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The aim of the present study was to quantify with a uniform technique the rates of conversion of ANG I to ANG II in the lung and kidney and the degradation of both peptides to biologically inactive products in the pulmonary, renal, and systemic circulation. We infused the peptides intravenously, into the left ventricle, and into the left renal artery of rats and compared their effects on renal blood flow. The measured change in renal blood flow was used as a bioassay parameter to estimate the concentration of circulating ANG II. Mathematical analysis of our data allowed us to calculate conversion and degradation rates. Furthermore, the role of aminopeptidases A (EC 3.4.11.7) and N (EC 3.4.11.2) in the degradation of the peptides in the kidney was investigated by intrarenal infusion of the inhibitor amastatin. Our results show that the conversion rate of ANG I is 75% in the pulmonary and 21% in the renal circulation. Both peptides are degraded by 5% in the pulmonary, by 67% in the systemic, and by 93% in the renal circulation. Amastatin prevented 60% of the renal degradation of the peptides to inactive products, and this effect could be attributed to inhibition of aminopeptidase N. The results indicate that the converting capacity of the kidney is of minor importance for endocrine generation of ANG II but could be useful for the paracrine production.

Using extracellular electrophysiological recording in an in vitro slice preparation, we investigated whether ANG I can be locally converted to the functionally active ANG II within the rat subfornical organ (SFO). ANG I and ANG II (10(-8)-10(-7) M) excited approximately 75% of all neurons tested with both peptides (n = 25); the remainder were insensitive. The increase in firing rate and the duration and the latency of the responses of identical neurons, superfused with equimolar concentrations of ANG I and ANG II, were not different. The threshold concentrations of the ANG I- and ANG II-induced excitations were both 10(-9) M. Inhibition of the angiotensin-converting enzyme by captopril (10(-4) M; n = 8) completely blocked the ANG I-induced excitation, a 10-fold lower dose was only effective in two of four neurons. The AT1-receptor antagonist losartan (10(-5) M; n = 6) abolished the excitation caused by ANG I and ANG II. Subcutaneous injections of equimolar doses of ANG I and ANG II (200 microliters; 2 x 10(-4) M) in water-sated rats similarly increased water intake by 2.4 +/- 0.5 (n = 16) and 2. 7 +/- 0.4 ml (n = 20) after 1 h, respectively. Control rats receiving saline drank 0.07 +/- 0.06 ml under these conditions. Pretreatment with a low dose of captopril (2.3 x 10(-3) M) 10 min before the injection of ANG I caused a water intake of 2.8 +/- 0.5 ml (n = 10), whereas a high dose of captopril (4.6 x 10(-1) M) suppressed the dipsogenic response of ANG I entirely (n = 11). These data provide direct functional evidence for an SFO-intrinsic renin-angiotensin system (RAS) and underline the importance of the SFO as a central nervous interface connecting the peripheral with the central RAS.

We evaluated in rats the time course of changes in cardiac versus plasma ANG I and II postmyocardial infarction (MI) and the effects of nephrectomy and angiotensin-converting enzyme (ACE) inhibitors on the early changes post-MI. Acute coronary artery ligation was induced in conscious rats using the two-stage model, and plasma and cardiac tissue were obtained shortly (6 h, 1 and 3 days) and chronically (1, 4, and 8-9 wk) after MI. In an additional group of rats, bilateral nephrectomy was performed 18 h before the coronary artery ligation, and samples were obtained at 6 h post-MI. Furthermore, in two additional groups of rats, treatment with enalapril and quinapril was started 3 days before the ligation, and samples were obtained at 1 or 3 days post-MI. In these groups of rats, plasma and left ventricular (LV) (infarct and infarct free) ANG I and II were measured by RIA after separation on HPLC. In control rats, plasma ANG I and II showed a clear increase at 6 h post-MI but subsequently only minor increases were observed. In contrast, LV ANG II showed major increases at 6 h and 1 day post-MI, which had returned to normal by 3 days in the infarct-free LV and after 1(-2) wk in the infarct LV. LV ANG I showed a more gradual increase and remained elevated in the infarct up to 8-9 wk. Nephrectomy preceding the MI lowered ANG I and II in plasma but enhanced their increases in the heart at 6 h post-MI. Both ACE inhibitors decreased plasma ANG II associated with large increases in plasma ANG I. They also inhibited the increases in LV ANG II in both the infarct and infarct-free LV at 1 and 3 days post-MI with however no significant increase in LV ANG I. In conclusion, induction of a MI in conscious rats leads to rapid and marked, but only short-lived, increases in cardiac tissue ANG II in both the infarct and infarct-free parts of the LV. Pretreatment with ACE inhibitors, but not nephrectomy, blocks this increase. Local production appears to play a major role in the increases in cardiac ANG II post-MI.

