Neurochemical pharmacology of the cholinergic system in rat retina.

摘要

In retina, there is strong evidence that acetylcholine is a neurotransmitter based upon anatomical, electrophysiological, and pharmacological studies. The neurochemical response to acetylcholine has been relatively unexplored. The objectives were to measure the neurotransmitter, muscarinic binding sites, and neurochemical response to cholinergic stimulation. In addition modulation of this neurochemical response was examined.; Acetylcholine and choline content was found to be 179 {dollar}pm{dollar} 28.0 and 728 {dollar}pm{dollar} 50.0 pmol/mg protein, respectively, by high pressure liquid chromatography with electrochemical detection. Saturation binding studies with L- (benzilic-4,4{dollar}spprime{dollar}-{dollar}sp{lcub}3{rcub}{dollar}H(N)) quinuclidinyl benzilate demonstrated muscarinic binding sites with a Bmax of 123 fmol/mg protein and a K{dollar}sb{lcub}rm d{rcub}{dollar} of 0.09 nM. Agonist displacement curves demonstrated the presence of high and low affinity binding sites.; Radiotracer experiments with myo- ({dollar}sp3{dollar}H) inositol revealed that stimulation of muscarinic type 1 receptors, but not nicotinic receptors, led to accumulation of ({dollar}sp3{dollar}H) IP{dollar}sb1{dollar}. Pertussis toxin attenuated this response, which supports the presence of a G protein coupling muscarinic receptors to phospholipase C. The cholinotoxin, AF64A, depleted retinal acetylcholine and caused up-regulation of muscarinic binding sites and a supersensitive response to acetylcholine.; This neurochemical response was also modulated by streptozotocin-induced hyperglycemia. Retinae from streptozotocin treated animals had significantly greater cholinergic-mediated ({dollar}sp3{dollar}H) IP{dollar}sb1{dollar} accumulation compared to control animals. {dollar}beta{dollar}PMA led to a significant attenuation of acetylcholine-mediated ({dollar}sp3{dollar}H) IP{dollar}sb1{dollar} accumulation. This finding suggested that protein kinase C stimulation with {dollar}beta{dollar}PMA caused desensitization at the level of the receptor or effector system. The cholinergic response was also modulated by increasing intracellular cAMP. Forskolin, isobutylmethylxanthine, cholera toxin, or the stable cAMP analogues attenuated ({dollar}sp3{dollar}H) IP{dollar}sb1{dollar} accumulation following acetylcholine stimulation. This finding suggests that there is an interaction between the adenylate cyclase and phospholipase C systems.; In summary, these experiments demonstrate the presence of acetylcholine and muscarinic receptor binding sites in the rat retina. Stimulation of these muscarinic receptors led to phosphoinositide hydrolysis. This receptor is coupled to phospholipase C by a pertussis toxin sensitive G protein. Furthermore, this cholinergic response was modulated by AF64A, hyperglycemia, phorbol ester, and increased intracellular levels of cAMP. The functional implications of acetylcholine in visual transduction is presently unknown.