Bunodosine 391: An Analgesic Acylamino Acid from the Venom of the Sea Anemone Bunodosoma cangicum

A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors:

Thyroid hormone stimulates NO production via activation of the PI3K/Akt pathway in vascular myocytes

Aims Thyroid hormone (TH) rapidly relaxes vascular smooth muscle cells (VSMCs). However, the mechanisms involved in this effect remain unclear. We hypothesize that TH-induced rapid vascular relaxation is mediated by VSMC-derived nitric oxide (NO) production and is associated with the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signalling pathway. Methods and results NO levels were determined using a NO-specific fluorescent dye (DAF-2) and nitrite (NO(2)) levels. Expression of NO synthase (NOS) isoforms and proteins of the PI3K/Akt pathway was determined by both western blotting and immunocytochemistry. Myosin light chain (MLC) phosphorylation levels were also investigated by western blotting. Exposure of cultured VSMCs from rat thoracic aortas to triiodothyronine (T3) resulted in a significant decrease of MLC phosphorylation levels. T3 also induced a rapid increase in Akt phosphorylation and increased NO production in a dose-dependent manner (0.001-1 mu M). VSMCs stimulated with T3 for 30 min showed an increase in the expression of all three NOS isoforms and augmented NO production, effects that were prevented by inhibitors of PI3K. Vascular reactivity studies showed that vessels treated with T3 displayed a decreased response to phenylephrine, which was reversed by NOS inhibition. These data suggest that T3 treatment induces greater generation of NO both in aorta and VSMCs and that this phenomenon is endothelium independent. In addition, these findings show for the first time that the PI3K/Akt signalling pathway is involved in T3-induced NO production by VSMCs, which occurs with expressive participation of inducible and neuronal NOS. Conclusion Our data strongly indicate that T3 causes NO-dependent rapid relaxation of VSMC and that this effect is mediated by the PI3K/Akt signalling pathway.

Na plus -Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na(+)-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.

Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium

Leite-Dellova DC, Oliveira-Souza M, Malnic G, Mello-Aires M. Genomic and nongenomic dose-dependent biphasic effect of aldosterone on Na(+)/H(+) exchanger in proximal S3 segment: role of cytosolic calcium. Am J Physiol Renal Physiol 295: F1342-F1352, 2008. First published August 20, 2008; doi:10.1152/ajprenal.00048.2008.-The effects of aldosterone on the intracellular pH recovery rate (pHirr) via Na(+)/H(+) exchanger and on the [Ca(2+)](i) were investigated in isolated rat S3 segment. Aldosterone [10(-12), 10(-10), or 10(-8) M with 1-h, 15- or 2-min preincubation (pi)] caused a dose-dependent increase in the pHirr, but aldosterone (10(-6) M with 1-h, 15- or 2-min pi) decreased it (these effects were prevented by HOE694 but not by S3226). After 1 min of aldosterone pi, there was a transient and dose-dependent increase of the [Ca(2+)](i) and after 6-min pi there was a new increase of [Ca(2+)](i) that persisted after 1 h. Spironolactone, actinomycin D, or cycloheximide did not affect the effects of aldosterone (15 -or 2-min pi) but inhibited the effects of aldosterone (1-h pi) on pHirr and on [Ca(2+)](i). RU 486 prevented the stimulatory effect of aldosterone (10(-12) M, 15 -or 2-min pi) on both parameters and maintained the inhibitory effect of aldosterone (10(-6) M, 15- or 2-min pi) on the pHirr but reversed its stimulatory effect on the [Ca(2+)](i) to an inhibitory effect. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on [Ca(2+)](i) and on the basolateral NHE1 and are compatible with stimulation of the NHE1 by increases in [Ca(2+)](i) in the lower range (at 10(-12) M aldosterone) and inhibition by increases at high levels (at 10(-6) M aldosterone) or decreases in [Ca(2+)](i) (at 10(-6) M aldosterone plus RU 486).

Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells

Coordinated proliferation and differentiation of progenitor cells is the base for production of appropriate numbers of neurons and glia during neuronal development in order to establish normal brain functions. We have used murine embryonal carcinoma P19 cells as an in vitro model for early differentiation to study participation of nicotinic (nAChR) and muscarinic acetylcholine (mAChR) receptors in the proliferation of neural progenitor cells and their differentiation to neurons. We have previously shown that functional nicotinic acetylcholine receptors (nAChRs) already expressed in embryonic cells mediate elevations in cytosolic free calcium concentration ([Ca2+](i)) via calcium influx through nAChR channels whereas intracellular stores contribute to nAChR- and mAChR-mediated calcium fluxes in differentiated cells [Resende et al., Cell Calcium 43 (2008) 107-121]. In the present study, we have demonstrated that nicotine provoked inhibition of proliferation in embryonic cells as determined by BrdU labeling. However, in neural progenitor cells nicotine stimulated proliferation which was reversed in the presence of inhibitors of calcium mobilization from intracellular stores, indicating that liberation of intracellular calcium contributed to this proliferation induction. Muscarine induced proliferation stimulation in progenitor cells by activation of G alpha(q/11)-coupled M-1, M-3 and M-5 receptors and intracellular calcium stores, whereas G alpha(i/o)-protein coupled M-2 receptor activity mediated neuronal differentiation. (C) 2008 Elsevier Inc. All rights reserved.

Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Transdural motor cortex stimulation reverses neuropathic pain in rats: A profile of neuronal activation

Motor cortex stimulation (MCS) has been used to treat patients with neuropathic pain resistant to other therapeutic approaches; however, the mechanisms of pain control by MCS are still not clearly understood. We have demonstrated that MCS increases the nociceptive threshold of naive conscious rats, with opioid participation. In the present study, the effect of transdural MCS on neuropathic pain in rats subjected to chronic constriction injury of the sciatic nerve was investigated. In addition, the pattern of neuronal activation, evaluated by Fos and Zif268 immunolabel, was performed in the spinal cord and brain sites associated with the modulation of persistent pain. MCS reversed the mechanical hyperalgesia and allodynia induced by peripheral neuropathy. After stimulation, Fos immunoreactivity (Fos-IR) decreased in the dorsal horn of the spinal cord and in the ventral posterior lateral and medial nuclei of the thalamus, when compared to animals with neuropathic pain. Furthermore, the MCS increased the Fos-IR in the periaqueductal gray, the anterior cingulate cortex and the central and basolateral amygdaloid nuclei. Zif268 results were similar to those obtained for Fos, although no changes were observed for Zif268 in the anterior cingulate cortex and the central amygdaloid nucleus after MCS. The present findings suggest that MCS reverts neuropathic pain phenomena in rats, mimicking the effect observed in humans, through activation of the limbic and descending pain inhibitory systems. Further investigation of the mechanisms involved in this effect may contribute to the improvement of the clinical treatment of persistent pain. (c) 2010 European Federation of International Association for the Study of Pain Chapters. Published by Elsevier Ltd. All rights reserved.

Nutrient Selection in the Absence of Taste Receptor Signaling

When allowed to choose between different macronutrients, most animals display a strong attraction toward carbohydrates compared with proteins. It remains uncertain, however, whether this food selection pattern depends primarily on the sensory properties intrinsic to each nutrient or, alternatively, metabolic signals can act independently of the hedonic value of sweetness to stimulate elevated sugar intake. Here we show that Trpm5(-/-) mice, which lack the cellular mechanisms required for sweet and several forms of L-amino acid taste transduction, develop a robust preference for D-glucose compared with isocaloric L-serine independently of the perception of sweetness. Moreover, a close relationship was found between glucose oxidation and taste-independent nutrient intake levels, with animals increasing intake as a function of glucose oxidation rates. Furthermore, microdialysis measurements revealed nutrient-specific dopaminergic responses in accumbens and dorsal striatum during intragastric infusions of glucose or serine. Specifically, intragastric infusions of glucose induced significantly higher levels of dopamine release compared with isocaloric serine in both ventral and dorsal striatum. Intragastric stimulation of dopamine release seemed to depend on glucose utilization, because administration of an anti-metabolic glucose analog resulted in lower dopamine levels in striatum, an effect that was reversed by intravenous glucose infusions. Together, our findings suggest that carbohydrate-specific preferences can develop independently of taste quality or caloric load, an effect associated with the ability of a given nutrient to regulate glucose metabolism and stimulate brain dopamine centers.

T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats

Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30 min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-1 mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration

Serrano-Nascimento C, Calil-Silveira J, Nunes MT. Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration. Am J Physiol Cell Physiol 298: C893-C899, 2010. First published January 27, 2010; doi:10.1152/ajpcell.00224.2009.-Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 mu g/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid.