Like human chymase, hamster chymase is an ANG II-forming enzyme, but pathophysiological roles of chymase are still unknown. We determined the functional conversion of ANG I and [Pro11, D-Ala12]ANG I, a chymase-selective substrate, to ANG II in the hamster cardiovascular system. ANG I and [Pro11, D-Ala12]ANG I produced similar dose-dependent pressor responses in conscious hamsters. Captopril and CV-11974, an ANG II type 1 (AT1)-receptor antagonist, inhibited the responses to ANG I; in contrast, the pressor responses to [Pro11, D-Ala12]ANG I were suppressed only by CV-11974. In the isolated aorta, captopril suppressed ANG I-induced contraction by 84%; administration of captopril with either chymostatin or aprotinin eliminated the contraction. [Pro11, D-Ala12]ANG I-induced contraction was not affected by captopril but was attenuated by chymostatin (71%) and aprotinin (57%). CV-11974 abolished the responses to both substrates, whereas PD-123319, an AT2-receptor antagonist, had no effect. In homogenates of the aorta and heart, soybean trypsin inhibitor-inhibitable ANG II formation predominated over captopril- or aprotinin-inhibitable ANG II formation. These data suggest that [Pro11,D-Ala12]ANG I and part of ANG I were functionally converted to ANG II by chymase and other serine protease(s) in hamster vessels, inducing AT1-receptor-mediated vasoconstriction. Biochemical data supported a role for chymase in the alternative pathway.

Gene targeting using homologous recombination in embryonic stem cells provides an avenue for the direct application of precise molecular genetic interventions to the study of complex systems in whole animals. As such, it represents a powerful approach for physiological investigation. Although its applications in physiology were initially limited because of technical difficulties in performing whole animal experiments in mice, these difficulties have been rapidly overcome, and gene targeting has been used productively in physiological experimentation. Studies have been performed using mice in which genes in the renin-angiotensin system (RAS) have been altered by gene targeting, and these studies illustrate both the feasibility and the utility of this technique for addressing physiological issues. These studies have demonstrated novel roles for the RAS in the development and maintenance of kidney structure and have added to the understanding of how RAS gene products regulate blood pressure and renal function. Finally, these experiments may contribute to understanding how naturally occurring mutations in RAS genes cause hypertension.

Angiotensin (ANG) II plays a vital role in blood pressure regulation and body fluid homeostasis. Although many peripheral tissues synthesize components of the renin-ANG system, very few synthesize all of the major components involved in the generation ofANG II. This study used interscapular brown adipose tissue (ISBAT) as a model system to evaluate the mechanism of ANG II generation in an extrarenal tissue. Polymerase chain reaction analysis of DNA from ISBAT demonstrated angiotensinogen gene expression; however, renin gene expression was not detected. Renin activity that was not completely derived from the residual blood pool was detected in ISBAT homogenates. Kinetic parameters for renin activity were similar in ISBAT and adrenal gland. Renin activity was partially inhibited by anti-renin antibody and completely inhibited by a specific rat renin inhibitor. Bilateral nephrectomy did not decrease renin activity in ISBAT. Western blot analysis, employing two species-specific renin antibodies, indicated the presence of a variety of isoforms of renin in ISBAT. The presence of renin activity in isolated brown adipocytes demonstrated that the enzyme is localized to adipocytes. The release of immunoreactive ANG peptides from ISBAT slices over 3 h indicated de novo synthesis. These studies support the existence of a local renin-ANG system in ISBAT and suggest involvement of renin in the formation of ANG II.

This study investigated the roles of serotonin (5-HT) receptors in the lateral parabrachial nucleus (LPBN) and brain angiotensin type 1 (AT1) receptors in the intake of 0.3 M NaCl and water induced by angiotensin II (ANG II). Rats were implanted with stainless steel cannulas for injections into the subfornical organ (SFO) and into the LPBN. Bilateral LPBN pretreatment with the nonselective serotonergic 5-HT1/5-HT2 receptor antagonist methysergide (4 micrograms/200 nl) markedly enhanced 0.3 M NaCl intake induced by injections of ANG II (20 ng/200 nl) into the SFO. Pretreatment of the SFO with the AT1 receptor antagonist losartan (1 microgram/200 nl) blocked the intake of 0.3 M NaCl induced by ANG II in combination with LPBN methysergide injections. These results suggest that serotonergic mechanisms associated with the LPBN inhibit the expression of salt appetite induced by ANG II injections into the SFO. In addition, the results indicate that the enhanced NaCl intake generated by central administration of ANG II in the presence of LPBN 5-HT blockade is mediated by brain AT1 receptors.

