In vitro method of studying the angiotensin activity of chemical compounds.

We developed and tested an experimental model to study in vitro the type 1 angiotensin antagonistic activity of compounds on the isolated portal vein of rats. The reliability of this method was confirmed in tests with saralasin (nonselective antagonist of angiotensin receptors) and losartan (selective antagonist of type 1 angiotensin receptors) in concentrations of 10(-9)-10(-5) mol/liter. The half-maximal inhibitory concentrations (IC50) of these substances were calculated.

Receptor-mediated nonproteolytic activation of prorenin and induction of TGF-ß1 and PAI-1 expression in renal mesangial cells.

While elevated plasma prorenin levels are commonly found in diabetic patients and correlate with diabetic nephropathy, the pathological role of prorenin, if any, remains unclear. Prorenin binding to the (pro)renin receptor [(p)RR] unmasks prorenin catalytic activity. We asked whether elevated prorenin could be activated at the site of renal mesangial cells (MCs) through receptor binding without being proteolytically converted to renin. Recombinant inactive rat prorenin and a mutant prorenin that is noncleavable, i.e., cannot be activated proteolytically, are produced in 293 cells. After MCs were incubated with 10(-7) M native or mutant prorenin for 6 h, cultured supernatant acquired the ability to generate angiotensin I (ANG I) from angiotensinogen, indicating both prorenins were activated. Small interfering RNA (siRNA) against the (p)RR blocked their activation. Furthermore, either native or mutant rat prorenin at 10(-7) M alone similarly and significantly induced transforming growth factor-ß(1), plasminogen activator inhibitor-1 (PAI-1), and fibronectin mRNA expression, and these effects were blocked by (p)RR siRNA, but not by the ANG II receptor antagonist, saralasin. When angiotensinogen was also added to cultured MCs with inactive native or mutant prorenin, PAI-1 and fibronectin were further increased significantly compared with prorenin or mutant prorenin alone. This effect was blocked partially by treatment with (p)RR siRNA or saralasin. We conclude that prorenin binds the (p)RR on renal MCs and is activated nonproteolytically. This activation leads to increased expression of PAI-1 and transforming growth factor-ß(1) via ANG II-independent and ANG II-dependent mechanisms. These data provide a mechanism by which elevated prorenin levels in diabetes may play a role in the development of diabetic nephropathy.

Intracellular angiotensin II activates rat myometrium.

Angiotensin II is a modulator of myometrial activity; both AT(1) and AT(2) receptors are expressed in myometrium. Since in other tissues angiotensin II has been reported to activate intracellular receptors, we assessed the effects of intracellular administration of angiotensin II via microinjection on myometrium, using calcium imaging. Intracellular injection of angiotensin II increased cytosolic Ca(2+) concentration ([Ca(2+)](i)) in myometrial cells in a dose-dependent manner. The effect was abolished by the AT(1) receptor antagonist losartan but not by the AT(2) receptor antagonist PD-123319. Disruption of the endo-lysosomal system, but not that of Golgi apparatus, prevented the angiotensin II-induced increase in [Ca(2+)](i). Blockade of AT(1) receptor internalization had no effect, whereas blockade of microautophagy abolished the increase in [Ca(2+)](i) produced by intracellular injection of angiotensin II; this indicates that microautophagy is a critical step in transporting the peptide into the endo-lysosomes lumenum. The response to angiotensin II was slightly reduced in Ca(2+)-free saline, indicating a major involvement of Ca(2+) release from internal stores. Blockade of inositol 1,4,5-trisphosphate (IP(3)) receptors with heparin and xestospongin C or inhibition of phospholipase C (PLC) with U-73122 abolished the response to angiotensin II, supporting the involvement of PLC-IP(3) pathway. Angiotensin II-induced increase in [Ca(2+)](i) was slightly reduced by antagonism of ryanodine receptors. Taken together, our results indicate for the first time that in myometrial cells, intracellular angiotensin II activates AT(1)-like receptors on lysosomes and activates PLC-IP(3)-dependent Ca(2+) release from endoplasmic reticulum; the response is further augmented by a Ca(2+)-induced Ca(2+) release mechanism via ryanodine receptors activation.

