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We investigated the involvement of the nitric oxide pathway in the inhibitory mechanisms of 5-hydroxytryptamine (5-HT) in the pressor responses induced by stimulation of sympathetic vasopressor outflow in diabetic pithed rats. Diabetes was induced in male Wistar rats by a single s.c. injection of alloxan. Four weeks later, the animals were anaesthetized, pretreated with atropine, and pithed. Electrical stimulation of the sympathetic outflow from the spinal cord (0.1, 0.5, 1 and 5 Hz) resulted in frequency-dependent increases in blood pressure. The inhibition of electrically induced pressor responses by 5-HT (10 microg/kg/min) in diabetic pithed rats could not be elicited after i.v. treatment with 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) (10 microg/kg), a guanylyl cyclase inhibitor, or N-omega-L-Arginine methyl ester hydrochloride (L-NAME) (10 mg/kg), a nitric oxide synthase (NOS) inhibitor. The inhibitory effect produced by infusion of the selective 5-HT(1A) receptor agonist 8-hydroxydipropylaminotretalin hydrobromide (8-OH-DPAT) (20 microg/kg/min) was abolished in the presence of ODQ (10 microg/kg), or L-NAME (10 mg/kg) in diabetic pithed rats. The administration of L-Arginine (100 mg/kg) 30 min after L-NAME reproduced the inhibitory effect caused by 5-HT (10 microg/kg/min) and 8-OH-DPAT (20 microg/kg/min) on the electrically induced pressor responses, whereas in the presence of D-Arginine (100 mg/kg)+L-NAME the 5-HT or 8-OH-DPAT inhibitory effect on the pressor responses was abolished. In conclusion, in diabetic pithed rats, the inhibition produced by prejunctional 5-HT(1A) activation on electrically induced sympathetic pressor responses is mediated by the NO synthesis/pathway.

The effects of oxytocin on renin secretion by denervated kidney were investigated in vivo, by infusing the peptide directly into the renal artery of anaesthetized rats. Renin secretion was calculated by the renal veno-arterial difference in plasma renin activity multiplied by renal plasma flow. The intra-renal arterial (i.r.a.) infusion of oxytocin (1.5 or 15 ng/kg/min, 10 min) induced a sixfold increase in renin secretion as compared to vehicle-treated controls, without effects on renal blood flow, mean arterial blood pressure, glomerular filtration rate or natriuresis. The effect of oxytocin (1.5 ng/kg/min) was prevented by pretreatment with an oxytocin receptor antagonist, desGly-NH(2),d(CH(2))(5)[D-Tyr(2),Thr(4),Orn(8)]vasotocin] (5.6 microg/kg bolus i.v. 20 min before oxytocin infusion, followed by 2.8 microg/kg/min i.r.a.). Nadolol (2.5 mg/kg i.v.), a beta-adrenoceptor antagonist, also blocked the oxytocin-induced increase in renin secretion. These results show that oxytocin is able to stimulate renin release by activating oxytocin receptors but that beta-adrenoceptors also seem to be involved.

We evaluated how renal vascular reactivity to vasopressin changes when nitric oxide (NO) synthesis varies, as has been reported to occur in the course of insulin-dependent diabetes mellitus. Renal vasoconstrictor responses to vasopressin were obtained in young and older Sprague-Dawley control rats (3 and 10 months old) and in age-matched diabetic rats that had been treated with streptozotocin (60 mg/kg i.v.) at the age of 2 months. In young rats, vasopressin (3-1000 ng/kg/min i.v.) induced in vivo a dose-dependent decrease in renal blood flow, which was diminished in streptozotocin diabetic rats (P0.05). Similarly, in in vitro perfused kidneys, the concentration-response curve for vasopressin (0.03-10 nM) was shifted 3-fold to the right in kidneys isolated from young diabetic rats (P0.05). This shift was abolished in the presence of an inhibitor of nitric oxide synthesis, N(G)-nitro-L-arginine (100 microM), in the perfusate. In 10-month-old rats, the in vivo renal vasoconstrictor dose-response curve to vasopressin was shifted 10-fold to the left as compared to that for young rats (P0.001). This shift was similar in both control and diabetic rats. In conclusion, the present study documented the existence of hyporesponsiveness to vasopressin in the early stage of diabetes, possibly related to nitric oxide overproduction. In contrast, renal vascular hyperreactivity to vasopressin occurs with aging, whether the rats are diabetic or not.

