A novel polymorphism in the coding region of the vasopressin type 2 receptor gene

Nephrogenic diabetes insipidus (NDI) is a rare disease characterized by renal inability to respond properly to arginine vasopressin due to mutations in the vasopressin type 2 receptor (V2(R)) gene in affected kindreds. In most kindreds thus far reported, the mode of inheritance follows an X chromosome-linked recessive pattern although autosomal-dominant and autosomal-recessive modes of inheritance have also been described. Studies demonstrating mutations in the V2(R) gene in affected kindreds that modify the receptor structure, resulting in a dys- or nonfunctional receptor have been described, but phenotypically indistinguishable NDI patients with a structurally normal V2(R) gene have also been reported. In the present study, we analyzed exon 3 of the V2(R) gene in 20 unrelated individuals by direct sequencing. A C®T alteration in the third position of codon 331 (AGC®AGT), which did not alter the encoded amino acid, was found in nine individuals, including two unrelated patients with NDI. Taken together, these observations emphasize the molecular heterogeneity of a phenotypically homogeneous syndrome

Human fetal malformations associated with the use of an angiotensin II receptor antagonist: Case Report

Introduction: The potential risks related to drug exposure during pregnancy represent a vast chapter in modern obstetrics and data regarding the safety of antihypertensive drugs during pregnancy are relatively scarce. Case report: A 37-year-old patient discovered her fifth pregnancy at our hospital after 26 weeks and 4 days of gestation. She reported a history of hypertension and was currently being treated with Losartan. Hospitalization was recommended for the patient and further evaluation of fetal vitality was performed. On the fourth day an ultrasound was performed, resulting in a severe oligohydramnios, fetal centralization and abnormal ductus venosus. After 36 hours, the newborn died. Pathologic evaluation: At autopsy, the skullcap had large fontanels and deficient ossification. The kidneys were slightly enlarged. A microscopic examination detected underdevelopment of the tubules and the presence of some dilated lumens. Immunohistochemical detection of epithelial membrane antigen was positive. Immunoreactivity of CD 15 was also assayed to characterize the proximal tubules, and lumen collapse was observed in some regions. Discussion: Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor antagonists (ARAs) are among the most widely prescribed drugs for hypertension. They are often used by hypertensive women who are considering become pregnant. While their fetal toxicity in the second or third trimesters has been documented, their teratogenic effect during the first trimester has only recently been demonstrated. Conclusion: Constant awareness by physicians and patients should be encouraged, particularly in regard to the prescription of antihypertensive drugs in women of childbearing age who are or intend to become pregnant.

Functional coupling of the human dopamine D-2 receptor with G alpha(i1), G alpha(i2), G alpha(i3) and G alpha(o) G proteins: evidence for agonist regulation of G protein selectivity

1 The human dopamine D-2long (D-2L) receptor was expressed with four different G proteins in Sf9 cells using the baculovirus expression system. When co-expressed with G(i)/G(o) G proteins (G(i1)alpha, G(i2)alpha, G(i3)alpha, or G(o)alpha, plus Gbeta(1) and Ggamma(2)) the receptor displayed a high-affinity binding site for the agonists (dopamine and NPA), which was sensitive to GTP (100 mum), demonstrating interaction between the receptor and the different G proteins. 2 The receptor to G protein ratio (R: G ratio) was evaluated using [H-3]-spiperone saturation binding (R) and [S-35]-GTPgammaS saturation binding (G). R: G ratios of 1: 12, 1: 3, 1: 14 and 1: 5 were found for G(i1), G(i2), G(i3), and Go preparations, respectively. However, when R:G ratios of 1:2 and 1: 12 were compared for G(i2) and G(o), no difference was found for the stimulation of [S-35]-GTPgammaS binding. 3 Several agonists were tested for their ability to stimulate [S-35]-GTPgammaS binding to membranes co-expressing the receptor and various G proteins. All the compounds tested showed agonist activity in preparations expressing G(i3) and G(o). However, for G(i2) and G(i1) preparations, compounds such as S-(-)-3-PPP and p-tyramine were unable to stimulate [S-35]-GTPyS binding. 4 Most of the compounds showed higher relative efficacies (compared to dopamine) and higher potencies in the preparation expressing G(o). Comparison of the effects of different agonists in the different preparations showed that each agonist differentially activates the four G proteins. 5 We conclude that the degree of selectivity of G protein activation by the D-2L receptor can depend on the conformation of the receptor stabilised by an agonist.

