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.

Nitric Oxide Inhibits the Rate and Strength of Cardiac Contractions in the LobsterHomarus americanusby Acting on the Cardiac Ganglion

“…The most widely recognized downstream effector for NO is the soluble, heme-containing form of guanylate cyclase, whose activity is strongly enhanced by NO (Hanafy et al, 2001). Consistent with this idea, NO has been shown to elevate cGMP levels when applied to isolated cardiac ganglia (Scholz et al, 2002), an effect whose potential significance is underscored by the observation that the NO-sensitive guanylate cyclase enzyme is not, in general, very abundant in the crustacean nervous system (Scholz et al, 1996;Prabhakar et al, 1997). Linkage between NO and cGMP in the lobster heart would also be consistent with the intertwined inhibitory roles for these signaling molecules in a variety of contexts in the mammalian cardiovascular system (Ignarro et al, 1999;Kone, 2001).…”

“…Although NO is normally considered to be quite labile in biological fluids, serum proteins that form stable complexes with NO have been identified in both mammals (Jai et al, 1996) and arthropods (Ribeiro et al, 1993). Although such NO-binding proteins have not yet been identified in crustaceans, it remains an intriguing possibility that other NOS-expressing organs, such as the hepatopancreas (Scholz et al, 2002), might be able to regulate cardiac performance by delivering NO to the heart through the circulation. In addition, it is also possible that NO produced by the heart Figure 13.…”

“…In a recent study of NOS expression in a crab (Cancer productus), Scholz et al (2002) demonstrated that, compared with other tissues, crab heart muscle extracts are enriched for a calciumactivated form of NOS. The high levels of enzyme activity observed in this previous study correlated well with the presence of an immunoreactive protein that was detected on Western blots of crab heart muscle cytosol, using a well characterized anti-NOS antibody directed against a strongly conserved core catalytic region of the enzyme.…”

“…Extracts of crustacean heart muscle contain high levels of a calcium-sensitive form of nitric oxide synthase (NOS) (Scholz et al, 2002), suggesting the possibility that contraction of the crustacean heart may be accompanied by NO production. A likely target for NO produced by cardiac muscle would be the cardiac ganglion, situated within the lumen of the heart and well within the diffusional radius of the short-lived NO molecule.…”

“…A likely target for NO produced by cardiac muscle would be the cardiac ganglion, situated within the lumen of the heart and well within the diffusional radius of the short-lived NO molecule. Indeed, NO is known to trigger a biochemical response (increased cGMP metabolism) when applied to the isolated cardiac ganglion of the crab (Scholz et al, 2002). Because the function of the cardiac ganglion is to provide the synaptic input that drives the contractions of the heart (Alexandrowicz, 1932;Cooke, 2002), it is attractive to speculate that NO may provide feedback information about the status of cardiac muscle that could be used to modulate the activity of the ganglion.…”