Hormonal and cardiovascular effects of losartan (DuP753), an angiotensin receptor antagonist, in nonhuman primates

Isometric quadriceps strength was remeasured in 14 healthy survivors of a group of elderly people first studied 8 years previously. There were 4 men (median age 81 years, range 79 to 84) and 10 women (82 years, range 79 to 89). They were selected for their health, not their levels of physical activity. Nevertheless, they were active when first studied and, with 1 exception, had maintained or increased their levels of physical activity. Isometric quadriceps strength was well preserved; the median change in the strength of the stronger quadriceps was only -0.3% per annum (95% confidence interval = -1.4 to +0.8).     

Differential gene expression of angiotensin II receptor subtypes in the epididymides of mature and immature rats

Electrospray Ionization Mass Spectrometry can be applied to detect aberrant proteins using intact molecules. Direct examination of hemolysate might well facilitate rapid ascertainment of a variant hemoglobin (Hb) provided that the mass difference between normal and abnormal chains is larger than the resolution power of standard instruments (i.e. = 10 Da). We propose immunoprecipitation as a preparation method of plasma and cell proteins other than Hb prior to MS. Amino acid sequences of various variants detected by MS were determined by MS/MS. Some of these variants were new. These new variants were; 1: Hb Sagami[beta 139(H17)Asn-->Thr]. 2: Hb Hokusetsu[beta 52(D3)Asp-->Gly]. 3: a variant transthyretin, amyloidogenic, [38Asp-->Ala]. 4: a variant transthyretin, non-amyloidogenic, [101Gly-->Ser]. The abundance of ion peaks showed the approximate ratio of each component, which was in agreement with the ratio obtained by chromatography and by ESIMS in the analyses of glycated hemoglobin. Samples with low kidney function (BUN > 50 mg/dl, creatinine > 2.5 mg/dl) showed higher values of glycated Hb on routine HPLC than the MS method. Samples containing high carbamylated Hb might cause this discrepancy.     

Human plasma protein binding of the angiotensin II receptor antagonist losartan potassium (DuP 753/MK 954) and its pharmacologically active metabolite EXP3174

Immunological tolerance can be induced by the oral administration of antigen. We induced oral tolerance rats to experimental allergic conjunctivitis by ovalbumin and investigated the suppression of inflammation. In groups which had started eating antigen both before and after the immunization, the serum anti-ovalbumin IgE level measured by passive cutaneous anaphylaxis reaction was significantly lower than in a group that had not been fed antigen. The intensity of experimental allergic conjunctivitis in the group which had started eating antigen before immunization, was significantly suppressed in regard to the leakage of Evans Blue from conjunctival vessels 30 minutes after the challenge and the neutrophil infiltration 6 hours after. The dye leakage of the group which had started feeding after immunization was also significantly suppressed. There was a positive correlation between the serum IgE level and the leakage of Evans Blue (r = 0.90, p < 0.001). These results suggest that the suppression of antigen-specific IgE antibody production caused by oral tolerance affected the decrease of local inflammation on the conjunctiva.     

Effects of angiotensin II and its blockers Sar1-Ile8-angiotensin II and DuP 753 on drinking in ducks in relation to properties of subfornical organ neurons

Properties of systemically applied angiotensin II in stimulating water intake of normally hydrated ducks were studied and the results compared with properties of angiotensin II-responsive neurons of the subfornical organ which are considered as targets for circulating angiotensin II acting as a dipsogen. Following intravenous infusion of hypertonic saline (2000 mosmol.kg-1 at 0.3 ml.min-1 for 1 h), intravenous infusion of 0.3 ml.min-1 isotonic saline with angiotensin II (200 ng.min-1), starting 1 h later, stimulated drinking in each case at an angiotensin II plasma level of about 1400 pg.ml-1. Without hypertonic priming, the same angiotensin II infusion did not stimulate drinking in each experiment; however, if effective, repeated infusions of ANGII induced stable dipsogenic responses. Angiotensin II infusions did not alter plasma levels of antidiuretic hormone. Sar1-Ile8-angiotensin II, a non-selective angiotensin II antagonist, acted weakly as a partial agonist when infused at a dose 200-fold higher than angiotensin II and effectively blocked the dipsogenic action of angiotensin II; this corresponds to the inhibition of angiotensin II-induced excitation by Sar1-Ile8-angiotensin II observed in duck subfornical organ neurons. DuP 753 (losartan), an angiotensin II antagonist specifically blocking AT1 receptors in mammals, had equivocal effects on angiotensin II-induced drinking in ducks at rates 50- and 200-fold higher than angiotensin II, which corresponds to the weak inhibitory action of this compound on angiotensin II-induced neuronal excitation in the duck SFO. Blood pressure was only marginally elevated by the applied angiotensin II dose and Sar1-Ile8-angiotensin II had no effect.     

