Deregulation and epigenetic modification of BCL2-family genes cause resistance to venetoclax in hematologic malignancies.

The BCL2 inhibitor venetoclax has been approved to treat different hematological malignancies. Because there is no common genetic alteration causing resistance to venetoclax in chronic lymphocytic leukemia (CLL) and B-cell lymphoma, we asked if epigenetic events might be involved in venetoclax resistance. Therefore, we employed whole-exome sequencing, methylated DNA immunoprecipitation sequencing, and genome-wide clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 screening to investigate venetoclax resistance in aggressive lymphoma and high-risk CLL patients. We identified a regulatory CpG island within the PUMA promoter that is methylated upon venetoclax treatment, mediating PUMA downregulation on transcript and protein level. PUMA expression and sensitivity toward venetoclax can be restored by inhibition of methyltransferases. We can demonstrate that loss of PUMA results in metabolic reprogramming with higher oxidative phosphorylation and adenosine triphosphate production, resembling the metabolic phenotype that is seen upon venetoclax resistance. Although PUMA loss is specific for acquired venetoclax resistance but not for acquired MCL1 resistance and is not seen in CLL patients after chemotherapy-resistance, BAX is essential for sensitivity toward both venetoclax and MCL1 inhibition. As we found loss of BAX in Richter's syndrome patients after venetoclax failure, we defined BAX-mediated apoptosis to be critical for drug resistance but not for disease progression of CLL into aggressive diffuse large B-cell lymphoma in vivo. A compound screen revealed TRAIL-mediated apoptosis as a target to overcome BAX deficiency. Furthermore, antibody or CAR T cells eliminated venetoclax resistant lymphoma cells, paving a clinically applicable way to overcome venetoclax resistance.

Olea Europea-derived phenolic products attenuate antinociceptive morphine tolerance: an innovative strategic approach to treat cancer pain.

Morphine and related opioid drugs are currently the major drugs for severe pain. Their clinical utility is limited in the management of severe cancer pain due to the rapid development of tolerance. Restoring opioid efficacy is therefore of great clinical importance. A great body of evidence suggests the key role of free radicals and posttranslational modulation in the development of tolerance to the analgesic activity of morphine. Epidemiological studies have shown a relationship between the Mediterranean diet and a reduced incidence of pathologies such as coronary heart disease and cancer. A central hallmark of this diet is the high consumption of virgin olive oil as the main source of fat which contains antioxidant components in the non-saponifiable fraction, including phenolic compounds absent in seed oils. Here, we show that in a rodent model of opiate tolerance, removal of the free radicals with phenolic compounds of olive oil such as hydroxytyrosol and oleuropein reinstates the analgesic action of morphine. Chronic injection of morphine in mice led to the development of tolerance and this was associated with increased nitrotyrosin and malondialdehyde (MDA) formation together with nitration and deactivation of MnSOD in the spinal cord. Removal of free radicals by hydroxytyrosol and oleuropein blocked morphine tolerance by inhibiting nitration and MDA formation and replacing the MnSOD activity. The phenolic fraction of virgin olive oil exerts antioxidant activities in vivo and free radicals generation occurring during chronic morphine administration play a crucial role in the development of opioid tolerance. Our data suggest novel therapeutic approach in the management of chronic cancer pain, in particular for those patients who require long-term opioid treatment for pain relief without development of tolerance.

A molecular phylogenetic framework for the Muricidae, a diverse family of carnivorous gastropods.

With over 1600 extant described species, the Muricidae are one of the most species-rich and morphologically diverse families of molluscs. As predators of molluscs, polychaetes, anthozoans barnacles and other invertebrates, they form an important component of many benthic communities. Traditionally, the classification of muricids at specific and generic levels has been based primarily on shells, while subfamilies have been defined largely by radular morphology, although the composition and relationships of suprageneric groups have never been studied exhaustively. Here we present the phylogenetic relationships of 77 muricid species belonging to nine of the ten currently recognized subfamilies, based on Bayesian inference and Maximum Likelihood analyses of partial sequences of three mitochondrial (12S, 16S and COI) and one nuclear (28S) genes. The resulting topologies are discussed with respect to traditional subfamilial arrangements, and previous anatomical and molecular findings. We confirm monophyly of each of the subfamilies Ergalataxinae, Rapaninae, Coralliophilinae, Haustrinae, Ocenebrinae and Typhinae as previously defined, but earlier concepts of Muricinae, Trophoninae and Muricopsinae are shown to be polyphyletic. Based on our phylogenetic hypothesis, a new arrangement of these subfamilies is proposed.

