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.

Angiotensin II signaling promotes follicle growth and dominance in cattle.

It is generally understood that angiotensin II (AngII) promotes follicle atresia in rats, although recent data suggested that this may not be true in cattle. In this study, we aimed to determine in vivo whether AngII alters follicle development in cattle, using intrafollicular injection of AngII or antagonist into the growing dominant follicle or the second largest subordinate follicle. Injection of saralasin, an AngII antagonist, into the growing dominant follicle inhibited follicular growth, and this inhibitory effect was overcome by systemic FSH supplementation. Injection of AngII into the dominant follicle did not affect follicular growth, whereas injection of AngII into the second largest follicle prevented the expected atresia of this subordinate follicle, and the treated follicle grew at the same rate as the dominant follicle for the next 24 h. Inhibition of AngII action in the dominant follicle decreased estradiol concentrations in follicular fluid and the abundance of mRNA encoding aromatase, 3ß-hydroxysteroid dehydrogenase, LH receptor, and cyclinD2 in granulosa cells, with minimal effects on theca cells. The effect of AngII on aromatase mRNA levels was confirmed using an in vitro granulosa cell culture system. In conclusion, these data suggest that AngII signaling promotes follicle growth in cattle and does so by regulating genes involved in estradiol secretion and granulosa cell proliferation and differentiation.

[Identification of metabolic pathways of brain angiotensin II and angiotensin III: predominant role of angiotensin III in the control of vasopressin secretion]. / Identification des voies métaboliques de l'angiotensine II et de l'angiotensine III cérébrales: rôle prédominant de l'angiotensine III dans le contrôle de la sécrétion de vasopressine.

Angiotensin (Ang) II and AngIII 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 AngII and AngIII, respectively. Mice received [3H]AngII intracerebroventricularly (i.c.v.) in the presence or absence of the APA inhibitor, EC33 ((S)-3-amino-4-mercapto-butylsulfonate de sodium) or the APN inhibitor, EC27 ((S)-2-amino-pentan-1,5-dithiol). [3H]AngII and [3H]AngIII levels were evaluated from hypothalamus homogenates by HPLC. EC33 increased the half-life of [3H]AngII 2.6-fold and completely blocked the formation of [3H]AngIII, whereas EC27 increased the half-life of [3H]AngIII 2.3-fold. In addition, the effects of EC33 and EC27 on Ang- induced vasopressin release were studied in mice. AngII 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 AngII, EC33 inhibited AngII-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) AngII. These results demonstrate for the first time that i) APA and APN are involved in vivo in the metabolism of brain AngII and AngIII, respectively, and that ii) the action of AngII on vasopressin release depends upon the prior conversion of AngII to AngIII. This shows that AngIII behaves as one of the main effector peptides of the brain renin-angiotensin system in the control of vasopressin release.

Angiotensin II induces plasminogen activator inhibitor-1 and -2 expression in vascular endothelial and smooth muscle cells.

Angiotensin II (AII)- and Arg8-vasopressin (AVP)-regulated gene expression in vascular cells has been reported to contribute to vascular homeostasis and hypertrophy. In this report, AVP-induced expression of plasminogen activator inhibitor (PAI)-2 mRNA in rat microvessel endothelial (RME) cells was identified using differential mRNA display. Further characterization of vasoactive peptide effects on PAI expression revealed that AII stimulated a 44.8 +/- 25.2-fold and a 12.4 +/- 3.2-fold increase in PAI-2 mRNA in RME cells and rat aortic smooth muscle cells (RASMC), respectively. AII also stimulated a 10- and 48-fold increase in PAI-1 mRNA in RME cells and RASMC, respectively. These AII effects were inhibited by either Sar1, Ile8-angiotensin or the AT1 antagonist DuP 735, but were not significantly altered in the presence of the AT2 antagonist PD123319. AII stimulation of RASMC and RME cells also significantly increased both PAI-1 protein and PAI activity released to the culture medium. Inhibition of protein kinase C completely blocked PMA-stimulated induction of PAI-2 mRNA in both cell types and inhibited the AII-stimulated increase in RASMC by 98.6 +/- 2.8%. In contrast, protein kinase C inhibition only partially decreased the AII-stimulated PAI-2 expression in RME cells by 68.8 +/- 11.1%, suggesting that a protein kinase C-independent mechanism contributes to a 6.9 +/- 1.5-fold AII induction of PAI-2 expression in endothelial cells. AII and PMA also stimulated protein tyrosine phosphorylation in RME cells, and the tyrosine kinase inhibitor genistein partially blocked their induction of PAI-2 mRNA. These findings suggest that AII may regulate plasminogen activation in the vasculature by inducing both PAI-1 and PAI-2 expression.

