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

Abstract

ABSTRACT-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 Ca 2+ 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 mM), both of which were abolished by pretreatment with atropine (10 mM) or intracellular perfusion of GDPbS (500 mM). Z-338 (³1 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 GDPb S, but not by pertussis toxin (PTX) pretreatment (500 ng /ml). ACh (³1 m M) 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 m M), AF-DX116 (1 mM), nor oxybutynin (100 nM) could prevent Z-338 (1 mM) and ACh (10 nM) from enhancing I Ca , 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 I Ca , and conversely, pipette inclusion of PKC inhibitor peptide (150 mM) abolished the effects of ACh and Z-338 on I Ca . These results collectively suggest that although contribution of the M 3 receptor is not excluded, the major actions of Z-338 on gastric myocytes are potentiation of I Ca through activation of M 5 -like receptor.Keywords: Z-338, M5 muscarinic receptor, L-type calcium channel, Gastric smooth muscle 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 significan...

Effects of Z-338, a novel gastroprokinetic agent, on the actions of excitatory and inhibitory neurotransmitters on neurons in area postrema

“…All these findings are consistent with the view that Z-338 facilitates excitatory neuroeffector transmission in the stomach by increasing ACh release from cholinergic nerve terminals via inhibition of pre-junctional M1-and M2-mAChR or nAChR. In addition to these pre-junctional actions, Z-338 potentiates L-type voltage-dependent Ca 2+ currents (ICa) possibly through activation of M5-like-mAChR in guinea pig gastric myocytes (Morita et al, 2002). These pre-and post-synaptic effects taken together would explain the actions of Z-338 to enhance spontaneous contractions without changing the basal muscle tone of the stomach, and suggest that Z-338 could improve gastric emptying in patients with functional dyspepsia.…”

“…Furthermore, our recent study indicates that rat stomach receives efferent neural input from AP and electrical stimulation of AP neurons relaxes the stomach in rat, thereby suggesting that some functional roles of AP neurons in the regulation of gastric motility (Kawachi et al, 2008). Z-338 has well documented excitatory but not inhibitory action on stomach motility through the peripheral autonomic nervous system (Ueki et al, 1998;Nakajima et al, 2000;Kanemoto et al, 2002;Morita et al, 2002). The actions of Z-338 on central and peripheral autonomic nervous system, taken together, would therefore explain the observations that Z-338 induces postmeal relaxation and improve postprandial fullness in patients with functional dyspepsia (Tack et al, 2002).…”

“…has significant benefit over placebo for global symptom score including postprandial fullness and mean and maximum post-meal relaxation. It is puzzling, however, how Z-338 is able to enhance the post-meal relaxation, since several lines of evidence obtained so far indicate that Z-338 facilitates excitatory neuro-effector transmission through pre-and post-junctional mechanisms, whereas there are no significant changes in peripheral inhibitory neuro-effector transmission and basal muscle tone of the stomach (Nakajima et al, 2000;Ogishima et al, 2000;Morita et al, 2002).…”

Acetylcholine induces Ca 2+ oscillations via m3/m4 muscarinic receptors in the mouse oocyte

“…The expression rate of m5 is less than 2-5% of total brain mAChR protein, and its physiological role in vivo is still indistinct [11]. A few studies have shown that m5 is involved in an increase of [Ca 2+ ] i in rat glomerular podocytes [33], the contraction of ciliary muscles in dog eye [7], and an enhancing action on the L-type Ca 2+ currents [32].…”

Acotiamide, a novel gastroprokinetic for the treatment of patients with functional dyspepsia: postprandial distress syndrome

“…The researchers concluded that such selective stimulation by acotiamide of postjunctional M 5 but not M 2 or M 3 receptors would explain how it enhances spontaneous rhythmic contractions of smooth muscle with little accompanying increase in the basal tension [18].…”