pituitrin and chelerythrine

17β-Estradiol (E2) has been shown to modulate the renin-angiotensin system in hydromineral and blood pressure homeostasis mainly by attenuating angiotensin II (ANGII) actions. However, the cellular mechanisms of the interaction between E2 and angiotensin II (ANGII) and its physiological role are largely unknown. The present experiments were performed to better understand the interaction between ANGII and E2 in body fluid control in female ovariectomized (OVX) rats. The present results are the first to demonstrate that PKC/p38 MAPK signaling is involved in ANGII-induced water and sodium intake and oxytocin (OT) secretion in OVX rats. In addition, previous data from our group revealed that the ANGII-induced vasopressin (AVP) secretion requires ERK1/2 signaling. Therefore, taken together, the present observations support a novel concept that distinct intracellular ANGII signaling gives rise to distinct neurohypophyseal hormone release. Furthermore, the results show that E2 attenuates p38 MAPK phosphorylation in response to ANGII but not PKC activity in the hypothalamus and the lamina terminalis, suggesting that E2 modulates ANGII effects through the attenuation of the MAPK pathway. In conclusion, this work contributes to the further understanding of the interaction between E2 and ANGII signaling in hydromineral homeostasis, as well as it contributes to further elucidate the physiological relevance of PKC/p38 MAPK signaling on the fluid intake and neurohypophyseal release induced by ANGII.

Topics: Angiotensin II; Animals; Benzophenanthridines; Brain; Drinking; Drug Interactions; Estradiol; Female; Homeostasis; Imidazoles; MAP Kinase Signaling System; Ovariectomy; Oxytocin; p38 Mitogen-Activated Protein Kinases; Protein Kinase C-alpha; Pyridines; Rats, Wistar; Vasopressins

The UT-A1 urea transporter plays a critical role in the production of concentrated urine. Both vasopressin and hypertonicity increase urea permeability in rat terminal inner medullary collecting ducts (IMCD). Each agonist independently increases UT-A1 phosphorylation and apical plasma membrane accumulation. Vasopressin activates PKA and phosphorylates UT-A1 at serines 486 and 499. Hypertonicity stimulates urea permeability through protein kinase C (PKC) and intracellular calcium. To determine whether the hypertonic stimulation of urea permeability results from a PKC-mediated phosphorylation of UT-A1, rat IMCDs were metabolically labeled with [(32)P]. Hypertonicity stimulated UT-A1 phosphorylation, and this increase was blocked by preincubation with a PKC inhibitor. IMCDs were biotinylated to assess plasma membrane UT-A1. Hypertonicity increased biotinylated UT-A1, and this increase was blocked by preincubation with a PKC inhibitor. When PKC was directly activated using a phorbol ester, total UT-A1 phosphorylation increased, but phosphorylation at serine 486 was not increased, indicating that PKC did not phosphorylate UT-A1 at the same residue as PKA. Since PKC-α is a calcium-dependent PKC isoform and PKC-α knockout mice have a urine-concentrating defect, it suggested that PKC-α may mediate the response to hypertonicity. Consistent with this hypothesis, hypertonicity increased phospho-PKC-α in rat IMCDs. Finally, PKC-α knockout mice were used to determine whether hypertonicity could stimulate UT-A1 phosphorylation in the absence of PKC-α. Hypertonicity significantly increased UT-A1 phosphorylation in wild-type mice but not in PKC-α knockout mice. We conclude that PKC-α mediates the hypertonicity-stimulated increase in UT-A1 phosphorylation in the IMCD.

Topics: Animals; Benzophenanthridines; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Glucose Solution, Hypertonic; Kidney Tubules, Collecting; Membrane Transport Proteins; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Animal; Phosphorylation; Protein Kinase C; Rats; Rats, Sprague-Dawley; Urea Transporters; Vasopressins

The current-voltage relationship of rat hepatocytes was investigated and its linear dependence was shown. On liver slices, we have investigated the effects of vasopressin on membrane potential of rat hepatocytes using microelectrode technique. Vasopressin evoked membrane hyperpolarization to -50 mV, suggesting that vasopressin activated synthetic processes in hepatocytes. Vasopressin application together with the inhibitor of protein kinase C chelerytrine also caused hyperpolarization of hepatocytes, but membrane potential changes were of smaller amplitude. This fact suggests that although protein kinase C plays an important role in the realization of effects of vasopressin, there is, however, another signal transduction pathway of vasopressin in rat hepatocytes. If that pathway activates adenylatecyclase, vasopressin can stimulate bile formation in hepatocytes. This assumption should be verified.

