RESUMO
The neuroendocrine bag cells of Aplysia provide an excellent model system for exploring the roles of second-messenger pathways regulating peptide hormone secretion. Both the cAMP and diacylglycerol second-messenger systems and their associated protein kinases (PKA and PKC) are involved in regulating membrane excitability in bag-cell neurons of Aplysia. The purpose of the present set of experiments was to determine if PKA and PKC also play roles in regulating egg laying hormone (ELH) secretion from bag-cell neurons. Abdominal ganglia with attached bag-cell clusters and connective nerves were dissected from reproductively mature Aplysia, and ELH secretion in response to electrically stimulated afterdischarges was measured by RIA. ELH secretion from bag cells treated with protein-kinase inhibitors (Rp-cAMPS to inhibit PKA; H-7 to inhibit PKC) was compared to that from untreated controls. Our experiments showed that 100 microM Rp-cAMPS significantly attenuated ELH secretion during the nonbreeding seasons (winter and spring) of 2 consecutive years. This suggested a role for PKA in regulating ELH secretion. However, Rp-cAMPS had no effect on ELH secretion during the breeding seasons (summer and fall) of 2 consecutive years, even when the dose of Rp-cAMPS was increased to 200 microM. These findings indicate that there is a seasonal fluctuation in responsiveness to PKA inhibition. We also investigated if there was a seasonal fluctuation in the ability of the PKC inhibitor H-7 to suppress ELH secretion. During the nonbreeding season, 10-100 microM H-7 significantly inhibited ELH secretion, but during the breeding season, only the highest dose (100 microM) of H-7 inhibited ELH release. These results confirm that PKC plays a role in regulating ELH secretion and indicate that there is a seasonal fluctuation in responsiveness to PKC inhibition. Overall, our findings suggest that both the cAMP and diacylglycerol second-messenger pathways are regulated on a seasonal basis.