Synthetic cDNA encoding the rat AT1a receptor: a useful tool for structure-function relationship analysis.

To carry out systematic structure-function studies of the rat angiotensin II receptors by site directed mutagenesis, or production of chimeric receptors, we have produced a synthetic cDNA coding for the AT1a receptor. The synthetic cDNA is 1101 base pairs long, and contains 49 unique restriction sites that are on the average 23 base pairs apart, allowing replacement of specific restriction fragments by synthetic counterparts containing the desired modified sequence. The total cDNA was assembled in the expression vector pECE. After stable expression in Chinese Hamster Ovary cells, the protein encoded by this synthetic cDNA presents a pharmacological profile and a signal transduction mechanism indistinguishable from the wild type rat AT1a receptor.

Angiotensin II is retained in gonadotrophs of pituitary cell aggregates cultured in serum-free medium but does not mimic the effects of exogenous angiotensins and luteinizing-hormone-releasing hormone on growth hormone release.

Angiotensin II (AII)-like immunoreactivity (LIR) was detected by immunostaining in 7.5 +/- 1.1% of cells obtained by redispersion of pituitary cell aggregates from 15- to 20-day-old female rats, cultured for 5-7 days in serum-free medium supplemented with thyroid hormone and dexamethasone. Also, renin-LIR was retained in these cultures. As shown by double immunostaining of paraffin-embedded sections of the aggregates, this AII-LIR was localized only in gonadotrophs. AII-LIR was detected at least up to 5 weeks in culture. On reversed-phase, high-performance liquid chromatography (HPLC), this AII-LIR co-migrated with authentic AII. In perifused aggregate cell cultures of 15- to 20-day-old female rat pituitary maintained in serum-free medium supplemented with dexamethasone (DEX) and triiodothyronine (T3), AII stimulated GH release. AI and AIII had a similar effect. To evaluate the possible involvement of endogenous AII in the local regulation of GH release, gonadotrophs were stimulated with luteinizing hormone-releasing hormone (LHRH). LHRH displayed a transient inhibitory effect on GH release, which was followed by a rebound of GH release after withdrawal of the peptide. Treatment of aggregates with pertussis toxin reversed this inhibitory effect into a significant stimulation of GH release. In aggregates cultured in serum-supplemented medium, LHRH provoked a significant stimulation of GH release which was still followed by a post-stimulus rebound release. In hemipituitaries from 5-day-old rats, a significant stimulatory effect of LHRH on GH release was found without rebound secretion. To evaluate the possible involvement of endogenous AII in the effects of LHRH on GH release, the influence of (Sar1,Ala8)AII, a peptide AII receptor antagonist, and of DUP753, a non-peptide AII receptor blocker was tested in various in vitro conditions. The effect of LHRH on GH release in aggregates cultured either in serum-free medium supplemented with DEX and T3 or in serum-supplemented medium was not affected by (Sar1,Ala8)AII, not even after enhancing the LHRH-induced GH release by treatment of the aggregates with pertussis toxin. A hundred times lower concentration of (Sar1,Ala8)AII, however, abolished the AII-induced changes in GH release. Also DUP753 (10 microM) failed to block LHRH-induced GH release in aggregates. (Sar1,Ala8)AII also failed to block the effect of LHRH on GH release from hemipituitaries. It is concluded that LHRH has inhibitory and stimulatory effects on GH release in cultured pituitary cell aggregates.(ABSTRACT TRUNCATED AT 400 WORDS)

Stimulation of prolactin secretion from rat pituitary by luteinizing hormone-releasing hormone: evidence against mediation by angiotensin II acting through a (Sar1-Ala8)-angiotensin II-sensitive receptor.