To characterize the renin secretory profile in Lyon hypertensive (LH) rats, renin responses to reductions of arterial pressure and beta-adrenoceptor stimulation were assessed in conscious unrestrained LH (n = 13) and Lyon normotensive (LN, n = 14) rats under normal-salt diet. Mean arterial pressure (MAP) in the infrarenal aorta was recorded beat to beat for 3 h. Then, plasma renin concentration (PRC) was measured 1) in basal conditions, 2) during 10-mmHg stepwise reductions of MAP down to 60 mmHg using a chronically implanted aortic inflatable cuff, and 3) during isoprenaline infusion (62.5, 125, and 250 ng.kg-1.min-1 iv). Compared with LN, LH rats had an elevated MAP (146 +/- 3 vs. 111 +/- 1 mmHg, P < 0.001) and decreased PRC [4.2 +/- 0.6 vs. 8.2 +/- 0.8 ng angiotensin (ANG) I.ml-1.h-1, P < 0.001] and kidney renin content (216 +/- 14 vs. 1,149 +/- 103 micrograms ANG I.h-1.g-1, P < 0.001). Pressure-dependent renin release occurred below 90 mmHg in LN rats and below 80 mmHg in LH rats, and its sensitivity in the low-pressure range did not differ between strains. Isoprenaline-induced increases in PRC were weaker (P < 0.01) in LH than in LN rats. In additional LH and LN rats (n = 6-8), acute ANG II AT1-receptor blockade with losartan (20 mg/kg, followed by 10 mg.kg-1.h-1 iv for 2 h) induced lesser (P < 0.001) PRC increases in LH than in LN rats. Renin responses to isoprenaline remained blunted (P < 0.01) during losartan infusion in LH rats. We conclude that, in LH rats, renin secretion is independent of MAP in the range of its spontaneous variations and is poorly responsive to beta-adrenoceptor stimulation, the alteration of which cannot be explained by an enhanced feedback inhibition by ANG II.

The renin-angiotensin system (RAS) has been implicated in the pathogenesis of diabetic nephropathy. In diabetes, renal RAS components are dysregulated, potentially increasing renal RAS effects. To explore the renal RAS, studies were conducted in control and diabetic rats. In both groups, intravenous angiotensin (ANG) I and ANG II produced similar increases in mean arterial pressure (MAP). In contrast, glomerular filtration rate defined only in diabetic rats. Renal plasma flow fell in both groups but decreased more in diabetic rats. Additional groups were given the same dose of ANG I directly into the left renal artery, and hemodynamics were studied in the treated and untreated kidneys. In contrast to the intravenous studies, intra-arterial ANG I had no effect on MAP in either group. The renal hemodynamic effects were similar to those in intravenous studies. Additionally, diabetic rats exhibited enhanced hemodynamic sensitivity in the untreated kidney, suggesting that renal effects could occur at nonpressor concentrations of circulating ANG II. Thus renal (but not systemic) responsiveness to angiotensins is enhanced in diabetic rats.

The purpose of the present investigation was to compare the effects of angiotensin II (ANG II) other growth factors implicated to play a role in ventricular hypertrophy on cardiac fibroblast changes associated with cardiac remodeling. These changes included induction of early growth response (Egr-1) gene and increases in message levels of extracellular matrix proteins. ANG II treatment (10(-10)-10(-6) M) of rat cardiac fibroblasts induced 1) Egr-1 and 2) a fourfold (P < 0.02) increase in fibronectin and a twofold (P = 0.05) increase in laminin mRNA levels but no increases in that of collagens I, III, or IV at 24-48 h, and 3) a decrease in AT1-receptor mRNA levels to 26% (P < 0.001) of basal at 4-6 h. These effects were all inhibited by the AT1-receptor blocker, losartan, but not AT2-receptor blockers. Immunostaining of cultured cells with antibody against rat fibronectin demonstrated positive staining of cells in serum-free medium; staining was more intense in cells treated with ANG II (10(-6) M, 48 h). Fluorescent-activated cell sorting using an antibody against rat AT1 receptor demonstrated a receptor signal in cells maintained in serum-free medium; however, the receptor signal was not detectable in ANG II-treated cells. Serum and epidermal growth factor (EGF) also induced Egr-1, but norepinephrine (NE) and endothelin (ET) had no effect. Serum increased fibronectin mRNA levels by twofold (P < 0.05). EGF, NE, and ET had no effect on matrix gene expression. Serum, EGF, and NE also transiently downregulated AT1-receptor mRNA levels at 4-6 h of treatment. These results demonstrate that 1) ANG II both induces protooncogene expression and enhances fibronectin mRNA levels in cultured cardiac fibroblasts, whereas EGF only induces Egr-1, and NE and ET have no effects on either function; 2) ANG II effects are primarily mediated by the AT1 receptor; and 3) growth factors can regulate AT1-receptor mRNA levels. Thus ANG II, relative to NE, ET, and EGF, appears to play a prominent and direct role in fibroblast changes associated with cardiac hypertrophy.