Angiotensin II stimulates H⁺-ATPase activity in intercalated cells from isolated mouse connecting tubules and cortical collecting ducts.

Intercalated cells in the collecting duct system express V-type H(+)-ATPases which participate in acid extrusion, bicarbonate secretion, and chloride absorption depending on the specific subtype. The activity of H(+)-ATPases is regulated by acid-base status and several hormones, including angiotensin II and aldosterone. Angiotensin II stimulates chloride absorption mediated by pendrin in type B intercalated cells and this process is energized by the activity of H(+)-ATPases. Moreover, angiotensin II stimulates bicarbonate secretion by the connecting tubule (CNT) and early cortical collecting duct (CCD). In the present study we examined the effect of angiotensin II (10 nM) on H(+)-ATPase activity and localization in isolated mouse connecting tubules and cortical collecting ducts. Angiotensin II stimulated Na(+)-independent intracellular pH recovery about 2-3 fold, and this was abolished by the specific H(+)-ATPase inhibitor concanamycin. The effect of angiotensin II was mediated through type 1 angiotensin II receptors (AT(1)-receptors) because it could be blocked by saralasin. Stimulation of H(+)-ATPase activity required an intact microtubular network--it was completely inhibited by colchicine. Immunocytochemistry of isolated CNT/CCDs incubated in vitro with angiotensin II suggests enhanced membrane associated staining of H(+)-ATPases in pendrin expressing intercalated cells. In summary, angiotensin II stimulates H(+)-ATPases in CNT/CCD intercalated cells, and may contribute to the regulation of chloride absorption and bicarbonate secretion in this nephron segment.

Angiotensin modulates long-term memory expression but not long-term memory storage in the crab Chasmagnathus.

Memory reconsolidation is a dynamic process in which a previously consolidated memory becomes labile following reactivation by a reminder. In a previous study in the crab Chasmagnathus memory model, we showed that a water-shortage episode, via angiotensin modulation during reconsolidation, could reveal a memory that otherwise remains unexpressed: weakly trained animals cannot reveal long-term memory (LTM) except when an episode of noticeable ethological meaning, water deprivation, is contingent upon reconsolidation. However, these results are at variance with two of our previous interpretations: weak training protocols do not build LTM and angiotensin II modulates the strength of the information storing process. A parsimonious hypothesis is that in Chasmagnathus angiotensins regulate LTM expression, but not LTM storage. Here, we tested three predictions of this hypothesis. First, the well-known retrograde amnesic effect of the angiotensin II antagonist saralasin is not due to interference on memory storage, but to modulation of memory expression. Second, the recovery of the LTM memory expression of the apparently amnesic retrograde effect produced by saralasin, through the water-shortage episode contingent upon reconsolidation, must be reconsolidation specific. Consequently, summation-like effects and retrieval deficits cannot explain these results because of the parametric conditions of reconsolidation. Third, weak training protocols build an unexpressed LTM that requires mRNA transcription and translation, a diagnostic characteristic of LTM. Results show that angiotensin modulates LTM expression but not LTM memory storage in the crab Chasmagnathus. The results lead us to suggest that, in Chasmagnathus, LTM expression - the process of gaining appreciable control over behavior of the reactivated trace in the retrieval session - may be considered a distinct attribute of its long-term storage. This strategy, a positive modulation during reconsolidation, is proposed to distinguish between memories that can be reactivated, labilized and are not expressed, and memories that are not stored long term, obliterated or altered in other retrieval mechanisms.

Brain renin-angiotensin system modifies the blood pressure response to intracerebroventricular cadmium in rats.