In aggregate cell cultures of 15- to 20-day-old rat pituitary maintained in serum-free medium, luteinizing hormone-releasing hormone (LHRH) (10 nM) stimulated prolactin (PRL) release, confirming our previous results and those of others with serum-supplemented medium. Since angiotensin II (AII) stimulates PRL release and a renin-angiotensin system is expressed in gonadotrophs, LHRH stimulation of PRL release might be mediated by AII. To evaluate this hypothesis, the influence of (Sar1,Ala8)AII and (Sar1,Ile8)AII two peptide AII receptor antagonists, of DUP753, a nonpeptide and stable AII receptor antagonist, of a converting enzyme inhibitor, and of angiotensinogen on LHRH-induced PRL release was tested in various in vitro conditions of 15- to 20-day-old female rat pituitary. In aggregates maintained in serum-free medium with or without dexamethasone (DEX) and triiodothyronine (T3), or maintained in serum-supplemented medium, the effect of LHRH on PRL release was not affected by (Sar1Ala8)AII (0.1 microM), (Sar1,Ile8)AII (10 microM) or DUP753 (10 microM). Only a high dose (10 microM) of (Sar1,Ala8)AII attenuated the LHRH-induced PRL release. The latter attenuation was seen only with aggregates cultured in the DEX/T3 medium and not with aggregates cultured in the presence of serum. A dose of 1 or 10 nM (Sar1,Ala8)AII also failed to block the effect of LHRH used at 1 nM. In contrast, (Sar1,Ala8)AII dose dependently as well as DUP753 (10 microM) abolished the AII-induced PRL release. (Sar1,Ala8)AII also failed to affect the LHRH-induced PRL release in pituitary cell aggregates from 6-week-old male rats. However, in aggregates from both immature and 6-week-old rats, (Sar1,Ala8)AII provoked a small and statistically significant attenuation of the LHRH-induced PRL release when a 100 nM dose of LHRH was used. In freshly isolated hemipituitaries from 5-day-old rats, (Sar1,Ala8)AII (1 or 10 microM) did not affect the LHRH- (10 nM) induced PRL release. In single cells obtained by redispersion of aggregates and mounted in a Biogel P2 column, LHRH still stimulated PRL release. Again this effect could not be blocked by DUP753. Treatment of aggregate cell cultures with the angiotensin-converting enzyme inhibitor captopril or with angiotensinogen did not alter the LHRH-induced PRL release. It is concluded that AII is not the paracrine factor mediating the effect of LHRH at low nanomolar doses on PRL release, at least not through the classical AII receptor. The involvement of AII acting on a non-(Sar1,Ala8)AII-sensitive receptor cannot be excluded and warrants further investigation.

Distribution of angiotensin II receptor subtypes in rat brain nuclei.

Angiotensin II (ANG II) receptor subtypes in rat brain were characterized and quantified by competitive radioligand binding using [125I]Sar1 Ile8 angiotensin II ([125I]sarilesin) as a tracer and ANG II, sarilesin and the subtype selective ligands DuP 753 (AT1) and CGP 42112A (AT2) as competitors. The distribution of AT1 and AT2 receptors was determined in midbrain, brainstem, hypothalamus as well as in individual hypothalamic and periventricular nuclei. Whereas in midbrain and brainstem the AT1: AT2 ratio was 40%: 60% and 70%: 30% respectively, the AT1 receptors were by far predominant in hypothalamus and in the nuclei investigated. Interestingly, we found that approximately 25% of the ANG II receptors in hypothalamus did not bind DuP 753 even at 0.1 mM. These sites which bind CGP 42112A, ANG II and sarilesin may represent a third ANG II receptor subtype.

Angiotensin II increases the corticotropin-releasing factor messenger ribonucleic acid level in the rat hypothalamus.

Angiotensin II (AII) has an important role in the regulation of CRF release. In the present study, the effect of centrally administered AII on CRF messenger RNA (mRNA) levels in the rat hypothalamus was examined. Administration of 0.1 nmol and 1 nmol AII into the lateral ventricle increased the levels of plasma ACTH 20 min and 45 min after administration and those of proopiomelanocortin mRNA in the anterior pituitary (AP) and CRF mRNA in the hypothalamus 2 h after administration. On the other hand, ACTH levels in AP and CRF levels in the median eminence temporarily decreased 45 min after the administration of 1 nmol AII, but it returned to the control level at 90 min. Administration of 10 nmol saralacin, an AII antagonist, blocked 1 nmol AII-induced increase in the levels of plasma ACTH, proopiomelanocortin mRNA in AP, and CRF mRNA in the hypothalamus. These results indicate that central administration of AII increases the CRF mRNA level in the hypothalamus in a receptor-specific manner and also increases CRF release. Therefore, AII seems to have an important role in the regulation of the release and synthesis of CRF in the hypothalamus.

[Angiotensin mechanism of auriculo-acupuncture dental analgesia]. / Angiotenzinovyi mekhanizm aurikulo-akupunkturnoi dental'noi analgezii.