Angiotensin II (ANG II) regulates whole kidney ion transport, yet its effects in the collecting duct are unknown. The purpose of these studies was to determine whether ANG II regulates luminal alkalinization and acidification in the rabbit cortical collecting duct (CCD). The rate of luminal alkalinization or acidification was measured as the rate of change of luminal fluid pH under stop-flow conditions using in vitro microperfused CCD segments. Outer CCD alkalinized the luminal fluid, consistent with net HCO3- secretion. Addition of ANG II, 10(-7) M, to the peritubular solution for 30 min significantly stimulated luminal alkalinization. The stimulatory effect of ANG II was not due to time-dependent effects and was blocked by peritubular addition of the ANG II type 1 (AT1) receptor antagonist, losartan, at 10(-6) M. Losartan, 10(-6) M, when added to the peritubular solution, did not alter the rate of luminal alkalinization independent of ANG II. In contrast, peritubular ANG II, 10(-7) M, did not alter inner CCD luminal acidification. Addition of ANG II to the peritubular solution at the lower concentration of 10(-10) M did not alter the rates of luminal alkalinization and acidification in the outer and inner CCD, respectively. Peritubular ANG II, 10(-7) M, but not vehicle, stimulated B cell apical HCO3- secretion occurring in response to peritubular Cl- removal. These studies demonstrate that ANG II acts through a basolateral AT1 receptor to stimulate outer CCD luminal alkalinization via, at least in part, B cell stimulation.

The aim of the present study was to investigate whether a pathway for conversion of angiotensin I (ANG I) to angiotensin II (ANG II) other than that via angiotensin-converting enzyme (ACE) is present in the smooth muscle of the human detrusor. Isolated detrusor strips from 11 patients were contracted by ANG I (1 microM) in the absence or presence of enalaprilat (10 microM), soybean trypsin inhibitor (STI, 200 micrograms/ml), or both. The metabolic activity in detrusor membranes from four patients was studied separately using Hip-Gly-Gly or ANG I as a substrate, with or without various protease inhibitors. The contractile response to ANG I (1 microM) was depressed by enalaprilat from 66 +/- 22 (mean +/- SD) to 39 +/- 13% of the K+ (124 mM)-induced response (P < 0.01, n = 11), and the combination of enalaprilat and STI resulted in a further reduction in contractile amplitude to 25 +/- 14% (P < 0.01 vs. K+, and P < 0.05 vs. enalaprilat alone) and a significantly slower developing contraction with a time to peak of 3.7 +/- 1.7 vs. 1.1 +/- 0.3 min for ANG I alone (P < 0.01). In detrusor membranes, a low ACE activity, inhibitable by captopril, was demonstrated by the formation of hippuric acid (0.70 nmol.min-1.mg protein-1) from the synthetic ACE substrate, Hip-Gly-Gly. However, the conversion of ANG I (166 nmol.min-1.mg protein-1) to ANG II was not affected by ACE inhibition, while serine protease inhibitors, e.g., STI and chymostatin, completely prevented ANG II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

The inhibitory effects of losartan (Dup 753), a nonpeptide angiotensin (ANG) II-type 1 receptor antagonist, on responses to ANG I, II, and III were investigated in the isolated perfused lung of the rat. Under conditions of constant pulmonary blood flow and left atrial pressure, injections of ANG I, II, and III into the pulmonary arterial perfusion circuit caused dose-related increases in pulmonary arterial pressure. Responses to ANG I, II, and III were reproducible with respect to time, and Dup 753, in a dose of 3 nM/ml injected into the perfusion circuit, decreased responses to the three peptides. Dup 753 had no significant effect on pressor responses to norepinephrine, serotonin, or BAY K 8644, and Dup 753 had no significant effect on mean baseline pulmonary arterial or airway pressure. Captopril and enalaprilat, ANG II-converting enzyme inhibitors, decreased the pressor response to ANG I while increasing the pressor response to ANG II and III. In addition, responses to ANG I, II, and III were similar when compared on a molar basis, suggesting the three peptides had similar pressor activity in the isolated perfused rat lung. Pressor responses to the angiotensin peptides were greater compared with responses to pressor agents that increase vascular resistance by various mechanisms, and Dup 753 did not significantly alter the pressor response to ventilatory hypoxia.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II (ANG II) is a stimulus for positive chronotropic and inotropic effects, protein synthesis, and hypertrophic growth in cardiac tissue. These short- and long-term effects of ANG II are mediated through specific plasma membrane receptors. Indirect evidence suggests that ANG II synthesized in the myocardium may be important in regulating cardiac function. The cell types in the myocardium that produce components of the renin-angiotensin system have not been determined. In this study, we evaluated whether cultured cardiomyocytes and fibroblasts obtained from ventricles of neonatal rat hearts were capable of synthesizing ANG I and II. Both cardiomyocytes and fibroblasts were found to have immunofluorescent staining for ANG I, ANG II, and angiotensin-converting enzyme (ACE). The amounts of ANG I and II in cell extracts and conditioned media obtained from cardiomyocytes and fibroblasts were quantified by radioimmunoassay. The amounts of ANG I and II detected in cardiomyocyte cultures (1.48 x 10(6) cells/dish) were 32.2 +/- 16.2 (n = 4) and 6.2 +/- 2.9 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II detected in the media conditioned by a 48-h exposure to cardiomyocytes were 5.2 +/- 1.2 (n = 3) and 2.1 +/- 1.2 (n = 3) ng/10(6) cells, respectively. The amounts of ANG I and II detected in fibroblast cultures (5.38 x 10(6) cells/dish) were 34.8 +/- 4.9 (n = 4) and 8.0 +/- 3.5 (n = 4) ng/10(6) cells, respectively. The amounts of ANG I and II obtained from media conditioned by a 48-h exposure to fibroblasts were 4.7 +/- 0.6 (n = 4) and 3.3 +/- 2.1 (n = 4) ng/10(6) cells, respectively. The identity of the radioimmunoassayable materials as ANG I and II peptides was confirmed in cardiomyocytes using an in vitro bioassay based on displacement of 125I-ANG II from receptor binding sites in cardiac membranes prepared from neonatal pig heart. Identification of ANG I and II and ACE in vitro in cultures of cardiac myocytes and fibroblasts supports the hypothesis that there is an intracardiac renin-angiotensin system that produces these peptides.

