Child feeding and care behaviors are associated with xerophthalmia in rural Nepalese households

OBJECTIVE: To compare the blood-pressure-lowering effects of an angiotensin converting enzyme inhibitor, perindopril, with those of an angiotensin II type 1 receptor antagonist, L-158,809, for adult spontaneously hypertensive rats. DESIGN: A cross-over design was used, to treat adult spontaneously hypertensive rats with one drug for 10 weeks, and then with the other for 5 weeks. METHODS: Adult, male spontaneously hypertensive rats (aged 15 weeks) were treated daily by gavage for 10 weeks with perindopril (P group) or L-158,809 (L group), then treatment was crossed over so that rats in the P group were treated with L-158,809 (P/L group) and rats in the L group were treated with perindopril (L/P group) for 5 weeks. Blood pressure was measured weekly. Plasma angiotensin converting enzyme activity, renal angiotensin receptor density, and arterial structure and functioning were measured after the single and crossover treatment periods. RESULTS: Treatment lowered the blood pressure from 206 +/- 2 mmHg in rats in the control group, to 126 +/- 2 in rats in the P group and 150 +/- 2 in rats in the L group. After the cross-over period, blood pressure decreased further from 150 +/- 2 to 129 +/- 3 mmHg in rats in the L/P group, whereas blood pressure of spontaneously hypertensive rats in the P/L group increased from 126 +/- 2 to 148 +/- 2 mmHg. Perindopril treatment almost abolished plasma angiotensin converting enzyme activity, whereas L-158,809 treatment had no effect. Renal angiotensin II receptor density was decreased versus baseline in rats in the P and L groups. The affinity of binding was decreased versus baseline in rats in the L group. A positive correlation to blood pressure was found for mesenteric artery wall thickness and wall: lumen ratio. Concentration for half-maximal effect for the response of mesenteric arteries from rats in the P group to norepinephrine was lower than that of the control group rats. Angiotensin II potentiated the norepinephrine-stimulated contraction of arteries from rats in the control and P groups, but not that of arteries from rats in the groups treated with L-158,809. CONCLUSION: Perindopril was more effective than was L-158,809 at lowering the blood pressure of adult spontaneously hypertensive rats, and at altering the structure and functioning of the arteries.     

Cardiovascular effects produced by microinjection of angiotensins and angiotensin antagonists into the ventrolateral medulla of freely moving rats

In this study we determined the cardiovascular effects produced by microinjection of angiotensin peptides [Angiotensin-(1-7) and Angiotensin II] and angiotensin antagonists (losartan, L-158,809, CGP 42112A. Sar1-Thr8-Ang II, A-779) into the rostral ventrolateral medulla of freely moving rats. Microinjection of angiotensins (12.5-50 pmol) produced pressor responses associated to variable changes in heart rate, usually tachycardia. Unexpectedly, microinjection of both AT1 and AT2 ligands produced pressor effects at doses that did not change blood pressure in anesthetized rats. Conversely, microinjection of Sar1-Thr8-Ang II and the selective Ang-(1-7) antagonist, A-779, produced a small but significant decrease in MAP an HR. These findings suggest that angiotensins can influence the tonic activity of vasomotor neurons at the RVLM. As previously observed in anesthetized rats, our results further suggest a role for endogenous Ang-(1-7) at the RVLM. The pressor activity of the ligands for AT1 and AT2 angiotensin receptor subtypes at the RVLM, remains to be clarified.     

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.     