In normotensive rats, we have previously demonstrated a role of brain mineralocorticoid receptors in blood pressure and renal function control. In the present study, the coordinate cardiovascular and renal effects of brain mineralocorticoid receptor blockade were examined by intracerebroventricular (i.c.v.) administration of a selective mineralocorticoid receptor antagonist (RU28318; 3,3-oxo-7 propyl-17-hydroxy-androstan-4-en-17yl-propionic acid lactone) in rats with hypertension induced by deoxycorticosterone acetate (DOCA) and salt. DOCA pellets were implanted s.c. in male Wistar rats given 0.9% NaCl as drinking solution 3 or 5 weeks before assessment of the effects of i.c.v. injection of RU28318 on cardiovascular and renal functions. Changes in expression of brain angiotensinogen, atrial natriuretic peptide (ANP) and mineralocorticoid receptor mRNA in specific brain areas in 3-week DOCA–salt rats were evaluated by in situ hybridization. The rise in systolic blood pressure induced by DOCA–salt treatment was most marked during the first 3 weeks. At 3 and 5 weeks after implantation of the DOCA-pellets a single i.c.v. injection of 10 ng of RU28318 significantly decreased systolic blood pressure during 24 h as assessed at 2, 8 and 24 h, while heart rate was not altered. Increased urinary excretion of water and electrolytes was observed in 3- and 5-week DOCA–salt rats during the period 0–8 h after i.c.v. injection of RU28318 while the suppressed plasma renin activity was not affected. The expression of brain angiotensinogen, ANP and mineralocorticoid receptor mRNA was not altered by 3-week DOCA–salt treatment, but 3 h after i.c.v. injection of RU28318, mineralocorticoid receptor mRNA expression in hippocampal cell fields responded with an increase of about 40%. In conclusion, these results demonstrate that in rats with hypertension induced by DOCA–salt, brain mineralocorticoid receptor blockade affects renal function and blood pressure regulation.

Kinins in the circulation are rapidly metabolized by several different peptidases. The purpose of this study was to evaluate the contribution of membrane-bound peptidases to kinin metabolism in the renal circulation. Experiments were performed in vitro, in isolated rat kidneys perfused at a constant flow rate (8 ml/min) with Tyrode's solution. The effects of peptidase inhibitors were evaluated on the functional vasodilator response caused by bradykinin (30 nM) or [Tyr(Me)(8)]bradykinin (10 nM) via activation of bradykinin B2 receptors in kidneys precontracted with prostaglandin F2alpha. Angiotensin converting enzyme inhibitors, enalaprilat (3 microM), ramiprilat (1 microM) or lisinopril (1 microM), increased the bradykinin-induced renal vasodilation by 40% or more. Inhibitors of neutral endopeptidase (thiorphan or phosphoramidon, 10 microM), basic carboxypeptidase (DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid or MGTPA, 10 microM) and aminopeptidase P (apstatin, 20 microM) however did not enhance the renal vasodilator response elicited by kinins, whatever tested alone or in the presence of lisinopril. These findings indicate that angiotensin converting enzyme is the major peptidase whose inhibition potentiates the renal bradykinin B2 receptor mediated vasodilator response of kinins. The relative contribution in this potentiation of inhibition of kinin inactivation and of cross-talk of angiotensin converting enzyme with bradykinin B2 receptor remains however to be clarified.

The renal vascular effects of [Arg8]vasopressin (vasopressin) were investigated in the isolated perfused rat kidney. Vasopressin (0.01-3 nM) elicited a dose-dependent vasoconstriction in kidneys from Sprague Dawley rats, with a EC50 value of 0.206 +/- 0.044 nM. Inhibition of nitric oxide synthase by N omega-nitro-L-arginine (100 microM) shifted the vasopressin-induced vasoconstrictor response curve to the left. Inhibition of cyclooxygenase by indomethacin (10 or 30 microM) blunted the constriction induced by low concentrations of the peptide. Vasopressin, like angiotensin II but not noradrenaline, induced tachyphylaxis, SR 49059 ((2S)1-[(2R,3S)-5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzene- sulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2- carboxamide) (1-30 nM), a new potent and selective non-peptide vasopressin V1A receptor antagonist, shifted the concentration-response curve for vasopressin to the right without decreasing the maximum contraction. Antagonism became competitive with a pA2 value (+/- S.D.) of 9.72 +/- 0.20 during inhibition of nitric oxide release. [Mpa1,D-Arg8]Vasopressin (desmopressin; 0.1-100 nM), or vasopressin (0.01-1 nM) after blockade of the vasopressin V1A receptor by SR 49059, induced no vasopressin V2 receptor-related renal relaxation in kidneys with vascular tone previously restored by noradrenaline or prostaglandin F2 alpha. These findings indicate that in the isolated perfused rat kidney vasopressin is a potent renal vasoconstrictor. The constriction depends on activation of smooth muscle vasopressin V1A receptors and is modulated by endothelial nitric oxide but not by prostacyclin or vasopressin V2 receptor-related vasodilation.