Angiotensin is generated within the kidney, but the precise loci for the formation of angiotensin I (ANG I) and angiotensin II (ANG II) have not been demonstrated. We performed electron microscopy immunocytochemistry in kidney sections of 10-day-old (newborn) and adult Wistar-Kyoto (WKY) rats using specific antibodies to renin, ANG I, ANG II, and angiotensinogen (AO). Renin, ANG I, ANG II, and AO were present in juxtaglomerular (JG) cells. Renin was largely confined to cytoplasmic granules; ANG I and ANG II were colocalized to these granules but also were present in the cytoplasm; AO was distributed throughout the cytoplasm. AO also was present in a renal cortical distribution in proximal tubular cells. Northern blot analysis demonstrated AO mRNA in total kidney and liver but not in renal microvessels. Using the reverse hemolytic plaque assay, we demonstrated release of ANG I and renin from individual renocortical cells of adult WKY rats. Under control conditions, the number of releasing cells was 11 +/- 1 for ANG I and 10 +/- 1 for renin. Addition of rat renin inhibitor (RI) (1 x 10(-5) M), which inhibited renin activity in the medium from 37 to 9 pg ANG I.ml-1.h-1, did not alter ANG I plaque number. Addition of rat AO increased ANG I plaque number to 17 +/- 2 (P less than 0.05). Incubation with both RI and AO prevented the increase in ANG I plaque number obtained with AO alone. Enalapril treatment (7 days; n = 5) increased the number of plaque-forming cells to 22 +/- 2 for ANG I (P less than 0.0005) and to 39 +/- 7 for renin (P less than 0.001). The results suggest an intracellular location for AO and angiotensin and release of renin and ANG I by renal cortical cells and suggest that released angiotensin is produced intracellularly and that secretion of ANG I is augmented by converting enzyme inhibition.

To quantify regional conversion of angiotensin (ANG) I to ANG II and its degradation to peptides other than ANG II, monoiodinated 125I-labeled ANG I was given to anesthetized pigs by constant infusion into the left cardiac ventricle. At steady state, blood samples were taken from the aorta and various regional veins. Distribution volume of ANG I appeared to be 24% of body weight. After angiotensin-converting enzyme (ACE) inhibitor treatment, fractional ANG I metabolism (fraction of arterially delivered ANG I that was metabolized during a single passage of blood) was 10% in the lungs (conversion 4%), compared with 56% in the combined systemic vascular beds (conversion 1%). Fractional ANG I metabolism during ACE inhibition was 93% in the kidney; 50-70% in myocardium, skeletal muscle, head, and skin; 21% in the left cardiac cavity; and 14% in the right cardiac cavity. Without ACE inhibition, fractional ANG I metabolism was 29% in the lungs (conversion 25%); 49% in the combined systemic vascular beds (conversion 10%); 38% in the left cardiac cavity (conversion 11%); and 14% in the right cardiac cavity (conversion 0%). It may thus be concluded that 1) extrapulmonary vascular beds make an important contribution to the conversion of circulating ANG I and 2) there is rapid extrapulmonary ANG I degradation that does not depend on ANG I-II conversion.

This study was designed to improve and validate methods for the accurate and consistent quantitation of angiotensin (ANG) I and II levels in rat kidney and to determine the effects on renal ANG I and II of changes in dietary sodium intake and ANG-converting enzyme (ACE) inhibition. Kidneys from pentobarbital-anesthetized rats were rapidly removed and homogenized in methanol before extraction and purification of ANG peptides by solid-phase extraction and high-performance liquid chromatography (HPLC). Recoveries of 125I-ANG I and II were greater than 80%. Reversed-phase HPLC of the partially purified methanol extract showed that greater than 75% of the ANG I- and greater than 82% of the ANG II-like immunoreactivity coeluted with ANG I and II, respectively. Dietary sodium deprivation (0.003 meq/g) and excess (1.34 meq/g) for 7 days significantly (P less than 0.01) increased and decreased renal ANG I (296 +/- 30 and 82.6 +/- 15.8 vs. 161 +/- 18 fmol/g) and ANG II (216 +/- 16 and 45.6 +/- 11.8 vs. 98 +/- 16 fmol/g) contents, respectively. Plasma ANG I and II levels showed similar changes. ACE activity was significantly upregulated by sodium deprivation in both kidney (44% increase) and plasma (30% increase). In rats fed normal chow, infusion of enalaprilat for 1 h abolished plasma ACE activity but decreased renal ACE activity by only 58%. ACE inhibition increased renal and plasma ANG I levels 2.8- and 12-fold, respectively, and decreased renal and plasma ANG II levels 75-78%.(ABSTRACT TRUNCATED AT 250 WORDS)