In vivo pharmacology of an angiotensin AT1 receptor antagonist with balanced affinity for AT2 receptors

L-163,017 (6-[benzoylamino]-7-methyl-2-propyl-3-[[2'-(N-(3-methyl-1-butoxy) carbonylaminosulfonyl)[1,1']-biphenyl-4-yl]methyl]-3H-imidazo[4,5- b]pyridine) is a potent, orally active, nonpeptide angiotensin II receptor antagonist. Conscious rats and dogs were dosed p.o. and i.v.; in both species the plasma bioequivalents are similar at the angiotensin AT1 and AT2 receptor sites indicating balanced activity is maintained in vivo. L-163,017 prevents the pressor response to intravenous (i.v.) angiotensin II in the conscious rat, dog, and rhesus monkey. L-163,017 also significantly reduces blood pressure in a renin-dependent model of hypertension, similar to an angiotensin converting enzyme inhibitor (Enalapril) and an angiotensin AT1 receptor-selective antagonist (L-159,282). These studies indicate that neither the angiotensin AT2 receptor nor bradykinin is important in the acute antihypertensive activity of angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists.     

Comparison of inversion-recovery gradient- and spin-echo and fast spin-echo techniques in the detection and characterization of liver lesions

For several G protein-coupled receptors, amino acids in the seventh transmembrane helix have been implicated in ligand binding and receptor activation. The function of this region in the AT1 angiotensin receptor was further investigated by mutation of two conserved polar residues (Asn294 and Asn295) and the adjacent Phe293 residue. Analysis of the properties of the mutant receptors expressed in COS-7 cells revealed that alanine replacement of Phe293 had no major effect on AT1 receptor function. Substitution of the adjacent Asn294 residue with alanine (N294A) reduced receptor binding affinities for angiotensin II, two nonpeptide agonists (L-162,313 and L-163,491), and the AT1-selective nonpeptide antagonist losartan but not that for the peptide antagonist [Sar1, Ile8]angiotensin II. The N294A receptor also showed impaired G protein coupling and severely attenuated inositol phosphate generation. In contrast, alanine replacement of Asn295 decreased receptor binding affinities for all angiotensin II ligands but did not impair signal transduction. Additional substitutions of Asn295 with a variety of amino acids did not identify specific structural elements for ligand binding. These findings indicate that Asn295 is required for the integrity of the intramembrane binding pocket of the AT1a receptor but is not essential for signal generation. They also demonstrate the importance of transmembrane helices in the formation of the binding site for nonpeptide AT1 receptor agonists. We conclude that the Asn294 residue of the AT1 receptor is an essential determinant of receptor activation and that the adjacent Asn295 residue is required for normal ligand binding.     

Captopril and angiotensin II receptor antagonist therapy in a pacing model of heart failure

Twenty-four splenectomized dogs were subjected to rapid right ventricular pacing (RRVP) at 250 beats/min for five weeks. During the final three weeks, four groups six dogs were untreated or treated with captopril alone, with the angiotensin II type 1 (AT1) receptor antagonist L158,809 alone or with the two drugs combined by constant intravenous infusion. Hemodynamic studies were carried out during light anesthesia at baseline, and after two and five weeks of pacing. Total vascular capacitance and stressed blood volume were calculated from the mean circulatory filling pressure during transient circulatory arrest after acetylcholine administration at three different circulating volumes. Central blood volume and cardiac output were measured by thermodilution. Severe heart failure was present in the untreated group after five weeks of RRVP, characterized by low cardiac output and total vascular capacitance, high right atrial and pulmonary capillary wedge and mean circulatory filling pressure, plus increased stressed and central blood volumes. While L158,809 had not effect, captopril alone or combined with L158,809 ameliorated the reduction in total vascular capacitance, and reduced right atrial and mean circulatory pressure and stressed blood volumes. Combined therapy reduced pulmonary capillary wedge pressure. Thus, angiotensin-converting enzyme inhibition with captopril was effective in this model of chronic low output heart failure, whereas AT1 receptor antagonism was not.     