Divergent functions of angiotensin II receptor isoforms in the brain.

The renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein-coupled receptors of two pharmacological classes, AT(1) and AT(2). AT(1) receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT(1) receptor isoforms, termed AT(1A) and AT(1B) receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT(1A) expression generally exceeding AT(1B) expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT(1A) and AT(1B) receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT(1A) receptor. However, the drinking response requires the presence of AT(1B) receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT(1B) receptors.

Angiotensin II regulates cellular immune responses through a calcineurin-dependent pathway.

The renin-angiotensin system (RAS) is a key regulator of vascular tone and blood pressure. In addition, angiotensin II also has a number of cellular effects that may contribute to disease pathogenesis. Using Agtr1a(-/-) mice, which lack AT(1A) receptors for angiotensin II, we have identified a novel function of the RAS to modulate the immune system. We find that angiotensin II, acting through type 1 (AT(1)) receptors on immune cells, triggers the proliferation of splenic lymphocytes. These actions contribute to the vigor of cellular alloimmune responses. Within lymphoid organs, sufficient components of the RAS are present to activate AT(1) receptors during an immune response, promoting cell growth. These actions require activation of calcineurin phosphatase. In an in vivo model of cardiac transplantation, the absence of AT(1) signaling accentuates the immunosuppressive effects of the calcineurin inhibitor cyclosporine. We conclude that inhibition of AT(1) receptor signaling should be useful as an anti-inflammatory and immunosuppressive therapy. Furthermore, the actions of the RAS to promote lymphocyte activation may contribute to inflammation that characterizes a number of diseases of the heart and the vascular system.

Synthesis and antioxidant evaluation of lipophilic oleuropein aglycone derivatives.

Oleuropein is the most important phenolic compound present in olive cultivars, but it is scarcely present in extra virgin olive oil (EVOO) due to its high hydrophilicity and degradability. Thus, a set of oleuropein aglycone derivatives were synthesized by transacetylation under mild conditions with the aim of circumventing these drawbacks and making the active moiety in oleuropein suitable to be added to food fats. The oleuropein aglycone (closed ring form) is obtained by hydrolyzing oleuropein using Lewis acid catalysis. Then, the permeation profiles as well as the antioxidant capacity of the oleuropein aglycone derivatives were evaluated by ORAC, DPPH assays and by ROS formation using the SH-SY5Y cell line. The biological activities of the obtained compounds exhibited a dependence on their level of lipophilicity.

Insights into the functions of type 1 (AT1) angiotensin II receptors provided by gene targeting.

The renin-angiotensin system (RAS) has a wide range of actions in biological processes ranging from development and reproduction to cardiovascular and renal functions. Most of these actions are mediated by the octapeptide hormone angiotensin II. The identified family of angiotensin II receptors is divided into two pharmacological classes: type 1 (AT1) and type 2 (AT2). The classically recognized actions of the RAS are primarily mediated by the AT1 subtype of angiotensin receptors, and these receptors are the targets of a new class of anti-hypertensive agents. In recent years, our understanding of the physiological functions of AT1 receptors has been advanced through the use of gene-targeting technology. In this review, we will summarize the emerging picture of AT1 receptor functions that has been provided by gene-targeting experiments.

Characterization of Brown spider (Loxosceles intermedia) hemolymph: cellular and biochemical analyses.

This is the first study on the hemolymph from a spider of the Loxosceles genus. These animals are responsible for a great number of envenomation cases worldwide. Several studies on Loxosceles venoms have been published, and the knowledge about the venom and its toxins is considerable, not only regarding the biological and biochemical characterization, but also regarding structural, genetic and phylogenetic approaches. However, the literature on Loxosceles hemolymph is nonexistent. The main goal of the present study was to characterize biochemically the hemolymph content, and especially, to identify its different hemocytes. Moreover, many papers have already shown molecules whose source is the hemolymph and their very interesting activities and biomedical applications, for example, antifungal and antibacterial activities. A 2D-SDS-PAGE of brown spider hemolymph showed approximately 111 spots for pH 3-10 and 150 spots for pH 4-7. A lectin-blotting assay showed that hemolymph carbohydrate residues were similar to those found in venom. Several types of TAG and DAG phospholipids were found in the hemolymph and characterized by HPTLC and mass spectrometry. Four different hemocytes were characterized in Loxosceles intermedia hemolymph: prohemocyte, plasmatocyte, granulocyte and adipohemocyte. This paper opens new possibilities on toxinology, studying an unknown biological material, and it characterizes a source of molecules with putative biotechnological applications.

