Saccharomyces boulardii for Clostridium difficile-associated enteropathies in infants

Angiotensin II has been shown to act prejunctionally to facilitate sympathetic neutrotransmission in various tissues including the iris-ciliary body. In the present study, we characterized the prejunctional angiotensin II receptor subtype and its signal transduction pathway in the rabbit iris-ciliary body. Angiotensin II caused concentration-dependent facilitation of electrically evoked [3H]-norepinephrine overflow from the isolated, superfused rabbit iris-ciliary body without affecting basal tritium efflux. Responses to angiotensin II were antagonized by saralasin and DuP753 but not by PD123177 indicating that prejunctional angiotensin II receptors of the AT1-subtype mediate the facilitation of evoked [3H]-norepinephrine release. The non-selective cyclic nucleotide phosphodiesterase inhibitor, isobutylmethyl xanthine enhanced the angiotensin II response whereas the cAMP-specific phosphodiesterase inhibitor, RO-20-1724 had no effect. In the presence of 8-bromo-cGMP, responses elicited by angiotensin II were significantly (P < 0.01) greater than that caused in the absence of 8-bromo-cGMP. In contrast, 8-bromo-cAMP had no effect on the angiotensin II-induced response. Guanylate cyclase inhibitors, methylene blue and LY83583 abolished angiotensin II-induced enhancement of [3H]-norepinephrine overflow without affecting basal tritium efflux. Taken together, these results suggest that cGMP could be involved in the angiotensin II response. Neither phospholipase C inhibitors (neomycin, 2-nitro-4-carboxyphenyl-N,N-diphenyl carbamate and phenylmethylsulfonyl fluoride) nor an inhibitor of protein kinase C (staurosporine) had any significant effect on the angiotensin II response, indicating that metabolites of inositol phospholipid metabolism or activation of protein kinase C are not involved in the response to this peptide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pulsed dexamethasone therapy on tolerance of intravenously administered lipids in extremely low birth weight infants

The effects of angiotensin II (AngII), the AngII analogues saralasin--[Sar1, Ala8]AngII, sarmesin--[Sar1Tyr(Me)4]AngII, the nonpeptide AngII receptor antagonists DuP753 (losartan) (for AT1 receptor subtype) and PD123319 (for AT2 receptor subtype), as well as combinations of AngII and each of its analogues and receptor antagonists, administered intracerebroventricularly (ICV), were studied on mice using the acetic acid-induced abdominal constrictions test (acetic acid 1% intraperitoneally, IP). The abdominal constrictions were counted at 5-min intervals for 30 min. AngII at doses of 0.05, 0.1, and 1 microg exerted a dose-dependent antinociceptive effect. Saralasin, sarmesin, losartan, and PD123319 exhibited a dose-dependent effect on nociception: they either increased or decreased it. PD123319 antagonized the antinociceptive effect of AnglI while losartan was ineffective. The importance of AT2 receptor subtype for the nociception reducing effect of AngII is considered.     

Role of extracellular signal-regulated kinases in angiotensin II-stimulated contraction of smooth muscle cells from human resistance arteries

BACKGROUND: We assessed the role of extracellular signal-regulated kinases (ERKs) in Ang II-stimulated contraction and associated signaling pathways in vascular smooth muscle cells (VSMCs) from human small arteries. METHODS AND RESULTS: VSMCs derived from resistance arteries (<300 microm in diameter) from subcutaneous gluteal biopsies of healthy subjects (n=8) were used to assess Ang II-stimulated [Ca2+]i, pHi, and contractile responses. [Ca2+]i and pHi were measured with fura 2-AM and BCECF-AM, respectively, and contraction was measured photomicroscopically in cells grown on Matrigel matrix. To determine whether tyrosine kinases and ERKs influence Ang II-stimulated responses, cells were pretreated with 10(-5) mol/L tyrphostin A-23 (tyrosine kinase inhibitor) and PD98059 (MEK inhibitor). Ang II-stimulated MEK activity was determined by tyrosine phosphorylation of ERKs. The angiotensin receptor subtypes (AT1 and AT2) were assessed with [Sar1,Ile8]Ang II (a nonselective subtype antagonist), losartan (a selective AT1 antagonist), and PD123319 (a selective AT2 antagonist). Ang II dose-dependently increased [Ca2+]i (pD2=8.4+/-0.36, Emax=541+/-55 nmol/L), pHi (pD2=9. 4+/-0.29, Emax=7.19+/-0.01), and contraction (pD2=9.2+/-0.21, Emax=36+/-2.2%). Ang II induced rapid tyrosine phosphorylation of ERKs, which was inhibited by PD98059. Tyrphostin A-23 and PD98059 attenuated (P<0.05) Ang II-stimulated second messengers, and PD98059 reduced Ang II-induced contraction by >50%. [Sar1,Ile8]Ang II and losartan, but not PD123319, blocked Ang II-stimulated responses. CONCLUSIONS: These data demonstrate that in VSMCs from human peripheral resistance arteries, functional Ang II receptors of the AT1 subtype are coupled to signaling cascades involving Ca2+ and pHi pathways that are partially dependent on tyrosine kinases and ERKs. ERKs, the signaling cascades characteristically associated with cell growth, may play an important role in Ang II-stimulated contraction of human VSMCs.     

