Hypertension and angiotensin II hypersensitivity in aminopeptidase A-deficient mice

Local concentrations of the vasopressor peptide, angiotensin II (AngII), depend upon the balance between synthesis and degradation. Previous studies of blood pressure (BP) regulation have focused primarily on the generation of AngII and its receptors, and less attention has been devoted to angiotensin degradation. Aminopeptidase A (APA, EC 3.4.11.7) is responsible for the N-terminal cleavage of AngII, a hydrolytic event that serves as a rate-limiting step in angiotensin degradation. To evaluate the physiological role of APA, we examined BP homeostasis in APA-deficient mice. We measured basal BP and BP with continuous infusion of AngII in APA mutant mice by tail-cuff method. We also evaluated the development and histology of AngII-targeted organs as well as urine excretion in these mice. Homozygous APA mutant mice were found to have elevated basal systolic BP when compared with heterozygous mutant and wild-type littermate mice. Infusion of AngII led to an enhanced systolic BP response in the APA-deficient mice. Despite the sustained elevation of BP in APA knockout mice, neither their renal and cardiac sizes nor their histological appearances were not different from control mice. Moreover, the volume, osmolality, and electrolyte content of the urine were normal in APA-deficient mice. APA deficiency increased baseline BP and enhanced the hypertensive response to increased levels of AngII. These findings indicate a physiological role for APA in lowering BP and offer novel insight into the mechanisms for developing hypertension.     

Acute abdominal vagotomy reduces drinking to peripheral but not central angiotensin II

Rats were tested two or three days after bilateral abdominal vagotomy or a laparotomy control procedure for their drinking responses to subcutaneous (1 mg-kg-1) or intracerebroventricular (100 ng) injections of angiotensin II. Vagotomy delayed the initiation of drinking and decreased 60-min water intake after subcutaneous, but not after intracerebroventricular, angiotensin II. This is the shortest postoperative interval in which the decrease in drinking after systemic injection of angiotensin II by abdominal vagotomy has been observed. The failure of vagotomy to decrease the response to intracerebroventricular angiotensin II demonstrates that the deficit after subcutaneous injection was not a nonspecific effect of recent vagotomy. These results, therefore, suggest that the abdominal vagus is necessary for normal drinking in response to circulating angiotensin II. Furthermore, the selective and acute onset of the deficit is consistent with the loss of a specific, rather than tonic facilitatory, vagal mechanism for drinking after elevation of circulating angiotensin II levels. Finally, the results imply that the physiological mechanisms which mediate the drinking responses to central and peripheral angiotensin are not identical.     

Endothelin-1 and angiotensin II contribute to BNP but not c-fos gene expression response to elevated load in isolated mice hearts

The early events in the cardiac hypertrophic process induced by hemodynamic load include activation of B-type natriuretic peptide (BNP) and c-fos gene expression. However, it is unknown whether stretch acts directly or through local paracrine factors to trigger changes in cardiac gene expression. Herein we studied the involvement of endothelin-1 (ET-1) and angiotensin II (Ang II) in load-induced activation of left ventricular BNP and c-fos gene expression using an in vitro stretch model in isolated perfused adult mice hearts. Two-hour stretch induced by increasing coronary flow rate from 2 to 5 ml/min increased the expression of BNP and c-fos genes by 1.9- and 1.5-fold, respectively (P<0.001 and P<0.05). A mixed ET(A/B) receptor antagonist bosentan attenuated the BNP gene expression response to load by 58% (P<0.005). A similar 53% inhibition was observed with the selective ET(A) receptor blocker BQ-123 (P<0.05). Type 1 Ang II receptor antagonist CV-11974 decreased the activation of BNP gene expression by 50% (P<0.05). In contrast, the activation of c-fos gene expression was not inhibited by antagonists of ET(A/B) and AT(1) receptors. Our results show that ET-1 and Ang II play a key role in the induction of BNP, but not c-fos gene expression in response to load in intact adult murine hearts.     

