6533b860fe1ef96bd12c3a2f

RESEARCH PRODUCT

Functional coupling of nitric oxide synthase and soluble guanylyl cyclase in controlling catecholamine secretion from bovine chromaffin cells

subject

description

This study was designed to evaluate whether the enzymes of the nitric oxide/cyclic-GMP pathway, nitric oxide synthase and soluble guanylyl cyclase, are functionally coupled in controlling catecholamine secretion in primary cultures of bovine chromaffin cells. In immunocytochemical studies, 80-85% of the tyrosine hydroxylase-positive chromaffin cells also possessed phenylethanolamine-N-methyltransferase, f1p4cating their capability to synthesize epinephrine. Immunoreactivity for neuronal-type nitric oxide synthase was found in over 90% of all chromaffin cells. Reverse transcription-polymerase chain reaction also demonstrated neuronal-type nitric oxide synthase messenger RNA. Immunoreactivity for soluble guanylyl cyclase was detectable in over 95% of chromaffin cells. Double-labeling immunofluorescence studies co-localized neuronal-type nitric oxide synthase and soluble guanylyl cyclase with tyrosine hydroxylase and phenylethanolamine-N-methyltransferase in the majority of chromaffin cells. Chromaffin cells possessed basal nitric oxide synthase activity which could be stimulated by acetylcholine and inhibited by NG-nitro-L-arginine methyl ester. Activation of soluble guanylyl cyclase by endogenously synthesized nitric oxide or the nitric oxide donor compound sodium nitroprusside was blocked by the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Catecholamine release and the increase in cytosolic Ca2+ concentration evoked by acetylcholine were enhanced by inhibitors of the endogenous nitric oxide/cyclic-GMP pathway such as NG-nitro-L-arginine methyl ester, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one and the protein kinase G inhibitor Rp-8-pCPT-cGMPS. These data indicate that chromaffin cells possess an autocrine nitric oxide/cyclic-GMP pathway tonically controlling the inhibition of catecholamine release.