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More than 1 billion people worldwide suffer from hypertension; therefore, hypertension management has been categorized as a global health priority. Losartan potassium (LP) is an antihypertensive drug with a limited oral bioavailability of about 33% since it undergoes the initial metabolic cycle. Thus, nasal administration is a unique route to overcome first-pass metabolism. The investigation focused on the potential effects of LP-loaded spanlastic vesicles (SNVs) on LP pharmacodynamics and pharmacokinetic parameters, utilizing a thin-film hydration methodology established on a 3 1 2 2 full factorial design. Entrapment efficiency (EE%) ranged from 39.8 ± 3.87.8 to 83.8 ± 2.92% for LP-SNVs. Vesicle size (VS) varied from 205.5 ± 6.5.10 to 445.1 ± 13.52 nm, and the percentage of LP released after 8 h (Q 8h ) ranged from 30.8 ± 3.10 to 68.8 ± 1.45%. LP permeated through the nasal mucosa during 24 h and flocculated from 194.1 ± 4.90 to 435.3 ± 13.53 µg/cm2. After twenty-four hours, the optimal LP-SNVs in-situ gel showed 2.35 times more permeation through the nasal mucosa than the LP solution. It also lowered systolic blood pressure, so it is thought to be better than the reference formulation in terms of pharmacodynamics. The pharmacokinetics studies demonstrated that the intranasal LP-SNVs gel boosted its bioavailability approximately 6.36 times compared to the oral LP solution. Our research showed that intranasal LP-SNVs could be a good nanoplatform because they are well-tolerated and have possible pharmacokinetics and pharmacodynamics., (© 2024. The Author(s).)

Objective: To review and summarize data on angiotensin II (AT-II), approved by the Food and Drug Administration (FDA) in December 2017 to increase blood pressure in adults with septic or other distributive shock., Data Sources: A PubMed/MEDLINE search was conducted using the following terms: (angiotensin ii OR angiotensin 2) AND (shock) from 1966 to February 2018., Study Selection and Data Extraction: A total of 691 citations were reviewed with only relevant clinical data extracted., Data Synthesis: AT-II is a peptide hormone with a multitude of physiological effects-namely, vasoconstriction of venous and arterial smooth muscle. The priority approval granted by the FDA was secondary to a phase 3 study of patients receiving at least 0.2 µg/kg/min of norepinephrine or equivalent for vasodilatory shock. Compared with placebo, AT-II had a significantly higher rate of response, defined as a mean arterial pressure of 75 mm Hg or an increase of 10 mm Hg. No significant difference was found in death by day 28., Conclusions: AT-II is a newly available vasoactive agent with a novel mechanism for the treatment of distributive shock. Further research is needed to define its exact role in therapy of shock states, identify patients most likely to benefit, and further study its safety profile in critical illness.

This article is one of ten reviews selected from the Annual Update in Intensive Care and Emergency Medicine 2018. Other selected articles can be found online at https://www.biomedcentral.com/collections/annualupdate2018 . Further information about the Annual Update in Intensive Care and Emergency Medicine is available from http://www.springer.com/series/8901 .

The present study was designed to determine the effects of senescence and angiotensin II (Ang II) on expression and processing of amyloid precursor protein (APP) in human brain microvascular endothelial cells (BMECs). Senescence caused a decrease in APP expression thereby resulting in reduced secretion of soluble APPα (sAPPα). In contrast, β-site APP cleaving enzyme (BACE1) expression and production of amyloid β (Aβ)40 were increased in senescent endothelium. Importantly, in senescent human BMECs, treatment with BACE1 inhibitor IV inhibited Aβ generation and increased sAPPα production by enhancing a disintegrin and metalloprotease (ADAM)10 expression. Furthermore, Ang II impaired expression of ADAM10 and significantly reduced generation of sAPPα in senescent human BMECs. This inhibitory effect of Ang II was prevented by treatment with BACE1 inhibitor IV. Our results suggest that impairment of α-processing and shift to amyloidogenic pathway of APP contribute to endothelial dysfunction induced by senescence. Loss of sAPPα in senescent cells treated with Ang II exacerbates detrimental effects of senescence on APP processing. Notably, inhibition of BACE1 has beneficial effects on senescence induced endothelial dysfunction. Reported findings may help to explain contributions of senescent cerebral microvascular endothelium to development of cerebral amyloid angiopathy and Alzheimer's disease (AD) pathology.

Low efficacy of cationic polymer-based formulations (polyplexes) for systemic gene delivery to tumors remains the crucial concern for their clinical translation. Here we show that modulating the physiological state of a tumor using clinically approved pharmaceuticals can improve delivery of intravenously injected polyplexes to murine melanoma tumors with different characteristics. Direct comparison of drugs with different mechanisms of action has shown that application of nitroglycerin or losartan improved extravasation and tumor uptake of polyplex nanoparticles, whereas angiotensin II had almost no effect on polyplex accumulation and microdistribution in the tumor tissue. Application of nitroglycerin and losartan caused from 2- to 6-fold enhanced efficacy of polyplex-mediated gene delivery depending on the tumor model. The results obtained on polyplex behavior in tumor tissues depending on physiological state of the tumor can be relevant to optimize delivery of polyplexes and other nanomedicines with similar physicochemical properties., (Copyright © 2016. Published by Elsevier B.V.)

Recently, the kelch-like protein 3 (KLHL3)-Cullin3 complex was identified as an E3 ubiquitin ligase for with no lysine (WNK) kinases, and the impaired ubiquitination of WNK4 causes pseudohypoaldosteronism type II (PHAII), a hereditary hypertensive disease. However, the involvement of WNK kinase regulation by ubiquitination in situations other than PHAII has not been identified. Previously, we identified the WNK3-STE20/SPS1-related proline/alanine-rich kinase-Na/K/Cl cotransporter isoform 1 phosphorylation cascade in vascular smooth muscle cells and found that it constitutes an important mechanism of vascular constriction by angiotensin II (AngII). In this study, we investigated the involvement of KLHL proteins in AngII-induced WNK3 activation of vascular smooth muscle cells. In the mouse aorta and mouse vascular smooth muscle (MOVAS) cells, KLHL3 was not expressed, but KLHL2, the closest homolog of KLHL3, was expressed. Salt depletion and acute infusion of AngII decreased KLHL2 and increased WNK3 levels in the mouse aorta. Notably, the AngII-induced changes in KLHL2 and WNK3 expression occurred within minutes in MOVAS cells. Results of KLHL2 overexpression and knockdown experiments in MOVAS cells confirmed that KLHL2 is the major regulator of WNK3 protein abundance. The AngII-induced decrease in KLHL2 was not caused by decreased transcription but increased autophagy-mediated degradation. Furthermore, knockdown of sequestosome 1/p62 prevented the decrease in KLHL2, suggesting that the mechanism of KLHL2 autophagy could be selective autophagy mediated by sequestosome 1/p62. Thus, we identified a novel component of signal transduction in AngII-induced vascular contraction that could be a promising drug target., (Copyright © 2015 by the American Society of Nephrology.)

The recent identification of a novel binding site for angiotensin (Ang) II as the peptidase neurolysin (E.C. 3.4.24.16) has implications for the renin-angiotensin system (RAS). This report describes the distribution of specific binding of 125I-Sarcosine1, Isoleucine8 Ang II (125I-SI Ang II) in neurolysin knockout mouse brains compared to wild-type mouse brains using quantitative receptor autoradiography. In the presence of p-chloromercuribenzoic acid (PCMB), which unmasks the novel binding site, widespread distribution of specific (3 µM Ang II displaceable) 125I-SI Ang II binding in 32 mouse brain regions was observed. Highest levels of binding >700 fmol/g initial wet weight were seen in hypothalamic, thalamic and septal regions, while the lowest level of binding <300 fmol/g initial wet weight was in the mediolateral medulla. 125I-SI Ang II binding was substantially higher by an average of 85% in wild-type mouse brains compared to neurolysin knockout brains, suggesting the presence of an additional non-AT1, non-AT2, non-neurolysin Ang II binding site in the mouse brain. Binding of 125I-SI Ang II to neurolysin in the presence of PCMB was highest in hypothalamic and ventral cortical brain regions, but broadly distributed across all regions surveyed. Non-AT1, non-AT2, non-neurolysin binding was also highest in the hypothalamus but had a different distribution than neurolysin. There was a significant reduction in AT2 receptor binding in the neurolysin knockout brain and a trend towards decreased AT1 receptor binding. In the neurolysin knockout brains, the size of the lateral ventricles was increased by 56% and the size of the mid forebrain (-2.72 to +1.48 relative to Bregma) was increased by 12%. These results confirm the identity of neurolysin as a novel Ang II binding site, suggesting that neurolysin may play a significant role in opposing the pathophysiological actions of the brain RAS and influencing brain morphology.