In order to elucidate the involvement of the brain renin-angiotensin system (RAS) in cadmium intracerebroventricular (ICV) hypertension, we evaluated the effects of a pretreatment with different drugs: clonidine, an alpha(2) adrenergic agonist, enalapril and captopril, both ACE inhibitors, and saralasin, a competitive nonselective AT(1) and AT(2) receptor antagonist. We used a rat strain with low levels of kallikrein (LKR) that was more sensitive to ICV cadmium hypertension, compared with normal kallikrein rats (NKRs), the control strain. The interplay between the kallikrein-kinin system and the RAS in the LKR strain caused various hemodynamic alterations, which we believe were the result of elevated RAS activity in these animals. Moreover, we suggest that the defective kallikrein-kinin system in LKR may also cause an alteration in the activation of brain RAS in these animals. The LKR displayed elevated concentrations of plasma AII, hypertrophy of the myocardium, and initial alterations in the renal glomerulotubular system. With the exception of clonidine, all of the other drugs showed greater antihypertensive effects of differing statistical significance in LKR, compared with NKR. Both ACE inhibitors were able to significantly reduce pressor response to cadmium ICV in LKR throughout the experiment, whereas in NKR, they were only able to reduce the hypertensive peak of cadmium. A significant protective effect was also observed in LKR pretreated with saralasin, while no effect was observed in NKR. These findings confirm the presence of brain RAS activation in LKR and its contribution to the central control of pressor response to cadmium ICV.

Blockage of ovulation by an angiotensin antagonist.

Angiotensin II (Ang II) is present in high concentrations in preovulatory follicular fluid, and ovarian follicular cells have specific Ang II receptors. To investigate the possible direct involvement of Ang II in ovulation the specific receptor antagonist of Ang II, saralasin, was administered by intraperitoneal injection to immature rats in which follide development and ovulation had been induced with pregnant mare serum gonadotrophin (PMSG) and human chorionic gonadotrophin (hCG), respectively. Saralasin halved the number of oocytes found in the fallopian tubes 17 to 20 hours after administration of hCG. The antiovulatory effect was observed when saralasin was given 1 hour before hCG or 1 or 3 hours after hCG but not when given 5 hours after hCG. Simultaneous administration of Ang II reversed the saralasin blockage of ovulation. These results indicate a direct, obligate role for Ang II in ovulation and raise the possibility of contraceptive and profertility applications for agonists or antagonists of the renin-angiotensin system that are aimed at the ovulatory process.

Reciprocation of renin dependency with sodium volume dependency in renal hypertension.

An angiotensin II inhibitor was administered to rats with two-kidney Goldblatt hypertension. The inhibitor produced a marked drop in blood pressure after 5 weeks but no significant change after 15 weeks of hypertension. However, even after 15 weeks of hypertension, following sodium depletion by either diuretics or a low sodium diet, the animals again became renin dependent as readministration of the inhibitor induced a significant fall in blood pressure. The data indicate that two-kidney Goldblatt hypertension is initially renin dependent but subsequently becomes sodium volume dependent in a way similar, although more protracted, to that already described for one-kidney Goldblatt hypertension.

Angiotensin: physiological role in water-deprivation-induced thirst of rats.

Cerebroventricular infusion of P-113, the blocking agent of angiotensin II, into rats for 75 minutes prior to their being allowed to drink, significantly attenuated their water intake when they had been deprived of water for 30 hours. However, a similar infusion had no effect on the food intake in rats fasted for 30 hours. The results indicate a physiological role for angiotensin II in the drinking response of rats deprived of water.

Sodium appetite in sheep induced by cerebral ventricular infusion of angiotensin: comparison with sodium deficiency.

Intraventricular administration of supraphysiological amounts of renin, nerve growth factor preparation, or angiotensin II greatly increased the consumption of water and hypertonic sodium bicarbonate solution by sheep. These effects were antagonized by intraventricular administration of drugs that prevent the formation of angiotensin II or block its receptors. The fact that these angiotensin-blocking drugs did not change the sodium intake of sodium-deficient sheep challenges the idea that central angiotensin action is involved in sodium appetite due to a deficiency.

Angiotensin II type 1 receptor blockade: high hopes sent back to reality?