Impaired pulmonary conversion of angiotensin I to angiotensin II in rats exposed to chronic hypoxia

The effects of exposing rats to hypoxia at normal atmospheric pressure for periods of 21-24 days on intrapulmonary conversion of angiotensin I (ANG I) to angiotensin II (ANG II) were examined using an isolated rat lung preparation perfused at constant flow. 125I-ANG I (160 fmol) was injected alone and with graded doses (0.1, 1.0, and 100 nmol) of unlabeled ANG I into the pulmonary artery, and the effluent was collected for measurement of ANG I, ANG II, and metabolites. At low doses of injected ANG I (125I-ANG I alone or with 0.1 or 1.0 nmol unlabeled ANG I), the percent conversion of ANG I to ANG II was 67.5 +/- 2.1 (SE), 65.1 +/- 2.0, and 62.5 +/- 1.6 in 21-day hypoxia-exposed animals and 83.8 +/- 2.7, 81.4 +/- 3.9, and 79.6 +/- 2.3 (P less than 0.01) in control rats maintained under normoxic conditions. At the highest dose (100 nmol) of injected ANG I, percent conversion was reduced in both hypoxic and control groups to 46.8 +/- 5.0 and 64.0 +/- 6.0, respectively (P less than 0.05). Mean transit times of labeled material through the pulmonary circulation were not significantly different in hypoxic vs. normoxic lungs at any ANG I load, suggesting that the decreased conversion seen in hypoxic lungs was not related to altered kinetics of substrate exposure. Thus chronic hypoxia is associated with significant inhibition of transpulmonary ANG I conversion that is independent of perfusate flow. We postulate that this phenomenon is due to alterations at the endothelial membrane level.     

Lys40 but not Arg143 influences selectivity of angiotensin conversion by human alpha-chymase

Human alpha-chymase is an efficient angiotensin (AT) converting enzyme, selectively hydrolyzing AT I at Phe8 to generate bioactive AT II, which can promote cardiac hypertrophy, vascular stenosis, and hypertension. Some related enzymes, such as rat beta-chymase 1, are much less selective, destroying AT by cleaving at Tyr4. Comparisons of chymase structure and activity led to speculation that interaction between AT and the side chain of Lys40 or Arg143 accounts for the human enzyme's marked preference for Phe8 over Tyr4. To test these hypotheses, we compared AT hydrolysis by wild-type chymase with that by mutants changing Lys40 or Arg143 to neutral residues. Lys40 was exchanged for alanine, the residue found in canine alpha- and rat beta-chymase 1, the latter being dramatically less selective for hydrolysis at Phe8. Arg143 was exchanged for glutamine found in rat beta-chymase 1. The Lys40Ala mutant is a dog-like enzyme retaining strong preference for Phe8 but with Tyr4 hydrolytic rates enhanced 16-fold compared to wild-type human enzyme. Thus, of 40 residues mismatched between dog and human enzymes, a single residue accounts for most of the difference in specificity between them. The Arg143Gln mutant, contrary to prediction, remains highly Phe8-selective. Therefore, Lys40, but not Arg143, contributes to human chymase's remarkable preference for AT II generation over destruction.     

Osmoregulatory fluid intake but not hypovolemic thirst is intact in mice lacking angiotensin

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.     

Nitrate increases in soil water following conversion of chaparral to grass

An Arizona watershed converted from chaparral to grass, released high concentrations of nitrate to stream water. The nitrate originated from the rooting zone of the decomposing shrubs. High nitrate concentrations (44–373 ppm) were found in soil solutions from 1.5-, 3.0-, and 4.6-m depths on the converted watershed as compared with low nitrate concentrations (0.2–6.2 ppm) found in an adjacent undisturbed area. Soil solution nitrate concentrations at the 0.3-m depth were generally low, especially in the untreated area. High nitrate concentrations were balanced mainly by relative decreases in bicarbonate anions in the soil solutions and in the stream water. Multiple stepwise regression analyses showed improvement in the regression of bicarbonate on nitrate when chloride and sulfate anions were entered as variables.     

Effects of platelet-activating factor on conversion of angiotensin I to II

We have studied the effects of platelet-activating factor (alkyl-acetyl-GPC) on conversion of angiotensin I to II. Platelet-activating factor (PAF) had a novel effect on angiotensin I conversion. Pulmonary artery endothelial cells converted 30% of [125I]angiotensin I to angiotensin II in the absence of PAF, but their activity was greatly stimulated in its presence. When PAF was added to pulmonary artery endothelial cells, the conversion of angiotensin I to II was enhanced up to 68%. Maximal stimulation without cellular damage was achieved at 10(-5) M PAF. LysoPAF did not stimulate the conversion of angiotensin I to angiotensin II at any concentration used.     