Localization of angiotensin AT1 receptors in the rat heart conduction system

We characterized angiotensin II receptor subtypes in the conduction system of the rat heart using quantitative autoradiography. In both the sinoatrial and atrioventricular nodes, binding could be totally displaced by losartan, and was insensitive to PD 123177, indicating that angiotensin II receptors in the conduction system of the rat heart belong to the AT1 subtype. Angiotensin AT1 receptors could play a direct role in the regulation of the heart chronotropic properties.     

Vasodepressor actions of angiotensin-(1-7) unmasked during combined treatment with lisinopril and losartan

Blockade of angiotensin II (Ang II) function during 8 days of oral therapy with lisinopril (20 mg/kg) and losartan (10 mg/kg) normalized the arterial pressure (112+/-3/70+/-3 mm Hg) and raised the plasma concentrations of the vasodilator peptide angiotensin-(1-7) [Ang-(1-7)] of 21 male spontaneously hypertensive rats (SHR). Treated animals were then given a 15-minute infusion of either mouse immunoglobulin G1 or a specific monoclonal Ang-(1-7) antibody while their blood pressure and heart rate were recorded continuously in the awake state. The concentrations of Ang II and Ang-(1-7) in arterial blood were determined by radioimmunoassay. Infusion of the Ang-(1-7) antibody caused significant elevations in mean arterial pressure that were sustained for the duration of the infusion and were accompanied by transient bradycardia. Although the hemodynamic effects produced by infusion of the Ang-(1-7) antibody had no effect on plasma levels of Ang II, they caused a twofold rise in the plasma concentrations of Ang-(1-7). A pressor response of similar magnitude and characteristics was obtained in a separate group of SHR treated with the combination of lisinopril and losartan for 8 days during an infusion of [Sar1-Thr8]Ang II. The pressor response induced by the administration of this competitive, non-subtype-selective Ang II receptor blocker was not modified by pretreatment of the rats with an angiotensin type-2 (AT2) receptor blocker (PD123319). Plasma concentrations of Ang II and Ang-(1-7) were not changed by the administration of [Sar1-Thr8]Ang II either in the absence or in the presence of PD123319 pretreatment. These results are the first to indicate an important contribution of Ang-(1-7) in mediating the vasodilator effects caused by combined inhibition of angiotensin-converting enzyme and AT1 receptors. The comparable results obtained by administration of [Sar1-Thr8]Ang II suggest that the vasodepressor effects of Ang-(1-7) during the combined treatment is modulated by a non-AT1/AT2 angiotensin subtype receptor.     

AT2-antagonist sensitive potentiation of angiotensin II-induced vasoconstrictions by blockade of nitric oxide synthesis in rat renal vasculature