Neuroendocrine effects of dehydration in mice lacking the angiotensin AT1a receptor.

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.

Circadian rhythms of activity and drinking in mice lacking angiotensin II 1A receptors.

Angiotensin II (ANG II) type 1 receptors are found in the mouse suprachiasmatic nucleus (SCN), the site of the circadian pacemaker, but their significance for circadian timekeeping is unknown. We examined circadian rhythms of wheel running and drinking in angiotensin AT(1a) receptor knockout (KO) mice. Mean daily running and drinking activity were elevated in KO mice under a light-dark (LD) cycle and in constant dark (DD). These increases were confined to the usual active (dark) period, thus, the 'amplitude' of running and drinking rhythms was higher in KO mice. The phase of entrainment to LD (measured by the onset of the daily active period) did not differ between groups, either in LD or on the first day of DD ('unmasked' phase). KO mice showed a modestly shorter free-running period (tau) in DD. The direction and magnitude of phase shifts to light pulses at two circadian times (CTs) in DD did not differ between groups. Core functions of the circadian system appear intact following AT(1a) receptor KO. The modestly shorter tau and increased rhythm amplitude in KO mice may be secondary to an effect of the mutation on the level of running and drinking activity.

Angiotensin-II-receptors: new targets for antihypertensive therapy.

The renin-angiotensin system regulates blood pressure and sodium homeostasis through a series of coordinated substrate-enzyme interactions. These interactions result in the production of angiotensin II (AII), which exerts a number of diverse biologic effects mediated through AII cell-surface receptors. Dysregulation of this system is implicated in the pathogenesis of various forms of hypertension. Traditional therapy for hypertension has included angiotensin-converting enzyme inhibitors, which block the production of AII. However, a new class of drugs, AT1-receptor blockers, now offers a number of benefits by specifically blocking the effects of AII at its physiologically relevant receptor.

Tolerance of hemodialysis: a randomized prospective trial of high-flux versus conventional high-efficiency hemodialysis.

Hemodialysis is frequently complicated by hypotension and associated symptoms. It has been suggested that these symptoms may be related to the biochemical changes caused by cellulosic dialysis membranes. In this study, a prospective randomized crossover trial was conducted comparing the incidence of hypotension and acute symptoms during dialysis with large-surface-area (1.6 m2) cellulosic (cuprophane [CUP]) and noncellulosic (polyacrylonitrile [PAN], AN69) membranes. Dialyzers were used for a single use only. There was no difference in predialysis BUN, predialysis blood pressure, intradialytic weight gain, blood flow, dialysis efficiency (urea reduction), dialysis duration, hematocrit, or erythropoietin dose between the two study phases. When these clinical characteristics were matched, there was no difference in the number of episodes of hypotension (CUP, 19 +/- 3; PAN, 22 +/- 3; P = not significant [NS]). The incidence of symptomatic hypotension, as reflected by the number of episodes of hypotension requiring more than 100 mL of saline for correction, was also not different between study phases (CUP, 10 +/- 1; AN69, 11 +/- 2; P = NS). The incidence of intradialytic symptoms, including emesis, cramping, headache, angina, pruritus, and bronchospasm, was similar during the two study phases (CUP, 11 +/- 2; AN69, 10 +/- 1; P = NS). It was concluded that noncellulosic membranes do not offer any significant advantage over cellulosic membranes in reducing the acute complications of hemodialysis.

Regulation of sodium balance and blood pressure by the AT(1A) receptor for angiotensin II.