Chronic activation of the renin-angiotensin system (RAS) plays a crucial role in the development of various cardiovascular diseases (CVD). Thus, effective RAS inhibition has been a major achievement to improve the treatment of patients at risk for CVDs, such as myocardial infarction, heart failure and stroke. Three substance classes that block RAS-activation are currently available, angiotensin converting enzyme (ACE) inhibitors, angiotensin II type 1 receptor blockade (ARB) and renin inhibitors. Although the overall goal of these drugs remains the blockade of RAS activation, their individual targets in this system vary and may substantially influence the clinical benefit derived from the long term use of these substances. Here, we summarize the evidence available for the use of ARBs in different cardiovascular pathologies and the impact of this evidence on current treatment guidelines for patients at risk for CVD. Today, ARBs represent a good alternative in case of ACE-inhibitor intolerance due to their outstanding tolerability. ARBs in comparison to ACE-inhibitors have been proven to exert similar effective in the treatment of systolic heart failure, primary prevention of stroke, new onset of diabetes mellitus (DM) type 2 and DM type 2 dependent macroalbuminuria. ARBs should be considered as alternatives to ACE-inhibitors in subjects post-myocardial infarction. Overall however, there is no profound proof for a specific cardiovascular protection by blockade of the angiotensin II Type 1 (AT1) receptor that exceeds the impact of ACE-inhibition or synergises with ACE-blockade. In fact, combination of ARBs and ACE-inhibitor result in an increased rate of adverse effects and, therefore, this combination should not be encouraged. To summarize, the initial hope for a more specific impact on cardiovascular diseases by inhibition of the AT1-receptor in comparison to ACE-inhibition has not come true. However, ARBs have been proven to be equally effective as ACE-blockade in a large variety of clinical settings.

Angiotensin II and myosin light-chain phosphorylation contribute to the stretch-induced slow force response in human atrial myocardium.

AIMS: Stretch is an important regulator of atrial function. The functional effects of stretch on human atrium, however, are poorly understood. Thus, we characterized the stretch-induced force response in human atrium and evaluated the underlying cellular mechanisms. METHODS AND RESULTS: Isometric twitch force of human atrial trabeculae (n = 252) was recorded (37 degrees C, 1 Hz stimulation) following stretch from 88 (L88) to 98% (L98) of optimal length. [Na(+)](i) and pH(i) were measured using SBFI and BCECF epifluorescence, respectively. Stretch induced a biphasic force increase: an immediate increase [first-phase, Frank-Starling mechanism (FSM)] to approximately 190% of force at L88 followed by an additional slower increase [5-10 min; slow force response (SFR)] to approximately 120% of the FSM. FSM and SFR were unaffected by gender, age, ejection fraction, and pre-medication with major cardiovascular drugs. There was a positive correlation between the amplitude of the FSM and the SFR. [Na(+)](i) rose by approximately 1 mmol/L and pH(i) remained unchanged during the SFR. Inhibition of Na(+)/H(+)-exchange (3 microM HOE642), Na(+)/Ca(2+)-exchange (5 microM KB-R7943), or stretch-activated channels (0.5 microM GsMtx-4 and 80 microM streptomycin) did not reduce the SFR. Inhibition of angiotensin-II (AngII) receptors (5 microM saralasin and 0.5 microM PD123319) or pre-application of 0.5 microM AngII, however, reduced the SFR by approximately 40-60%. Moreover, stretch increased phosphorylation of myosin light chain 2 (MLC2a) and inhibition of MLC kinase (10 microM ML-7 and 5 microM wortmannin) decreased the SFR by approximately 40-85%. CONCLUSION: Stretch elicits a SFR in human atrium. The atrial SFR is mediated by stretch-induced release and autocrine/paracrine actions of AngII and increased myofilament Ca(2+) responsiveness via phosphorylation of MLC2a by MLC kinase.

Evidence that the effect of angiotensin II on bovine oocyte nuclear maturation is mediated by prostaglandins E2 and F2alpha.