Topics: Adenylyl Cyclases; Animals; Benzophenanthridines; Cells, Cultured; Enzyme Activation; Hepatocytes; Membrane Potentials; Microelectrodes; Protein Kinase C; Rats; Vasopressins

Hypertonicity increases urea transport independently of, as well as synergistically with, vasopressin in the inner medullary collect duct (IMCD). We previously showed that hypertonicity does not increase the level of cAMP in the IMCD, but it does increase the level of intracellular calcium. Since we also showed that hypertonicity increases both the phosphorylation and biotinylation of the urea transporters UT-A1 and UT-A3, this would suggest involvement of a calcium-dependent protein kinase in the regulation of urea transport in the inner medulla. In this study, we investigated whether protein kinase C (PKC), which is present in the IMCD, is a regulator of urea permeability. We tested the effect of PKC inhibitors and activators on urea permeability in the isolated, perfused rat terminal IMCD. Increasing osmolality from 290 to 690 mosmol/kgH(2)O significantly stimulated (doubled) urea permeability; it returned to control levels on inhibition of PKC with either 10 μM chelerythrine or 50 μM rottlerin. To determine the potential synergy between vasopressin and PKC, phorbol dibutyrate (PDBu) was used to stimulate PKC. Vasopressin stimulated urea permeability 247%. Although PDBu alone did not change basal urea permeability, in the presence of vasopressin, it significantly increased urea permeability an additional 92%. The vasopressin and PDBu-stimulated urea permeability was reduced to AVP alone levels by inhibition of PKC. We conclude that hypertonicity stimulates urea transport through a PKC-mediated phosphorylation. Whether PKC directly phosphorylates UT-A1 and/or UT-A3 or phosphorylates it as a consequence of a cascade of activations remains to be determined.

Topics: Acetophenones; Animals; Benzophenanthridines; Benzopyrans; Kidney Tubules, Collecting; Male; Membrane Transport Proteins; Osmolar Concentration; Permeability; Phorbol 12,13-Dibutyrate; Protein Kinase C; Rats; Rats, Sprague-Dawley; Urea; Urea Transporters; Vasopressins

Experiments were conducted to characterize the effects of oxytocin (OT) and vasopressin (VP) on epithelial cells isolated from human (1 degree HVD) and porcine (1 degree PVD) vas deferens and an immortalized epithelial cell line derived from porcine vas deferens (PVD9902 cells). Cultured monolayers were assessed in modified Ussing flux chambers and the OT- or VP-induced change in short circuit current (I(SC)) was recorded. All cell types responded to basolateral OT or VP with a transient increase in I(SC) that reached a peak of 3-5 microA cm(-2). Concentration-response curves constructed with 1 degree PVD and PVD9902 cells revealed that the apparent K(D) (k(app)) for OT was approximately 100-fold less than the k(app) for VP. Amplicons for the OT receptor (OXTR) and vasopressin type 2 and type 1a receptors (AVPR2 and AVPR1A) were generated with RT-PCR and the identification of each amplicon confirmed by sequence analysis. A selective antagonist for OXTR and AVPR1A fully blocked the effects of OT and partially blocked the effects of VP when assessed in both 1 degree PVD and PVD9902 monolayers. APVR2 antagonists blocked the effects of low (< or =30 nM) but not high concentrations of VP, indicating that VP was affecting both AVPR2 and a second receptor subtype, likely OXTR or AVPR1A. Experiments employing chelerythrine demonstrated that OT stimulation of vas deferens monolayers requires PKC activity. Alternatively, VP (but not OT) increased the accumulation of cytosolic cAMP in vas deferens epithelial cells. Results from this study demonstrate that OT and VP can modulate ion transport across vas deferens epithelia by independent mechanisms. OT and VP have the potential to acutely change the environment to which sperm are exposed and thus, have the potential to affect male fertility.

Topics: Alkaloids; Animals; Anions; Antidiuretic Hormone Receptor Antagonists; Benzophenanthridines; Cell Line; Cyclic AMP; Electrophysiology; Epithelial Cells; Humans; Ion Transport; Male; Oxytocin; Piperidines; Quinolones; Receptors, Oxytocin; Receptors, Vasopressin; Reverse Transcriptase Polymerase Chain Reaction; Swine; Vas Deferens; Vasopressins

Chelerythrine and sanguinarine, two benzophenanthridine alkaloids, have been isolated from a crude methanolic extract of Eschscholtzia californica cell suspension cultures by successive fractionations. These two molecules exhibited affinity for rat liver vasopressin V1 receptors and are competitive inhibitors of [3H]-vasopressin binding within the micromolar range (Ki). Chelerythrine and sanguinarine represent two of the first non-peptidic structures providing original chemical leads for the design of synthetic vasopressin compounds.

Topics: Alkaloids; Animals; Benzophenanthridines; Isoquinolines; Liver; Phenanthridines; Plants; Rats; Receptors, Angiotensin; Receptors, Vasopressin; Vasopressins