Differential effects of Ca2+ channel blockers on Ca2+ transients and cell cycle progression in vascular smooth muscle cells

The predominant angiotensin II receptor expressed in the human myometrium is the angiotensin AT2 receptor. This preparation was used for a structure-activity relationship study on angiotensin II analogues modified in positions 1 and 8. The angiotensin AT2 receptor present on human myometrium membranes displayed a high affinity (pKd = 9.18) and was relatively abundant (53-253 fmol/mg of protein). The pharmacological profile was typical of an angiotensin AT2 receptor with the following order of affinities: (angiotensin III > or = angiotensin II > angiotensin I > PD123319 > angiotensin-(1-7) > angiotensin-(1-6) approximately angiotensin IV > Losartan). Modifications of the N-terminal side chain and of the primary amine of angiotensin II were evaluated. Neutralisation of the methylcarboxylate (Asp) to a methylcarboxamide (Asn) or to a hydroxymethyl (Ser) or substitution for a methylsulfonate group (cysteic acid) improved the affinity. Extension from methylcarboxylate (Asp) to ethylcarboxylate (Glu) did not affect the affinity. Introduction of larger side chains such as the bulky p-benzoylphenylalanine (p-Bpa) or the positively charged Lys did not substantially affect the affinity. Complete removal of the side chain (angiotensin III), however, resulted in a significant affinity increase. Removal or acetylation of the primary amine of angiotensin II did not noticeably influence the affinity. Progressive alkylation of the primary amine significantly increased the affinity, betain structures being the most potent. It appears that quite important differences exist between the angiotensin AT1 and AT2 receptors concerning their pharmacological profile towards analogues of angiotensin II modified in position 1. On position 8 of angiotensin II, a structure-activity relationship on the angiotensin AT2 receptor was quite similar to that observed with angiotensin AT1 receptor. Bulky, hydrophobic aromatic residues displayed affinities similar to or even better than [Sarcosine1]angiotensin II. Aliphatic residues, especially those of reduced size, caused a significant decrease in affinity especially [Sarcosine1, Gly8]angiotensin II who showed a 30-fold decrease. Introduction of a positive charge (Lys) at position 8 reduced the affinity even further. Stereoisomers in position 8 (L-->D configuration) also induced lower affinities. The angiotensin AT2 receptor display a structure-activity relationship similar to that observed on the AT1 receptor for the C-terminal position of the peptide hormone. Position 1 structure-activity relationships are however fundamentally different between the angiotensin AT1 and AT2 receptor.     

Assessment of the role of the renin-angiotensin system in cardiac contractility utilizing the renin inhibitor remikiren

1. The role of the renin-angiotensin system in the regulation of myocardial contractility is still debated. In order to investigate whether renin inhibition affects myocardial contractility and whether this action depends on intracardiac rather than circulating angiotensin II, the regional myocardial effects of systemic (i.v.) and intracoronary (i.c.) infusions of the renin inhibitor remikiren, were compared and related to the effects on systemic haemodynamics and circulating angiotensin II in open-chest anaesthetized pigs (25-30 kg). The specificity of the remikiren-induced effects was tested (1) by studying its i.c. effects after administration of the AT1-receptor antagonist L-158,809 and (2) by measuring its effects on contractile force of porcine isolated cardiac trabeculae. 2. Consecutive 10 min i.v. infusions of remikiren were given at 2, 5, 10 and 20 mg min-1. Mean arterial pressure (MAP), cardiac output (CO), heart rate (HR), systemic vascular resistance (SVR), myocardial oxygen consumption (MVO2) and left ventricular (LV) dP/dtmax were not affected by remikiren at 2 and 5 mg min-1, and were lowered at higher doses. At the highest dose, MAP decreased by 48%, CO by 13%, HR by 14%, SVR by 40%, MVO2 by 28% and LV dp/dtmax by 52% (mean values; P < 0.05 for difference from baseline, n = 5). The decrease in MVO2 was accompanied by a decrease in myocardial work (MAP x CO), but the larger decline in work (55% vs. 28%; P < 0.05) implies a reduced myocardial efficiency ((MAP x CO)/MVO2). 3. Consecutive 10 min i.c. infusions of remikiren were given at 0.2, 0.5, 1, 2, 5 and 10 mg min-1. MAP, CO, MVO2 and LV dP/dtmax were not affected by remikiren at 0.2, 0.5 and 1 mg min-1, and were reduced at higher doses. At the highest dose, MAP decreased by 31%, CO by 26%, MVO2 by 46% and LV dP/dtmax by 43% (mean values; P < 0.05 for difference from baseline, n = 6). HR and SVR did not change at any dose. 4. Thirty minutes after a 10 min i.v. infusion of the AT1 receptor antagonist, L-158,809 at 1 mg min-1, consecutive 10 min i.c. infusions (n = 5) of remikiren at 2, 5 and 10 mg min-1 no longer affected CO and MVO2, and decreased LV dP/dtmax by maximally 27% (P < 0.05) and MAP by 14% (P < 0.05), which was less than without AT1-receptor blockade (P < 0.05). HR and SVR remained unaffected. 5. Plasma renin activity and angiotensin I and II were reduced to levels at or below the detection limit at doses of remikiren that were not high enough to affect systemic haemodynamics or regional myocardial function, both after i.v. and i.c. infusion. 6. Remikiren (10(-10) to 10(-4) M) did not affect contractile force of porcine isolated cardiac trabeculae precontracted with noradrenaline. In trabeculae that were not precontracted no decrease in baseline contractility was observed with remikiren in concentrations up to 10(-5) M, whereas at 10(-4) M baseline contractility decreased by 19% (P < 0.05). 7. Results show that with remikiren i.v., at the doses we used, blood pressure was lowered primarily by vasodilation and with remikiren i.c. by cardiac depression. The blood levels of remikiren required for its vasodilator action are lower than the levels affecting cardiac contractile function. A decrease in circulating angiotensin II does not appear to be the sole explanation for these haemodynamic responses. Data support the contention that myocardial contractility is increased by renin-dependent angiotensin II formation in the heart.     