Angiotensin II (AngII) prevents the inhibitory effect of follicular cells on oocyte maturation, but its involvement in LH-induced meiotic resumption remains unknown. The aim of this study was to assess the involvement of AngII in LH-induced meiotic resumption and of prostaglandins (PGs) in the action of AngII. In the experiment I, seven cows were superovulated, intrafollicularly injected with 10 muM saralasin (a competitive AngII antagonist) or saline when the follicles reached a diameter larger than 12 mm, and challenged with a GnRH agonist to induce an LH surge. Fifteen hours after GnRH, the animals were ovariectomized and the oocytes were recovered to determine the stage of meiosis. The oocytes from follicles that received saline were in germinal vesicle (GV) breakdown (30.8%) or metaphase I (MI; 69.2%) stage while those that received saralasin were in the GV stage (100%; P<0.001) 15 h after GnRH agonist. In another experiment, oocytes were co-cultured with follicular hemisections for 15 h to determine whether PGs mediate the effect of AngII on meiotic resumption. Indomethacin (10 microM) inhibited AngII-induced meiotic resumption (13.4 vs 77.5% MI without indomethacin; P<0.001). Furthermore, the GV oocytes progressed to MI at a similar rate when PGE(2), PGF(2alpha) or AngII was present in the co-culture system with follicular cells (PGE(2) 77.4%, PGF(2alpha) 70.0%, and AngII 75.0% MI). In conclusion, our results provide strong evidence that AngII mediates the resumption of meiosis induced by an LH surge in bovine oocytes and that this event is dependent on PGE(2) or PGF(2alpha) produced by follicular cells.

Peripheral mechanisms involved in the pressor and bradycardic effects of centrally administered arachidonic acid.

In the current study, we aimed to determine the cardiovascular effects of arachidonic acid and peripheral mechanisms mediated these effects in normotensive conscious rats. Studies were performed in male Sprague Dawley rats. Arachidonic acid was injected intracerebroventricularly (i.c.v.) at the doses of 75, 150 or 300 microg and it caused dose- and time-dependent increase in mean arterial pressure and decrease in heart rate in normal conditions. Maximal effects were observed 10 min after 150 and 300 microg dose of arachidonic acid and lasted within 30 min. In order to evaluate the role of main peripheral hormonal mechanisms in those cardiovascular effects, plasma adrenaline, noradrenaline, vasopressin levels and renin activity were measured after arachidonic acid (150 microg; i.c.v.) injection. Centrally injected arachidonic acid increased plasma levels of all these hormones and renin activity. Intravenous pretreatments with prazosin (0.5 mg/kg), an alpha1 adrenoceptor antagonist, [beta-mercapto-beta,beta-cyclopentamethylenepropionyl1, O-Me-Tyr2-Arg8]-vasopressin (10 microg/kg), a vasopressin V1 receptor antagonist, or saralasin (250 microg/kg), an angiotensin II receptor antagonist, partially blocked the pressor response to arachidonic acid (150 microg; i.c.v.) while combined administration of these three antagonists completely abolished the effect. Moreover, both individual and combined antagonist pretreatments fully blocked the bradycardic effect of arachidonic acid. In conclusion, our findings show that centrally administered arachidonic acid increases mean arterial pressure and decreases heart rate in normotensive conscious rats and the increases in plasma adrenaline, noradrenaline, vasopressin levels and renin activity appear to mediate the cardiovascular effects of the drug.

Endogenous angiotensin stimulation of vasopressin in the newborn lamb.

