The characterization of muscarinic receptors inhibitory to adenylate dyclase in eight day post-natal rat cerebellar granule cells in primary culture

Abstract

In membranes from primary cultures of rat cerebellar granule cells, muscarinic (M2) agonists such as oxotremorine (3 x 10ˉ⁵M), carbachol (1 x 10ˉ⁴M), arecoline (1 x 10ˉ⁴M), and muscarine (3 x 10ˉ⁴M) inhibited basal adenylate cyclase by 60%. This muscarinic receptor mediated inhibition of adenylate cyclase is thought to result from agonist interaction with the muscarinic M2 receptor subtype. A two hour exposure with carbachol (1 x 10ˉ⁴M), resulted in a 50-60% desensitization of the inhibition of adenylate cyclase (the M2 receptor mediated response). Oxotremorine (3 x 10ˉ⁵M), produced a 30-40% desensitization of the M2 response. The EC₅₀ for the desensitization of the carbachol mediated inhibition of adenylate cyclase was 2 x 10ˉ⁶M. This action of carbachol was abolished with 1 x 10ˉ⁶M atropine. The desensitization was maximal after treating the cells in culture for 30 min. with 1 x 10ˉ⁴M carbachol. The resensitization of the M2 response reached control values one hour after treating the carbachol exposed cells in culture with 1 x 10ˉ⁶M atropine. Since muscarinic receptors can possibly stimulate guanylate cyclase, 1 x 10ˉ⁶M dibutryl-cGMP was tested, but no effect was observed. Since these cells are glutamatergic, DL-2-aminophosphonovaleric acid (1 x 10ˉ⁶M), an antagonist for one subtype of glutamate receptor, was tested, and no effect on the desensitization of the M2 receptor was evident. Various compounds which mimic the actions of second messengers such as cGMP, or diacylglycerol were also tested. Both forskolin (a cAMP analog, 1 x 10ˉ⁵M) and phorbol-12-myristate, 13-acetate (PMA, which mimics the action of diacylglycerol, 3 x 10ˉ⁷M) had no effect on the desensitization of the M2 receptor mediated response. The cultures were exposed to 1 x 10ˉ⁴M nicotine or to nicotinic antagonists such as hexamethonium (up to 3 x 10ˉ⁴M) and d-tubocurarine (1 x 10ˉ⁵M). No effect on the desensitization of the M2 receptor was observed with these nicotinic compounds themselves, or when exposed in conjunction with muscarinic agonists.