Muscarinic receptor subtypes in bovine adrenal medulla

Catecholamine secretion in the bovine adrenal medulla is evoked largely by nicotinic receptor activation. However, bovine adrenal medulla also contain muscarinic receptors that mediate several cell responses. To understand the physiological role of muscarinic receptors in the bovine adrenal medulla it is important to identify the pharmacological subtypes present in this tissue. For this, we analyzed the abilities of differnt selective muscarinic antagonists in displacing the binding of the non-selective antagonist [3H] quinuclidinyl benzylate to an enriched plasma membrane fraction prepared from bovine adrenal medulla. All the selective antagonists bind at least two bindings sites with different affinities. The binding profile of the sites with high proportion is similar to the M2 subtype and those present in low proportion have a M1 profile. However, some variation in the proportion of the sites for the different ligands suggest the presence of the third pharmacological subtype (M3). We conclude that the sites in high proportion (60–80%) correspond to M2 muscarinic subtypes, and the rest is constitute by M1 plus M3 subtypes. The presence of multiplicity of subtypes in the adrenal medulla membranes suggests a diversity of functions of muscarinic receptors in the adrenal gland.Abbreviations [3H]QNB [3H]quinuclidinyl benzylate - HHSiD hexahydro-siladifenidol-hydrochloride - AF-DX 116 11-[[2-(diethylamino)methyl]]-1-piperidinyl]-5,11-dihydro-6H-pyrido[2,3,-b][1,4]benzodiazepin-6-one - 4-DAMP 4-diphenylacetoxy-N-methyl piperidine methobromide     

Muscarinic receptor subtypes modulating smooth muscle contractility in the urinary bladder

Normal physiological voiding as well as generation of abnormal bladder contractions in diseased states is critically dependent on acetylcholine-induced stimulation of contractile muscarinic receptors on the smooth muscle (detrusor) of the urinary bladder. Muscarinic receptor antagonists are efficacious in treating the symptoms of bladder hyperactivity, such as urge incontinence, although the usefulness of available drugs is limited by undesirable side-effects. Detrusor smooth muscle is endowed principally with M2 and M3 muscarinic receptors with the former predominating in number. M3 muscarinic receptors, coupled to stimulation of phosphoinositide turnover, mediate the direct contractile effects of acetylcholine in the detrusor. Emerging evidence suggests that M2 muscarinic receptors, via inhibition of adenylyl cyclase, cause smooth muscle contraction indirectly by inhibiting sympathetically (beta-adrenoceptor)-mediated relaxation. In certain diseased states, M2 receptors may also contribute to direct smooth muscle contraction. Other contractile mechanisms involving M2 muscarinic receptors, such as activation of a non-specific cationic channel and inactivation of potassium channels, may also be operative in the bladder and requires further investigation. From a therapeutic standpoint, combined blockade of M2 and M3 muscarinic receptors would seem to be ideal since this approach would evoke complete inhibition of cholinergically-evoked smooth muscle contractions. However, if either the M2 or M3 receptor assumes a greater pathophysiological role in disease states, then selective antagonism of only one of the two receptors may be the more rational approach. The ultimate therapeutic strategy is also influenced by the extent to which pre-junctional M1 facilitatory and M2 inhibitory muscarinic receptors regulate acetylcholine release and also which subtypes mediate the undesirable effects of muscarinic receptor blockade such as dry mouth. Finally, the consequence of muscarinic receptor blockade in the central nervous system on the micturition reflex, an issue which is poorly studied and seldom taken into consideration, should not be ignored.     

Muscarinic receptor subtypes: M1 and M2 biochemical and functional characterization

