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The involvement of nitric oxide (NO) in the regulation of goldfish growth hormone (GH) secretion was further characterized using primary cultures of dispersed goldfish pituitary cells. Western blots revealed the presence of an inducible nitric oxide synthase (iNOS)-like protein of approximately 120 kDa in cytosol/plasma membrane extracts. By contrast, brain NOS-immunoreactive proteins of approximately 120–140 kDa were occasionally detected in a cytoskeleton/organelle fraction but were absent from cytosol/plasma membrane extracts. The NO donor sodium nitroprusside (SNP) acutely increased GH secretion but this response was not observed in the presence of either a NO scavenger (PTIO) or a soluble guanylate cyclase inhibitor (ODQ). SNP also significantly increased the levels of cyclic (c) GMP in somatotrope-enriched cell populations. Treatments with 1400W (iNOS inhibitor), PTIO and rutin hydrate (NO scavengers) and ODQ abolished the acute GH-release response to two endogenous gonadotropin-releasing hormones (GnRH). 1400W, rutin hydrate, PTIO and ODQ alone did not significantly alter basal GH secretion. Together, these results establish that an iNOS-like peptide is constitutively present in the pituitary of the goldfish. Furthermore, these data suggest that NO, most likely through the generation of cGMP, is a necessary signal transduction component of GnRH-induced GH secretion.