1. Although the actions of angiotensin II (Ang II) on renal haemodynamics appear to be mediated by activation of the AT1 receptor subtype, AT2 binding sites have also been evidenced in the adult kidney vasculature. As NO is known to mask part of the renal effects of vasoconstrictor drugs, we queried whether the Ang II-induced vasoconstrictions could occur via multiple receptor subtypes during inhibition of NO synthesis. We explored the effect of AT1 and AT2 receptor (AT-R) antagonists on Ang II-induced pressure increases during NO synthase or soluble guanylyl cyclase inhibition in rat isolated kidneys perfused in the presence of indomethacin at constant flow in a single-pass circuit. 2. In the absence of NO blockade, the AT1-R antagonist L-158809 (500 nM) antagonized the Ang II-induced vasoconstrictions, while the AT2-R antagonist PD-123319 (500 nM) had no effect. 3. Perfusing kidneys in the presence of either NO synthase inhibitors, L-NAME (100 microM) or L-NOARG (1 mM), or soluble guanylyl cyclase inhibitor, LY-83583 (10 microM), significantly increased both molar pD2 (from 9.40+/-0.25 to 10.36+/-0.11) and Emax values (from 24.9+/-3.1 to 79.9+/-4.9 mmHg) of the concentration-response curve for Ang II-induced vasoconstriction. 4. In the presence of L-NAME, 500 nM L158809 abolished the Ang II-induced vasoconstrictions whatever the concentration tested. On the other hand, 500 nM PD-123319 reversed the left shift of the concentration-response curve for Ang II (molar pD2 value 9.72+/-0.13) leaving Emax value unaffected (91.3+/-7.6 mmHg). 5. In the presence of L-NAME, the potentiated vasoconstriction induced by 0.1 nM and the augmented vasoconstriction induced by 10 nM Ang II were fully inhibited in a concentration-dependent manner by L-158809 (0.05-500 nM). By contrast, PD-123319 (0.5-500 nM) did not affect the 10 nM Ang II-induced vasoconstriction and concentration-dependently decreased the 0.1 nM Ang II-induced vasoconstriction plateauing at 65% inhibition above 5 nM antagonist. 6. Similar to PD-123319, during NO blockade the AT2-R antagonist CGP-42112A at 5 nM decreased by 50% the 0.1 nM Ang II-induced vasoconstriction and at 500 nM had no effect on 10 nM Ang II-induced vasoconstriction. 7. In conclusion, the renal Ang II-induced vasoconstriction, which is antagonized only by AT1-R antagonist in the presence of endogenous NO, becomes sensitive to both AT1- and AT2-R antagonists during NO synthesis inhibition. While AT1-R antagonist inhibited both L-NAME-potentiated and -augmented components of Ang II-induced vasoconstriction, AT2-R antagonists inhibited only the L-NAME-potentiated component.     

bcl-2 expression in the spectrum of preinvasive breast lesions

This study was designed to define more precisely the relationship between specific angiotensin receptors and the growth of vascular smooth muscle cells in response to angiotensin II. These experiments employed quiescent A10 cells which were characterized as smooth muscle by the expression of specific contractlle proteins. Cell growth was monitored by measuring the incorporation of metabolic precursors into RNA or DNA. The treatment of A10 cells with angiotensin II (1 microM) stimulated a hypertrophic response as indicated by an increase in RNA synthesis and protooncogene expression in the absence of DNA synthesis. This increase in RNA synthesis could be blocked by PD123319, an AT2 antagonist, but not by losartan, an AT1 antagonist. RT-PCR analysis demonstrated that quiescent A10 cells express only the AT2 receptor while proliferating A10 cells express the AT1a and AT1b receptors in addition to the AT2 receptor. In addition, induction of AT2 receptor-mediated RNA synthesis was prevented by indomethacin, a cyclooxygenase inhibitor. These studies therefore support a direct connection between the AT2 receptor and smooth muscle growth that is mediated, in part, by prostaglandin synthesis.     

The hyperglycemia induced by angiotensin II in rats is mediated by AT1 receptors

We have shown that the renin-angiotensin system (RAS) is involved in glucose homeostasis during acute hemorrhage. Since almost all of the physiological actions described for angiotensin II were mediated by AT1 receptors, the present experiments were designed to determine the participation of AT1 receptors in the hyperglycemic action of angiotensin II in freely moving rats. The animals were divided into two experimental groups: 1) animals submitted to intravenous administration of angiotensin II (0.96 nmol/100 g body weight) which caused a rapid increase in plasma glucose reaching the highest values at 5 min after the injection (33% of the initial values, P < 0.01), and 2) animals submitted to intravenous administration of DuP-753 (losartan), a non-peptide antagonist of angiotensin II with AT1-receptor type specificity (1.63 mumol/100 g body weight as a bolus, i.v., plus a 30-min infusion of 0.018 mumol 100 g body weight-1 min-1 before the injection of angiotensin II), which completely blocked the hyperglycemic response to angiotensin II (P < 0.01). This inhibitory effect on glycemia was already demonstrable 5 min (8.9 +/- 0.28 mM, angiotensin II, N = 9 vs 6.4 +/- 0.22 mM, losartan plus angiotensin II, N = 11) after angiotensin II injection and persisted throughout the 30-min experiment. Controls were treated with the same volume of saline solution (0.15 M NaCl). These data demonstrate that the angiotensin II receptors involved in the direct and indirect hyperglycemic actions of angiotensin II are mainly of the AT1-type.     

