title: Muscarinic acetylcholine receptor-mediated phosphoinositide turnover in cultured cerebellar granule cells: desensitization by receptor agonists.
abstract: Cultured granule cells prepared from 8-day postnatal rats were used for the study of carbachol-induced phosphoinositide turnover. The addition of carbachol induced a 20- to 30-fold increase in [3H]inositol monophosphate (IP1) accumulation within 30 min in the presence of 20 mM LiCl in granule cells prelabeled with [3H]myoinositol. Carbachol also stimulated the formation of [3H]inositol bisphosphate and [3H]inositol trisphosphate assayed either in the presence or in the absence of lithium; the increase in [3H]inositol triphosphate and [3H]inositol biphosphate synthesis appeared to be faster in the time course but much smaller in amount when compared with the formation of [3H]IP1. The EC50 of carbachol was approximately 7 microM, and the saturation concentration was about 100 microM. This carbachol-induced response was completely blocked by two muscarinic acetylcholine receptor (mAChR) antagonists, atropine and pirenzepine, with a Ki of 0.5 and 120 nM, respectively. Saturation studies of the binding of [3H]N-methylscopolamine and [3H]quinuclidinyl benzilate to mAChRs in intact granule cells revealed the presence of a single class of binding sites with a Kd of 140 and 126 pM, respectively, and a Bmax of approximately 70 fmol/10(6) cells for both ligands. Within 1 hr after pre-exposure of cells to carbachol, the subsequent carbachol-induced [3H]IP1 accumulation was reduced by about 50%, whereas mAChR binding, assessed by using either [3H]N-methylscopolamine or [3H]quinuclidinyl benzilate, was unchanged. A second slower phase of desensitization was associated with a loss of mAChR binding sites; at 18 hr, the decrease of responsiveness was about 80%, whereas the loss of mAChR sites was about 60%.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008297:Male; D019832:N-Methylscopolamine; D010095:Oxotremorine / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D010890:Pirenzepine / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000502:physiology*; D012602:Scopolamine Derivatives / Q000378:metabolism; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
chemical_list: D010703:Phorbol Esters; D010716:Phosphatidylinositols; D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; D010566:Virulence Factors, Bordetella; D010890:Pirenzepine; D010095:Oxotremorine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002217:Carbachol; D019832:N-Methylscopolamine