The heterogeneity of muscarinic receptors was examined in sympathetic ganglia and atria by “in vitro” binding techniques and functional studies. As tools we have used the classical antagonist atropine, the selective antagonist pirenzepine and the unique muscarinic agonist McN-A-343. In binding studies atropine showed similar affinities to muscarinic sites in ganglionic and atrial membranes with dissociation constants of 1.1 and 3.2 nM, respectively. In contrast, pirenzepine displayed a distinctly different binding profile. In atria it bound to an homogenous population of low affinity sites (diss. const. 620 nM) while in ganglia it revealed the presence of two sites: a major population of high affinity sites (diss. const. 11 nM) and a minor one of lower affinity (diss. const. 280 nM). The functional correlate of the receptor properties in the two tissues was studied in the pithed rat by measuring A) the increase of arterial pressure evoked by McN-A-343 through selective activation of muscarinic receptors in ganglia and B) the bradycardia elicited by acetylcholine release in the heart through vagal stimulation. Mirroring the “in vitro” binding data atropine inhibited both muscarinic responses in the same narrow range of doses (2–30 μg/kg i.v.) whereas pirenzepine showed similar potency to atropine in inhibiting ganglionic stimulation (ED50 4.1 μg/kg i.v.) but was almost two orders of magnitude weaker in blocking vagal bradycardia (ED50 172 μg/kg i.v.). These data suggest that McN-A-343 and pirenzepine act selectively on a common muscarinic receptor subtype, a finding which agrees with the view that muscarinic receptors are heterogenous and that excitatory ganglionic receptors (Ml) are distinguishable from those (M2) present in effector organs like smooth muscle and heart.     

Muscarinic receptor activation inhibits both release and synthesis of noradrenaline in rat hippocampal synaptosomes

Rat hippocampal synaptosomes were used to investigate the effects of muscarinic cholinergic drugs on the release of [³H]noradrenaline and the rate of noradrenaline synthesis. Oxotremorine and acetylcholine caused depression of K⁺-evoked release of [³H]noradrenaline; these effects were reversed by atropine and pirenzepine. Muscarinic agonists depressed 2-chloroadenosine- and isoprenaline-stimulated noradrenaline synthesis but had no effect on 8-Br-cyclic AMP-stimulated synthesis. Oxotremorine also depressed the K⁺-acceleration of noradrenaline synthesis. The action of pirenzepine suggests that the inhibition of release and synthesis are mediated by separate muscarinic receptor subtypes.     

Muscarinic receptor subtypes mediating central and peripheral antinociception studied with muscarinic receptor knockout mice: a review

To gain new insight into the physiological and pathophysiological roles of the muscarinic cholinergic system, we generated mutant mouse strains deficient in each of the five muscarinic acetylcholine receptor subtypes (M(1)-M(5)). In this chapter, we review a set of recent studies dealing with the identification of the muscarinic receptor subtypes mediating muscarinic agonist-dependent analgesic effects by central and peripheral mechanisms. Most of these studies were carried out with mutant mouse strains lacking M(2) or/and M(4) muscarinic receptors. It is well known that administration of centrally active muscarinic agonists induces pronounced analgesic effects. To identify the muscarinic receptors mediating this activity, wild-type and muscarinic receptor mutant mice were injected with the non-subtype-selective muscarinic agonist, oxotremorine (s.c., i.t., and i.c.v.), and analgesic effects were assessed in the tail-flick and hot-plate tests. These studies showed that M(2) receptors play a key role in mediating the analgesic effects of oxotremorine, both at the spinal and supraspinal level. However, studies with M(2)/M(4) receptor double KO mice indicated that M(4) receptors also contribute to this activity. Recent evidence suggests that activation of muscarinic receptors located in the skin can reduce the sensitivity of peripheral nociceptors. Electrophysiological and neurochemical studies with skin preparations from muscarinic receptor mutant mice indicated that muscarine-induced peripheral antinociception is mediated by M(2) receptors. Since acetylcholine is synthesized and released by different cell types of the skin, it is possible that non-neuronally released acetylcholine plays a role in modulating peripheral nociception. Our results highlight the usefulness of muscarinic receptor mutant mice to shed light on the functional roles of acetylcholine released from both neuronal and non-neuronal cells.     

Muscarinic receptor subtypes mediate stimulatory and paradoxical inhibitory effects on an insulin-secreting beta cell line