Cows that were normally hydrated or deprived of water were given intravenous or intracerebroventricular (icv) infusions of angiotensin I converting-enzyme inhibitors (CEI) or angiotensin II antagonists. Normally hydrated Na-deficient cows increased water intake in a dose-related manner in response to icv infusion of angiotensin I (n = 5). The response to 3 micrograms/h angiotensin I was abolished by concurrent icv infusion of the CEI captopril at 3 mg/h but not by intravenous infusion of captopril at 120 mg/h, which reduced Na appetite (n = 5). The icv infusion of captopril at 12 mg/h did not reduce the water intake of cows that were water restricted for 26.5 h (n = 4) or water restricted and Na deficient (n = 4). The icv infusion of the more lipophilic CEI ramipril at 3 mg/h (n = 7) did not reduce the water intake of normally hydrated or dehydrated cows but reduced the "need-free" intake of Na solution by dehydrated cows. The icv infusion of the nonpeptide antagonist Du Pont 753 at 3 mg/h (n = 7) reduced water intake in dehydrated cows. The results indicate that brain angiotensin may be involved in thirst in cattle. The data suggest that this brain angiotensin II may be formed by a pathway that does not include converting enzyme and that is sited inside the blood brain barrier, possibly in the median preoptic nucleus.

Extrarenal renin has been a subject of considerable interest. Chiefly, studies have focused on brain and vascular renin activity in large arteries. A method now exists for the isolation from the cerebrum of microvascular tissues consisting of arterioles, capillaries and venules. The present study has demonstrated reninlike enzymatic activity within the cerebral microvasculature of the rat that is distinct from plasma renin activity. Maximal activity was observed at pH 4.5 with no measurable activity at pH 7.4. Activity with homologous renin substrate was only 13% of that measured with hog substrate whereas a 400-fold increase in reninlike specific activity was observed when microvessel homogenates were incubated with synthetic tetradecapeptide renin substrate. Bilateral nephrectomy did not affect microvascular reninlike activity. Pepstatin 15 nM, abolished reninlike activity in microvessel homogenates. Mean specific microvessel reninlike activity was 1.15 +/- 0.20 pg angiotensin I . microgram protein-1 . h-1 in control animals. Neither sodium depletion nor DOC-saline administrations caused a significant change in microvascular reninlike activity. It is suggested that the reninlike activity observed in microvessels is an acid protease, perhaps cathepsin D derived from lysosomes.

The pulmonary vasculature has been implicated in the clearance of several vasoactive substances from the circulation including angiotensin I (AI). In view of the previously reported age-related differences in angiotensin-converting enzyme (ACE) activity of lung homogenates, it was of interest to examine the ability of intact perfused lungs to metabolize AI. Lungs from newborn and adult rats were perfused with Krebs bicarbonate buffer containing 1.0 ng/ml AI in a single-pass, nonrecirculating system. The rate of perfusion was normalized to lung mass. Removal of AI was determined from the transpulmonary difference in radioimmunoassayable AI. Lungs from 7-day-old rats removed a smaller fraction of AI from the circulation than did adult lungs. The age-related increase in AI clearance was accompanied by an increase in pulmonary ACE content; however, enzyme content alone could not account for the observed differences. The increased metabolism of AI by the pulmonary vasculature during development may contribute to the age-related increase in circulating angiotensin II concentrations.

The effects of [des-Asp1]angiotensin I and angiotensin III on mesenteric blood flow were compared in 15 pentobarbital-anesthetized dogs. These agonists were administered as bolus injections directly into the vasculature supplied by the superior mesenteric artery. Both [des-Asp1]angiotensin I and angiotensin III produced dose-dependent decreases in mesenteric blood flow, with angiotensin III being more potent than [des-Asp1]angiotensin I at all doses tested. The constrictor responses to [des-Asp1]angiotensin I were markedly attenuated in the presence of an angiotensin-converting enzyme inhibitor (SQ20881); SQ20881 did not alter responses to angiotensin III or norepinephrine. The administration of [Ile7]angiotensin III (an angiotensin III antagonist) attenuated the responses to both [des-Asp1]angiotensin I and angiotensin III, without altering the responses to norepinephrine. These results suggest that the decrease in mesenteric blood flow produced by [des-Asp1]angiotensin I is largely caused by its local enzymatic conversion to angiotensin III. This conversion in one transit through the mesenteric vasculature is approximately 24%.