In unanaesthetized rabbits auriculo-acupuncture electrostimulation with frequency of 15 Hz decreased the amplitude of somatosensory EP second component in response to the tooth pulp electrostimulation which was blocked by intravenous injection of naloxone but not by intraventricular injection of saralasin. The same effect of auriculo-acupuncture electrostimulation with frequency 100 Hz was blocked by saralasin, was increased by angiotensin II, was diminished by methysergide but wasn't changed by naloxone. It's suggested that there is angiotensinergic antinociceptive mechanism of dental pain which is activated by auriculo-acupuncture electrostimulation with frequency 100 Hz.

[Angiotensin II in the mechanisms of hypothalamic pressor reactions]. / Angiotenzin II v mekhanizmakh pressornykh gipotalamicheskikh reaktsii.

The influence of intracerebroventricular injections of the angiotensin II antagonist--saralasin on the cardiovascular reactions elicited by electrical stimulation of different structures of the hypothalamus in rabbit was studied. The saralasin in doses from 1 to 1.5 mg reduced arterial pressure by 9 +/- 0.2 mm Hg and decreased the amplitude of the hypertensive reactions elicited by electrical stimulation of the paraventricular, supraoptic, ventromedial, supramammillary nucleus of the hypothalamus, area hypothalamic anterior and lateral. It has been shown that most suppressing influence of the saralasin on amplitude of the hypertensive reactions elicited by electrical stimulation of the paraventricular (68%) and supraoptic (76%) nucleus of the hypothalamus which contain magnocellular neurosecretory neurons.

[Effect of angiotensin II and naloxone on the nociceptive sensitivity of rabbits undergoing electrostimulation of the skin and dental pulp]. / Vliianie angiotenzina II i naloksona na notsitseptivnuiu chuvstvitel'nost' krolikov pri élektrostimuliatsii kozhi i pul'py zuba.

In unanesthetized rabbits intraventricular and intravenous administration of angiotensin II resulted in a decrease of the somatosensory evoked potential amplitude in response to nociceptive electrodental stimulation but not nociceptive electrocutaneous stimulation. Saralasin administered intraventricularly abolished the effect of angiotensin II. Naloxone injected by the same route increased the evoked potential amplitude in response to electrodental but not to electrocutaneous stimulation and also reversed the analgesic effect of angiotensin II. The selectivity of the antinociceptive effect of angiotensin II is probably due to the presence of different specific peptide pain mechanisms of varying origin.

Altered angiotensin II sensitivity of neurons in the organum vasculosum lamina terminalis region of the spontaneously hypertensive rat.

Using in vitro hypothalamic brain slices, differences in angiotensin II (AII) sensitivity of neurons in the organum vasculosum lamina terminalis (OVLT) region were compared between spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar-Kyoto rats (WKY). AII, the AII competitive antagonist saralasin, and L-glutamate were micropressure-applied onto OVLT neurons. AII excitation of SHR neurons was blocked or antagonized by simultaneous application of saralasin, evoked at significantly lower thresholds and displayed exaggerated periods of postactivity compared to OVLT neurons in preparations taken from WKY controls. Neuronal responses to L-glutamate were similar between the two rat strains. Differences in neuronal sensitivity to AII may be causally linked to hypertension in SHR.

Comparative neuroanatomy of angiotensin II receptor localization in the mammalian hypothalamus.

1. The distribution of angiotensin II (AII) receptor binding sites in the hypothalamus of rat, rabbit, sheep and human was determined by in vitro autoradiography using 125I-[Sar1,Ile8]-AII as radioligand. 2. High receptor binding levels were observed in the continuum of tissue comprising the anterior wall of the third ventricle, including the subfornical organ, the median pre-optic nucleus and the organum vasculosum of the lamina terminalis. 3. High levels of binding sites were also found in the paraventricular and supra-optic nuclei, the median eminence and the arcuate nucleus. 4. These findings demonstrate sites in the hypothalamus of rat, rabbit, sheep and human where AII could exert its known actions on fluid and electrolyte balance, pituitary hormone release and cardiovascular function.

Naloxone reversal of hemorrhagic hypotension in the conscious guinea-pig is impeded by inhibition of the renin-angiotensin II system.