Acetylcholine (ACh), a major neurotransmitter from the autonomic nervous system, regulates the cholinergic stimulation of insulin secretion, through interactions with muscarinic receptors. The present study has characterised the individual involvement of muscarinic receptor subtypes in ACh-induced insulin secretion, using clonal beta cells and selective muscarinic receptor antagonists. BRIN BD11 cells clearly expressed mRNA encoding m1--m4 whereas m5 was not detected by RT-PCR. Insulin release was measured from BRIN BD11 cells treated with ACh in the presence of muscarinic receptor antagonists at concentrations ranging from 3 nM to 1 microM. 300 nM of muscarinic toxin-3 (M4 antagonist) and 1 microM of methoctramine (M2 antagonist) increased ACh (100 microM) stimulated insulin secretion by 168% and 50% respectively (ANOVA, P<0.05). The antagonists alone had no effect on insulin secretion. In contrast, 300 nM of pirenzepine (M1 antagonist) and 30 nM of hexahydro-sila-difenidol p-fluorohydrochloride (M3 antagonist) inhibited ACh stimulation by 91% and 84% respectively (ANOVA, P<0.01). It is concluded that ACh acts on different receptor subtypes producing both a stimulatory and an inhibitory action on insulin release.     

Distinct in vitro interaction pattern of dopamine receptor subtypes with adaptor proteins involved in post-endocytotic receptor targeting

The sequences contributing to the catalytic site of protein kinases are not all comprised within the highly conserved catalytic core. Thus, in mammalian cAMP-dependent protein kinase (PKA), the C-terminal sequence participates in substrate binding. Using synthetic peptides mimicking the FxxF motif present at most C-termini of AGC kinases, we have raised highly specific antibodies which are potent and specific inhibitors of the catalytic activity of the cognate protein kinase. Taking into account the structure of PKA, these results point to the potential of the C-terminal region of protein kinases as a target for designing specific protein kinase inhibitors.     

Muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom: interaction with subtypes of human mAChR.

Snake venoms can contain a variety of well-studied neurotoxins, especially nicotinic acetylcholine receptor inhibitor, normally called postsynaptic neurotoxin. Karlsson first reported muscarinic acetylcholine receptor (mAChR) inhibitor from snake venom. In a previous study in our laboratory, we found a mAChR inhibitor from Naja naja sputatrix venom that bound to rat brain synaptosomes. Brain synaptosomes contain all subtypes of mAChRs, and thus the exact selectivity of the inhibitor could not be determined. mAChR inhibitor from N. naja sputatrix venom was purified and the binding to all human mAChR subtypes (M1, M2, M3, M4, and M5) was investigated and is reported in this communication. The inhibitor bound to all subtypes of the human mAChR, but showed considerably high selectivity for the M5 subtype. It was also found that the reduction of disulfide bonds in the inhibitor eliminated the binding to the mAChR. This suggests that a specific tertiary conformation maintained by disulfide bonds is essential for binding to the mAChR. An oligo peptide, QIHDNCYNE, comparable to a part of the inhibitor molecule, was synthesized and studied for its binding to the mAChR. The synthetic peptide did not show any binding activity, suggesting this portion of the inhibitor molecule is not involved in mAChR binding. The selective binding of the M5 mAChR subtype to antagonists has not yet been reported. Therefore, the purified inhibitor reported in this communication may be a useful tool to clarify the mechanism of muscarinic cholinergic transmission.     

Muscarinic receptor sites in human foetal brain

The specific binding of the muscarinic cholinergic ligand N-methylscopolamine to human foetal brain has been measured. A level of binding of 64 pmol/g protein was found with a dissociation constant, K_d of 0.27 nM. Values of 0.17 nM min^?1 and 0.048 min^?1 for the association rate constant, K_on, and the dissociation rate constant K_off respectively, were obtained. The pharmacological properties of the binding site were found to be very similar to those reported for muscarinic receptors from adult mammalian brain except that the binding of pirenzepine and the nicotinic antagonists d-tubocurarine and decamethonium shows differences from that seen in adult brain.     

Mossy fiber Zn2+ spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3 circuits

Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non-N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor-mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.     

Muscarinic excitation in grasshopper song control circuits is limited by acetylcholinesterase activity