The presence of a renin-angiotensin system was demonstrated in the poikilotherm Ambystoma tigrinum, commonly called the tiger salamander. Standard radioimmunoassay techniques were employed to measure the intrarenal renin activity (IRA) and the plasma renin activity (PRA) of A. tigrinum kept at either 5 or 20 degrees C. Basal IRA and PRA values were not affected by the temperature at which the animals were maintained. Intraperitoneal injection of the beta-adrenergic agonist isoproterenol, however, increased PRA only in those animals maintained at 20 degrees C. This is consistent with the hypothesis of a temperature sensitivity of the renal adrenergic system in vivo. In addition, we were able to demonstrate the existence of a contractile response of Ambystoma vascular smooth muscle to angiotensin II that was blocked by the competitive inhibitor saralasin.

Recent studies have suggested that the lung plays an important role in modifying concentrations of circulating vasoactive compounds. Isolated lungs of spontaneously hypertensive rats (SHR) were examined for their ability to modify concentrations of perfused norepinephrine, 5-hydroxytryptamine, or angiotensin I. Lungs from 4- or 14-wk-old SHR removed significantly more perfused 0.1 microM norepinephrine (NE) than lungs of normotensive controls (Wistar-Kyoto rats, WKY) of the same age. For example, lungs of 14-wk-old WKY removed 18.5 +/- 1.8% of perfused NE in a single pass through the pulmonary vasculature, whereas lungs from SHR removed 31.7 +/- 1.0%. Lungs of 14-wk-old SHR also produced more O-methylated NE metabolite than did lungs of WKY. Conversely, lungs of 14-wk-old SHR removed slightly less perfused 5-hydroxytryptamine and produced less 5-hydroxyindoleacetic acid metabolite than those of WKY. Monoamine oxidase activity, determined in 600 g supernatant fractions of lung homogenates, was the same in SHR and WKY. In addition, no difference in the ability of lungs of WKY and SHR to remove perfused angiotensin I was found either in animals 4 or 14 wk of age. These results suggest that lung may contribute to differences in concentrations of circulating biogenic amines observed in SHR and WKY.

The acceptability of radioimmunoassay to determine the levels of compounds antigenic to anti-angiotensin antibodies, in unprocessed methanolic blood extracts, was established for sheep blood. This approach was used to follow the clearance of antigenic compounds after administration of angiotensins I and II and fragments of angiotensin II in anesthetized sheep. The organs supplied by the systemic circulation and also the lungs effectively removed angiotensin I, but the removal of octapeptide occurred only in the peripheral tissues. The blood concentrations of compounds reacting with the anti-angiotensin II antibody always increased with passage of angiotensin I through the pulmonary circulation but not with passage of angiotensin II. The results indicate that factors other than efficiency of removal by the tissue is important in establishing blood levels. The sites of administration and of sampling were shown to be important in relation to ratios of the concentrations of antigenic material. There was a similar uptake of both hormones in the kidney; the relative inability of angiotensin I to reduce renal blood flow, therefore, does not result from a failure of uptake.

In small (0.5 mg/kg) subcutaneous doses, the angiotensin-converting enzyme inhibitor, captopril, greatly enhanced drinking in response to caval ligation in the rat. Drinking was not secondary to urinary water loss since the rats developed a substantial positive fluid balance. High (50 mg/kg) subcutaneous doses of captopril reduced drinking to a level below that following caval ligation alone. This effect could be mimicked by giving repeated intracerebroventricular injections of captopril (total amount 110 micrograms) to rats treated with the lower subcutaneous dose of captopril. With this combination, therefore, not only did the lower dose enhancement disappear, the basal caval ligation drinking response was also reduced with a total dose of captopril of less than 2% of the higher subcutaneous dose alone. These results show that, when conversion of angiotensin I to angiotensin II is prevented in the brain as well as systemically, drinking in response to caval ligation is reduced although not entirely prevented. The original report that such drinking is multifactorial, depending on angiotensin as well as nonangiotensin mechanisms, is confirmed.

Angiotensin II (ANG II) reversibly depolarizes renin-containing juxtaglomerular epithelioid cells (JGECs) of the hydronephrotic mouse kidney afferent arteriole. This depolarizing response was utilized to assess changes in ANG II concentration in the vicinity of JGECs in order to test whether ANG II is generated from ANG I and artificial renin substrate (ARS) in this preparation. Depolarizations were also produced by the application of ANG I and ARS in the superfusing medium. These responses to ANG I and ARS were completely blocked by saralasin. Hence, our findings are indicative for an intrarenal, local generation of ANG II. As opposed to saralasin, several converting enzyme and renin inhibitors only diminished but generally did not abolish the actions of ANG I and ARS, respectively. These results suggest an alternative, nonrenin and non-converting enzyme-dependent pathway of ANG II generation in renal tissue.