Pre-meal water consumption reduces meal energy intake in older but not younger subjects

Objective: To determine whether the consumption of water 30 minutes before an ad libitum meal reduces meal energy intake in young and older adults. Research Methods and Procedures: Healthy, non‐obese young (n = 29; age, 21 to 35 years) and older (n = 21; age, 60 to 80 years) individuals were provided with an ad libitum lunch meal on two occasions. Thirty minutes before the lunch meals, subjects were given either a water preload (WP: 375 mL, women; 500 mL, men) or no preload (NP). Energy intake at the two lunch meals was measured. Visual analog scales were used to assess changes in hunger, fullness, and thirst during the meal studies. Results: There was no significant difference in meal energy intake between conditions in the young subjects (892 + 51 vs. 913 ± 54 kcal for NP and WP, respectively; p = 0.65). However, meal energy intake after the WP was significantly reduced relative to the NP condition in the older subjects (682 + 53 vs. 624 ± 56 kcal for NP and WP, respectively; p = 0.02). This effect was caused primarily by the reduction in meal energy intake after water consumption in older men. Hunger ratings were lower and fullness ratings were higher in older compared with younger adults (p < 0.01). Fullness ratings were higher in the WP condition compared with the NP condition for all subjects (p = 0.01). No age differences in thirst were detected during the test meals. Discussion: Under acute test meal conditions, pre‐meal water consumption reduces meal energy intake in older but not younger adults. Because older adults are at increased risk for overweight and obesity, intervention studies are needed to determine whether pre‐meal water consumption is an effective long‐term weight management strategy for the aging population.     

Conversion of brain angiotensin II to angiotensin III is critical for pressor response in rats

The present investigation measured the relative pressor potencies of intracerebroventricularly infused ANG II, ANG III, and the metabolically resistant analogs d-Asp(1)ANG II and d-Arg(1)ANG III in alert freely moving rats. The stability of these analogs was further facilitated by pretreatment with the specific aminopeptidase A inhibitor EC33 or the aminopeptidase N inhibitor PC18. The results indicate that the maximum elevations in mean arterial pressure (MAP) were very similar for each of these compounds across the dose range 1, 10, and 100 pmol/min during a 5-min infusion period. However, d-Asp(1)ANG II revealed significantly extended durations of pressor effects before return to base level MAP. Pretreatment intracerebroventricular infusion with EC33 blocked the pressor activity induced by the subsequent infusion of d-Asp(1)ANG II, whereas EC33 had no effect on the pressor response to subsequent infusion of d-Arg(1)ANG III. In contrast, pretreatment infusion with PC18 extended the duration of the d-Asp(1)ANG II pressor effect by about two to three times and the duration of d-Arg(1)ANG III's effect by approximately 10 to 15 times. Pretreatment with the specific AT(1) receptor antagonist losartan blocked the pressor responses induced by the subsequent infusion of both analogs indicating that they act via the AT(1) receptor subtype. These results suggest that the brain AT(1) receptor may be designed to preferentially respond to ANG III, and ANG III's importance as a centrally active ligand has been underestimated.     

Ciglitazone is not itself thermogenic but increases the potential for thermogenesis in lean mice

Chronic dietary administration of the oral hypoglycaemic ciglitazone (3 g/day for 14–28 days) to lean, non-diabetic CD1 mice resulted in increased brown adipose tissue mitochondrial GDP binding and a marked increase in the thermic effect of the beta-adrenoceptor agonist BRL 26830A. However, ciglitazone was not itself thermogenic after an acute dose, nor did it raise resting metabolic rate during chronic dietary dosing.     

Protein restriction to pregnant rats increases the plasma levels of angiotensin II and expression of angiotensin II receptors in uterine arteries

Whether gestational protein restriction affects the renin-angiotensin system (RAS) in uterine artery remains unknown. In this study, we hypothesized that gestational protein restriction alters the expression of RAS components in uterine artery. In study one, time-scheduled pregnant Sprague Dawley rats were fed a normal or low-protein (LP) diet from Day 3 of pregnancy until they were killed at Days 19 and 22. The uterine arteries were collected and used for gene expression of Ace, Ace2, Agtr1a, Agtr1b, Agtr2, Esr1, and Esr2 by quantitative real-time PCR and/or Western blotting. LP increased plasma levels of angiotensin II in pregnant rats. In the uterine artery, the expressions of Agtr1a, Agtr1b, and Esr1 were increased by LP at Days 19 and 22 of pregnancy, whereas the abundance of AGTR1 and AGTR2 was increased by LP at Day 19 of pregnancy. The expression of Ace2 was not detectable in rat uterine artery. In study two, virgin female rats were ovariectomized and implanted with either 17beta-estradiol (E2), progesterone (P4), both E2 and P4, or placebo pellets until they were killed 7 days later. In rat uterine artery, E2 and P4 reduced the expression of Agtr1a, and E2 increased the expression of Agtr1b and Agtr2, but neither E2 nor P4 regulated the expression of Ace. These results indicate that gestational protein restriction induces an increase in Agtr1 expression in uterine artery, and thus may exacerbate the vasoconstriction to elevated angiotensin II present in maternal circulation, and that female sex hormones also play a role in this process.     