Cardiac senescence is associated with enhanced expression of angiotensin II receptor subtypes

Recent studies have pointed out the differential role of angiotensin II (Ang II) receptor subtypes, AT1 and AT2, in cardiac hypertrophy and fibrosis during pathological cardiac growth. Because senescence is characterized by an important cardiovascular remodeling, we examined the age-related expression of cardiac Ang II receptors in rats. AT1 and AT2 receptor subtype messenger RNA (mRNA) levels were quantitated by RT-PCR. In parallel, specific Ang II densities were determined in competition binding experiments using specific antagonists. AT1a and AT1b mRNA levels were markedly up-regulated (5.6-fold) in the left ventricle of 24-month-old rats compared with 3-month-old rats, but not in the right ventricle. In contrast, AT2 gene expression was increased in both ventricles of senescent rats (4.2- and 2.8-fold in the left and right ventricles, respectively). Similarly, AT1 and AT2 gene expression was increased 2.3- and 2-fold, respectively, in freshly isolated cardiomyocytes from aged rats. Furthermore, AT1 and AT2 specific binding was increased in the aged left ventricular myocardium. Even though the mechanistic pathway of this up-regulation of Ang II receptor subtype gene expression might be intrinsic to developmental gene reprogramming, the up-regulation of AT1 mRNA accumulation in the left ventricle during aging could also be secondary to age-related hemodynamic changes, whereas increased AT2 gene expression in both ventricles may depend upon hormonal and humoral factors.     

Differential roles of AT1 and AT2 receptor subtypes in vascular trophic and phenotypic changes in response to stimulation with angiotensin II

The essential reaction in the widely accepted proton-motive Q-cycle mechanism of the bc1 complex is the bifurcation of the electron flow during hydroquinone oxidation at the hydroquinone oxidation (Q(P)) site formed by the 'Rieske' iron sulfur protein and by the heme bL domain of cytochrome b. The 'Rieske' [2Fe-2S] cluster has a unique structure containing two exposed histidine ligands, which are the binding site for quinones. The affinity of the 'Rieske' cluster for quinones increases several orders of magnitude upon reduction; this will stabilize semiquinone at the Q(P) site. Based on this affinity change, a reaction scheme is presented which can explain the bifurcation of the electron flow without invoking highly unstable semiquinone species.     

High-performance liquid chromatographic determination of angiotensin II receptor antagonists in human plasma and urine. I. DuP 532 (L-694,492)

A sensitive reversed-phase high-performance liquid chromatographic method with ultraviolet detection was developed for the analysis of a new angiotensin II receptor antagonist, DuP 532 (L-694,492), in human plasma and urine. The analyte and internal standard are extracted from plasma and urine at a pH between 3.3 to 3.6 by liquid-liquid extraction and analyzed on a C6 column with ultraviolet detection at 254 nm. The mobile phase is composed of acetonitrile and phosphate buffer at pH 2.5. The limits of quantification are 6 and 7.5 ng/ml for plasma and urine, respectively.     