Tumor necrosis factor-alpha mediates endotoxin induced suppression of gonadotropin-releasing hormone pulse generator activity in the rat

Little is known regarding the developmental regulation of the cardiac angiotensin type 1 (AT1) and type 2 (AT2) receptor genes or their role in normal cardiac growth. Regulation of AT1 and AT2 receptor genes were examined using total and poly A + RNA isolated from whole Sprague-Dawley rat hearts. AT1 mRNA levels were 3.5-fold higher in the 19-day-old fetal heart compared to the 90-day-old adult as detected with 2 or 5 microg of poly A + RNA. AT2 mRNA was only detectable with 20 microg of poly A + RNA. AT2 mRNA levels were highest in the 19-day-old fetal heart with no detectable message in the 90-day-old adult heart. Qualitative PCR for AT2 mRNA also could not detect AT2 mRNA in the adult heart. Treatment with the AT1 receptor antagonist losartan for 3 weeks in the 21-day-old rat or for 4 days in the 38-day-old rat resulted in a significant decrease in heart/body weight in both groups and body weight in the 3-week treatment group. AT2 blockade for 4 days with PD123319 or beta-receptor blockade with propranolol for 3 weeks did not alter heart/body weights. Losartan treatment also resulted in a three-fold increase in cardiac AT1 mRNA levels in both the 4-day and 3-week treatment groups compared to controls. We conclude that Ang II, acting primarily, if not exclusively via the AT1 receptor plays a significant role in the regulation of normal cardiac growth in the young rat.     

The subtype-2 (AT2) angiotensin receptor regulates renal cyclic guanosine 3', 5'-monophosphate and AT1 receptor-mediated prostaglandin E2 production in conscious rats

