h-89 and 1-2-dioctanoylglycerol

Urinary acidification in the collecting duct is mediated by the activity of H(+)-ATPases and is stimulated by various factors including angiotensin II and aldosterone. Classically, aldosterone effects are mediated via the mineralocorticoid receptor. Recently, we demonstrated a nongenomic stimulatory effect of aldosterone on H(+)-ATPase activity in acid-secretory intercalated cells of isolated mouse outer medullary collecting ducts (OMCD). Here we investigated the intracellular signaling cascade mediating this stimulatory effect. Aldosterone stimulated H(+)-ATPase activity in isolated mouse and human OMCDs. This effect was blocked by suramin, a general G protein inhibitor, and GP-2A, a specific G(αq) inhibitor, whereas pertussis toxin was without effect. Inhibition of phospholipase C with U-73122, chelation of intracellular Ca(2+) with BAPTA, and blockade of protein kinase C prevented the stimulation of H(+)-ATPases. Stimulation of PKC by DOG mimicked the effect of aldosterone on H(+)-ATPase activity. Similarly, aldosterone and DOG induced a rapid translocation of H(+)-ATPases to the luminal side of OMCD cells in vivo. In addition, PD098059, an inhibitor of ERK1/2 activation, blocked the aldosterone and DOG effects. Inhibition of PKA with H89 or KT2750 prevented and incubation with 8-bromoadenosine-cAMP mildly increased H(+)-ATPase activity. Thus, the nongenomic modulation of H(+)-ATPase activity in OMCD-intercalated cells by aldosterone involves several intracellular pathways and may be mediated by a G(αq) protein-coupled receptor and PKC. PKA and cAMP appear to have a modulatory effect. The rapid nongenomic action of aldosterone may participate in the regulation of H(+)-ATPase activity and contribute to final urinary acidification.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Aldosterone; Animals; Chelating Agents; Diglycerides; Egtazic Acid; Enzyme Inhibitors; Estrenes; Flavonoids; GTP-Binding Proteins; Humans; Isoquinolines; Kidney Tubules, Collecting; Male; Mice; Mice, Inbred C57BL; Pertussis Toxin; Protein Kinase C; Pyrrolidinones; Signal Transduction; Sulfonamides; Suramin; Type C Phospholipases; Vacuolar Proton-Translocating ATPases

Ghrelin (GRLN) and its receptor have been identified and characterised in goldfish brain and the pituitary, and recent evidence shows that goldfish (g)GRLN(19) induces both growth hormone (GH) and maturational gonadotrophin (LH) release through an extracellular Ca(2+) -dependent mechanism in goldfish. To further understand the role of GRLN in hormone release, the present study examined the involvement of protein kinase C (PKC) and protein kinase A (PKA) in gGRLN(19) -induced GH and LH release and corresponding Ca(2+) signals in primary cultures of goldfish pituitary cells. Treatments with PKC inhibitors, Bis-II and Gö 6976, significantly reduced gGRLN(19) -induced GH and LH release and their corresponding intracellular Ca(2+) signals in identified somatotrophs and gonadotrophs, respectively. gGRLN(19) was unable to further stimulate hormone release or Ca(2+) signals when cells were pretreated with the PKC agonist, DiC8. PKA inhibitors, H-89 and KT 5720, inhibited gGRLN(19) -induced LH release and Ca(2+) signals in gonadotrophs but not GH release or Ca(2+) signals in somatotrophs. Interestingly, pretreatment of pituitary cells with the adenylate cyclase activator forskolin potentiated gGRLN(19) -induced GH, but not LH, release, although it had no effect on intracellular Ca(2+) signals in either cell type. Taken together, the results suggest that PKC is an important intracellular component in gGRLN(19) -induced GH and LH release, whereas PKA is involved in gGRLN(19) -elicited LH release. Furthermore, the PKA pathway potentiates gGRLN(19) -induced GH release via a Ca(2+) -independent mechanism. Overall, the present study provides insight into the neuroendocrine regulation of GH and LH release by elucidating the mechanistic aspects of GRLN, a hormone involved in many critical physiological processes, including pituitary functions.

Topics: Animals; Calcium; Carbazoles; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Female; Ghrelin; Goldfish; Gonadotropins; Growth Hormone; Isoquinolines; Male; Pituitary Gland; Protein Kinase C; Protein Kinase Inhibitors; Sulfonamides