The circulating, endocrine renin-angiotensin system (RAS) is important to circulatory homeostasis, while ubiquitous tissue and cellular RAS play diverse roles, including metabolic regulation. Indeed, inhibition of RAS is associated with improved cellular oxidative capacity. Recently it has been suggested that an intra-mitochondrial RAS directly impacts on metabolism. Here we sought to rigorously explore this hypothesis. Radiolabelled ligand-binding and unbiased proteomic approaches were applied to purified mitochondrial sub-fractions from rat liver, and the impact of AngII on mitochondrial function assessed. Whilst high-affinity AngII binding sites were found in the mitochondria-associated membrane (MAM) fraction, no RAS components could be detected in purified mitochondria. Moreover, AngII had no effect on the function of isolated mitochondria at physiologically relevant concentrations. We thus found no evidence of endogenous mitochondrial AngII production, and conclude that the effects of AngII on cellular energy metabolism are not mediated through its direct binding to mitochondrial targets.

Overactivity of the sympathetic nervous system has long been implicated in the hypertensive response to elevated angiotensin II (Ang II) levels. Although recent studies suggest that high dietary salt may alter cardiovascular responses to Ang II, direct evidence demonstrating chronic activation of sympathetic nerve activity is lacking. The objective of this study was to determine whether a low dose of Ang II, on a background of high salt intake, would result in a chronic increase in renal sympathetic nerve activity (RSNA). Arterial pressure and RSNA were recorded via telemetry. Two groups of rabbits were studied: 1 group drank a 0.9% NaCl solution and received Ang II (20 ng/kg per minute for 21 days, Salt+Ang), and the other drank tap water throughout and was not infused with Ang II (Control). In the Salt+Ang group, mean arterial pressure increased over the first week and remain elevated by 18.5±4.1 mm Hg at day 21. RSNA was not significantly different between groups on day 7 but was significantly elevated in the Salt+Ang group on day 21 (13.5±3.2% compared with 6.8±0.8% in the Control group; P<0.05). Baroreflex control of RSNA showed a rightward shift on day 21, but not day 7, and baroreflex responses indicated that RSNA could not be completely suppressed when arterial pressure was increased. No changes were observed in either mean arterial pressure or RSNA variables in the Control group. Our results support the hypothesis that elevated Ang II levels, in conjunction with a high salt diet, have the ability to chronically increase RSNA and, thus, potentially contribute to the maintenance of hypertension.

Intracerebroventricular (i.c.v.) administration of angiotensin IV (Ang IV) or Leu-Val-Val-haemorphin 7 (LVV-H7) improves memory performance in normal rats and reverses memory deficits in rat models for cognitive impairment. These memory effects were believed to be mediated via the putative 'AT4 receptor'. However, this binding site was identified as insulin-regulated aminopeptidase (IRAP). Correspondingly, Ang IV and LVV-H7 were characterised as IRAP inhibitors. This study investigates whether and how IRAP may be involved in the central effects of Ang IV and LVV-H7. We determined the effects of i.c.v. administration of Ang IV or LVV-H7 on hippocampal neurotransmitter levels using microdialysis in rats. We observed that Ang IV modulates hippocampal acetylcholine levels, whereas LVV-H7 does not. This discrepancy was reflected in the observation that Ang IV binds with micromolar affinity to the AT1 receptor whereas no binding affinity was observed for LVV-H7. Correspondingly, we demonstrated that the AT1 receptor is involved in the effects of Ang IV on hippocampal neurotransmitter levels and on spatial working memory in a plus maze spontaneous alternation task. However, the AT1 receptor was not involved in the spatial memory facilitating effect of LVV-H7. Finally, we demonstrated that Ang IV did not diffuse to the hippocampus following i.c.v. injection, suggesting an extrahippocampal site of action. We propose that AT1 receptors are implicated in the neurochemical and cognitive effects of Ang IV, whereas LVV-H7 may mediate its effects via IRAP.

Accumulation of specific deposits and extracellular molecules under the retinal pigment epithelium (RPE) has been previously observed in eyes with age-related macular degeneration (AMD) and may play a role in the pathogenesis of AMD. Even though age is the major determinant for developing AMD, clinical studies have revealed hypertension (HTN) as another systemic risk factor. Angiotensin II (Ang II) is considered the most important hormone associated with HTN. To evaluate the relationship of Ang II to AMD, we studied whether mouse RPE expresses functional Ang II receptor subtypes and whether HTN-induced Ang II regulates expression of these receptors as well as critical ECM molecules (MMP-2 and type IV collagen) involved in ECM turnover in RPE. We used 9-month-old C57BL/6 male mice infused with Ang II alone or Ang II in combination with the AT1 receptor antagonist candesartan or the AT2 receptor antagonist PD123319 for 4 weeks to determine whether HTN-associated Ang II was important for ECM regulation in RPE. We found that mouse RPE expressed both Ang II receptor subtypes at the mRNA and protein levels. Infusion with Ang II induced HTN and elevated plasma and ocular Ang II levels. Ang II also regulated AT1a and AT1b receptor mRNA expression, the intracellular concentration of calcium [Ca(2+)](i), MMP-2 activity, and type IV collagen accumulation. Concurrent administration of Ang II with the AT1 receptor blocker prevented the increase in blood pressure and rise in ocular Ang II levels, as well as the calcium and MMP-2 responses. In contrast, the type IV collagen response to Ang II was prevented by blockade of AT2 receptors, but not AT1 receptors. Plasma Ang II levels were not modified by the AT1 or AT2 receptor blockade. Since the effects of Ang II on MMP-2 and type IV collagen require inhibition of both Ang II receptor subtypes, these receptors may play a role as a potential therapeutic targets to prevent ECM turnover dysregulation in the RPE basement membrane, suggesting a pathogenic mechanism to explain the link between HTN and AMD.

A transient reduction in the barrier nature of the skin can be a pre-requisite for successful (trans)dermal delivery of some drugs. The aim of this present study was to investigate and effect of a dermal abrading "rotating brush" device on percutaneous absorption and skin integrity. In vitro experiments were conducted using excised human epidermal membrane. The effect of device parameters (bristle type, treatment duration and applied pressure) on skin permeability of model solutes (methyl paraben, butyl paraben, caffeine, acyclovir and angiotensin II) with varying physicochemical properties was examined and compared to established methods of skin penetration enhancement (positive controls). The device parameter which was found to have the most marked effect on permeability of the compounds was bristle type. Profound changes (2- to 100-fold increase) were observed in the epidermal permeability of the hydrophilic penetrants (caffeine, acyclovir and angiotensin II), when the brush device was employed compared to positive controls (ethanol enhancement, delipidisation, iontophoresis and tape-stripping). Findings from this present study support the effectiveness of a rotating brush applied to the skin in enhancing epidermal permeability. Further optimization of operational parameters is required to exploit this simple and effective delivery device.