Inhibition of sympathetic outflow by the angiotensin II receptor antagonist, eprosartan, but not by losartan, valsartan or irbesartan: relationship to differences in prejunctional angiotensin II receptor blockade

It is well established that angiotensin II can enhance sympathetic nervous system function by activating prejunctional angiotensin II type I (AT1) receptors located on sympathetic nerve terminals. Stimulation of these receptors enhances stimulus-evoked norepinephrine release, leading to increased activation of vascular alpha 1-adrenoceptors and consequently to enhanced vasoconstriction. In the present study, the effects of several chemically distinct nonpeptide angiotensin II receptor antagonists were evaluated on pressor responses evoked by activation of sympathetic outflow through spinal cord stimulation in the pithed rat. Stimulation of thoracolumbar sympathetic outflow in pithed rats produced frequency-dependent pressor responses. Infusion of sub-pressor doses of angiotensin II (40 ng/kg/min) shifted leftward the frequency-response curves for increases in blood pressure, indicating augmented sympathetic outflow. Furthermore, pressor responses resulting in spinal cord stimulation were inhibited by the peptide angiotensin II receptor antagonist, Sar1, Ile8 [angiotensin II] (10 micrograms/kg/min). These results confirm the existence of prejunctional angiotensin II receptors at the vascular neuroeffector junction that facilitate release of norepinephrine. The nonpeptide angiotensin II receptor antagonist, eprosartan (0.3 mg/kg i.v.), inhibited the pressor response induced by spinal cord stimulation in a manner similar to that observed with the peptide antagonist, Sar1, Ile8[angiotensin II]. In contrast, equivalent doses (0.3 mg/kg i.v.) of other nonpeptide angiotensin II receptor antagonists, such as losartan, valsartan, and irbesartan, had no effect on spinal cord stimulation of sympathetic outflow in the pithed rat. Although the mechanism by which eprosartan, but not the other nonpeptide angiotensin II receptor antagonists, inhibits sympathetic outflow in the pithed rat is unknown, one possibility is that eprosartan is a more effective antagonist of prejunctional angiotensin II receptors that augment neurotransmitter release. Because eprosartan is more effective in inhibiting sympathetic nervous system activity compared to other chemically distinct nonpeptide angiotensin II receptor antagonists, eprosartan may be more effective in lowering systolic blood pressure and in treating isolated systolic hypertension.     

Angiotensin II inhibits glomerular adenylate cyclase via the angiotensin II receptor subtype 1 (AT1)

We examined the role of angiotensin II (AII) receptor subtypes in the regulation of hormone-stimulated cyclic AMP (cAMP) accumulation in isolated rat glomeruli. All inhibited cAMP formation induced by histamine, serotonin and parathyroid hormone, but not by prostaglandin E2 or calcitonin gene-related peptide. Angiotensin III but not the angiotensin fragments (1-7) and (3-8) also showed inhibitory activity. The inhibition of histamine-induced cAMP accumulation by AII was concentration-dependent and was absent in glomeruli isolated from pertussis toxin-treated rats. The effect of AII on histamine-induced cAMP levels was not mimicked by the protein kinase C activator, phorbol-12-myristate-13-acetate, nor was the effect of AII inhibited by the protein kinase C inhibitors, staurosporine and H-7. The angiotensin II receptor subtype 1 (AT1) antagonists, SK&F 108566 and losartan, attenuated the inhibitory effect of AII on histamine-stimulated cAMP accumulation, whereas the AT2 selective antagonists, CGP 42112A, WL-19 and PD 123319, had no effect. Displacement of [125I]AII from glomerular membrane using the subtype-selective antagonists confirmed that the glomerular AII receptor has characteristics of an AT1 subtype. The results suggest that AII, through activation of the AT1 receptor, may act to maintain the contractile state of glomerular mesangial cells by attenuating the increase in cAMP levels induced by some hormones.     

