|dc.description.abstract||Stimulation of urine production by the Malpighian (renal) tubules in Rhodnius prolixus is regulated by at least two diuretic hormones, CRF-related peptide and serotonin, that have traditionally been believed to function through the activation of cAMP-mediated intracellular second messenger pathways. In this study I demonstrate that serotonin stimulation triggered, in addition to cAMP, intracellular Ca2+ waves in the Malpighian tubule cells of R. prolixus. Treatment with the intracellular Ca2+ chelator BAPTA-AM blocked the intracellular Ca2+ waves and reduced serotonin-stimulated fluid secretion by 75%. This suggests a role for intracellular Ca2+ signaling in the excretory system of R. prolixus. Serotonin stimulated Malpighian tubules (MTs) exposed to Ca2+-free saline plus BAPTA-AM secreted an abnormal fluid, showing: increased K+ concentration, reduced Na+ concentration and lower pH. These results along with measurement of transepithelial potential (TEP) suggest that the basolateral Na+:K+:2Cl- cotransporter (NKCC) activity is reduced in tubule cells treated with BAPTA-AM, suggesting that Ca2+ is required to modulate the activity of the basolateral NKCC.
Treatment with the non-hydrolysable cell-permeable cAMP analog, 8Br-cAMP, produced fluid with the same K+ and Na+ concentration and at the same secretion rate as serotonin-stimulated tubules. In addition, 8Br-cAMP triggered intracellular Ca2+ oscillations similar to those obtained with serotonin. 8Br-cAMP-stimulated tubules treated with BAPTA-AM decreased their fluid secretion by about 40% and increased Na+ concentration, similar to the effect observed on serotonin-stimulated tubules. Therefore, I conclude that the intracellular Ca2+ waves triggered by serotonin are mediated by cAMP.
The role of inositol-3-phospate (InsP3) in Ca2+ release was tested by treating the tubules with the InsP3 receptor blocker xestospongin. The treatment decreased fluid secretion rate as well as the amplitude of Ca2+ waves in serotonin-stimulated tubules. These results suggest that serotonin activates the production of InsP3 and, most likely, diacylglycerol (DAG). Thus, I decided to test whether the protein kinase C (PKC) may be involved in serotonin-stimulated secretion.
The PKC inhibitors chelerythrine and bisindolylmaleimide (BIM) decreased secretion fluid rate in serotonin-stimulated tubules by 50% and 70%, respectively. Fluid secreted by tubules treated with BIM showed no differences in K+ and Na+ concentrations compared to controls, however both ion fluxes decreased. The evidence suggests that PKC is involved in serotonin stimulated secretion; the mechanism is still not understood.
Taken together, the results suggest that cAMP, Ca2+ and PLC-PKC pathway are involved in serotonin stimulated secretion. However cAMP stimulation is enough for maximal secretion rate. Therefore PLC-PKC must act downstream of cAMP. Based on those results we hypothesize that serotonin binds a GPCR, increasing cAMP by activation of an adenylate cyclase (AC). Subsequently, cAMP is somehow able to activate PLC, which finally produces Ca2+ release, PKC activation and NKCC upregulation.||en_US