Background: The response of proximal convoluted tubules (PCTs) to angiotensin II is mediated by specific type 1 receptors found on both apical and basolateral surface membrane cells. After ligand association with type 1 receptors, different signaling pathways are triggered and determine changes in fluid absorption (Jv). The presence of AT1 and actin cytoskeleton, which are directly related to Jv, can undergo changes in distribution based on the actions of AngII and losartan., Methods: Using a microperfusion technique and immunohistochemistry analysis, we investigated the basolateral action in PCTs, of AngII and/or losartan on Jv in rabbits, with regard to AT1 and actin cytoskeleton., Results: AngII increased Jv, while in contrast, losartan and combined AngII + losartan led to its decrease. AngII did not change fluorescence intensity of AT1 receptors on tubular membranes, while losartan and AngII + losartan demonstrated a slight increase after treatment. On the other hand, AngII increased the fluorescence intensity of actin cytoskeleton, while losartan induced a decrease. AngII + losartan led actin cytoskeleton having a higher fluorescence intensity than in the control group., Conclusions: In the present study, we demonstrated that treatment of the basolateral side of PCT cells with AngII and losartan could lead to changes in absorptive tubular function. Important alterations were detected in AT1 receptor fluorescence on the luminal and basolateral membranes, and changes in F-actin cytoskeleton were verified by fluorescence following these protocols.

Endothelium-derived steroidogenic factor (EDSF) is an endothelial peptide that stimulates aldosterone release from bovine adrenal zona glomerulosa (ZG) cells. The regulation of aldosterone release by combinations of EDSF and angiotensin II (AII) or EDSF and ACTH was investigated. Endothelial cells (ECs) and EC-conditioned media (ECCM) increased aldosterone release from ZG cells, an activity attributed to EDSF. AII (10(-12) to 10(-8) M) and ACTH (10(-12) to 10(-9) M) also stimulated the release of aldosterone from ZG cells. The stimulation by AII, but not ACTH, was greatly enhanced when ZG cells were coincubated with ECs. AII was metabolized by ECs to peptides identified by mass spectrometry as angiotensin (1-7) and angiotensin IV. There was very little metabolism of AII by ZG cells. Neither of these two AII metabolites altered aldosterone release from ZG cells, so they could not account for the enhanced response with ECs. AII-induced aldosterone release from ZG cells was enhanced by ECCM but not cell-free conditioned medium. This enhanced response was not due to increased EDSF release from ECs by AII. The synergistic effect of EDSF and AII was apparent when AII was added during or after the generation of ECCM and not observed when the AII component of the enhancement was blocked by the AII antagonist, losartan. These studies indicate that EDSF enhances the steroidogenic effect of AII. In the adrenal gland, ECs are in close anatomical relationship with ZG cells and may sensitize ZG cells to the steroidogenic action of AII by releasing EDSF.

We set out to confirm the long-held, but untested, assumption that dietary salt affects proximal reabsorption through reciprocal effects on the renin-angiotensin system in a way that facilitates salt homeostasis. Wistar rats were fed standard or high-salt diets for 7 days and then subjected to renal micropuncture for determination of single-nephron GFR (SNGFR) and proximal reabsorption. The tubuloglomerular feedback (TGF) system was used as a tool to manipulate SNGFR in order to distinguish primary changes in net proximal reabsorption (Jprox) from changes due to glomerulotubular balance. The influence of Ang II over Jprox was determined by the sensitivity of Jprox to the AT1 receptor antagonist, losartan. Plasma, whole kidneys, and fluid from midproximal tubules were assayed for Ang II content by radioimmunoassay. In rats on the standard diet, losartan reduced Jprox by 25% and reduced the maximum range of the TGF response by 50%. The high-salt diet suppressed plasma and whole-kidney Ang II levels. But the high-salt diet failed to reduce the impact of losartan on Jprox or the TGF response and actually caused tubular fluid Ang II content to increase. The persistent effect of Ang II on Jprox prevented a major rise in late proximal flow rate in response to the high-salt diet. These observations challenge the traditional model and indicate that the role of proximal tubular Ang II in salt-replete rats is to stabilize nephron function rather than to contribute to salt homeostasis.

The renal roles of physiologically relevant doses of angiotensin II (Ang II) and C-type natriuretic peptide (CNP) were investigated in the dogfish, Scyliorhinus canicula, using an in situ perfused trunk preparation. Perfusion with 10(-9) M Ang II resulted in a glomerular antidiuresis and decreases in perfusate flow rate, transport maxima for glucose and the proportion of filtering glomeruli. In addition, the renal clearances and excretion of urea, sodium, and chloride were significantly reduced, whereas the relative clearances of these parameters remained unchanged. In contrast, perfusion of 10(-9) M CNP caused a glomerular diuresis, an increase in transport maxima for glucose, but no significant change in the proportion of filtering glomeruli. In addition, the renal clearances of urea, sodium, and chloride were significantly increased but there was no effect on the relative clearances of urea, sodium, or chloride. Perfusion with 10(-10) M Ang II or CNP had no significant renal effects. Our results suggest that these hormones act at the level of the glomeruli rather than at a tubular level.

Background: The aim of the present study was to compare glomerular permeability alterations associated with experimental hypertension models known to have different effects on the circulating renin-angiotensin system (RAS)., Methods: Five groups, 10 animals each, were studied. One group served as a nonhypertensive control. The other four groups of hypertensive animals were composed of spontaneously hypertensive rats, deoxycorticosterone acetate hypertensive rats, Goldblatt two-kidney, one-clip rats, and a group of Wistar rats infused with angiotensin II (200 ng/kg/min). Tail-cuff sphygmomanometric systolic blood pressure (BP), albumin permeability determined in isolated glomeruli exposed to oncotic gradients (P(alb)), glomerular filtration rate (GFR, iopamidol method), plasma renin activity (PRA), and albuminuria were evaluated., Results: Alterations in P(alb) and albumin excretion rate were more evident in the experimental models with an activation of the RAS despite similar levels of systolic BP and GFR. A positive correlation was found between P(alb) and albuminuria (r = 0.51; P < .001) and between systolic BP and albuminuria (r = 0.37; P < .01). No relation was found between systolic BP and P(alb)., Conclusions: The present study indicates that the activation of the RAS plays a significant role in the development of glomerular albumin permeability defects in hypertensive models and may contribute to the mechanisms that lead to target organ damage in hypertension.

SSR149744C (SSR, 2-butyl-3-[4-[3-(dibutylamino)pro-pyl]benzoyl]-1-benzofuran-5-carboxylate isopropyl fumarate), is a new non-iodinated benzofuran derivative. The aim of this study was to evaluate in vivo its electrophysiological, hemodynamic, and anti-adrenergic properties and to determine its mechanism of action using in vitro studies. In chloralose-anesthetized dogs, SSR149744C (1-10 mg/kg i.v.) prolonged the sinus cycle length, A-H interval, Wenckebach cycle length, atrial effective refractory period (ERP), and atrio-ventricular node ERP in a dose-dependent manner without change of ventricular ERP and HV, QRS, or QTc intervals. Arterial blood pressure and ventricular inotropism were slightly decreased. SSR149744C, which has no or low affinity for alpha 1 and beta 1 adrenergic and angiotensin II AT1 receptors, reduced isoproterenol-induced tachycardia and phenylephrine- or angiotensin II-induced hypertension in anaesthetized dogs. In guinea pig papillary muscle, SSR149744C did not modify the resting potential, action potential amplitude and duration, but reduced the dV/dt max of the depolarization phase in a frequency-dependent manner. In isolated guinea pig cardiomyocytes and transfected CHO cells, SSR149744C (0.01-30 microM) inhibited several potassium currents: IKr (IC50 approximately 10 microM), IKs (IC50 approximately 30 microM), IK(ACh) (IC50 = 0.09 microM), and IKv1.5 (IC50 = 2.7 microM), the L-type calcium current: ICa(L) (IC50 approximately 5 microM) and also the amplitude of [Ca2+]i transient and cell shortening. Therefore, SSR149744C appears to have a multifactorial mechanism of action, which combines the blockade of several ion channels with the inhibition of responses of alpha 1 and beta 1 adrenergic as well as AT1 receptor stimulation. Like amiodarone, SSR149744C possesses the pharmacological effects of class I, II, III, and IV antiarrhythmic agents, which may confer upon this new drug a strong antiarrhythmic potential without ventricular proarrhythmia and iodine-related amiodarone-like side-effects.