Effects of Ischemia and Reperfusion on P2X(2) Receptor Expressing Neurons of the Rat Ileum Enteric Nervous System

Purpose We investigated the effects of ischemia/reperfusion in the intestine (I/R-i) on purine receptor P2X(2)-immunoreactive (IR) neurons of the rat ileum. Methods The superior mesenteric artery was occluded for 45 min with an atraumatic vascular clamp and animals were sacrificed 4 h later. Neurons of the myenteric and submucosal plexuses were evaluated for immunoreactivity against the P2X(2) receptor, nitric oxide synthase (NOS), choline acetyl transferase (ChAT), calbindin, and calretinin. Results Following I/R-i, we observed a decrease in P2X(2) receptor immunoreactivity in the cytoplasm and surface membranes of neurons of the myenteric and submucosal plexuses. These studies also revealed an absence of calbindin-positive neurons in the I/R-i group. In addition, the colocalization of the P2X(2) receptor with NOS, ChAT, and calretinin immunoreactivity in the myenteric plexus was decreased following I/R-i. Likewise, the colocalization between P2X(2) and calretinin in neurons of the submucosal plexus was also reduced. In the I/R-i group, there was a 55.8% decrease in the density of neurons immunoreactive (IR) for the P2X(2) receptor, a 26.4% reduction in NOS-IR neuron, a 25% reduction in ChAT-IR neuron, and a 47% reduction in calretinin-IR neuron. The density of P2X(2) receptor and calretinin-IR neurons also decreased in the submucosal plexus of the I/R-i group. In the myenteric plexus, P2X(2)-IR, NOS-IR, ChAT-IR and calretinin-IR neurons were reduced in size by 50%, 49.7%, 42%, and 33%, respectively, in the I/R-i group; in the submucosal plexus, P2X(2)-IR and calretinin-IR neurons were reduced in size by 56% and 72.6%, respectively. Conclusions These data demonstrate that ischemia/reperfusion of the intestine affects the expression of the P2X(2) receptor in neurons of the myenteric and submucosal plexus, as well as density and size of neurons in this population. Our findings indicate that I/R-i induces changes in P2X(2)-IR enteric neurons that could result in alterations in intestinal motility.

The role of interleukin-10 in the differential expression of interleukin-12p70 and its beta 2 receptor on patients with active or treated paracoccidioidomycosis and healthy infected subjects

Paracoccidioidomycosis patients present an antigen-specific Th1 immunosuppression. To better understand this phenomenon, we evaluated the interleukin (IL)-12 pathway by measuring IL-12p70 production and CD3(+) T cell expression of the IL-12 receptor (IL-12R)beta1/beta2 chains, induced with the main fungus antigen (gp43) and a control antigen, from Candida albicans (CMA). We showed that gp43-induced IL-12p70 production and IL-12Rbeta2 expression were significantly decreased in acute and chronic patients as compared to healthy subjects cured from PCM or healthy infected subjects from endemic areas. Interestingly, the healthy infected Subjects had higher gp43-induced IL12p70 production and beta2 expression than the cured subjects. The addition of a neutralizing anti-IL-10 antibody to the cultures increased IL12p70 levels and beta2 expression in acute and chronic patients to levels observed in Cured subjects. Conversely, addition of the cytokine IL-10 strongly inhibited both parameters in the latter group. In conclusion, we have shown that paracoccidioidomycosis-related Th1 immunosuppression is associated with down-modulation of the IL-12 pathway, that IL-10 may participate in this process, and that patients cured from paracoccidioidomycosis may not fully recover their immune responsiveness. (C) 2004 Elsevier B.V. All rights reserved.