Deferential roles of angiotensin receptor subtypes in adrenocortical function in mice

The functional significance of angiotensin II (Ang II) receptor subtypes in adrenals remains unknown. Ang II receptor type 1a (AT1a) expression was localized by in situ hybridization to the zona glomerulosa and zona fasciculata, while AT1b was localized to the zona glomerulosa. Plasma aldosterone and corticosterone levels were measured after injection with Ang II or the type 2 receptor (AT2) agonist CGP-42112 in wild-type and AT1a deficient mice. Aldosterone and corticosterone levels were lower in AT1a deficient mice. Ang II increased plasma aldosterone levels in AT1a deficient mice, but to a lesser extent in mice pretreated with nonselective AT1a/AT1b antagonist, CV-11974. CGP-42112 did not affect aldosterone levels. Ang II increased corticosterone levels in wild-type mice but not in AT1a deficient mice. Results suggest Ang II stimulates aldosterone secretion via AT1a and AT1b in the zona glomerulosa and corticosterone secretion via AT1a in the zona fasciculata, and provide first evidence for differential roles of AT1a and AT1b in the adrenals.     

The effect of central angiotensin II receptor blockade on interleukin-1beta- and prostaglandin E-induced fevers in rats: possible involvement of brain angiotensin II receptor in fever induction

We investigated the role of the brain angiotensin II (Ang II) receptor subtypes AT1 and AT2 in the development of fever induced in freely moving rats by administration of interleukin-1beta (IL-1beta) or prostaglandin E2 (PGE2). Intraperitoneal (i.p.) injection of IL-1beta (2 microg/kg) induced a marked fever of rapid onset. Intracerebroventricular (i.c.v.) administration, immediately before IL-1beta injection, of a selective AT2 receptor antagonist, CGP42112A (5 or 20 microg), reduced the fever in a dose-related manner. Rats given an i.c.v. injection of PGE2 (200 ng) developed a monophasic fever response that was attenuated by i.c.v. treatment with CGP42112A (10 or 20 microg) in a dose-related manner. The IL-1beta (2 microg/kg i.p.)- and PGE2 (200 ng i.c.v.)-induced fevers were unchanged by the selective AT1 receptor antagonist losartan (60 microg i.c.v.). Treatment with exogenous Ang II (100 ng i.c.v.), which itself had no effect on resting body temperature, resulted in an enhancement of the PGE2 (50 ng i.c.v.)-induced fever. The administration of CGP42112A (2 and 5 microg) into the rostral hypothalamus (preoptic/anterior hypothalamic region) reduced fevers induced by IL-1beta (2 microg/kg i.p.) or intrahypothalamic (i.h.) PGE2 (100 ng). Moreover, i.h. injection of Ang II (25 ng) augmented the PGE2 (25 ng i.h.)-induced fever. Finally, the i.h. administration, 15 min before i.h. PGE2 (100 ng), of the angiotensin-converting enzyme (ACE) inhibitor lisinopril (5 and 10 microg) attenuated the PGE2-induced fever. These results suggest that brain AT2 receptors contribute to the induction of such febrile responses in rats.     

The effect of angiotensin II receptor antagonism with losartan on glucose metabolism and insulin sensitivity

OBJECTIVE: To investigate the metabolic effects of losartan (Cozaar) in patients with essential hypertension. METHODS: Twenty patients with mild hypertension (office blood pressure > 140/95 mmHg and home diastolic blood pressure > 90 mmHg) were examined in a double-blind, placebo-controlled cross-over study of 4 weeks of treatment with 50-100 mg losartan. The effects on glucose metabolism were assessed by euglycaemic glucose clamp examinations [glucose disposal rate (GDR, mg/kg per min)] and oral glucose-tolerance tests (OGTT). RESULTS: Supine blood pressure was reduced from 146 +/- 3/90 +/- 3 mmHg on placebo to 134 +/- 4/83 +/- 3 mmHg on losartan and the difference was maintained during 120 min of insulin infusion and glucose clamping. GDR was 6.2 +/- 0.5 mg/kg per min on placebo and 6.4 +/- 0.5 mg/kg per min on losartan. The glucose and insulin responses (the area under the curve) during OGTT were similar with placebo and losartan (0.86 +/- 0.3 versus 0.88 +/- 0.4 and 341 +/- 60 versus 356 +/- 60, respectively; arbitary units). Serum cholesterol was 5.3 +/- 0.2 mmol/l on placebo and 5.1 +/- 0.2 mmol/l losartan treatment. High-density lipoprotein cholesterol and triglycerides were, respectively, 1.1 +/- 0.1 and 1.5 +/- 0.2 mmol/l with placebo, and 1.1 +/- 0.1 and 1.4 +/- 0.1 mmol/l with losartan treatment. CONCLUSION: In mildly hypertensive patients, selective angiotensin II receptor antagonism with losartan for 4 weeks lowers blood pressure at rest and during 120 min of glucose clamping, and has neutral effects on insulin sensitivity, glucose metabolism and serum lipids.     