The role of mineralocorticoids as sodium retaining hormones has been recently enlarged to include their function as modulators of cardiovascular function and injury. This study evaluated the contribution of possible functional changes in resistance vessels to the additional BP increase produced by the chronic administration of DOCA to SHR. The flow-pressure curve and renal responses to vasoconstrictors (phenylephrine [Phe] and angiotensin II [AII]) and vasodilators (acetylcholine [ACh] and nitroprusside [NP]) were characterized in isolated kidneys from Wistar Kyoto (WKY) and SHR treated or untreated with DOCA for nine weeks. DOCA increased BP in SHR but did not modify BP in WKY rats. Kidneys from SHR showed enhanced reactivity to Phe and AII that was not increased by DOCA. DOCA reduced sensitivity to AII in SHR. Responsiveness to ACh was increased in SHR and was not attenuated by DOCA in WKY or SHR. Vasodilator response to NP was not significantly affected by DOCA in WKY or SHR. The flow-pressure curve was markedly up-shifted in SHR when compared with kidneys from WKY rats. DOCA administration did not modify the flow-pressure curve in WKY but produced attenuation at low flow levels in SHR. Our results demonstrate that DOCA increases BP in SHR but does not increase the flow-pressure curve or renal vascular reactivity to vasoconstrictors, and does not reduce responsiveness to endothelium-dependent and independent vasodilators in SHR or WKY rats. Therefore, our data suggest that the BP increase produced by DOCA in SHR is not related to abnormalities in vascular function in resistance vessels.

The renin-angiotensin system (RAS), in addition to controlling blood pressure and the sodium-water balance, may be involved in cell growth and/or death in the brain. In order to address this issue, we compared the expression of RAS components in surgical specimens of human brain tumors and adjacent tissue. Human brain tumor cells and rat brain cells in culture were used to evaluate RAS functions. We found evidence that the RAS may be involved in maintaining the functions of the cerebral vasculature (the blood-brain-barrier) by controlling the ratio between angiotensin II and angiotensin III production, and by playing a more direct role in the survival of astroglial cells.

Oxidative stress is known to contribute to numerous cardiac disease processes. However, the contribution of reactive oxygen species to cardiac hypertrophy has not yet been fully investigated. The aim of the present study was therefore to determine whether levels of reactive oxygen species were increased in angiotensin II-induced cardiac hypertrophy. We continuously administered angiotensin II (1.1 mg/kg per day) into wild-type and angiotensin II type-1a receptor knockout mice for 2 weeks. The angiotensin II treatment increased blood pressure and heart weight/body weight ratio in wild-type mice but not in knockout mice. The generation of hydroxyl radicals in heart tissue homogenate was directly assessed with electron spin resonance spectroscopy using a spin trapping agent, alpha-phenyl-N-tert butylnitrone. Angiotensin II significantly increased hydroxyl radical production 2.2-fold (p < 0.01) in the hearts of wildtype mice but not in knockout mice. The present study provided direct evidence for increased production of hydroxyl radicals in angiotensin II-induced cardiac hypertrophy through angiotensin II type-1a receptor. These findings in this study may provide important insights into the development of hypertrophy and the transition of hypertrophy to heart failure.

Evidence of biological activity of fragments of ANG II is accumulating. Fragments considered being inactive degradation products might mediate actions previously attributed to ANG II. The study aimed to determine whether angiotensin fragments exert biological activity when administered in amounts equimolar to physiological doses of ANG II. Cardiovascular, endocrine, and renal effects of ANG II, ANG III, ANG IV, and ANG-(1-7) (6 pmol.kg-1.min-1) were investigated in conscious dogs during acute inhibition of angiotensin I-converting enzyme (enalaprilate) and aldosterone (canrenoate). Furthermore, ANG III was investigated by step-up infusion (30 and 150 pmol.kg-1.min-1). Arterial plasma concentrations [ANG immunoreactivity (IR)] were determined by an ANG II antibody cross-reacting with ANG III and ANG IV. Metabolic clearance rates were higher for ANG III and ANG IV (391 +/- 19 and 274 +/- 13 ml.kg-1.min-1, respectively) than for ANG II (107 +/- 13 ml.kg-1.min-1). ANG II increased ANG IR by 60 +/- 7 pmol/ml, blood pressure by 30%, increased plasma aldosterone markedly (to 345 +/- 72 pg/ml), and plasma vasopressin transiently, while reducing glomerular filtration rate (40 +/- 2 to 33 +/- 2 ml/min), sodium excretion (50 +/- 7 to 16 +/- 4 micromol/min), and urine flow. Equimolar amounts of ANG III induced similar antinatriuresis (57 +/- 8 to 19 +/- 3 micromol/min) and aldosterone secretion (to 268 +/- 71 pg/ml) at much lower ANG IR increments ( approximately 1/7) without affecting blood pressure, vasopressin, or glomerular filtration rate. The effects of ANG III exhibited complex dose-response relations. ANG IV and ANG-(1-7) were ineffective. It is concluded that 1) plasma clearances of ANG III and ANG IV are higher than those of ANG II; 2) ANG III is more potent than ANG II in eliciting immediate sodium and potassium retention, as well as aldosterone secretion, particularly at low concentrations; and 3) the complexity of the ANG III dose-response relationships provides indirect evidence that several effector mechanisms are involved.

Background: Angiotensin II (Ang II)-dependent hypertension is associated with augmented intrarenal concentrations of Ang II; however, the distribution of the increased intrarenal Ang II has not been fully established., Objective: To determine the changes in renal interstitial fluid Ang II concentrations in Ang II-induced hypertension and the consequences of treatment with an angiotensin II type 1 (AT1) receptor blocker., Design and Methods: Rats were selected to receive vehicle (5% acetic acid subcutaneously; n = 6), Ang II (80 ng/min subcutaneously, via osmotic minipump; n = 7) or Ang II plus an AT1 receptor antagonist, candesartan cilexetil (10 mg/kg per day, in drinking water; n = 6) for 13-14 days, at which time, experiments were performed on anesthetized rats. Microdialysis probes were implanted in the renal cortex and were perfused at 2 microl/min. The effluent dialysate concentrations of Ang I and Ang II were measured by radioimmunoassay and reported values were corrected for the equilibrium rates at this perfusion rate., Results: Ang II-infused rats developed greater mean arterial pressures (155 +/- 7 mmHg) than vehicle-infused rats (108 +/- 3 mmHg). Ang II-infused rats showed greater plasma (181 +/- 30 fmol/ml) and kidney (330 +/- 38 fmol/g) Ang II concentrations than vehicle-infused rats (98 +/- 14 fmol/ml and 157 +/- 22 fmol/g, respectively). Renal interstitial fluid Ang II concentrations were much greater than plasma concentrations, averaging 5.74 +/- 0.26 pmol/ml in Ang II-infused rats - significantly greater than those in vehicle-infused rats (2.86 +/- 0.23 pmol/ml). Candesartan treatment prevented the hypertension (87 +/- 3 mmHg) and led to increased plasma Ang II concentrations (441 +/- 27 fmol/ml), but prevented increases in kidney (120 +/- 15 fmol/g) and renal interstitial fluid (2.15 +/- 0.12 pmol/ml) Ang II concentrations., Conclusions: These data indicate that Ang II-infused rats develop increased renal interstitial fluid concentrations of Ang II, which may contribute to the increased vascular resistance and reduced sodium excretion. Furthermore, the augmentation of renal interstitial fluid Ang II is the result of an AT1 receptor-mediated process and can be dissociated from the plasma concentrations.

The inhibitory effect of lipopolysaccharides (LPS) on alpha-adrenergic contraction is quite well known, but molecular mechanism of this inhibition is unclear. In the present study, the interaction between alpha-adrenoceptor and vasopressin receptor response, and LPS in rat tail artery was investigated using chemical stimulation. In the presence of LPS, noradrenaline, phenylephrine and arginine-vasopressin, concentration-response curves (CRCs) were shifted to the right with the change in maximal responses. The K(A) and K(B) values calculated in the presence and absence of LPS did not differ significantly. The results strongly suggest that LPS did not change the receptors affinity. The changes in the relationship between receptor occupancy and response to an agonist in the presence of LPS and reduction of K(A)/ED(50) value suggest reduction of receptor reserve. In the presence of angiotensin II (Ang II), CRCs were shifted to the right with significant increase in receptor reserve. Moreover, this effect was still present in LPS-pretreated arteries. The receptor reserve reduced by LPS significantly increased in the presence of Ang II. It suggests that inhibitory effect of LPS is partially reversible. The results strongly suggest that in early endotoxemia, inhibitory effect of LPS may by partially reverted by an increase in activity of renin-angiotensin-aldosterone system.