Swimming training exacerbates pathological cardiac hypertrophy in kinin B(2) receptor-deficient mice

Kallikrein-kinin system exerts cardioprotective effects against pathological hypertrophy. These effects are modulated mainly via B(2) receptor activation. Chronic physical exercise can induce physiological cardiac hypertrophy characterized by normal organization of cardiac structure. Therefore, the aim of this work was to verify the influence of kinin B(2) receptor deletion on physiological hypertrophy to exercise stimulus. Animals were submitted to swimming practice for 5 min or for 60 min, 5 days a week, during 1 month and several cardiac parameters were evaluated. Results showed no significantly difference in heart weight between both groups, however an increased left ventricle weight and myocyte diameter were observed after the 60 min swimming protocol, which was more pronounced in B(2)(-/-) mice. In addition, sedentary B(2)(-/-) animals presented higher left ventricle mass when compared to wild-type (WT) mice. An increase in capillary density was observed in exercised animals, however the effect was less pronounced in B(2)(-/-) mice. Collagen, a marker of pathological hypertrophy, was increased in B(2)(-/-) mice submitted to swimming protocol, as well as left ventricular thickness, suggesting that these animals do not respond with physiological hypertrophy for this kind of exercise. In conclusion, our data suggest an important role for the kinin B(2) receptor in physiological cardiac hypertrophy. (c) 2007 Elsevier B.V. All rights reserved.

Endothelial and neuronal nitric oxide synthase inhibitors influences angiotensin II pressor effect in central nervous system

The present study investigated the central role of angiotensin II and nitric oxide on arterial blood pressure (MAP) in rats. Losartan and PD123349 AT 1 and AT 2 (selective no peptides antagonists angiotensin receptors), as well as FK 409 (a nitric oxide donor), N W-nitro-L-arginine methyl ester (L-NAME) a constituve nitric oxide synthase inhibitor endothelial (eNOSI) and 7-nitroindazol (7NI) a specific neuronal nitric oxide synthase inhibitor (nNOSI) were used. Holtzman strain, (Rattus norvergicus) weighting 200-250 g were anesthetized with zoletil 50 mg kg -1 (tiletamine chloridrate 125 mg and zolazepan chloridrate 125 mg) into quadriceps muscle anda stainless steel cannula was stereotaxically implanted into their Lateral Ventricle (LV). Controls were injected with a 0.5 μl volume of 0.15 M NaCl. Angiotensin II injected into LV increased MAP (19±3 vs. control 3±1 mm Hg), which is potentiated by prior injection of L-NAME in the same site 26±2 mm Hg. 7NI injected prior to ANG II into LV also potentiated the pressor effect of ANG II but with a higher intensity than L-NAME 32±3 mm Hg. FK 409 inhibited the pressor effect of ANG II (6±1 mm Hg). Losartan injected into LV before ANG II influences the pressor effect of ANG II (8±1 mm Hg). The PD 123319 decreased the pressor effects of ANG II (16±1 mm Hg). Losartan injected simultaneously with FK 409 blocked the pressor effect of ANG II (3±1 mm Hg). L-NAME produced an increase in the pressor effect of ANG II, may be due to local vasoconstriction and all at once by neuronal NOS inhibition but the main effect is of the 7-NIT an specific nNOS inhibitor. The AT 1 antagonist receptors improve basal nitric oxide (NO) production and release. These data suggest the involvement of constitutive and neuronal NOS in the control of arterial blood pressure induced by ANG II centrally, evolving AT 1 receptor-mediated vasoconstriction and AT 2 receptor-mediated vasodilatation. These results were confirmed by the experiment using FK 409. © 2006 Asian Network for Scientific Information.

In situ expression of mononuclear cell markers and interleukin-2 receptor in renal allograft biopsies of acute rejection and borderline cases

The correct diagnosis of renal allograft rejection may be difficult using only clinical and/or histopathological criteria. Immunological assays should be considered in order to evaluate the phenotype of inflammatory infiltrate in renal allograft biopsies. Immunohistochemical studies were performed to detect mononuclear cells, CD4 and CD8 T lymphocytes, B lymphocytes, macrophages, null cells, and positive cells for interleukin-2 receptors. A total of 41 allograft biopsies classified into three groups were studied: acute cellular rejection (28 biopsies/22 patients), borderline (7 biopsies/5 patients) and control (6 biopsies/6 patients). In the rejection group (RG), increased cellularity was found mainly at the tubulo-interstitial level. Expression of CD8 positive cells was higher in RG when compared to borderline (BG) and control (CG) groups, respectively (0.9 vs. 0.0 vs. 0.35 cells/mm2; p < 0.001). Expression of macrophages was not statistically significant among the three groups (RG = 0.6 vs. BG = 0.2 vs. CG = 0.0 cells/mm2; p < 0.02). In the BG, CD4 + cells predominated (BG = 0.2 vs. RG = 0.05 vs. CG = 0.0 cells/mm2; p < 0.05). Clinically these patients were treated as cases of acute rejection. The numbers and different types of infiltrating cells did not correlate with patient's clinical outcome. Copyright © Informa Healthcare.