AT1 angiotensin receptor inhibition as a new therapeutic possibility

The octapeptide hormone, angiotensin II, binds to two major subtypes of cell surface receptors: the AT1 and the AT2 angiotensin receptors. The important physiological and pathophysiological effects of angiotensin II on cardiovascular regulation and salt-water balance are mediated by the AT1 receptor subtype. As a consequence of the outstanding clinical success of angiotensin-converting enzyme inhibitors, the appearance of AT1 receptor inhibitors in the therapy of hypertension and other cardiovascular diseases was preceded with great expectations. The available experimental and clinical data indicate that the first AT1 receptor inhibitor, losartan, has the same therapeutic potential as angiotensin-converting enzyme inhibitors, but it does not evoke the angiotensin-independent side-effects of ACE inhibitors, such as dry cough or angioedema. The physiological importance and the biochemical, molecular biological and pharmacological properties of AT1 and AT2 receptors are reviewed in this paper, and a summary of the available clinical data is presented.     

The role of G-regulatory proteins in the intracellular mechanism of cardiac angiotensin-II receptors

High-affinity angiotensin II receptors have been identified in cardiac tissue of many animal species. In the heart, angiotensin II exerts positive inotropic and chronotropic effects, constricts coronary vessels, and stimulates cell growth. In vascular smooth muscle and adrenal cortex angiotensin II interacts with guanidine nucleotide regulatory proteins because GTP-gamma-S causes dissociation of the radioligand from its receptor. To investigate whether angiotensin II interacts with guanidine nucleotide regulatory proteins also in cardiac tissue, we studied the effects of GTP-gamma-S on [Sar1, Ile8]-angiotensin II binding to angiotensin II receptor subtypes (AT1 and AT2) in hearts obtained from 16- to 20-week-old Sprague-Dawley rats. We employed an in situ technique performed on frozen tissue sections. Competition experiments performed with the nonpeptide inhibitors losartan and PD123177 allowed identification of both AT1 and AT2 angiotensin II receptors in rat heart. These receptors were present in comparable amounts. In a different set of experiments the effects of GTP-gamma-S (100 microM) on radioligand displacement from AT1 and AT2 receptors were studied. GTP-gamma-S caused a progressive dissociation of the radioligand from the AT1 receptor indicating that this receptor interacts with guanidine nucleotide regulatory proteins. In contrast, the AT2 receptor does not appear to directly interact with guanidine nucleotide regulatory proteins. In summary, the study shows that both angiotensin II receptor subtypes are present in rat heart and that guanidine nucleotide regulatory proteins are implicated in the signal transduction mechanism of the cardiac AT1 receptor.     

Bimodal effects of angiotensin II on migration of human and rat smooth muscle cells. Direct stimulation and indirect inhibition via transforming growth factor-beta 1

Angiotensin II may be an important mediator of neointima formation in vascular disease. This study was designed to examine the mechanisms involved in angiotensin II-stimulated migration of human and rat aortic vascular smooth muscle cells (VSMCs). VSMCs were seeded in one corner of Nunc four-well culture chambers; angiotensin II within filter paper was glued onto the wall of the opposite side. After 48 hours of incubation in serum-free medium containing growth-arresting factor, migrated cells were counted using a light microscope. Angiotensin II (2 x 10(-11) to 2 x 10(-8) mol/L) increased migration of VSMCs in a concentration-dependent manner. Interestingly, at higher concentrations of angiotensin II (up to 2 x 10(-6) mol/L), migration was reduced to levels comparable with control levels. Losartan, an AT1 receptor antagonist, prevented migration, while PD123319, an AT2 receptor antagonist, had no significant inhibitory effect. Transforming growth factor-beta 1 (TGF-beta 1; 0.01 to 10.0 pg/mL) inhibited migration induced by angiotensin II (2 x 10(-8) mol/L) in a concentration-dependent manner. A neutralizing TGF-beta antibody unmasked migratory effects of high concentrations of angiotensin II. Furthermore, angiotensin II (10(-6) mol/L) upregulated TGF-beta 1 mRNA levels fivefold in rat and fourfold in human VSMCs; this effect was prevented by losartan but not by PD123319. Thus, the effects of angiotensin II on migration of VSMCs are bimodal, ie, both migratory and antimigratory pathways are activated. Autocrine release of TGF-beta 1 induced by angiotensin II exerts an antimigratory effect in rat and human VSMCs. The AT1 receptor is involved in regulation of both pathways.     