Bioavailability of apocynin through its conversion to glycoconjugate but not to diapocynin

Apocynin (4-hydroxy-3-methoxyacetophenone) is a major active ingredient from the rhizomes of Picrorhiza kurroa, a botanical plant used as an herbal medicine for treatment of a number of inflammatory diseases. Recently, apocynin is regarded as a specific inhibitor for NADPH oxidase in cell and animal models. In vitro studies indicated conversion of apocynin to diapocynin in the presence of peroxidases, e.g., myloperoxidase, posing the possibility that diapocynin also contributes to the anti-oxidative action of apocynin. The objectives of this study are to examine the bioavailability of apocynin to plasma, liver and brain tissue after intraperitoneal (i.p.) injection, and to examine whether apocynin is converted to diapocynin in vivo. Diapocynin was chemically synthetized and characterized by NMR and IR. Apocynin (5 mg/kg body wt) was injected i.p. to adult male Sprague-Dawley rats and plasma, liver and brain were collected at different times (30 min, 1 and 2 h) after injection. Samples were treated with β-glucuronidase to hydrolyze the glycosyl linkage and analyzed by HPLC/MS. At 30 min and 1 h after injection, approximately 50% of apocynin was converted to its glycosyl derivative and was distributed in plasma, liver and brain. No diapocynin was detected in any samples. These results indicate rapid glycosylation of apocynin and its transport to blood and other organs but no apparent conversion to diapocynin in vivo.     

Kallikrein generates angiotensin II but not bradykinin in the plasma of the urodele,Amphiuma tridactylum

Incubation of heat-denatured plasma from the urodele, Amphiuma tridactylum (three-toed amphiuma) or from the anurans Rana ridibunda (European green frog) and Rana catesbeiana (American bullfrog) with either glass beads, porcine pancreatic kallikrein or trypsin did not generate bradykinin-like immunoreactivity. However, peptides were generated in kallikrein-treated amphiuma plasma that contracted vascular rings from the bullfrog systemic arch and had a spasmogenic action on the bullfrog urinary bladder. These peptides, which were not generated in trypsin-treated plasma, were purified to homogeneity by reverse-phase HPLC and their primary structures established as: Asp-Arg-Val-Tyr-Val-His-Pro-Phe ([Asp¹,Val⁵]angiotensin II) and Asn-Arg-Val-Tyr-Val-His-Pro-Phe ([Asn¹,Val⁵]angiotensin II). Incubation of synthetic [Asn¹,Val⁵]angiotensin II with amphiuma plasma resulted in deamidation to [Asp¹,Val⁵]angiotensin II. The data suggest, therefore, that amphiuma plasma contains an l-asparagine amidohydrolase (asparaginase), as previously described for the eel. Although bradykinin-related peptides have been isolated from frog skin, this study provides evidence that the kallikrein-kinin system may be absent from the blood of amphibia.     

Lack of vasopressin receptors in liver, but not in kidney, of ob/ob mice.

The activity of phosphorylase a was measured in isolated hepatocytes from fed lean and ob/ob mice after addition of vasopressin, angiotensin, phenylephrine and glucagon. The binding of these hormones to purified liver plasma membranes was also determined. In hepatocytes of ob/ob mice, no increase in phosphorylase a was measured after addition of vasopressin, whereas the other hormones promoted an increase in the activity of the enzyme. No specific vasopressin receptors could be measured on purified liver plasma membrane of ob/ob mice. A decrease in the number of receptors for angiotensin and glucagon, without modification of the affinity, was also observed. No restoration of the number of vasopressin receptors was observed in liver of ob/ob mice starved for 3 days or in younger (5-6 weeks) animals. Vasopressin receptors and vasopressin-stimulated adenylate cyclase, measured on purified kidney medulla membranes, were similar in both lean and ob/ob mice. The data indicate a selective lack of vasopressin receptors and metabolic response in liver of the ob/ob mouse.     