The effect of furosemide on plasma renin, vasopressin (AVP), and aldosterone concentrations was studied in 10 control and 6 nephrectomized lambs during the 1st 2 wk of life. In a separate study in 10 newborn lambs, 1-sarcosine-8-alanine-angiotensin II (saralasin acetate, 5 mug/kg per min) was infused alone for 40 min, after which furosemide 2 mg/kg i.v. was injected in association with continuing saralasin acetate infusion. Plasma renin activity increased from a mean (+/-SEM) of 21.3+/-3.4 ng/ml per h in the 10 control lambs to 39.4+/-8.2 ng/ml per h at 8 min (P < 0.001) and remained high through 120 min after furosemide. Plasma AVP and aldosterone concentrations increased from respective mean values of 2.1+/-0.4 muU/ml and 12.8+/-2.5 ng/dl to 9.8+/-2.0 muU/ml (P < 0.01) and 23.0+/-7.7 ng/dl (P < 0.05) at 35 min and 13.8+/-2.1 muU/ml and 23.0+/-4.4 ng/dl at 65 min after furosemide (each P < 0.01). There was an insignificant AVP response in the 10 lambs treated with angiotensin inhibitor: from a mean base line of 4.7+/-0.9 to 8.3+/-2.0 muU/ml at 35 min, and 7.4+/-2.0 muU/ml at 65 min after furosemide. There was no increase in AVP in the anephric lambs. The mean increment AVP response from base line in the newborn lambs without saralasin, Delta 10.8+/-2.0 muU/ml, was greater than in the lambs with saralasin, Delta4.0+/-1.9 (P < 0.05), and greater than in the anephric lambs, Delta3.3+/-2.1 muU/ml (P < 0.05). The mean blood pressure fell 6 mm Hg in the 10 control lambs (P < 0.05), 7 mm Hg in the anephric lambs (P < 0.05), and 16 mm Hg in the lambs treated with angiotensin inhibitor (P < 0.05) by 35 min after furosemide. However, the changes in plasma AVP were not related to the fall in blood pressure. These data support the view that the observed AVP response to furosemide in the newborn lamb was mediated through the renin-angiotensin system.

Role for intrarenal mechanisms in the impaired salt excretion of experimental nephrotic syndrome.

A unilateral model of puromycin aminonucleoside (PAN)-induced albuminuria was produced in Munich-Wistar rats to examine the mechanisms responsible for renal salt retention. 2 wk after selective perfusion of left kidneys with PAN (n = 8 rats) or isotonic saline (control, n = 7 rats), increases in albumin excretion and decreases in sodium excretion were demonstrated in PAN-perfused but not in nonperfused kidneys of PAN-treated rats although systemic plasma protein concentration remained at control level. Total kidney glomerular filtration rate (GFR) and superficial single nephron (SN) GFR were also reduced selectively in PAN-perfused kidneys, on average by approximately 30%, due primarily to a marked decline in the glomerular capillary ultrafiltration coefficient (Kf), which was also confined to PAN-perfused kidneys. Values for absolute proximal reabsorption (APR) were also selectively depressed in PAN-perfused kidneys, in keeping with a similarly selective decline in peritubular capillary oncotic pressure measured in these kidneys, the latter also a consequence of the fall in Kf. In a separate group of seven PAN-treated rats, however, no differences were detected between PAN-perfused and nonperfused kidneys in the absolute amount of sodium reaching the early (0.77 +/- 0.09 neq/min vs. 0.74 +/- 0.08, P greater than 0.40) and late portions of superficial distal tubules (0.31 +/- 0.02) neq/min vs. 0.32 +/- 0.05, P greater than 0.50), despite the lesser filtered load of sodium in PAN-perfused kidneys. Suppressed sodium reabsorption in both proximal convoluted tubules and short loops of Henle of PAN-perfused kidneys contributed to this equalization of sodium delivery rates to the late distal tubule, as did comparable reabsorption along distal convolutions. In two additional groups of PAN-treated rats, infusion of saralasin (0.3 mg/kg per h, i.v.) led to substantial increases in total kidney GFR and SNGFR in PAN-perfused but not in nonperfused kidneys. Despite these increases in total and SNGFR, urinary sodium excretion by PAN-perfused kidneys remained at a level far below that for nonperfused kidneys, again indicating that the antinatriuresis characterizing the PAN-perfused kidney is due to alterations in sodium handling by the tubules rather than changes in GFR. These results therefore indicate (a) that reductions in Kf and depressed sodium reabsorption by proximal tubules and Henle's loop segments in this model are brought about by intrarenal rather than circulating or systemic factors, and (b) assuming that superficial nephrons are representative of the entire nephron population, renal salt retention in this model is due primarily to intrarenal factor(s) acting beyond the distal convolution.