Background: Angiotensin II is a potent vasoconstrictor that is elevated after shock. Previous studies suggest that angiotensin II may directly modulate the endothelial barrier. Our hypothesis was that angiotensin II would increase microvascular hydraulic permeability in a dose-dependent fashion., Methods: Hydraulic permeability (Lp) is a measure of water flow across the endothelial barrier. Lp was measured in rat mesenteric venules using the modified Landis micro-occlusion technique. Venules were first perfused with Ringer's solution and baseline measurements of Lp were obtained. The venules were then recannulated and perfused with angiotensin II at 0.2 ng/mL (n = 5), 2.0 ng/mL (n = 5), 20 ng/mL (n = 8), and 200 ng/mL (n = 5), before final Lp measurements., Results: Baseline values for Lp averaged 1.35 +/- 0.12. The 20-ng/mL and 200-ng/mL concentrations of angiotensin II significantly increased Lp to 3.86 +/- 0.4 (p < 0.0008) and 7.94 +/- 1.1 (p < 0.005), respectively. The maximal effect of angiotensin II was seen at 15 minutes of perfusion. Units for Lp are x 10(-7) cm.s-1.cm H2O-1., Conclusion: Angiotensin II affects a dose-dependent increase in microvascular permeability. This suggests that angiotensin II is involved in modulating intravascular fluid flux across the vessel wall. This effect is opposite to that observed in other vasoconstrictors that are up-regulated after trauma.

The dopamine D(2)-like receptor agonist quinpirole has been reported to lower blood pressure. This effect appears to be mediated via activation of presynaptic D(2)-like receptors inhibiting the stimulated neural norepinephrine release. The aim of the present study was to investigate the role of renal nerves and the renin-angiotensin system (RAS) in the blood pressure lowering effect of quinpirole. Therefore, clearance experiments using different doses of quinpirole (0.3 to 100 microg/kg/min) were performed in thiopental-anesthetized rats with intact kidneys (INN) or 5 to 7 days after bilateral renal denervation (DNX). The functional involvement of the RAS in the blood pressure lowering effect of quinpirole was determined in rats pretreated with a subpressor dose of angiotensin II (10 microg/kg/min) or in rats pretreated with the angiotensin II (AT(1)) receptor antagonist losartan, in a subdepressor dose (10 microg/kg/min). Quinpirole dose-dependently decreased mean arterial blood pressure (MAP) by up to 29%. This blood pressure lowering effect of quinpirole was observed at lower doses in DNX rats when compared with INN animals (ED(50): 0.98 microg/kg/min in DNX vs. 6.02 microg/kg/min in INN animals). Quinpirole in a dose of 3 microg/kg/min, which did not affect MAP in vehicle treated INN rats, significantly reduced MAP in rats with losartan pretreatment. In DNX rats pretreated with angiotensin II the MAP-response to the infusion of 3 microg/kg/min quinpirole was clearly attenuated in comparison with untreated DNX animals. Our data show that stimulation of dopamine D(2)-like receptors dose-dependently decreased blood pressure, which was potentiated by both interruption of the renal innervation and AT(1) receptor blockade, while exogenous ANG II restored the enhancement of the blood pressure response to quinpirole. We conclude that the increased vasodilatory effect of quinpirole after renal denervation might depend on a decreased activity of the RAS.

We have examined the binding distribution of a selective AT(2) receptor ligand [125I] CGP42112 in the brain of adult Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). AT(2) receptor localization was also examined in the rat brainstem following unilateral nodose ganglionectomy. Specific [125I] CGP42112 binding was observed in discrete brain regions from both rat strains, including the nucleus of the solitary tract (NTS), and did not differ between WKY and SHR. [125I] CGP42112 binding in the NTS revealed an AT(2) receptor component that was displaceable by PD 123319 and Ang II (50-58%), as well as a non-angiotensin II receptor component (42-49%). Following unilateral nodose ganglionectomy, [125I] CGP42112 binding density on the denervated side of the NTS was increased approximately two-fold in both WKY and SHR. This increased [125I] CGP42112 binding density in the ipsilateral NTS was comprised of a greater non-angiotensin II component than that observed in the sham groups, since only approximately 30% was displaced by PD123319 and angiotensin II. Furthermore, [125I] CGP42112 also revealed high binding density on the denervated side in the dorsal motor nucleus and the nucleus ambiguus in both WKY and SHR. AT(2) receptor immunoreactivity was also visualised in the NTS of sham operated rats, but was not observed in the dorsal motor nucleus or the nucleus ambiguus, nor was it up-regulated following nodose ganglionectomy. These results demonstrate, for the first time, an AT(2) receptor binding site in the NTS, as well as a non-angiotensin II [125I] CGP42112 binding site. These studies also demonstrate that nodose ganglionectomy represents a useful model in which to study a non-angiotensin II [125I] CGP42112 binding site that is up-regulated following degeneration of afferent vagal nerves.

Acute experimental iron loading causes iron to accumulate in the renal tissue. The accumulation of iron may play a role in enhancing oxidant-induced tubular injury by producing increased amounts of reactive oxygen species. From findings in cells from heme oxygenase-1 (HO-1)-deficient mice, HO-1 is postulated to prevent abnormal intracellular iron accumulation. Recently, it has been reported that HO-1 is induced in the renal tubular epithelial cells, in which iron is deposited after iron loading, and that this HO-1 induction may be involved in ameliorating iron-induced renal toxicity. We previously showed that chronic administration of angiotensin II to rats induces HO-1 expression in the tubular epithelial cells. These observations led us to investigate whether there is a link between iron deposition and HO-1 induction in renal tubular cells in rats undergoing angiotensin II infusion. In the present study, rats were given angiotensin II for continuously 7 days. Prussian blue staining revealed the distinct deposits of iron in the proximal tubular epithelial cells after angiotensin II administration. Electron microscopy demonstrated that iron particles were present in the lysosomes of these cells. Histologic and immunohistochemical analyses showed that stainable iron and immunoreactive ferritin and HO-1 were colocalized in the tubular epithelial cells. Treatment of angiotensin II-infused rats with an iron chelator, deferoxamine, blocked the abnormal iron deposition in kidneys and also the induced expression of HO-1 and ferritin expression. Furthermore, deferoxamine treatment suppressed the angiotensin II-induced increase in the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase, a marker of tubular injury; however, deferoxamine did not affect the angiotensin II-induced decrease in glomerular filtration rate. These results suggest that angiotensin II causes renal injury, in part, by inducing the deposition of iron in the kidney.

Hypertension induced by long-term infusion of angiotensin II (Ang II) is associated with augmented intrarenal Ang II levels to a greater extent than can be explained on the basis of the circulating Ang II levels. Although part of this augmentation is due to AT(1) receptor-dependent internalization, the intracellular compartments involved in this Ang II accumulation remain unknown. In the present study, we sought to determine whether Ang II trafficking into renal cortical endosomes is increased during Ang II hypertension, and if so, whether the AT(1) receptor antagonist, candesartan, prevents this accumulation. Compared with controls (n=12; 114+/-2 mm Hg), Ang II-infused rats (n=12; 80 ng/kg/min, SC, for 13 days) developed hypertension with systolic blood pressure rising to 185+/-4 mm Hg by Day 12. In Ang II hypertensive rats, plasma renin activity was suppressed, whereas plasma and kidney Ang II levels were increased by 3-fold (348+/-58 versus 119+/-16 fmol/mL) and 2-fold (399+/-39 versus 186+/-26 fmol/g). Intracellular endosomal Ang II levels were increased by more than 10-fold (1100+/-283 versus 71+/-12 fmol/mg protein), whereas intermicrovillar cleft Ang II levels were increased by more than 2-fold (88+/-22 versus 37+/-7 fmol/mg protein). Flow cytometric analysis detected significant increases in AT(1A) receptor antibody binding in endosomal and intermicrovillar clefts of Ang II-infused rats. The hypertension induced by Ang II was prevented in rats treated concurrently with candesartan (2 mg/kg/d, 119+/-3 mm Hg). Candesartan treatment (n=8) also prevented increases in kidney (215+/-19 fmol/g), endosomal (96+/-29 fmol/mg protein), and intermicrovillar cleft Ang II levels (11+/-2 fmol/mg protein). These results indicate that there is substantial intracellular accumulation of angiotensin peptides in renal cortical endosomes during Ang II-dependent hypertension via an AT(1) receptor-mediated process.

