Identification of metabolic pathways of brain angiotensin II and III using specific aminopeptidase inhibitors: predominant role of angiotensin III in the control of vasopressin release.

Angiotensin (Ang) II and Ang III are two peptide effectors of the brain renin-angiotensin system that participate in the control of blood pressure and increase water consumption and vasopressin release. In an attempt to delineate the respective roles of these peptides in the regulation of vasopressin secretion, their metabolic pathways and their effects on vasopressin release were identified in vivo. For this purpose, we used recently developed selective inhibitors of aminopeptidase A (APA) and aminopeptidase N (APN), two enzymes that are believed to be responsible for the N-terminal cleavage of Ang II and Ang III, respectively. Mice received [3H]Ang II intracerebroventricularly (i.c.v.) in the presence or absence of the APN inhibitor, EC33 (3-amino-4-thio-butyl sulfonate) of the APN inhibitor, EC27 (2-amino-pentan-1,5-dithiol). [3H]Ang II and [3H]Ang III levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]Ang II 2.6-fold and completely blocked the formation of [3H]Ang III, whereas EC27 increased the half-life of [3H]Ang III 2.3-fold. In addition, the effects of EC33 and EC27 on Ang-induced vasopressin release were studied in mice. Ang II was injected i.c.v. in the presence or absence of EC33, and plasma vasopressin levels were estimated by RIA. While vasopressin levels were increased 2-fold by Ang II (5 ng), EC33 inhibited Ang II-induced vasopressin release in a dose-dependent manner. In contrast, EC27 injected alone increased in a dose-dependent manner vasopressin levels. The EC27-induced vasopressin release was completely blocked by the coadministration of the Ang receptor antagonist (Sar1-Ala8) Ang II. These results demonstrate for the first time that (i) APA and APN are involved in vivo in the metabolism of brain Ang II and Ang III, respectively, and that (ii) the action of Ang II on vasopressin release depends upon the prior conversion of Ang II to Ang III. This shows that Ang III behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Corticotropin-releasing hormone causes antidiuresis and antinatriuresis by stimulating vasopressin and inhibiting atrial natriuretic peptide release in male rats

In both normally hydrated and volume-expanded rats, there was a biphasic effect of corticotropin-releasing hormone (CRH) (1–10 μg, i.v.) on renal function. Within the first hour, CRH caused antidiuresis, antinatriuresis, and antikaliuresis together with reduction in urinary cGMP output that, in the fourth hour, were replaced by diuresis, natriuresis, and kaliuresis accompanied by increased cGMP output. Plasma arginine vasopressin (AVP) concentrations increased significantly within 5 min, reached a peak at 15 min, and declined by 30 min to still-elevated values maintained for 180 min. Changes in plasma atrial natriuretic peptide (ANP) were the mirror image of those of AVP. Plasma ANP levels were correlated with decreased ANP in the left ventricle at 30 min and increased ANP mRNA in the right atrium at 180 min. All urinary changes were reversed by a potent AVP type 2 receptor (V2R) antagonist. Control 0.9% NaCl injections evoked an immediate increase in blood pressure and heart rate measured by telemetry within 3–5 min. This elevation of blood pressure was markedly inhibited by CRH (5 μg). We hypothesize that the effects are mediated by rapid, direct vasodilation induced by CRH that decreases baroreceptor input to the brain stem, leading to a rapid release of AVP that induces the antidiuresis by direct action on the V2Rs in the kidney. Simultaneously, acting on V2Rs in the heart, AVP inhibits ANP release and synthesis, resulting in a decrease in renal cGMP output that is responsible for the antinatriuretic and antikaliuretic effects.

Isolated rat hepatocytes can signal to other hepatocytes and bile duct cells by release of nucleotides.

Intercellular communication among certain cell types can occur via ATP secretion, which leads to stimulation of nucleotide receptors on target cells. In epithelial cells, however, intercellular communication is thought to occur instead via gap junctions. Here we examined whether one epithelial cell type, hepatocytes, can also communicate via nucleotide secretion. The effects on cytosolic Ca2+ ([Ca2+]i) of mechanical stimulation, including microinjection, were examined in isolated rat hepatocytes and in isolated bile duct units using confocal fluorescence video microscopy. Mechanical stimulation of a single hepatocyte evoked an increase in [Ca2+]i in the stimulated cell plus an unexpected [Ca2+]i rise in neighboring noncontacting hepatocytes. Perifusion with ATP before mechanical stimulation suppressed the [Ca2+]i increase, but pretreatment with phenylephrine did not. The P2 receptor antagonist suramin inhibited these intercellular [Ca2+]i signals. The ATP/ADPase apyrase reversibly inhibited the [Ca2+]i rise induced by mechanical stimulation, and did not block vasopressin-induced [Ca2+]i signals. Mechanical stimulation of hepatocytes also induced a [Ca2+]i increase in cocultured isolated bile duct units, and this [Ca2+]i increase was inhibited by apyrase as well. Finally, this form of [Ca2+]i signaling could be elicited in the presence of propidium iodide without nuclear labeling by that dye, indicating that this phenomenon does not depend on disruption of the stimulated cell. Thus, mechanical stimulation of isolated hepatocytes, including by microinjection, can evoke [Ca2+]i signals in the stimulated cell as well as in neighboring noncontacting hepatocytes and bile duct epithelia. This signaling is mediated by release of ATP or other nucleotides into the extracellular space. This is an important technical consideration given the widespread use of microinjection techniques for examining mechanisms of signal transduction. Moreover, the evidence provided suggests a novel paracrine signaling pathway for epithelia, which previously were thought to communicate exclusively via gap junctions.

Oxytocin mediates atrial natriuretic peptide release and natriuresis after volume expansion in the rat.

Our previous studies have shown that stimulation of the anterior ventral third ventricular region increases atrial natriuretic peptide (ANP) release, whereas lesions of this structure, the median eminence, or removal of the neural lobe of the pituitary block ANP release induced by blood volume expansion (BVE). These results indicate that participation of the central nervous system is crucial in these responses, possibly through mediation by neurohypophysial hormones. In the present research we investigated the possible role of oxytocin, one of the two principal neurohypophysial hormones, in the mediation of ANP release. Oxytocin (1-10 nmol) injected i.p. caused significant, dose-dependent increases in urinary osmolality, natriuresis, and kaliuresis. A delayed antidiuretic effect was also observed. Plasma ANP concentrations increased nearly 4-fold (P < 0.01) 20 min after i.p. oxytocin (10 nmol), but there was no change in plasma ANP values in control rats. When oxytocin (1 or 10 nmol) was injected i.v., it also induced a dose-related increase in plasma ANP at 5 min (P < 0.001). BVE by intra-atrial injection of isotonic saline induced a rapid (5 min postinjection) increase in plasma oxytocin and ANP concentrations and a concomitant decrease in plasma arginine vasopressin concentration. Results were similar with hypertonic volume expansion, except that this induced a transient (5 min) increase in plasma arginine vasopressin. The findings are consistent with the hypothesis that baroreceptor activation of the central nervous system by BVE stimulates the release of oxytocin from the neurohypophysis. This oxytocin then circulates to the right atrium to induce release of ANP, which circulates to the kidney and induces natriuresis and diuresis, which restore body fluid volume to normal levels.

Extrapituitary expression of the rat V1b vasopressin receptor gene.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.