The renal effects of angiotensin II(AII) are attributed to AT1 receptors. In contrast, the function of renal AT2 receptors in unknown. Using a microdialysis technique, we monitored changes in renal interstitial fluid (RIF) prostaglandin E2 (PGE2) and cyclic guanosine 3', 5'-monophosphate (cGMP) in response to dietary sodium (Na) depletion alone, or Na depletion or normal Na diet combined with the AT1 receptor blocker, Losartan, the AT2 receptor blocker, PD 123319 (PD), or angiotensin II, individually or combined in conscious rats. Na depletion significantly increased PGE2 and cGMP. During Na depletion, Losartan decreased PGE2 and did not change cGMP. In contrast, PD significantly increased PGE2 and decreased cGMP. Combined administration of Losartan and PD decreased PGE2 and cGMP. During normal Na diet, RIF PGE2 and cGMP increased in response to angiotensin II. Neither Losartan nor PD, individually or combined, changed RIF PGE2 or cGMP. Combined administration of angiotensin II and Losartan or PD produced a significant decrease in response of PGE2 and cGMP to angiotensin II, respectively. These data demonstrate that activation of the reninangiotensin system during Na depletion increases renal interstitial PGE2 and cGMP. The AT1 receptor mediates renal production of PGE2. The AT2 receptor mediates cGMP. AT2 blockade potentiates angiotensin-induced PGE2 production at the AT1 receptor.     

Evidence that angiotensin II, endothelins and nitric oxide regulate mitogen-activated protein kinase activity in rat aorta

We measured the activity of mitogen-activated protein (MAP) kinases, enzymes believed to be involved in the pathway for cell proliferation, in rat aortic strips with or without endothelium, and examined effects of angiotensin receptor antagonists, endothelin receptor antagonists and nitric oxide (NO)-related agents. Endothelium removal produced an activation of MAP kinase activity in the strips, whereas the enzyme activity was not affected in the adventitia. The MAP kinase activation was inhibited by either the angiotensin AT1 receptor antagonist losartan or the endothelin ETA receptor antagonist BQ 123. The combination of both antagonists caused an additive inhibition. The angiotensin AT2 receptor antagonist PD 123,319 and the endothelin ETB receptor antagonist BQ 788 did not affect the MAP kinase activation. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) caused an activation of MAP kinase in the endothelium-intact aorta and the MAP kinase activation was inhibited by losartan or BQ123. The NO releaser nitroprusside inhibited the MAP kinase activation induced by endothelium removal or angiotensin II. These results suggest that even in isolated arteries, NO of endothelial origin tonically exert MAP kinase-inhibiting effects and endogenous angiotensin II and endothelins in the media are tonically released to cause MAP kinase-stimulating effects in medial smooth muscle.     

The role of angiotensin AT1 receptors in the diuretic, natriuretic, kaliuretic and blood pressure responses induced by angiotensin II activation of the median preoptic nucleus in conscious rats

We determined the effects of two classical angiotensin II (ANG II) antagonists, [Sar1, Ala8]-ANG II and [Sar1, Thr8]-ANG II, and losartan (a nonpeptide and selective antagonist for the AT1 angiotensin receptors) on diuresis, natriuresis, kaliuresis and arterial blood pressure induced by ANG II administration into the median preoptic nucleus (MnPO) of male Holtzman rats weighing 250-300 g. Urine was collected in rats submitted to a water load (5% body weight) 1 h later. The volume of the drug solutions injected was 0.5 microliters over 10-15 s. Pre-treatment with [Sar1, Ala8]-ANG II (12 rats) and [Sar1, Thr8]-ANG II (9 rats), at the dose of 60 ng reduced (13.7 +/- 1.0 vs 11.0 +/0 1.0 and 10.7 +/0 1.2, respectively), whereas losartan (14 rats) at the dose of 160 ng totally blocked (13.7 +/- 1.0 vs 7.6 +/- 1.5) the urine excretion induced by injection o 12 ng of ANG II (14 rats). [Sar1, Ala8]-ANG II impaired Na+ excretion (193 +/- 16 vs 120 +/- 19), whereas [Sar1, Thr8]-ANG II and losartan block Na+ excretion (193 +/- 16 vs 77 +/- 15 and 100 +/- 12, respectively) induced by ANG II. Similar effects induced by ANG II on K+ excretion were observed with [Sar1, Ala8]-ANG II, [Sar1, Thr8]- ANG II, and losartan pretreatment (133 +/- 18 vs 108 +/- 11, 80 +/- 12, and 82 +/- 15, respectively). The same doses as above of [Sar1, Ala8]-ANG II (8 rats), [Sar1, Thr8]-ANG II (8 rats), and losartan (9 rats) blocked the increase in the arterial blood pressure induced by 12 ng of ANG II (12 rats) (32 +/- 4 vs 4 +/- 2, 3.5 +/- 1, and 2 +/- 1, respectively. The results indicate that the AT1 receptor subtype participates in the increases of diuresis, natriuresis, kaliuresis and arterial blood pressure induced by the administration of ANG II into the MnPO.     

