Muscarinic receptor subtypes in rat pancreatic islets: binding and functional studies

Abstract

Cholinergie agents arepotent modulators of insulin release that aet via musearinie reeeptors. We now investigated the musearlnie receptor subtype present in rat panereatic islets in binding and funetional studies. Binding of 5 nM [ 3 H]N-methylscopolamine ([ 3 H]NMS) was half maximal at 30 min. At 60 min, the maximal total bindingwas 1.29% and the non-specifie binding (presence of 100 ,uM atropine) was 0.18% of the total radioaetivity per 10 f.'g islet protein. The high-affinity Kds were 8.5, 56, 1300 and 1300 nM, respectively. The high affinity Kd of the muscarinie receptor agonist, arecaidine propargyl ester (APE), was 8.1 nM. The EC 50 for the biologieal effects of APE on insulin and glucagon secretion was 3.2 and 2.3 nM. The rank order for the high-affinity biological effects of antagonists (inhibition of APE-mediated insulin/ glucagon release) was almost the same as for binding. The data indicate that rat pancreatie islets contain neither an MI subtype (high-affinity for pirenzepine) nor an M 2 subtype (high-affinity for methoctramine) receptor. However, the data evidence an M 3 receptor subtype, since SiHC in the absence of the M 1 receptor subtype shows a relatively high affinity to the receptors in rat panereatie islets.

Muscarinic Stimulation of Pancreatic Insulin and Glucagon Release Is Abolished in M3 Muscarinic Acetylcholine Receptor–Deficient Mice

“…5), indicating that this response is mediated by the M 3 receptor subtype in WT animals. In agreement with this finding, previous in vitro and in vivo studies using muscarinic antagonists of limited receptor subtype selectivity have also suggested that the M 3 subtype plays a key role in muscarinic receptorϪmediated glucagon release (26,27).…”

“…Classic pharmacological studies using different "subtype-preferring" muscarinic antagonists have suggested that the M 3 receptor subtype (previously also referred to as "glandular M 2 receptor subtype") (32) plays a key role in the control of insulin secretion (26,27,32). However, the proper interpretation of these experiments is complicated by the limited subtype selectivity of the muscarinic antagonists used in these studies.…”

“…Mice were killed by cervical dislocation, and whole pancreata were dissected and immediately frozen in liquid nitrogen. Protein extracts were prepared using the acid-ethanol method (26). In brief, pancreata weighing 200 -300 mg were homogenized in 1 ml acid-ethanol (95% ethanol and 10.2 N HCl at a ratio of 50:1) by an Ultrasonic Homogenizer (Biologic) for 2 min applying 20 pulses.…”

Non-Selectivity of the Monoclonal Antibody M35 for Subtypes of Muscarinic Acetylcholine Receptors

“…Medium spiny cells in the striatum, motorneurons of the mesencephalon, endocrine cells of the islets of Langerhans in the pancreas, and cells in the islands of Calleja are selectively enriched in m1, m2, m3, and m4 mAChR subtypes, respectively [12,13,20,42]. M35 immunoreactivity was observed in all four areas (Fig.…”

Protein kinase C isoform specificity of cholinergic potentiation of glucose-induced pulsatile 5-HT/ insulin release from mouse pancreatic islets

“…Although glucose is the primary physiological trigger and regulator of insulin release, this is also amenable to modulation by the parasympathetic nervous system, which relies mostly on acetylcholine release from islet nerve endings and its binding to specific β-cell muscarinic (M 3 ) receptors (Prentki and Matschinsky, 1987;Verspohl et al, 1990). Cholinergic stimulation of pancreatic β-cells involves phosphoinositide-specific phospholipase C (PLC) activation and the consequent generation of both inositol 1,4,5-trisphosphate (1,4,5-IP 3 ), which triggers Ca 2+ release from internal stores, and 1,2-sn-diacylglycerol (DAG) (Nishizuka, 1986;Wollheim and Biden, 1986), which may lead to the activation of either conventional (i.e., Ca 2+ -and DAG/ phorbol ester-sensitive) or novel (i.e., Ca 2+ -insensitive and DAG/phorbol estersensitive) protein kinase C (PKC) isoforms (cPKC and nPKC, respectively).…”