) did not alter the resting membrane potential or basal muscle tone (3), thus suggesting that Z-338 has no stimulatory effects on post-junctional M2 receptors. The lack of agonistic actions of Z-338 on post-junctional M3 receptors is also suggested by virtual absence of its inhibitory effects on L-type Ca 2+ channels which are evident at high concentrations of ACh (see above). Such selective stimulation by Z-338 of post-junctional M 5 but not M 2 or M3 receptors may serve advantageously to specifically enhance spontaneous rhythmic contractions of GI muscle. It is well known that the rhythmic contractions are closely associated with nifedipine-sensitive voltage-dependent Ca 2+ entry in smooth muscle cells which are activated by slow pace-making depolarizations (slow waves) originated from interstitial cells of Cajal (ICCs) (37,38). Thus, if this Ca 2+ entry would selectively be potentiated through stimulation of M 5 receptor, only the rhythmic contractions would be accentuated with little accompanying increase in the basal tension. This might be therapeutically beneficial, because an excessive basal tone increase caused by M 2 and /or M 3 receptor stimulation might be deleterious rather than promoting to propel a bolus through the gut. Furthermore, the other additional actions of Z-338, e.g., enhancement of ACh release via inhibition of pre-junctional M1 and M2 autoreceptors as well as blockade of post-junctional M2 receptor, would also help to more effectively amplify rhythmic contractions with little change in the resting tension.