To examine the role of the angiotensin II (AT)(1A) receptor in the regulation of blood pressure and sodium balance, we measured systolic blood pressure responses in AT(1A) receptor-deficient (Agtr1a-/-) and wild-type (Agtr1a+/+) mice while dietary sodium content was systematically altered. On a 0.4% sodium diet, systolic blood pressures were significantly lower in Agtr1a-/- than in +/+ mice. In Agtr1a+/+ mice, changing dietary sodium content did not affect blood pressure. In contrast, when Agtr1a-/- mice were fed a high-salt diet (6% NaCl), their systolic blood pressures increased significantly from 79+/-4 to 94+/-4 mm Hg (P<0.006). The low blood pressures of Agtr1a-/- mice decreased further while on a low-salt diet from 82+/-3 to 69+/-3 mm Hg (P<0.03). On the high-salt diet, urinary sodium excretion increased to similar levels in Agtr1a+/+ and -/- mice. Although urinary sodium excretion was substantially reduced in both groups during the low-salt diet, cumulative sodium balances became negative in Agtr1a-/- mice despite a 6-fold increase in urinary aldosterone. We infer, therefore, that the reduced blood pressures in Agtr1a-/- mice on a normal diet are caused by depletion of sodium and extracellular volume. Their "sodium sensitivity" suggests a critical role for renal AT(1A) receptors to modulate sodium handling.

Renal function in the AT1A receptor knockout mouse during normal and volume-expanded conditions.

BACKGROUND: Genetically altered mice lacking the AT1A angiotensin II (Ang II) receptor were used to examine the role of AT1A receptors in regulating renal hemodynamics, sodium excretion, glomerulotubular balance, and Ang II levels in plasma and kidney during normal and volume-expanded conditions. METHODS: AT1A receptor-deficient mice and their wild-type controls were anesthetized with inactin and ketamine, and were prepared to allow intravenous infusions of solutions and measurements of aortic pressure and urine collections. Inulin and para-aminohippurate (PAH) solutions were infused intravenously for clearance determinations under conditions of euvolemia (2.5 microliter/min infusion of isotonic saline) or volume-expansion conditions (12.5 microliter/min). After three 30-minute urine collections, blood samples were collected, and kidneys were harvested. Plasma and kidney Ang II measurements were made by radioimmunoassay. RESULTS: In the euvolemic state, mean arterial pressures (MAPs) were significantly lower in the AT1A receptor-deficient mice (68 +/- 4 mm Hg) compared with wild-type controls (89 +/- 3 mm Hg). Despite the lower MAP, the glomerular filtration rate (GFR), renal plasma flow (RPF), absolute sodium excretion, and fractional sodium excretion were not significantly different between wild-type and AT1A-/- mice. Volume expansion did not alter MAP in wild-type mice, but significantly increased MAP in the AT1A-/- mice (68 +/- 4 to 83 +/- 5 mm Hg). Similar increases in GFR, RPF, absolute sodium excretion, and fractional sodium excretion in AT1A+/+ and AT1A-/- mice were observed. Glomerulotubular balance was not disrupted by the absence of AT1A receptors. During euvolemia, plasma Ang II concentrations were significantly higher in the AT1A-/- mice compared with wild-type mice (536 +/- 172 vs. 198 +/- 36 fmol/ml). Although volume expansion had no effect on plasma Ang II levels in the AT1A+/+ group, plasma Ang II concentrations were markedly suppressed in the AT1A-/- mice to levels that were not different from those in wild-type mice. In contrast, kidney tissue Ang II contents were reduced in the AT1A-/- mice and were not significantly altered during volume expansion in either the AT1A-/- or the AT1A+/+ mice. CONCLUSIONS: The absence of AT1A receptors does not impair chronic regulation of renal blood flow, GFR, or glomerulotubular balance. The prompt restoration of MAP following volume expansion suggests that low blood pressure in the AT1A receptor-deficient mice is primarily due to reduced effective plasma and extracellular fluid volume. Normalization of plasma Ang II levels with volume expansion demonstrates a dominant effect of extracellular fluid volume and blood pressure over AT1A receptor-mediated short-loop feedback in the regulation of plasma Ang II levels.

Effects of candesartan on angiotensin II-induced renal vasoconstriction in rats and mice.