Angiotensin II receptor blockade and end-organ protection: pharmacological rationale and evidence

BACKGROUND: The renin-angiotensin system is a widely studied hormonal system that comprises substrate-enzyme interactions, the end result of which is the production of the active peptide angiotensin II. Because angiotensin II affects blood pressure control, sodium and water homeostasis, and cardiovascular function and structure, a great deal of research effort has been directed toward blocking the renin-angiotensin system. Angiotensin II also may be involved in end-organ damage in hypertension, heart failure, and vascular disease. ANGIOTENSIN II RECEPTORS: At least two subtypes of angiotensin II receptors have been identified, angiotensin type 1 (AT)1 and type 2 (AT2). The AT1 receptor mediates all the known actions of angiotensin II on blood pressure control. Additionally, research has indicated that the AT1 receptor modulates cardiac contractility and glomerular filtration, increases renal tubular sodium reabsorption, and cardiac and vascular hypertrophy. Less is known about the function of the AT2 receptor. Evidence suggests that the AT2 receptor inhibits cell proliferation and reverses the AT1-induced hypertrophy. Indeed, these receptors are thought to exert opposing effects. ANGIOTENSIN RECEPTOR ANTAGONISTS: This newly introduced class of drugs is able to inhibit the renin-angiotensin system at the receptor level by specifically blocking the AT1 receptor subtype. These drugs induce a dose-dependent blockade of angiotensin II effects, resulting in reduced blood pressure, urinary protein, and glomerular sclerosis. It is postulated that AT1 receptor antagonists may provide end-organ protection by blocking angiotensin II effects via the AT1 receptor, leaving the AT2 receptor unopposed. Consequently, these agents may reduce the morbidity and mortality that result from myocardial infarction and other conditions resulting from structural alterations in the heart, kidney, and vasculature.     

AT1 receptor-mediated stimulation by angiotensin II of rat aortic fibronectin gene expression in vivo

Fibronectin plays an important role in various vascular diseases. A subpressor (200 ng kg-1 min-1) or pressor (1000 ng kg-1 min-1) dose of angiotensin II was continuously infused into rats by osmotic minipump for various times, to investigate the effects on aortic fibronectin gene expression. In rats infused with a subpressor dose of angiotensin II in which blood pressure was normal for 3 days, aortic fibronectin mRNA levels started to increase by 1.4 fold at 12 h and reached the maximal levels (increased by 3.1 fold) at 3 days. Treatment with TCV-116 (3 mg kg-1 day-1), a non-peptide selective AT1 receptor antagonist, completely inhibited the angiotensin II-induced increase in aortic fibronectin mRNA, while hydralazine (10 mg kg-1 day-1) did not block this effect. Similar results were also obtained for a pressor dose of angiotensin II. Thus, angiotensin II directly stimulates aortic fibronectin gene expression in vivo, which is mediated by the AT1 receptor but not by blood pressure.     