Naloxone reverses hemorrhagic hypotension in the conscious guinea-pig. Captopril and saralasin impede this naloxone effect, suggesting that angiotensin II is involved in naloxone action. This is compatible with previous work which has shown that B-endorphin inhibits the centrally mediated pressor action of angiotensin II, and that naloxone blocks this effect. Naloxone may be interacting with the postulated brain renin-angiotension II system or may be blocking the action of shock-induced circulating angiotensin II on a centrally located area such as the hypothalamus.

Antihypertensive and aldosterone-lowering effects of synthetic atrial natriuretic factor in renin-dependent renovascular hypertension.

A 24-amino acid residue synthetic atrial natriuretic factor (ANF) antagonizes angiotensin II-induced vascular contractility and aldosterone production in isolated blood vessels and adrenal cells, respectively. To determine the significance of these effects in vivo, the blood pressure and aldosterone responses to synthetic ANF were evaluated in rats with two-kidney, one clip hypertension (n = 5) and in sham-operated controls (n = 4). In the latter, ANF caused a slight fall in mean blood pressure (-7 +/- 3%) and inconsistent changes in plasma renin and aldosterone. In hypertensive rats, ANF decreased blood pressure by 31 +/- 7 mmHg (17 +/- 3%), comparable to the effect of the angiotensin antagonist saralasin (31 +/- 4 mmHg). Plasma renin activity increased from 48 +/- 15 to 79 +/- 23 ng/ml/h. Despite this, ANF caused marked suppression of plasma aldosterone (from 97 +/- 28 to 20 +/- 8.9 ng/100 ml). These results show that ANF can exert potent antihypertensive and aldosterone-lowering effects in vivo, at least when the renin-angiotensin system is stimulated.

Central nervous system action of angiotensin during onset of renal hypertension in awake rats.

The increase in arterial pressure and vascular resistance during acute unilateral renal artery stenosis (RSt) in conscious rats is, in part, dependent on elevated neurogenic vascular tone produced by an indirect neural interaction of angiotensin II (ANG II) with the sympathetic nervous system. The present experiments examined whether this interaction occurs within the central nervous system. Conscious rats that had been chronically instrumented with miniaturized Doppler flow probes for measurement of regional vascular resistance were subjected to a 50% reduction in unilateral renal flow with an implanted pneumatic occluder. Arterial pressure increased by 35% after 60 min of RSt. In animals in which the pressor response to intracerebroventricular (icv) ANG II had been eliminated by prior surgical interruption of the "ANG II pressor pathway" in the anterior hypothalamus, the increase in blood pressure following RSt was attenuated by 44% (P less than 0.01). In a second series, a central action of ANG II during acute renal hypertension (RH) was assessed by central ANG II receptor blockade with icv saralasin. Unlike normotensive controls, acutely RH animals responded to saralasin with significant (P less than 0.01) decreases in arterial pressure (-32%) and hindquarters (-26%) and contralateral renal (-9%) resistance. These changes were accentuated (-57% decrease in pressure) in animals made areflexic by prior sinoaortic baroreceptor denervation. Thus activation of the sympathetic nervous system during the early high-renin phase of RH depends significantly on a central action of ANG II. This mechanism may account for some 40-50% of the pressure increase following acute RSt.

Influence of uranyl nitrate upon tubular reabsorption and glomerular filtration in blood perfused isolated dog kidneys.

The early changes in tubular reabsorption, glomerular filtration, blood flow and sodium excretion brought about by uranyl nitrate were investigated in isolated, blood-perfused dog kidneys during water diuresis. No significant changes in urine volume were observed; the decrease in fluid reabsorption was counterbalanced quantitatively by a reduction in glomerular filtration rate; only a small diminution of renal blood flow was found. The balance between reabsorption and filtration was observed as well when angiotensin action or prostaglandin synthesis were inhibited. The intrarenal venous pressure rose, suggesting that an increase in proximal intratubular hydrostatic pressure caused the decrease in filtration. Tubular back-leak of fluid, or back-diffusion, induced by the toxin, were excluded. The presence of natriuretic compounds in the urine was confirmed.

Angiotensin II facilitates the potassium-evoked release of 3H-noradrenaline from the rabbit hypothalamus.

Antiotensin II facilitated in a concentration-dependent manner the potassium-evoked 3H-noradrenaline over-flow from the rabbit hypothalamus. This effect which is probably mediated through presynaptic angiotensin facilitatory receptors on noradrenergic nerve terminals was blocked by the specific angiotensin receptor antagonist, saralasin. These results demonstrate that angiotensin II also facilitates the stimulation-evoked release of noradrenaline in the central nervous system.