Uricosuric effect of the angiotensin II receptor antagonist losartan in heart transplant recipients

BACKGROUND: The angiotensin II receptor antagonist losartan is an effective antihypertensive agent with unique uricosuric properties. This study evaluates the uricosuric effects of losartan in 10 hypertensive heart transplant patients with hyperuricemia. METHODS: The patients were randomized to receive losartan 50 mg once daily and enalapril 20 mg once daily for 4 weeks according to a double-blind crossover design. Office blood pressure, plasma uric acid levels, and urinary uric acid excretion were monitored throughout the study. RESULTS: Plasma uric acid levels decreased significantly after 4 weeks of treatment with losartan (P<0.05) but not with enalapril. On day 1 and after 1 month of treatment, a significant increase in uric acid excretion was observed only with losartan. Significant decreases in office systolic and diastolic blood pressures were obtained with enalapril but not with losartan. CONCLUSIONS: Losartan effectively lowers plasma uric acid levels in hyperuricemic heart transplant patients.     

The effect of angiotensin II receptor antagonists on kidney function in two-kidney, two-clip Goldblatt hypertensive rats

We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech, Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include alpha-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.     

Hemodynamic effects of angiotensin II and the influence of angiotensin receptor antagonists in pithed rabbits

We investigated the cardiovascular effects of angiotensin II (AII) and the influences of four angiotensin receptor antagonists: losartan, PD123177, BIBS 39, and BIBS 222 in the pithed rabbit preparation. AII (0.03-10 nmol/kg) elicited a dose-dependent increase in blood pressure (BP), left ventricular pressure (LVP), LV end-diastolic pressure (LVEDP), dP/dtmax, and heart rate (HR). The maximal hypertensive effect of AII is comparable to that of norepinephrine (NE), but its effects on LVEDP and HR are weaker than those of NE. On a molar base, AII is approximately 27 times more potent than NE. Propranolol (0.5 mg/kg i.v.) did not significantly influence the AII-induced increase in diastolic BP (DBP) and LVEDP, but it abolished AII-induced positive chronotropic effects over the entire dose range of angiotensin AII studied. Losartan, but not PD123177, shifted the dose-response curves for AII to the right in a parallel manner. BIBS 39 and BIBS 222 also caused rightward shifts of the AII dose-response curve. These experiments indicate that in propranolol-treated pithed rabbits AII causes vasoconstrictor effects in both resistance vessels and in the venous system, which are both mediated by AT1- but not by AT2-receptors. The AII-induced positive chronotropic effect is an indirect action mediated by the stimulation of postsynaptic beta 1-adrenoceptors. BIBS 39 and BIBS 222, two new nonpeptide angiotensin receptor blockers that have affinity for both AT1- and AT2-receptors are also potent antagonists of the cardiovascular effects of AII in pithed rabbits.     

Differential distribution of endothelin receptor subtypes in placentae from normal and growth-restricted pregnancies

The endothelins (ETs) are potent vasoconstrictor peptides that bind to two distinct receptors, ETA and ETB. This study compares the localization of ETA and ETB receptors in placentae complicated by intrauterine growth retardation (IUGR) and abnormal umbilical Doppler waveform, gestationally matched controls, fetuses that were small for gestational age (SGA), and normal term placentae. Quantitative autoradiography was performed using ETA and ETB subtype-selective ligands. Both ETA and ETB receptors were expressed in the human placenta. Gestational and fetal size effects on the receptor density within stem villi were found, but no effect of abnormal placental blood flow could be demonstrated. A distinct spatial distribution of receptor subtypes within the placenta was observed. Smooth muscle cells expressed both receptors with ETA expression predominant in the proximal regions of the villous tree and ETB abundant in the periphery and decidua. Both receptors were also expressed at lower density on paravascular stromal cells in stem villi. Although these data do not demonstrate aberrant localization of ET receptors in IUGR and SGA placentae, the spatially distinct distribution of ET receptors in the human placenta suggests that ETs play a role in modulation of placental blood flow.     

