Muscarinic-agonist and Guanine Nucleotide Stimulation of Myo-Inositol Trisphosphate Formation in Membranes Isolated from Bovine Iris Sphincter Smooth Muscle: Effects of Short-Term Cholinergic Desensitization richard E. Honkanen

Abstract

The effect of short-term cholinergic desensitization on muscarinic acetylcholine receptor (mAChR)-mediated activation of phospholipase C was investigated in membranes isolated from the bovine iris sphincter smooth muscle. Membranes prepared from normal or desensitized muscles, prelabeled with either [³H]myo-inositol or ³²P from [γ-³²P]ATP, were incubated with a hydrolysis-resistant analogue of GTP, GTPγS, or GTPγS plus carbachol (CCh), and the production of [³H]myo-inositol 1,4,5-trisphosphate (IP₃) and the breakdown of polyphosphoinositides were assessed. In normal membranes, GTP (≥1 mM), GTPγS (>70 µ) and GTPγS (1 µ) plus CCh (10 µ), but not GDP or GDPβS, increased phosphatidylinositol 4,5-bisphosphate (PIP₂) hydrolysis and IP₃ production. GTPγS increased IP₃ accumulation in a time- and dose-dependent manner, and CCh, which had no effect on phospholipase C activity in the absence of GTPγS, potentiated the effects of GTPγS. The effect of CCh plus GTPγS on IP₃ production was inhibited by atropine, had an absolute requirement for nM amounts of Ca²⁺ and was not affected by pertussis toxin. At higher concentrations (>7 µ), Ca²⁺ alone induced PIP₂ hydrolysis. Short-term exposure (< 60 min) of the muscle to CCh (100 µ) did not affect the total number (B_max) of mAChRs nor their affinity (K_D) for [³H]-N-methylscopolamine. Desensitization did, however, result in: (1) a loss of the CCh-high affinity binding state of the sphincter mAChRs in a manner analogous to that produced by GTPγS; (2) a loss of the ability of GTPγS to affect CCh binding to the receptors; and (3) an attenuation of the GTPγS plus CCh-stimulated PIP₂ hydrolysis. In conclusion, the data presented suggest that, in the iris smooth muscle, G-proteins are involved in the coupling of mAChRs to phospholipase C and that short-term cholinergic desensitization results in (1) the uncoupling of the receptor-G-protein complex and (2) the attenuation of mAChR-activation of phospholipase C.

Key Words:

• PIP₂
• phosphatidylinositol 4
• 5-bisphosphate
• PIP
• phosphatidylinositol 4-phosphate
• PI
• phosphatidylinositol
• IP₃
• myo-inositol 1,4,5-trisphosphate
• GTPγS
• guanosine 5–0-(3-thiotriphosphate)
• CCh
• carbamylcholine
• NMS
• N-methylscopolamine
• mAChRs
• muscarinic acetylcholine receptors