A non-needle syringe (jet injector) was utilized to increase skin permeation of drugs by iontophoresis. Briefly, physiological saline was initially flushed by the injector to make a pore in the stratum corneum of excised hairless rat skin, and the iontophoretic skin permeation of two model compounds, sodium diclofenac and angiotensin II, was followed using a 2-chamber diffusion cell. Constant voltage and constant current iontophoresis treatments were evaluated. Pretreatment using the jet injector alone resulted in about 13- and 22-fold increases in the steady-state flux of diclofenac and angiotensin II, respectively, through the skin, compared with non-treated controls. Jet injector pretreatment with constant voltage iontophoresis further enhanced skin permeation of diclofenac and angiotensin II, and the enhancement was also greater than that by constant voltage iontophoresis alone. Thus, a synergistic effect was observed. The ratio of enhancement was greater compared with the control. Jet injector pretreatment with constant current iontophoresis, however, did not always yield higher skin permeation of the drugs than injector pretreatment alone, although the lag time was shortened. The difference in the enhancement between the constant voltage- and constant current iontophoresis can be explained by the electric current through the excised skin. Constant current iontophoresis after a short period of constant voltage iontophoresis with multiple jet injector pretreatments may be the best way to increase drug permeability while preventing severe skin damage.

Selective competitive angiotensin AT(1) receptor antagonists exhibit diverse patterns of antagonism of angiotensin-II-mediated responses in functional assays. These range from the classical parallel rightward shift of agonist concentration-response curves with no depression of the maximum response to an apparently straightforward insurmountable antagonism with complete depression of the maximum response and no rightward shift. This article reviews some earlier equilibrium-based models that have been used to explain the insurmountable antagonism, and suggests that a kinetic model might provide a more satisfactory account of the observations. Such a model might provide deeper insights into the pharmacology of G-protein-coupled receptors than the more popular equilibrium models.

Recent studies have characterized a specific binding site for the C-terminal 3-8 fragment of angiotensin II (Ang IV). In the present study we looked at the internalization process of this receptor on bovine aortic endothelial cells (BAEC). Under normal culture conditions, BAEC efficiently internalized (125)I-Ang IV as assessed by acid-resistant binding. Internalization of (125)I-Ang IV was considerably decreased after pretreatment of cells with hyperosmolar sucrose or after pretreatment of BAEC with inhibitors of endosomal acidification such as monensin or NH(4)Cl. About 50% of internalized (125)I-Ang IV recycled back to the extracellular medium during a 2 h incubation at 37 degrees C. (125)I-Ang IV remained mostly intact during the whole process of internalization and recycling as assessed by thin layer chromatography. As expected, internalization of (125)I-Ang IV was completely abolished by divalinal-Ang IV, a known AT(4) receptor antagonist. Interestingly, (125)I-divalinal-Ang IV did not internalize into BAEC. These results suggest that AT(4) receptor undergoes an agonist-dependent internalization and recycling process commonly observed upon activation of functional receptors.

Studies have been performed to assess the possibility of using small unilamellar liposomes as therapeutic carriers to the brain of hypertensive rats. Rats were made temporal hypertensive by the infusion of angiotensin II (AII; 15 micrograms in 1 ml) through their right common carotid artery. Another control group was infused with physiological saline. Free 125iodine-BSA (125I-BSA) and 125I-BSA encapsulated liposomes (average diameter approximately equal to 100 nm) were injected in the tail vein 2 min after the infusion of AII or saline. Plasma radioactivity was monitored at different times up to 15 min when the cerebral uptake of 125I-BSA was determined. While a little variation in plasma clearance pattern of liposomes in hypertensive and control group was noticed, the uptake by cerebral tissues was markedly higher in hypertensive group. Analysis of pharmacokinetic parameters in relation to cerebral uptake indicated AII induced a short term opening of the blood-brain barrier (BBB) resulting in an increased cerebral uptake. Positively charged liposomes was found to be most effective in hypertensive state.

Background: Angiotensin II has been found to be a growth stimulating factor for myocardial cells. In humans, angiotensin II infusion causes vasoconstriction in systemic and renal vasculature and leads to aldosterone secretion. Our hypothesis was that hyper-responsiveness to angiotensin II is related to left ventricular mass in human essential hypertension., Methods and Results: In 30 normotensive individuals and 30 subjects with mild essential hypertension (white men, mean age 26+/-3 years), the responsiveness to angiotensin II was assessed by measuring changes in mean arterial pressure, renal blood flow, glomerular filtration rate and aldosterone secretion in response to i.v. angiotensin II infusion (0.5 and 3.0 ng/kg per min). The provoked changes to angiotensin II infusion were similar in the normotensive and hypertensive group with the exception of an exaggerated increase in mean arterial pressure in hypertensives (14+/-5 versus 10+/-5 mm Hg, P<0.001 at 3.0 ng/kg per min angiotensin II). The increase in mean arterial pressure was correlated with left ventricular mass in hypertensive subjects (angiotensin II 0.5 ng/kg per min: r = 0.49, P<0.005; angiotensin II 3.0 ng/kg per min: r = 0.35, P<0.05); no such correlation was found in the normotensive group. After taking into account baseline mean arterial pressure and body mass index, the increase in mean arterial pressure to angiotensin II 0.5 ng/kg per min was still correlated with left ventricular mass (partial r = 0.50, P<0.01). Similarly, the change of glomerular filtration rate but not of renal blood flow in response to angiotensin II 0.5 ng/kg per min was correlated with left ventricular mass, (r = 0.42, P<0.02) in the hypertensive group but not in the normotensive one. This relationship remained significant even after taking baseline glomerular filtration rate, mean arterial pressure and body mass index into account (partial r = 0.43, P<0.05)., Conclusion: Hyper-responsiveness to angiotensin II is related to an increased left ventricular mass in hypertensive subjects independent of blood pressure.

Angiotensin (Ang) type 1a (AT1a) receptors are critical in the control of blood pressure and water balance. Experiments were performed to determine the influence of dehydration on brain Ang receptors and plasma vasopressin (VP) in mice lacking this receptor. Control or AT1a knockout (AT1aKO) male mice were give water ad libitum or deprived of water for 48 hours. Animals were anesthetized with halothane, blood samples were collected by heart puncture, and brains were processed for Ang-receptor autoradiography with 125I-sarthran (0.4 nmol/L). Dehydration produced an increase in AT1 receptors in the paraventricular nucleus (PVN) and anterior pituitary (AP) in control mice (PVN: 70+/-16 versus 146+/-10 fmol/mg protein; AP: 41+/-7 versus 86+/-15 fmol/mg protein). No changes were noted in the median preoptic nucleus. The majority of the brain receptors were of the AT1 subtype. There was little or no specific Ang binding in AT1aKO mice and no effect of dehydration. Plasma VP levels were elevated in the halothane-anesthetized animals (>200 pg/mL) with no significant effect of dehydration. A separate experiment was performed with decapitated mice anesthetized with pentobarbital. Dehydration increased plasma VP in control mice, from 3.3+/-0.6 to 13.3+/-4.7 pg/mL, whereas no change was noted in the AT1aKO mice, 5.1+/-0.3 versus 6.1+/-0.7 pg/mL (water versus dehydration). These results demonstrate a differential response to dehydration in mice lacking AT1a receptors. There was no evidence for AT1 receptors of any subtype in the brain regions examined and no effect of dehydration on VP secretion or brain Ang receptors.