Angiotensin III as well as angiotensin II regulates water flow through aquaporins in a clam worm

Angiotensin III has been reported to exist in various animals and tissues. The physiological role, however, is still unclear except that brain angiotensin III is a central regulator of vasopressin release. In this study, angiotensin III as well as angiotensin II enhanced an increase in body weight of clam worms of Perinereis sp. under a hypo-osmotic condition and suppressed a decrease in body weight under a hyper-osmotic condition. When clam worms were treated with tetrachloroaurate (III) after angiotensin-treatment, these enhancing and suppressive effects of the angiotensins under hypo- and hyper-osmotic conditions were inhibited. In contrast, when clam worms were pretreated with tetrachloroaurate (III) before angiotensin-treatment, these effects of angiotensins were not inhibited. Since tetrachloroaurate (III) is a representative blocker of aquaporins, these results indicate that angiotensin III as well as angiotensin II regulates water flow through aquaporins in clam worms.     

Probiotic use decreases intestinal inflammation and increases bone density in healthy male but not female mice

Osteoporosis can result from intestinal inflammation, as is seen with inflammatory bowel disease. Probiotics, microorganisms that provide a health benefit to the host when ingested in adequate amounts, can have anti‐inflammatory properties and are currently being examined to treat inflammatory bowel disease. Here, we examined if treating healthy male mice with Lactobacillus reuteri ATCC PTA 6475 (a candidate probiotic with anti‐TNFα activity) could affect intestinal TNFα levels and enhance bone density. Adult male mice were given L. reuteri 6475 orally by gavage for 3×/week for 4 weeks. Examination of jejunal and ileal RNA profiles indicates that L. reuteri suppressed basal TNFα mRNA levels in the jejunum and ileum in male mice, but surprisingly not in female mice. Next, we examined bone responses. Micro‐computed tomography demonstrated that L. reuteri 6475 treatment increased male trabecular bone parameters (mineral density, bone volume fraction, trabecular number, and trabecular thickness) in the distal femur metaphyseal region as well as in the lumbar vertebrae. Cortical bone parameters were unaffected. Dynamic and static histomorphometry and serum remodeling parameters indicate that L. reuteri ingestion increases osteoblast serum markers and dynamic measures of bone formation in male mice. In contrast to male mice, L. reuteri had no effect on bone parameters in female mice. Taken together our studies indicate that femoral and vertebral bone formation increases in response to oral probiotic use, leading to increased trabecular bone volume in male mice. J. Cell. Physiol. 228: 1793–1798, 2013. © 2013 Wiley Periodicals, Inc.     

Increased plasma level of apelin with NYHA grade II and III but not IV

Amino Acids - The change in plasma apelin level in heart failure (HF) patients is controversial. We investigated the change in plasma apelin level in HF patients versus control and non-HF patients....     

The epidermal growth factor receptor is involved in angiotensin II but not aldosterone/salt-induced cardiac remodelling

Experimental and clinical studies have shown that aldosterone/mineralocorticoid receptor (MR) activation has deleterious effects in the cardiovascular system; however, the signalling pathways involved in the pathophysiological effects of aldosterone/MR in vivo are not fully understood. Several in vitro studies suggest that Epidermal Growth Factor Receptor (EGFR) plays a role in the cardiovascular effects of aldosterone. This hypothesis remains to be demonstrated in vivo. To investigate this question, we analyzed the molecular and functional consequences of aldosterone exposure in a transgenic mouse model with constitutive cardiomyocyte-specific overexpression of a mutant EGFR acting as a dominant negative protein (DN-EGFR). As previously reported, Angiotensin II-mediated cardiac remodelling was prevented in DN-EGFR mice. However, when chronic MR activation was induced by aldosterone-salt-uninephrectomy, cardiac hypertrophy was similar between control littermates and DN-EGFR. In the same way, mRNA expression of markers of cardiac remodelling such as ANF, BNF or β-Myosin Heavy Chain as well as Collagen 1a and 3a was similarly induced in DN-EGFR mice and their CT littermates. Our findings confirm the role of EGFR in AngII mediated cardiac hypertrophy, and highlight that EGFR is not involved in vivo in the damaging effects of aldosterone on cardiac function and remodelling.