Prolonged treatment with an angiotensin converting enzyme inhibitor produces an induction of plasma angiotensin converting enzyme. Induction of angiotensin converting enzyme in tissues during prolonged treatment with an angiotensin converting enzyme inhibitor is less well documented. We compared the effects of 1 h and 1 week treatment with ramipril (0.1, 0.3, 1 mg/kg) on angiotensin converting enzyme activity in the plasma, renal cortex and renal brush border membrane of Wistar rats. As an increase in activity could be masked by the inhibition due to the presence of ramiprilat which is the active form of ramipril, we eliminated the ramiprilat present in renal cortex homogenates with EGTA during brush border preparation. The 1-h treatment with ramipril induced a dose-dependent inhibition of plasma and renal cortex angiotensin converting enzyme activity. The 1-week treatment with ramipril produced an increase in plasma angiotensin converting enzyme activity, whereas renal cortex angiotensin converting enzyme activity decreased. The decrease in angiotensin converting enzyme activity persisted in the brush border membrane after elimination of residual ramiprilat with EGTA. Our results show that prolonged ramipril treatment produces opposite responses in plasma and renal cortex angiotensin converting enzyme activity, suggesting that plasma and epithelial angiotensin converting enzymes are subject to specific local regulatory factors.

The [3H]ramiprilat binding to the angiotensin-converting enzyme (ACE) in rat renal brush-border membranes was studied. At pH 7.9, and in the presence of 50 mM NaCl, specific binding of [3H]ramiprilat was saturable with a dissociation constant KD = 5.05 nM and maximum number of binding sites of 1.52 pmol/mg prot. [3H]Ramiprilat binding was completely inhibited by specific inhibitors of ACE: ramiprilat, ramipril, enalaprilat, enalapril and captopril. [3H]Ramiprilat binding was Zn2(+)- and Cl(-)-dependent. In the presence of EGTA, which chelates Zn2+ ions, ramiprilat binding was inhibited, but the addition of Zn2+ restored the initial binding. Binding was maximum in the presence of 10 mM NaCl while higher NaCl concentrations decreased the binding, corresponding to a decrease in affinity. The association and dissociation kinetics were also NaCl-dependent. In the absence of NaCl, association and dissociation kinetics were rapid and monophasic. Two-step dissociation kinetics appeared in the presence of 10, 50 and 300 mM NaCl and dissociation time increased with the NaCl concentration. These results confirmed the role of Cl- in the isomerisation and the stability of the membrane-associated ACE-inhibitor complex.

The hypercalcemia caused by malignancy factor, also called parathyroid hormone-related protein (PTHrP), exhibits most of the biological activities of parathyroid hormone (PTH) in kidney and bone. On the basis of the well-documented vascular action of PTH, we characterized the vasodilator action of human (h) PTHrP-(1–34) on a preparation of the isolated rat kidney, and its activity to stimulate adenylate cyclase in microvessels isolated from rabbit kidney cortex. Injection of sequential cumulative doses of hPTHrP-(1–34) into the isolated kidney preparation produced increasing vasodilation up to 10 −18 M (EC 50 of 3 × 10 −9 M) and decreasing responses thereafter. The maximal effect represented 26% of the reference relaxation induced by papaverine. Single injections of hPTHrP-(1–34) resulted in a greater (over 60%) vasodilatation. These results were reminiscent of the tachyphylaxis that occurs after repeated exposure to the peptide. The (3–34) PTH antagonist inhibited the hPTHrP-induced vasodilatation. Human PTHrP-(1– 34) was equipotent with hPTH-(1–34) (EC 50 values of 3 × 10 −9 M) but 5-fold less potent than rat (r) PTH-(1–34) in stimulating microvessel adenylate cyclase. GTP enhanced the enzyme responses to the peptides but reduced their potency. Both (3–34) and (7–34) PTH antagonists were inhibitors of hPTHrP- or PTH-stimulated microvascular adenylate cyclase. Synthetic hPTHrP-(1–16) had neither vasodilator nor adenylate cyclase-stimulating activity. This hPTHrP fragment exhibited some inhibitory effect on the hPTHrP-(1–34)-induced stimulation of microvessel adenylate cyclase. These results indicate that hPTHrP possesses PTH-like activity to cause vasorelaxation and to stimulate microvascular adenylate cyclase in the kidney.