Synergist interaction between angiotensin II and DOCA on sodium and water balance in rats

Blockade of central angiotensin receptors with the specific antagonist [Leu8]-ANG II abolished water ingestion and water and sodium excretion induced by infusion of angiotensin II (ANGII) into the lateral ventricle (LV) of rats. The antagonist reduced but did not suppress the salt appetite induced by ANGII infusion. Subcutaneous injection of deoxycorticosterone acetate (DOCA) caused increases in water and 3% NaCl ingestion and decreases in sodium excretion. When central ANGII infusion was combined with peripheral DOCA, the water intake was similar to that induced by ANGII alone and the ingestion of 3% NaCl was increased, whereas sodium excretion was inhibited. When ANGII was infused alone, a detailed temporal analysis of fluid and sodium balance showed a negative balance similar those saline controls that persisted throughout the experiment. Combined administration of ANGII and DOCA induce significant changes in water and sodium balance. Sodium and water maintained a positive balance through out the 8-h experiment. The data support an interaction of central ANGII and DOCA on sodium intake and water and sodium balance. © 1994.

Expression of the P2X(2) receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

AIM: To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. METHODS: The study examined the distribution of the P2X(2) receptor (P2X(2)R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X(2)R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm(2)) and area profile (mu m(2)) of P2X(2)R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. RESULTS: In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X(2)R, nNOS, ChAT and CaIR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X(2)R-IR was observed to co-localize 100% with that for nNOS, ChAT and CaIR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm(2)) of P2X(2)R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CaIR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (mu m(2)) of nNOS-IR, ChAT-IR and CaIR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the nnyenteric ganglia revealed an overall similarity between the two groups. CONCLUSION: We demonstrate increases in P2X(2)R expression and alterations in nNOS, ChAT and CaIR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls. (c) 2012 Baishideng. All rights reserved.

Atorvastatin Prevents the Downregulation of Aquaporin-2 Receptor After Bilateral Ureteral Obstruction and Protects Renal Function in a Rat Model

OBJECTIVE To assess the effects of atorvastatin (ATORV) on renal function after bilateral ureteral obstruction (BUO), measuring inulin clearance and its effect on renal hemodynamic, filtration, and inflammatory response, as well as the expression of Aquaporin-2 (AQP2) in response to BUO and after the release of BUO. METHODS Adult Munich-Wistar male rats were subjected to BUO for 24 hours and monitored during the following 48 hours. Rats were divided into 5 groups: sham operated (n = 6); sham + ATORV (n = 6); BUO (n = 6); BUO + ATORV (10 mg/kg in drinking water started 2 days before BUO [n = 5]; and BUO + ATORV (10 mg/kg in drinking water started on the day of the release of BUO [n = 5]). We measured blood pressure (BP, mm Hg); inulin clearance (glomerular filtration rate [GFR]; mL/min/100 g); and renal blood flow (RBF, mL/min, by transient-time flowmeter). Inflammatory response was evaluated by histologic analysis of the interstitial area. AQP2 expression was evaluated by electrophoresis and immunoblotting. RESULTS Renal function was preserved by ATORV treatment, even if initiated on the day of obstruction release, as expressed by GFR, measured by inulin clearance. Relative interstitial area was decreased in both BUO + ATORV groups. Urine osmolality was improved in the ATORV-treated groups. AQP2 protein expression decreased in BUO animals and was reverted by ATORV treatment. CONCLUSION ATORV administration significantly prevented and restored impairment in GFR and renal vascular resistance. Furthermore, ATORV also improved urinary concentration by reversing the BUO-induced downregulation of AQP2. These findings have significant clinical implication in treating obstructive nephropathy. UROLOGY 80: 485.e15-485.e20, 2012. (c) 2012 Elsevier Inc.