We determined the mechanism accounting for the removal and metabolism of angiotensin-(1-7) [Ang-(1-7)] in 21 anesthetized spontaneously hypertensive (SHR), 18 age-matched normotensive Sprague-Dawley (SD), and 36 mRen-2 transgenic (TG+) rats. Animals of all 3 strains were provided with tap water or tap water containing losartan, lisinopril, or a combination of lisinopril and losartan for 2 weeks. On the day of the experiment, Ang-(1-7) was infused for a period of 15 minutes at a rate of 278 nmol . kg-1 . min-1. After this time, samples of arterial blood were collected rapidly at regular intervals for the assay of plasma Ang-(1-7) levels by radioimmunoassay. Infusion of Ang-(1-7) had a minimal effect on vehicle-treated SD rats but elicited a biphasic pressor/depressor response in vehicle-treated SHR and TG+ rats. In lisinopril-treated rats, Ang-(1-7) infusion increased blood pressure, whereas losartan treatment abolished the pressor component of the response without altering the secondary fall in arterial pressure. Combined treatment with lisinopril and losartan abolished the cardiovascular response to Ang-(1-7) in all 3 strains. In vehicle-treated SD, SHR and TG+ the half-life (t1/2) of Ang-(1-7) averaged 10+/-1, 10+/-1, and 9+/-1 seconds, respectively. Lisinopril alone or in combination with losartan produced a statistically significant rise in the half-life of Ang-(1-7) in all 3 strains of rats. Plasma clearance of Ang-(1-7) was significantly greater in the untreated SD rats compared with either the SHR or TG+ rat. Lisinopril treatment was associated with reduced clearance of Ang-(1-7) in all 3 strains. Concurrent experiments in pulmonary membranes from SD and SHR showed a statistically significant inhibition of 125I-Ang-(1-7) metabolism in the presence of lisinopril. These studies showed for the first time that the very short half-life of Ang-(1-7) in the circulation is primarily accounted for peptide metabolism by ACE. These findings suggest a novel role of ACE in the regulation of the production and metabolism of the two primary active hormones of the renin angiotensin system.

Previous studies have demonstrated that augmentation of intrarenal angiotensin II (ANG II) levels during ANG II induced hypertension involves both endogenous formation and accumulation of circulating ANG II. The present work extends these findings and determines whether accumulation of infused ANG II in the kidney requires AT1 receptor activation by using Val5-ANG II as the infused peptide. Male Sprague-Dawley rats were uninephrectomized and divided into three groups: control (n = 6), Val5-ANG II (exogenous form) infused (n = 8), and Val5-ANG II infused rats treated with losartan (n = 8). Val5-ANG II, which has the same biological and immunoreactive properties as endogenous ANG II, was infused at 40 ng/min via an osmotic minipump implanted subcutaneously. By day 12, systolic blood pressure (SBP) increased significantly in Val5-ANG II infused rats (197 +/- 7 mm Hg). As previously shown, the development of hypertension in ANG II infused rats was prevented by losartan treatment. Blood and kidney samples were harvested, subjected to HPLC to separate Val5-ANG II (exogenous) from Ile5-ANG II (endogenous) and the fractions were measured by radioimmunoassay. In the Val5-ANG II infused rats treated with losartan, total plasma ANG II levels were elevated to a greater extent than in rats not treated with losartan (289 +/- 20 v 119 +/- 14 fmol/mL). However, losartan markedly decreased by 88% the enhancement of intrarenal Val5-ANG II content that occurred in the rats infused with Val5-ANG II alone. These results demonstrate that AT1 receptor blockade markedly reduces the intrarenal uptake of circulating ANG II that occurs in ANG II induced hypertension.

This study assessed the role of angiotensin (Ang) AT1 and AT2 receptors as modulators of the plasma clearance of Ang II. Groups of male spontaneously hypertensive rats (SHRs; n = 25) were given an intravenous injection of either saline, losartan, PD123319, losartan in combination with PD123319, or Sar1-Thr8-Ang II. One hour later, Ang II (0.5 microg/kg) was infused for 15 min into a vein. Immediately thereafter, arterial blood samples were collected at regular intervals for the assay of plasma Ang II levels by radioimmunoassay. The infusion of Ang II significantly increased baseline mean arterial pressure (MAP) in rats pretreated with either saline or PD123319 but not in those receiving losartan, losartan combined with PD123319, or Sar1-Thr8-Ang II. The plasma clearance of Ang II was significantly greater in rats injected with either PD123319, losartan combined with PD123319, or Sar1-Thr8-Ang II compared to those injected either saline or losartan. Furthermore, the half-life of Ang II in rats pretreated with saline or losartan was significantly greater than that measured in the other three groups. These results suggest that plasma clearance of Ang II in the SHRs is independent of an AT1 receptor, but plasma levels of the peptide are unexpectedly protected by an AT2 receptor-dependent mechanism.

We conducted studies in conscious chronically catheterized, trained young (3-5 months) and old (18-20 months) rats to assess the impact of aging on baseline renin activity (PRA) and metabolic clearance rate (MCR) of angiotensin II (ANG II). We observed that under unstressed conditions the baseline values of PRA and plasma ANG II were no different in young versus old rats (1.8 +/- 0.2 versus 1.5 +/- 0.2 ng Al/ml/h and 18 +/- 3 versus 15 +/- 2 fmol/ml, respectively). Values of PRA in the present study were similar to those reported by others for old rats, but our young rat values were lower than usually reported. This probably reflects our use of an unstressed preparation. We also observed a blunted increase in PRA in old rats in response to acute converting enzyme inhibition. Overall, our observations suggest that old rats may lose their ability to increase PRA in response to acute stimuli, including perhaps, the stress of blood drawing in emotionally or surgically stressed preparations. We also observed that the MCR of ANG II increased with age, despite similar baseline plasma ANG II concentrations in young and old. This suggests that with aging, an increase occurs in the rate of synthesis of ANG II. These results emphasize the importance of establishing true baseline values for indices of the renin-ANG II system in aging.

We have designed and tested specific peptide linkers for the glutathione-mediated reductive release of the angiotensin II analog N-acetyl-CGDKVYIHPF attached to recombinant human hemoglobin mutants by a disulfide bond. Inclusion of negatively charged residues decreased the rate of release by as much as 5-fold for the N-terminal linker DCD when compared to that of the control linker CG. Two different surface cysteine mutants in the second domain of the alpha,alpha-chain of recombinant human hemoglobin, D75C and K16C, were examined for their effect on the release of peptide by reduced glutathione. The reaction of the D75C-peptide conjugate with glutathione for release of the peptide is slow, with a second-order rate constant of 2.1 M-1 S-1, allowing the possibility of long term delivery. The rate of peptide release from the K16C vs the D75C mutant was decreased 15-fold. Thus, different peptide release rates can be obtained by changing both peptide linker residues and the surface location of peptide attachment. Kinetic modeling of this release using either measured or literature values for different parameters suggests boundary conditions for application to the in vivo release of peptidomimetics, small molecules, or other drugs bound to the hemoglobin surface.

This study was designed to assess in normal volunteers the potency, efficacy, and tolerability of the new nonpeptidic, orally active, angiotensin (Ang) II subtype 1 (AT1)-receptor antagonist SC-52458. After a randomized, single-blind, placebo-controlled protocol, two groups of eight healthy men ingested placebo or increasing single oral doses (10, 25, and 50 mg or 100, 150, and 200 mg) of SC-52458. Finger blood pressure (BP) was continuously monitored (Finapres), and BP response to repeated intravenous challenges with Ang II was compared with baseline BP response to the same dose of Ang II. Up to 24 h after drug intake, effects on plasma renin activity (PRA), Ang II, and aldosterone and pharmacokinetics were estimated. One, 4, and 10 h after the 200-mg dose, diastolic BP response to Ang II challenges was decreased from 30.3 to 2.6 mm Hg (mean +/- SEM; n = 8; i.e., to 8.3 +/- 1.1% of baseline response), 10.1 mm Hg (35.4 +/- 1.8%), and 17.5 mm Hg (58.7 +/- 1.8%), respectively. SC-52458 produced dose-related increases in PRA and Ang II concentrations < or = 10 h after drug intake. Plasma aldosterone concentrations tended to be decreased for < or = 24 h after SC-52458 doses of > or = 100 mg. No drug-related side effects were observed. The pharmacokinetics were linear over the dose range of 10-150 mg (t1/2 = 1.14-2.39 h). Efficacy was dose dependent, with a peak effect after 1 h. In conclusion, the novel AT1-receptor antagonist SC-52458 is well tolerated and orally active. It produces a rapid-onset inhibition of the renin-angiotensin system and reduces BP response to Ang II for > or = 10 h. These characteristics promise strong antihypertensive properties for SC-52458.

