Expression of nitric oxide synthase and nitric oxide-sensitive guanylate cyclase in the crustacean cardiac ganglion

Abstract

The cardiac ganglion is a simple central pattern-generating network that controls the rhythmic contractions of the crustacean heart. Enzyme assays and Western blots show that whole heart homogenates from the crab Cancer productus contain high levels of nitric oxide synthase (NOS), an enzyme that catalyzes the conversion of arginine to citrulline with concomitant production of the transmitter nitric oxide (NO). Crab heart NOS is calcium-dependent and has an apparent molecular weight of 110 kDa. In the cardiac ganglion, antibodies to NOS and citrulline indicate the presence of a NOS-like protein and NOS enzymatic activity in the four small pacemaker neurons and the five large motor neurons of the cardiac network. In addition, all cardiac neurons label positively with an antibody to cyclic guanosine monophosphate (cGMP). The NO donor sodium nitroprusside (SNP, 10 mM) stimulates additional cGMP production in the isolated ganglion. This increase is blocked by [(1)H](1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ, 50 microM), an inhibitor of the NO-sensitive soluble guanylate cyclase (sGC). Taken together, our data indicate that NO- and cGMP-mediated signaling pathways are enriched in the cardiac system relative to other crab tissues and that the cardiac network may be a target for extrinsic and intrinsic neuromodulation via NO produced from the heart musculature and individual cardiac neurons, respectively. The crustacean cardiac ganglion is therefore a promising system for studying cellular and synaptic mechanisms of nitrergic neuromodulation in a simple pattern-generating network.