Comparison of human immunodeficiency virus 1 DNA polymerase chain reaction and qualitative and quantitative RNA polymerase chain reaction in human immunodeficiency virus 1-exposed infants

The endothelial lining of the blood-brain barrier tightly controls the distribution of peptide hormones between the central nervous system and the circulation. By using primary cultures of brain microvessel endothelial cells, an in vitro model of the blood-brain barrier, we report here the uptake and transport of the octapeptide angiotensin II by a specific receptor population. With the angiotensin II antagonists losartan (AT1 specific) and PD 123,319 (AT2 specific), we showed that both the uptake and transport of angiotensin II were mediated by the AT1 receptor. Western blot analysis confirmed the existence of the AT1 receptor in our cell-culture model. Rhodamine 123 studies also suggested that both angiotensin II antagonists, but not angiotensin II, were substrates for the P-glycoprotein efflux system, thus restricting the transport of these compounds. These results suggest an AT1 receptor mediates uptake and transport of angiotensin II at the blood-brain barrier and may contribute to the regulation of cerebrovascular levels of the peptide.     

Angiotensin II stimulates proliferation of normal early erythroid progenitors

Angiotensin II exerts a mitogenic effect in several in vitro models, but a direct effect on erythroid progenitors has not been documented. Angiotensin-converting enzyme inhibitors and losartan, an angiotensin II type 1 receptor (AT1) antagonist, ameliorate posttransplant erythrocytosis, without altering serum erythropoietin levels. We studied erythroid differentiation and the effect of angiotensin II on proliferation of erythroid progenitors by culturing CD34+ hematopoietic progenitor cells in liquid serum-free medium favoring growth of erythroid precursors. Aliquots of cells were collected every third day, and were used for RNA preparation. AT1 mRNA was detected after 6 d. In these same samples, erythroid-specific mRNA (erythropoietin receptor) was also detected. AT1 protein was detected in 7-d-old burst-forming units-erythroid colonies by Western blotting. The CD34+ cell liquid cultures were used to incubate erythroid precursors with angiotensin II from days 6-9. After incubation, cells were transferred to semisolid medium and cultured with erythropoietin. Angiotensin II increased proliferation of early erythroid progenitors, defined as increased numbers of burst-forming units-erythroid colonies. Losartan completely abolished this stimulatory effect of angiotensin II. Moreover, we observed increased numbers of erythroid progenitors in the peripheral blood of posttransplant erythrocytosis patients. Thus, activation of AT1 with angiotensin II enhances erythropoietin-stimulated erythroid proliferation in vitro. A putative defect in the angiotensin II/AT1 pathway may contribute to the pathogenesis of posttransplant erythrocytosis.     

Angiotensin II AT1 receptor/signaling mechanisms in the biphasic effect of the peptide on proximal tubular Na+,K+-ATPase

The present study was designed to determine the cellular signaling mechanisms responsible for mediating the effects of angiotensin II on proximal tubular Na+,K+-ATPase activity. Angiotensin II produced a biphasic effect on Na+,K+-ATPase activity: stimulation at 10(-13) - 10(-10) M followed by inhibition at 10(-7) - 10(-5) M of angiotensin II. The stimulatory and inhibitory effects of angiotensin II were antagonized by losartan (1nM) suggesting the involvement of AT1 receptor. Angiotensin II produced inhibition of forskolin-stimulated cAMP accumulation at 10(-13) - 10(-10) M followed by a stimulation in basal cAMP levels at 10(-7) - 10(-5) M. Pretreatment of proximal tubules with losartan (1nM) antagonized both the stimulatory and inhibitory effects of angiotensin II on cAMP accumulation. Pretreatment of the proximal tubules with pertussis toxin (PTx) abolished the stimulation of Na+,K+-ATPase activity but did not affect the inhibition of Na+,K+-ATPase activity produced by angiotensin II. Pretreatment of the tubules with cholera toxin did not alter the biphasic effect of angiotensin II on Na+,K+-ATPase activity. Mepacrine (10microM), a phospholipase A2 (PLA2) inhibitor, reduced only the inhibitory effect of angiotensin II on Na+,K+-ATPase activity. These results suggest that the activation of AT1 angiotensin II receptors stimulates Na+,K+-ATPase activity via a PTx-sensitive G protein-linked inhibition of adenylyl cyclase pathway, whereas the inhibition of Na+,K+-ATPase activity following AT1 receptor activation involves multiple signaling pathways which may include stimulation of adenylyl cyclase and PLA2.     

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.