This study examined the effect of N-ethylmaleimide (NEM, 10(-7) M) on agonist-induced contraction and the relaxation following drug-washout, of vascular smooth muscle (VSM) segments derived from hypertensive rabbits. Mean blood pressure increase was produced either by renal constriction plus contralateral nephrectomy, or by cadmium acetate ingestion. Freely-ionized calcium (45Ca)flux, cyclic 3':5'-guanosine monophosphate (cGMP), and cyclic 3':5' adenosine monophosphate (cAMP), were analyzed. NEM was used as a stereoselective probe to clarify the role of sulfhydryl (SH) groups in hypertension. Contractile response to norepinephrine (NE, 5.9 x 10(-7) M), angiotensin II (AT, 9.8 x 10(-8) M), and potassium chloride (KCl, 2.2 x 10(-2) M) were significantly depressed in hypertensive tissue. Exposure to NEM, before agonist challenge, caused an even greater depression in contractile response. As for the normotensive group, an inhibition of relaxation occurred when NEM was added after the development of a maximal contractile response to NE, AT or KCl. Changes in contractile ability and in relaxation were attributed to specific alterations in calcium distribution. These alterations were examined by 45Ca washout components and were related to cAMP and cGMP metabolism. These results suggest a regulatory role of SH groups in contraction and relaxation and a modification of this role in hypertension.

To quantify the renal excretion of angiotensin II (AII) in birds, synthetic AII was intravenously infused into conscious Pekin ducks at rates of 5, 15 and 45 ng/kg/min for 30 min and the relationships between plasma and urine AII concentrations were monitored by radioimmunoassay. The infusions of AII produced dose-dependent elevations in the plasma concentrations of the hormone, however, urinary concentrations and excretion rates did not change significantly from the basal values of 11.6 +/- 1.2 pg/ml and 4.3 +/- 1.0 pg/min, respectively. The metabolic clearance rate of AII remained constant at each infusion dose (approximately 120 ml/min/kg). The urinary clearance rates (< 0.3 ml/min/kg) indicated that the fraction of AII cleared from the blood via the urine was less than 0.2%. Clearly, excretion of intact AII via this route is not a quantitatively important means of elimination for this hormone in birds.

Background: The presence of mRNA for the essential components of the renin-angiotensin system (RAS) has been found in animal and human hearts. The present study was designed to provide evidence for the existence of a (functional) cardiac RAS., Methods and Results: Twenty-four patients with atypical chest pain undergoing coronary angiography for diagnostic purposes were investigated. The cardiac production rate of angiotensins was estimated by measurement of the cardiac extraction of 125I-angiotensin I and 125I-angiotensin II associated with the determination of endogenous angiotensins in aortic and coronary sinus blood in normal, low, or high sodium diets. In a normal sodium diet, angiotensin I and II aorta-coronary sinus gradients were tendentially negative (-1.8 +/- 2.5 and -0.9 +/- 1.7 pg/mL, respectively), and the amounts of angiotensin I and II added by cardiac tissues were 6.5 +/- 3.1 and 2.7 +/- 1.3 pg/mL, respectively. The low sodium diet caused a significant increase in both plasma renin activity (PRA) and angiotensin I concentration in aortic but not in coronary sinus blood, resulting in a more negative aorta-coronary sinus gradient (-9.7 +/- 3.1 pg/mL, P < .01). Angiotensin formation by PRA in blood during transcardiac passage increased (P < .001), whereas angiotensin I formed by cardiac tissues decreased dramatically. Accordingly, in the low sodium diet, 125I-angiotensin II extraction did not change, the cardiac fractional conversion rate of 125I-angiotensin I to 125I-angiotensin II notably decreased (P < .01), and angiotensin II formation by cardiac tissues was undetectable. The high sodium diet caused a decrease in PRA and no changes in cardiac extraction of radiolabeled angiotensins; conversely, angiotensin I formed by cardiac tissues, cardiac Ang I fractional conversion rate, and angiotensin II formed during transcardiac passage significantly (P < .01 for all) increased., Conclusions: These results provide evidence for the existence of a functional cardiac RAS independent of but related to the circulating RAS.

Because angiotensin II (Ang II) has been found at high concentrations in the proximal tubule fluid and because tubular brush border membranes exhibit a marked capacity for degrading Ang II, we thought it of interest to examine the binding sites for Ang II (3-8) (referred to as Ang IV), a metabolite of Ang II, downstream in the nephron. We studied the binding of [125I]-Ang IV and also of [125I]-Sar1, Ala8, Ang II to SV-40 transformed human collecting duct cell (HCD) membranes. No specific binding site for [125I]-Sar1, Ala8, Ang II and no Ang II-dependent cytosolic calcium response could be observed. Moreover, no signal for the human type I Ang II receptor (hAT1) mRNA was present in HCD cells. In contrast, [125I]-Ang IV bound specifically to HCD cell membranes. Mean Kd and Bmax values derived from saturation binding studies were 5.6 +/- 2.0 nM and 1007.6 +/- 140.2 fmol/mg protein, respectively. The rank order of affinity for competitive Ang II-related peptides was: Ang IV > Ang III > Ang II > Ang II (4-8) > Ang II (1-7). [125I]-Ang IV binding was not modified by nonpeptide AT1 (losartan) or AT2 (PD123177) antagonists. GTP gamma S and dithiotreitol did not affect [125I]-Ang IV binding. Ang IV stimulated cAMP production by intact HCD cells in the presence of forskolin but did not modify cGMP production or cytosolic calcium concentration. Taken together, these results indicate that HCD cells represent a target site for Ang IV but do not possess Ang II receptors.

Chronic low-dose angiotensin II (Ang II) infusion for 13 days mimics two-kidney, one clip Goldblatt hypertension and increase intrarenal Ang II levels. We performed studies to determine the time course for the enhancement of intrarenal Ang II levels and whether the increased intrarenal Ang II is a tissue-specific event and requires a receptor-mediated step. Male Sprague-Dawley rats were uninephrectomized, and either vehicle or Ang II (40 ng/min) was infused via a subcutaneous osmotic minipump. Plasma and renal Ang II levels were measured 3, 7, 10, and 13 days after minipump implantation. Compared with controls (126 +/- 2 mm Hg), systolic pressure in Ang II-infused rats exhibited a detectable increase by day 6 (146 +/- 2 mm Hg) and continued to increase to 189 +/- 5 mm Hg by day 12. Plasma Ang II levels were elevated by day 3, whereas intrarenal Ang II levels were not significantly elevated until 10 days of Ang II infusion. Renal injury characterized by focal and segmental glomerulosclerosis was evident after 13 days of Ang II infusion. Losartan (30 mg/kg per day) prevented the development of hypertension in the Ang II-infused rats for the duration of the infusion period (125 +/- 1 mm Hg) and reduced the degree of glomerular injury. Plasma renin activity was suppressed in the Ang II-infused group but was elevated markedly in both losartan-treated groups. Plasma Ang II levels were elevated in the Ang II-infused rats and were even higher during losartan treatment. Intrarenal Ang II levels were enhanced significantly (354 +/- 60 versus 164 +/- 23 fmol/g) in the Ang II-infused rats. However, losartan treatment prevented the augmentation of intrarenal Ang II caused by Ang II infusion. Heart and adrenal Ang II levels were not significantly increased in the Ang II-infused rats but were significantly elevated during losartan treatment. These results suggest that the tissue-specific elevations of intrarenal Ang II levels caused by chronic Ang II infusion are mediated by angiotensin type 1 receptor activation, which leads to either receptor-mediated internalization of Ang II, enhancement of intrarenal Ang II formation, or both.