Mechanism of preservation of glomerular perfusion and filtration during acute extracellular fluid volume depletion. Importance of intrarenal vasopressin-prostaglandin interaction for protecting kidneys from constrictor action of vasopressin.

Glomerular circulatory dynamics were assessed in 60 adult anesthetized rats, which were either deprived or not deprived of water for 24-48 h. Water-deprived rats (n = 21) were characterized by a depressed level of single nephron glomerular filtration rate (SNGFR) when compared with nonwater-deprived controls (n = 8) (23.2 +/- 1.3 vs. 44.8 +/- 4.1 nl/min). This was primarily due to decreased glomerular plasma flow rate (71 +/- 5 vs. 169 +/- 23 nl/min) and glomerular capillary ultrafiltration coefficient (0.028 +/- 0.003 vs. 0.087 +/- 0.011 nl/[s . mmHg]). Infusion of saralasin to these water-deprived rats resulted in significant increases in plasma flow rate and ultrafiltration coefficient, and decline in arteriolar resistances. Consequently, SNGFR increased by approximately 50% from pre-saralasin levels. When water-deprived saralasin-treated rats were given a specific antagonist to the vascular action of arginine vasopressin (AVP), d(CH2)5Tyr(Me)AVP, a fall in systemic blood pressure occurred, on average from 102 +/- 5 to 80 +/- 5 mmHg, unaccompanied by dilation of renal arterioles, so that both plasma flow rate (129 +/- 8 vs. 85 +/- 13 nl/min) and SNGFR (31.0 +/- 2.9 vs. 18.2 +/- 4.4 nl/min) decreased. This more selective extrarenal constrictor action of AVP was further documented in additional studies in which cardiac output and whole kidney blood flow rate were simultaneously measured. In water-diuretic rats, administration of a moderately pressor dose of AVP (4 mU/kg per min) resulted in a significant rise in kidney blood flow rate (from 8.8 +/- 1.2 to 9.6 +/- 1.3 ml/min). The higher kidney blood flow rate occurred despite a fall in cardiac output (from 111 +/- 7 to 98 +/- 9 ml/min), and was associated with a significant increase in the ratio of systemic vascular to renal vascular resistance (on average from 0.083 +/- 0.014 to 0.106 +/- 0.019). Furthermore, infusion of d(CH2)5Tyr(Me)AVP to water-deprived animals (n = 6) to antagonize endogenous AVP resulted in systemic but not renal vasodilation, so that kidney blood flow rate fell (by approximately 30%), as did systemic-to-renal resistance ratio (by approximately 30%). When the above two experiments were repeated in indomethacin-treated animals, exogenous AVP administration in water-diuretic rats (n = 6) and antagonism of endogenous AVP in water-deprived rats (n = 7) caused, respectively, parallel constriction and dilation in systemic and renal vasculatures. The net effect was unaltered systemic to renal vascular resistance ratio in both cases. These results indicate that (1) unlike angiotensin II, AVP maintains glomerular perfusion and filtration in acute extracellular fluid volume depletion by a more selective constriction of the extrarenal vasculature. (2) The relative renal insensitivity to the vasoconstrictor action of AVP appears to be due to an AVP-induced release of a potent renal vasodilator, sensitive to indomethacin, presumably prostaglandins.

Angiotensin II: a potent regulator of acidification in the rat early proximal convoluted tubule.

The early proximal convoluted tubule (PCT) is the site of 50% of bicarbonate reabsorption in the nephron, but its control by angiotensin II has not been previously studied. In vivo microperfusion was used in both the early and late PCT in Munich-Wistar rats. Systemic angiotensin II administration (20 ng/kg X min) or inhibition of endogenous angiotensin II activity with saralasin (1 microgram/kg X min) caused profound changes in bicarbonate absorption in the early PCT (169 +/- 25 and -187 +/- 15 peq/mm X min, respectively). Because the bicarbonate absorptive capacity of the early PCT under free-flow conditions is 500 peq/mm X min, angiotensin II administration or inhibition affected greater than 60% of proton secretion in this segment. Both agents less markedly affected bicarbonate absorption in the late PCT (+/- 28 peq/mm X min) or chloride absorption (+/- 68-99 peq/mm X min) in both the early and late PCT. Because of its potential for controlling the majority of bicarbonate absorption in the early PCT (hence greater than or equal to 30% of bicarbonate absorption in the entire nephron), angiotensin II may be a powerful physiologic regulator of renal acidification.