Flowering-time genes modulate the response to LEAFY activity

Angiotensin 1-7 (Ang 1-7) has been reported to induce relaxation which is partially blocked by a kinin receptor antagonist. We investigated the relationship between kinins and angiotensin peptides with use of preconstricted isolated pig coronary arteries. Ang 1-7 alone (up to 10(-5) M) had no relaxant effect. Bradykinin (BK) (10(-10)-10(-7) M) induced transient relaxation, returning to basal tone, although BK remained in the bath. In these BK-stimulated rings, Ang 1-7 but not BK (both 5 x 10(-6) M) again relaxed the rings by approximately 50%. This relaxation was blocked by a BK B2 antagonist, a kininase, and a nitric oxide synthase inhibitor. Ang 1-7 inhibited purified angiotensin-converting enzyme (ACE) by 30 +/- 3.5% (n = 4) at 10(-6) M. However, in BK-pretreated rings, the ACE inhibitor ramiprilat did not induce relaxation, nor did it affect the relaxant response to Ang 1-7, which suggests that the effect of Ang 1-7 was not caused by ACE inhibition. Ang 1-7-induced vasodilation was reduced by 69.9 +/- 6.2% by an AT2 receptor blocker, PD-123319, and 29.3 +/- 7.3% by an AT1 antagonist, losartan. Neither the nonselective AT1/AT2 receptor antagonist sarthran nor saralasin inhibited the response to Ang 1-7. Ang II did not elicit relaxation either alone or in the presence of losartan, which suggests that activation of AT2 receptors does not cause relaxation. Thus, in the presence of bradykinin, Ang 1-7 relaxes pig coronary arteries via a PD-123319-sensitive mechanism involving nitric oxide, kinins and the BK B2 receptor. The kallikrein-kinin and renin-angiotensin systems may be linked through the interaction of Ang 1-7 and BK.     

Candesartan (CV-11974) dissociates slowly from the angiotensin AT1 receptor

The mechanisms of the insurmountable antagonism of 2-ethoxy-1-[[2'-(1H-tetrazol-5-yl)biphenyl-4-yl]methyl]1H-benzimid azole -7-carboxylic acid, candesartan (CV-11974), an angiotensin AT1 receptor antagonist, on angiotensin II-induced rabbit aortic contraction were examined in contraction and binding studies. Preincubation of the rabbit aorta with CV-11974 (0.1 nM) for 30 min reduced the maximal contractile response to angiostensin II by approximately 50%. This insurmountable antagonism of CV-11974 was reversed in the presence of losartan (1 microM), a surmountable angiotensin AT1 receptor antagonist. The inhibitory effect of CV-11974 on angiotensin II-induced contraction persisted longer after washing than did that of losartan but was irreversible. Scatchard analysis of [3H]CV-11974 binding in bovine adrenal cortical membranes indicated the existence of a single class of binding sites (Kd = 7.4 nM). Competition binding studies using angiotensin II receptor agonists and antagonists have demonstrated that [3H[CV-11974 binding sites may be identical to angiotensin AT1 receptors. The dissociation rate of [3H]CV-11974 binding (t1/2 = 66 min) was 5 times slower than that of [125I]angiotensin II binding (t1/2 = 12 min). These results suggest that the insurmountable antagonism by CV-11974 is due to its slow dissociation from angiotensin AT1 receptors.     

Cortical spreading depression protects against subsequent focal cerebral ischemia in rats

Specific, high-affinity angiotensin II (A II) receptors were observed on granulosa and thecal cells of preovulatory ovarian follicles from immature PMSG-treated rabbits. Scatchard analysis of 125I-[Sar1,Ile8]A II binding to freshly prepared cells was indicative of only one class of binding sites. Kd values were 0.26 +/- 0.11 nM and 0.18 +/- 0.02 nM, densities of A II receptors were 0.06 +/- 0.02 fmol/10(5) cells and 0.08 +/- 0.01 fmol/10(5) cells for granulosa and thecal cells, respectively. When cells were incubated for 48 h with hCG, Kd values were of the same order of magnitude, but the amount of A II receptors was increased 2-fold in granulosa and 4-fold in theca. Using subtype specific ligands (Losartan for AT1 and PD 123319 for AT2) in competitive binding experiments, A II receptors were found to be of the AT1 type on both granulosa and thecal cells freshly prepared or incubated 48 h in vitro. These results establishing the existence of high affinity AT1 receptors on the two cell types of the rabbit preovulatory follicles contrast with previous observations showing the presence of AT2 receptors on granulosa or theca from several species.