This study determined the inhibitory effect of the angiotensin II (AngII) type I (AT1) receptor blocker candesartan on renal vascular reactivity in vivo. Reactivity to AngII before and during candesartan administration was assessed by measuring (by electromagnetic or ultrasonic flowmetry) renal blood flow responses to AngII in rats and mice. AngII produced greater renal vasoconstriction in 7-wk-old, spontaneously hypertensive rats than in Wistar-Kyoto rats. After indomethacin treatment, AngII (2 ng) produced 40% reductions in renal blood flow in both rat strains, without affecting systemic arterial pressure. Coadministration of candesartan blocked AngII effects in a dose-dependent manner, with similar levels of inhibition in spontaneously hypertensive rats and Wistar-Kyoto rats; maximal inhibition was 80%. In rats that had been pretreated (for 30 min) with intravenous candesartan, AngII-induced renal vasoconstriction was inhibited dose dependently up to 98%. To evaluate receptor subtype mediation, responses were compared in mice with or without the AT1A receptor (deleted by gene targeting). Intrarenal AngII (1 ng) caused a 32% reduction of renal blood flow in wild-type mice and an 8% reduction of renal blood flow in AT1A receptor-knockout mice. Ten nanograms of AngII were required to elicit 20% renal vasoconstriction in these mutant mice. Concurrent injection of candesartan caused dose-dependent inhibition of AngII up to 80%. The candesartan IC50 values for percentage changes in renal blood flow did not differ in the two groups of mice. These studies establish that candesartan is an effective, highly selective, AT1 receptor blocker, inhibiting renal vasoconstriction in rodents in a concentration- and time-dependent manner. Candesartan effectively blocks AT1A and AT1B receptors in renal resistance vessels of rodents, with similar efficacies in rats and mice.

Renal vascular reactivity in mice: AngII-induced vasoconstriction in AT1A receptor null mice.

The present study describes methodology and its application to evaluate renal reactivity in acute studies on anesthetized mice. Renal blood flow (RBF) was measured using an ultrasonic transit-time flowmeter and a non-cannulating V-shaped probe. An intrarenal artery injection technique established feasibility and reproducibility of studies of renal vascular reactivity to angiotensin II (AngII) in adult wild-type mice. The study also examined whether AngII would affect RBF in mice lacking AT1A receptors due to gene targeting. Mean arterial pressure averaged 83 and 62 mmHg, respectively, in mice with and without AT1A receptors. The RBF was similar in both groups, averaging 7 ml/min per g kidney wt. AngII injection (10-microl bolus) into the renal artery produced transient, dose-dependent, selective reductions in RBF in AT1A knockout mice as well as wild-type mice. The response was considerably greater in mice with AT1A receptors: 10% for 0.1 ng, 30% for 1 ng, and 45% for 5 ng AngII in control animals versus respective decreases of 6, 15, and 17% in knockout mice. In other studies, angiotensin-converting enzyme (captopril) or renin (CP-71362-14) was inhibited. During inhibition of AngII formation, renal vascular reactivity to AngII increased twofold in both groups. Coadministration of the AT1 receptor antagonist losartan (1 to 1000 ng) elicited dose-dependent inhibition of AngII effects, with near maximum blockage of 80 to 90% in both groups of mice. The putative AT2 receptor antagonist PD 123319 inhibited 30 to 40% of AngII-induced vasoconstriction, whereas CGP 42112 had no effect in either group. In conclusion, AngII can elicit renal vasoconstriction, albeit attenuated, in AT1A knockout mice. The weaker RBF effects are most likely due to the absence of the AT1A receptor. Inhibition of the response by AT1 receptor antagonist suggests mediation by the AT1B receptor in these animals. The residual constrictor effect observed during AT1 receptor blockade and sensitive to PD 123319 appears to be mediated by a non-AT1 receptor.

Diagnosis of Legionnaires' disease by radioimmunoassay of Legionella antigen in pleural fluid.

We describe a case of culture-proven Legionnaires' disease (serogroup 1) in which a rapid diagnosis was made by detection of Legionella antigen in pleural fluid by use of the Binax radioimmunoassay for urine.

Enhanced central response to dehydration in mice lacking angiotensin AT(1a) receptors.

The objective was to determine the central nervous system (CNS) responses to dehydration (c-Fos and vasopressin mRNA) in mice lacking the ANG AT(1a) receptor [ANG AT(1a) knockout (KO)]. Control and AT(1a) KO mice were dehydrated for 24 or 48 h. Baseline plasma vasopressin (VP) was not different between the groups; however, the response to dehydration was attenuated in AT(1a) KO (24 +/- 11 vs. 10.6 +/- 2.7 pg/ml). Dehydration produced similar increases in plasma osmolality and depletion of posterior pituitary VP content. Neuronal activation was observed as increases in c-Fos protein and VP mRNA. The supraoptic responses were not different between groups. In the paraventricular nucleus (PVN), c-Fos-positive neurons (57.4 +/- 10.7 vs. 98.4 +/- 7.4 c-Fos cells/PVN, control vs. AT(1a) KO) and VP mRNA levels (1.0 +/- 0.1 vs. 1.4 +/- 0.1 microCi, control vs. AT(1a) KO) were increased with greater responses in AT(1a) KO. A comparison of 1- to 2-day water deprivation showed that plasma VP, brain c-Fos, and VP mRNA returned toward control on day 2, although plasma osmolality remained high. Data demonstrate that AT(1a) KO mice show a dichotomous response to dehydration, reduced for plasma VP and enhanced for PVN c-Fos protein and VP mRNA. The results illustrate the importance of ANG AT(1a) receptors in the regulation of osmotic and endocrine balance.