In vitro prostacyclin production by ovine uterine and systemic arteries. Effects of angiotensin II.

Normal pregnancy is associated with reduced systemic pressor responses to infused angiotensin II (ANG II); furthermore, the uterine vascular bed is even less responsive to vasoconstriction by ANG II than the systemic vasculature overall. The mechanism(s) for this refractoriness remains unknown. To determine if vessel production of prostacyclin may be responsible, uterine and omental artery segments were obtained from four groups of sheep, nonpregnant (NP), pregnant (P; 131 +/- 4 d), early postpartum (2.2 +/- 0.4 d), and late postpartum (16 +/- 2 d), and incubated in Krebs-Henseleit alone or with ANG II in the absence or presence of Saralasin. Prostacyclin was measured as 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha). Synthesis of 6-keto-PGF1 alpha was de novo, since aspirin inhibited its formation. P and early uterine arteries produced more 6-keto-PGF1 alpha than NP and late vessels (P less than 0.05): 386 +/- 60 (X +/- SE) and 175 +/- 23 vs. 32 +/- 5 and 18 +/- 4 pg/mg X h, respectively. A similar relationship was observed for omental arteries: 101 +/- 14 and 74 +/- 14 vs. 36 +/- 10 and 22 +/- 4 pg/mg X h, respectively. Furthermore, synthesis by arteries from P and early animals was greater in uterine than omental vessels (P less than 0.05); this was not observed in NP or late vessels. ANG II increased 6-keto-PGF1 alpha production 107 +/- 20% and 92 +/- 16% in P and early uterine arteries only; the threshold dose was between 5 X 10(-11) and 5 X 10(-9) M ANG II. This ANG II-induced increase in 6-keto-PGF1 alpha by uterine arteries was inhibited by Saralasin, which by itself had no effect. During pregnancy, the reduced systemic pressor response to ANG II and the even greater refractoriness of the uterine vascular bed may be reflective of vessel production of the potent vasodilator, prostacyclin. Furthermore, in the uterine vasculature, this antagonism may be potentiated by specific ANG II receptor-mediated increases in prostacyclin.

Effect of angiotensin II on ammonia production and secretion by mouse proximal tubules perfused in vitro.

The effects of angiotensin II on total ammonia (tNH3) production and net secretion were investigated using in vitro microperfused mouse S2 proximal tubule segments incubated in Krebs-Ringer bicarbonate buffer containing 0.5 mM L-glutamine. Basolateral exposure of mouse S2 segments to 10(-11), 10(-10), and 10(-9) M angiotensin II stimulated tNH3 production rates by 23, 52, and 49%, respectively. Addition of 10(-6) M angiotensin II inhibited the tNH3 production rate by 34%. 10(-10) M angiotensin II inhibited net luminal secretion of tNH3 in the presence of enhanced luminal acidification and in the absence of altered luminal tNH3 efflux rates. Measurements of intracellular pH (pHi) and intracellular calcium concentration [( Ca2+]i) suggested that the effects of angiotensin II on tNH3 production were not mediated by changes in pHi but by the stimulatory effect of angiotensin II correlated with increased [Ca2+]i. Inhibition of the calcium-calmodulin-dependent pathway with W-7 blocked the stimulatory effect of 10(-10) M angiotensin II on tNH3 production and luminal acidification. These results indicate that angiotensin II has concentration-dependent effects on tNH3 production; that its action to stimulate tNH3 production may be mediated by rises in [Ca2+]i and the calcium-calmodulin pathway; and that angiotensin II, at concentrations that stimulate tNH3 production, inhibits net luminal ammonia secretion by a mechanism that is not mediated by diminished luminal acidification or by changes in luminal ammonia efflux rates.