Absence of somatostatin SST(2) receptor internalization in vivo after intravenous SOM230 application in the AR42J animal tumor model

Among clinically relevant somatostatin functions, agonist-induced somatostatin receptor subtype 2 (sst(2)) internalization is a potent mechanism for tumor targeting with sst(2) affine radioligands such as octreotide. Since, as opposed to octreotide, the second generation multi-somatostatin analog SOM230 (pasireotide) exhibits strong functional selectivity, it appeared of interest to evaluate its ability to affect sst(2) internalization in vivo. Rats bearing AR42J tumors endogenously expressing somatostatin sst(2) receptors were injected intravenously with SOM230 or with the [Tyr(3), Thr(8)]-octreotide (TATE) analog; they were euthanized at various time points; tumors and pancreas were analyzed by immunohistochemistry for the cellular localization of somatostatin sst(2) receptors. SOM230-induced sst(2) internalization was also evaluated in vitro by immunofluorescence microscopy in AR42J cells. At difference to the efficient in vivo sst(2) internalization triggered by intravenous [Tyr(3), Thr(8)]-octreotide, intravenous SOM230 did not elicit sst(2) internalization: immunohistochemically stained sst(2) in AR42J tumor cells and pancreatic cells were detectable at the cell surface at 2.5min, 10min, 1h, 6h, or 24h after SOM230 injection while sst(2) were found intracellularly after [Tyr(3), Thr(8)]-octreotide injection. The inability of stimulating sst(2) internalization by SOM230 was confirmed in vitro in AR42J cells by immunofluorescence microscopy. Furthermore, SOM230 was unable to antagonize agonist-induced sst(2) internalization, neither in vivo, nor in vitro. Therefore, SOM230 does not induce sst(2) internalization in vivo or in vitro in AR42J cells and pancreas, at difference to octreotide derivatives with comparable sst(2) binding affinities. These characteristics may point towards different tumor targeting but also to different desensitization properties of clinically applied SOM230.

Characterization of a novel five-transmembrane domain cholecystokinin-2 receptor splice variant identified in human tumors

The cholecystokinin-2 receptor (CCK2R), is expressed in cancers where it contributes to tumor progression. The CCK2R is over-expressed in a sub-set of tumors, allowing its use in tumor targeting with a radiolabel ligand. Since discrepancies between mRNA levels and CCK2R binding sites were noticed, we searched for abnormally spliced variants in tumors from various origins having been previously reported to frequently express cholecystokinin receptors, such as medullary thyroid carcinomas, gastrointestinal stromal tumors, leiomyomas and leiomyosarcomas, and gastroenteropancreatic tumors. A variant of the CCK2R coding for a putative five-transmembrane domains receptor has been cloned. This variant represented as much as 6% of CCK2R levels. Ectopic expression in COS-7 cells revealed that this variant lacks biological activity due to its sequestration in endoplasmic reticulum. When co-expressed with the CCK2R, this variant diminished membrane density of the CCK2R and CCK2R-mediated activity (phospholipase-C and ERK activation). In conclusion, a novel splice variant acting as a dominant negative on membrane density of the CCK2R may be of importance for the pathophysiology of certain tumors and for their in vivo CCK2R-targeting.

Mechanisms of osteoclastogenesis inhibition by a novel class of biphenyl-type cannabinoid CB(2) receptor inverse agonists

The cannabinoid CB(2) receptor is known to modulate osteoclast function by poorly understood mechanisms. Here, we report that the natural biphenyl neolignan 4'-O-methylhonokiol (MH) is a CB(2) receptor-selective antiosteoclastogenic lead structure (K(i) < 50 nM). Intriguingly, MH triggers a simultaneous G(i) inverse agonist response and a strong CB(2) receptor-dependent increase in intracellular calcium. The most active inverse agonists from a library of MH derivatives inhibited osteoclastogenesis in RANK ligand-stimulated RAW264.7 cells and primary human macrophages. Moreover, these ligands potently inhibited the osteoclastogenic action of endocannabinoids. Our data show that CB(2) receptor-mediated cAMP formation, but not intracellular calcium, is crucially involved in the regulation of osteoclastogenesis, primarily by inhibiting macrophage chemotaxis and TNF-α expression. MH is an easily accessible CB(2) receptor-selective scaffold that exhibits a novel type of functional heterogeneity.