The species- and situation-specific sound production of grasshoppers can be stimulated by focal application of both nicotinic and muscarinic receptor agonists into the central body complex of the protocerebrum. Pressure injection of the intrinsic transmitter acetylcholine only elicits fast and short-lived responses related to nicotinic receptor-mediated excitation. Prolonged sound production that includes complex song patterns requires muscarinic receptor-mediated excitation. In addition, basal muscarinic excitation in the central body neuropil seems to determine the general motivation of a grasshopper to stridulate. To demonstrate that endogenous acetylcholinesterase limits the activation of muscarinic receptors by synaptically released acetylcholine in the central body of Chorthippus biguttulus, we investigated both its presence in the brain and effects on sound production resulting from inhibition of esterase activity. Acetylcholinesterase activity was detected in the upper and lower division of the central body. Both these neuropils known to be involved in the cephalic control of stridulation were also shown to contain muscarinic acetylcholine receptors expressed by columnar neurons suggested to serve as output neurons of the central complex. Pressure injection of the acetylcholinesterase inhibitor eserine into protocerebral control circuits of restrained male grasshoppers stimulated long-lasting stridulation that depended on scopolamine-sensitive muscarinic receptors. In restrained males, eserine released the typical response song by potentiating the stimulatory effect of the conspecific female song. Eserine-mediated inhibition of acetylcholinesterase in the central body prolongs the presence of synaptically released acetylcholine at its postsynaptic receptors and increases its potency to activate muscarinic receptor-initiated signaling pathways acting to promote grasshopper sound production.     

Architecture of spatial circuits in the hippocampal region

The hippocampal region contains several principal neuron types, some of which show distinct spatial firing patterns. The region is also known for its diversity in neural circuits and many have attempted to causally relate network architecture within and between these unique circuits to functional outcome. Still, much is unknown about the mechanisms or network properties by which the functionally specific spatial firing profiles of neurons are generated, let alone how they are integrated into a coherently functioning meta-network. In this review, we explore the architecture of local networks and address how they may interact within the context of an overarching space circuit, aiming to provide directions for future successful explorations.     

Prostaglandin E receptor subtypes involved in stimulation of gastroduodenal bicarbonate secretion in rats and mice.

We investigated prostaglandin E (EP) receptor subtypes responsible for the HCO3- stimulatory action of prostaglandin E2 (PGE2) in the gastroduodental mucosa, by examining the effects of various prostanoids with subtype specific EP receptor agonists in rats and those of PGE2 in knockout mice lacking EP1 or EP3 receptors. In rats, gastric HCO3- secretion was stimulated by i.v. administration of PGE2, 17-phenyl PGE2 the selective EP1 agonist as well as sulprostone the EP1 and EP3 agonist, but was not affected by other EP agonists such as butaprost the selective EP2 agonist, ONO-NT-012 the selective EP3 agonist or 11-deoxy PGE1 the EP3 and EP4 agonist. In contrast, the HCO3- secretion in rat duodenums was stimulated by PGE2, sulprostone, ONO-NT-012 as well as 11-deoxy PGE1 but not affected by either 17-phenyl PGE2 or butaprost. The HCO stimulatory effect of sulprostone in the stomach was significantly inhibited by ONO-AE-829, the selective EP1 antagonist. On the other hand, PGE2 applied topically to the mucosa for 10 min caused a dose-dependent increase of HCO3- secretion in both the stomach and duodenum of wild-type mice. The HCO3- stimulatory action of PGE2 in the stomach was also observed dose-dependently in knockout mice lacking EP3-receptors but was absent in EP1-receptor knockout mice, while the stimulatory effect in the duodenum was observed in EP1-receptor knockout mice, similar to wild-type animals, but not in knockout mice lacking EP3-receptors. These results indicate that PGE2 stimulates HCO3- secretion via different EP receptor subtypes in the stomach and duodenum; the former is mediated by EP1-receptors, while the latter mediated by EP3-receptors.     

Muscarinic induction of hippocampal gamma oscillations requires coupling of the M1 receptor to two mixed cation currents

Oscillatory network activity at gamma frequencies is assumed to be of major importance in cortical information processing. Whereas the synaptic mechanisms of gamma oscillations have been studied in detail, the ionic currents involved at the cellular level remain to be elucidated. Here we show that in vitro gamma oscillations induced by muscarine require activation of M1 receptors on hippocampal CA3 pyramidal neurons and are absent in M1 receptor-deficient mice. M1 receptor activation depolarizes pyramidal neurons by increasing the mixed Na(+)/K(+) current I(h) and the Ca(2+)-dependent nonspecific cation current I(cat), but not by modulation of I(M). Our data provide important insight into the molecular basis of gamma oscillations by unequivocally establishing a novel role for muscarinic modulation of I(h) and I(cat) in rhythmic network activity.