Regulation of blood pressure by the type 1A angiotensin II receptor gene.

The renin-angiotensin system plays a critical role in sodium and fluid homeostasis. Genetic or acquired alterations in the expression of components of this system are strongly implicated in the pathogenesis of hypertension. To specifically examine the physiological and genetic functions of the type 1A receptor for angiotensin II, we have disrupted the mouse gene encoding this receptor in embryonic stem cells by gene targeting. Agtr1A(-/-) mice were born in expected numbers, and the histomorphology of their kidneys, heart, and vasculature was normal. AT1 receptor-specific angiotensin II binding was not detected in the kidneys of homozygous Agtr1A(-/-) mutant animals, and Agtr1A(+/-) heterozygotes exhibited a reduction in renal AT1 receptor-specific binding to approximately 50% of wild-type [Agtr1A(+/+)] levels. Pressor responses to infused angiotensin II were virtually absent in Agtr1A(-/-) mice and were qualitatively altered in Agtr1A(+/-) heterozygotes. Compared with wild-type controls, systolic blood pressure measured by tail cuff sphygmomanometer was reduced by 12 mmHg (1 mmHg = 133 Pa) in Agtr1A(+/-) mice and by 24 mmHg in Agtr1A(-/-) mice. Similar differences in blood pressure between the groups were seen when intraarterial pressures were measured by carotid cannulation. These studies demonstrate that type 1A angiotensin II receptor function is required for vascular and hemodynamic responses to angiotensin II and that altered expression of the Agtr1A gene has marked effects on blood pressures.

Absence of tubuloglomerular feedback responses in AT1A receptor-deficient mice.

Experiments were performed in a recently generated strain of mice with an angiotensin II AT1A-receptor null mutation (M. Ito, M. I. Oliverio, P. J. Mannon, C. F. Best, N. Maeda, O. Smithies, and T. M. coffman. Proc. Natl. Acad. Sci. USA 92: 3521-3525, 1995) to examine the effects of chronic AT1A receptor deficiency on tubuloglomerular feeback (TGF) responses. All animals were genotyped by polymerase chain reaction using primers designed to amplify sequences from the deleted AT1A gene and from the neomycin resistance gene. Normal mice (AT1A +/+) and mice heterozygous (AT1A +/-) and homozygous (AT1A -/-) for the gene disruption were anesthetized, and stop-flow pressures (PSF) were determined during changes in loop perfusion rate with previously established micropuncture methods. In five AT1A +/+ mice (26 tubules) mean PSF at zero loop flow was 37.2 +/- 1.5 mmHg, falling to 28.2 +/- 1.9 mmHg at a flow of 45 nl/min (P < 0.0001). Flow rate causing the half-maximum response (V1/2) was 8.7 +/- 0.4 nl/min. In four AT1A +/- animals (19 tubules) mean PSF at zero flow was 39.9 +/- 2.4 mmHg, falling to 34.8 +/- 2.7 mmHg at 45 nl/min (mean V1/2 8.6 +/- 1.04 nl/min). In five AT1A -/- mice (24 tubules) PSF was not significantly affected by loop flow with PSF averaging 33.9 +/- 1.7 mmHg at zero flow and 33.2 +/- 1.6 mmHg at 45 nl/min (not significant). Mean arterial blood pressures in the anesthetized and laparotomized mice were 91.8 +/- 2.2, 97.1 +/- 3, and 80.7 +/- 3.2 mmHg in the AT1A +/+, AT1A +/-, and AT1A -/- animals, respectively. Blood pressure responses to exogenous angiotensin II were greatly blunted in the AT1A -/- mice. We conclude that AT1A receptor-mediated effects of angiotensin II are in essential component of TGF responsiveness under chronic conditions. Our studies show the feasibility of using complex micropuncture methods in mice, an approach that widens the potential of genetically altered mouse strains as experimental models.