Up-regulation of angiotensin type 2 receptor mRNA by angiotensin II in rat cortical cells

The present experiment demonstrates that the exposure of angiotensin II (AII) produced an up-regulation of the AT2 receptor mRNA level in rat cortical cells. AII (10(-9)-10(-5) M) exerted a marked increase of AT2 receptor mRNA in a dose-dependent manner. The maximum increase was observed at 3 hr of AII stimulation and lasted 3 hr. The up-regulation of AT2 receptor mRNA was antagonized by PD123319, an AT2 receptor antagonist, but not by SC-52458, an AT1 receptor antagonist, thus suggesting that the increase in AT2 receptor mRNA is mediated via AT2 receptor. This increase is blocked by serine/threonine phosphatase inhibitor okadaic acid, but not by the phosphotyrosine phosphatase inhibitor sodium vanadate, thus suggesting the involvement of serine/threonine phosphatase in this process. Protein kinase C inhibitor, H-7 and calphostin C, did not inhibit the AII-induced up-regulation significantly. In addition, calcium ionophore, A23187 had no effect. These findings suggest that the AT2 receptor mRNA expression by AII is regulated by the activity of serine/threonine phosphatase in the cortical neurons. This observation is also the first example concerning the regulation of AT2 receptor within the brain.     

Cardioprotective effect of enalapril in renovascular and angiotensin II hypertension

The influence of chronic treatment with enalapril or losartan (10 or 30 mg/kg/24h, respectively) on cardiac mass was evaluated in one-kidney, one clip (1K-1C) hypertensive rats submitted to sodium restriction 3 weeks after clipping and in rats infused for 10 days with angiotensin II (ANGII: 200 ng/kg/min). In 1K-1C hypertension, cardiac mass and arterial pressure were reduced to a similar extent by enalapril and losartan. In ANGII hypertension, enalapril and losartan blunted the increase in cardiac mass whereas losartan but not enalapril prevented the development of hypertension. The cardioprotective effect of enalapril was attenuated by concomitant blockade of bradykinin receptors (Hoe140: 300 micrograms/kg/24h) in both models. The beneficial influence of enalapril on cardiac mass appears to be independent of its effect on blood pressure and ANGII generation and seems partly mediated by endogenous bradykinin in these high ANGII models of hypertension.     

Influence of oxidized low-density lipoprotein on vascular angiotensin II receptor expression

OBJECTIVES: To investigate the effect of oxidized low-density lipoprotein (LDL) on angiotensin type 1 (AT1) receptor expression and on angiotensin-induced mitogenesis in vascular smooth muscle cells. DESIGN: Since both LDL and the AT1 receptor are thought to be involved in the pathogenesis of chronic vascular disease, we studied possible interactions between these two biological systems in cultured rat vascular smooth muscle cells. Recent studies have demonstrated that native LDL profoundly increases the AT1 receptor gene expression in vascular smooth muscle cells. The present study was designed to show whether an oxidative modification of LDL, which has been implicated as a major participant in atherogenesis, has similar effects on vascular smooth muscle cells. MATERIALS AND METHODS: Vascular smooth muscle cells isolated from rat thoracic aorta were cultured with oxidized LDL and assessed for their ability to synthesize DNA, and messenger (m)RNA expression, using Northern blot technology. RESULTS: Oxidized LDL had no significant effect on AT1 receptor mRNA steady-state levels when incubated for 0-24 h. Moreover, oxidized LDL itself had no stimulatory effect on DNA synthesis in vascular smooth muscle cells, nor was the angiotensin II-induced increase in DNA synthesis influenced by oxidized LDL. CONCLUSIONS: These data show that in contrast to native LDL, oxidized LDL has no effect on AT1 receptor mRNA expression and no effect on angiotensin II-induced mitogenesis in vascular smooth muscle cells.