The effects of high plasma renin levels on plasma levels of both total immunoreactive angiotensinogen (direct radioimmunoassay) and intact angiotensinogen measured by angiotensin I released by renin (indirect assay) were studied in sodium-depleted rats both with and without captopril treatment and in adrenalectomized rats. The direct assay measures both intact angiotensinogen and des-angiotensin I-angiotensinogen, its residue cleaved by renin. The indirect assay measures only intact angiotensinogen. Neither sodium depletion, captopril treatment, nor adrenalectomy modified the circulating levels of total angiotensinogen. However these treatments produced a decrease in intact angiotensinogen that was in proportion to the elevation of renin levels. The difference between the two assays for angiotensin represents the level of des-angiotensin I-angiotensinogen and correlated satisfactorily with the plasma levels of renin. Identical correlations were observed in adrenalectomized rats and captopril-treated rats. We conclude that des-angiotensin I-angiotensinogen levels are an index of activation of the renin-angiotensin system dependent on the circulating level of renin.

The in vivo and in vitro rates of renin secretion were measured in kidneys from five groups of dogs in which renal perfusion pressure, salt diet, and neural input were varied to cause large changes in renin secretion rates and renal renin content. It was found that both the in vivo and in vitro secretory rates were proportional to the renal renin content. However, in vitro secretion rates were dependent on content up to 100 ng angiotensin I/mg tissue per h. At higher renin contents no increment in in vitro secretion rate was seen. In vivo secretion rate did not appear to reach a maximum until renal renin content was above 250 ng AI/mg tissue per h. The data are interpreted to support the hypothesis that there exist at least two pools of renin. One releases renin at a fractional rate of about 1.5% of the total content per hour. The other releases renin at a rate dependent on the magnitude of the stimuli acting on the kidneys. It is also suggested that the rate of renin synthesis may be a determinant of the basla rate of renin secretion.

Plasma clearance and tissue accumulation of 125I-angiotensin I, [Asp1, Ile5]ANG I, and [14C]sucrose, an inert volume reference, were measured after a bolus injection into the dorsal aorta of rainbow trout, Salmo gairdneri. Retention and metabolism of ANG I to angiotensin II (ANG II) and their constituent 1-4 peptide by the gill were examined using an isolated perfused arch preparation in which outflow from the respiratory and central filamental (venous) pathways was separated. Clearance of ANG I from plasma is multiexponential, reflecting dilution and tissue extraction. Liver, bile, gonads, corpuscles of Stannius, and white skeletal muscle accumulate more 125I than 14C; gill tissue accumulates less 125I than 14C. ANG I and II are retained by the perfused gill longer than the inert vascular marker sucrose, even though the distribution volumes of the former are less. The gill respiratory pathway converts ANG I to ANG II whereas the venous pathway metabolizes either ANG I or II to the 1-4 peptide and other metabolites. The gill respiratory pathway is in series with the systemic vasculature, has a large blood-cell contact area, and, like the mammalian lung, is ideally suited to activate ANG I. The gill venous pathway is in parallel with the systemic vasculature and removes ANG II from the circulation. During stress, elevated plasma catecholamines may reduce venous perfusion and thereby help maintain elevated circulating ANG II levels through reduced venous metabolism.

The purpose of this investigation was to correlate the development of the various enzyme activities associated with the renin-angiotensin system with age-related differences in the steady-state concentrations of angiotensin I (AI) and II (AII). Angiotensin was quantified by radioimmunoassay. Plasma renin activity and concentration increased between birth and 3 wk of age, and declined thereafter to adult values. Renal renin content, on the other hand, increased throughout the first 6 wk of postnatal life. The concentration of AII in plasma also increased following birth; however, maximum concentrations were not attained until 5 wk of age. In contrast, plasma AI did not increase between 3 and 6 wk of age. These data suggest that the steady-state concentration of AII in neonatal rat plasma may be partially limited by the low plasma renin substrate concentration. The increase in AII between 3 and 6 wk of age may reflect the increasing converting enzyme activity.

Intrarenal conversion of angiotensin I (ANG I) to angiotensin II (ANG II) under conditions of normal and reduced renal blood flow (RBF) elicited by constriction of the renal artery was examined in pentobarbital-anesthetized dogs. In eight animals, tracer doses of 125I-ANG I (5-12 pmol) were injected into the renal artery and 125I-ANG I, 125I-ANG II, and 125I-labeled metabolites were measured in renal venous effluent by high-voltage paper electrophoresis. The mean conversion of ANG I to ANG II during a single passage through the kidney was 21.8 +/- 2.1% at control RBF. When RBF was decreased by 25 and 53%, percent ANG I conversion was not altered significantly. In six dogs percent conversion of 125I-[Sar1, Ile5]ANG I, an ANG I analogue refractory to hydrolysis by aminopeptidases, was 18.1 +/- 1.7% at control RBF and did not change significantly when the RBF was reduced by 55%. Although there were severalfold increases in renal renin secretion rate and net ANG I generation rate during reduced RBF, net renal ANG II formation rate did not change significantly. These data indicate that there is substantial conversion of ANG I in a single passage through the dog kidney and that intrarenal ANG I conversion is independent of RBF even under conditions in which renin secretion rate and ANG I generation rate are increased severalfold.