Obesity induced by neonatal treatment with monosodium glutamate impairs microvascular reactivity in adult rats: Role of NO and prostanoids

Background and aim: given that obesity is an independent risk factor for the development of cardiovascular diseases we decided to investigate the mechanisms involved in microvascular dysfunction using a monosodium glutamate (MSG)-induced model of obesity, which allows us to work on both normotensive and normoglycemic conditions. Methods and results: Male offspring of Wistar rats received MSG from the second to the sixth day after birth. Sixteen-week-old MSG rats displayed higher Lee index, fat accumulation, dyslipidemia and insulin resistance, with no alteration in glycemia and blood pressure. The effect of norepinephrine (NE), which was increased in MSG rats, was potentiated by L-nitro arginine methyl ester (L-NAME) or tetraethylammonium (TEA) and was reversed by indomethacin and NS-398. Sensitivity to acetylcholine (ACh), which was reduced in MSG rats, was further impaired by L-NAME or TEA, and was corrected by indomethacin, NS-398 and tetrahydrobiopterin (BH4). MSG rats displayed increased endothelium-independent relaxation to sodium nitroprusside. A reduced prostacyclin/tromboxane ratio was found in the mesenteric beds of MSG rats. Mesenteric arterioles of MSG rats also displayed reduced nitric oxide (NO) production along with increased reactive oxygen species (ROS) generation; these were corrected by BH4 and either L-NAME or superoxide dismutase, respectively. The protein expression of eNOS and cyclooxygenase (COX)-2 was increased in mesenteric arterioles from MSG rats. Conclusion: Obesity/insulin resistance has a detrimental impact on vascular function. Reduced NO bioavailability and increased ROS generation from uncoupled eNOS and imbalanced release of COX products from COX-2 play a critical role in the development of these vascular alterations (C) 2010 Elsevier B.V. All rights reserved.

Upregulation of gp91(phox) Subunit of NAD(P)H Oxidase Contributes to Erectile Dysfunction Caused by Long-term Nitric Oxide Inhibition in Rats: Reversion by Regular Physical Training

OBJECTIVES To test the hypothesis that glyco protein 91phox (gp91(phox)) subunit of nicotinamide adenine dinucleotide phosphate [NAD(P) H] oxidase is a fundamental target for physical activity to ameliorate erectile dysfunction (ED). Vascular risk factors are reported to contribute to ED. Regular physical exercise prevents cardiovascular diseases by increasing nitric oxide (NO) production and/or decreasing NO inactivation. METHODS Male Wistar rats received the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) for 4 weeks, after which animals were submitted to a run training program for another 4 weeks. Erectile functions were evaluated by in vitro cavernosal relaxations and intracavernous pressure measurements. Expressions of gp91(phox) subunit and neuronal nitric oxidase synthase in erectile tissue, as well as superoxide dismutase activity and nitrite/nitrate (NO(x)) levels were determined. RESULTS The in vitro acetylcholine-and electrical field stimulation-induced cavernosal relaxations, as well as the increases in intracavernous pressure were markedly reduced in sedentary rats treated with L-NAME. Run training significantly restored the impaired cavernosal relaxations. No alterations in the neuronal nitric oxidase synthase protein expression (and its variant penile neuronal nitric oxidase synthase) were detected. A reduction of NO(x) levels and superoxide dismutase activity was observed in L-NAME-treated animals, which was significantly reversed by physical training. Gene expression of subunit gp91(phox) was enhanced by approximately 2-fold in erectile tissue of L-NAME-treated rats, and that was restored to basal levels by run training. CONCLUSIONS Our study shows that ED seen after long-term L-NAME treatment is associated with gp91(phox) subunit upregulation and decreased NO bioavailability. Exercise training reverses the increased oxidative stress in NO-deficient rats, ameliorating the ED. UROLOGY 75: 961-967, 2010. (C) 2009 Elsevier Inc.

Ablation of primary afferent neurons by neonatal capsaicin treatment reduces the susceptibility of the portal hypertensive gastric mucosa to ethanol-induced injury in cirrhotic rats

Primary sensory afferent neurons modulate the hyperdynamic circulation in Cirrhotic rats with portal hypertension.The stomach of cirrhotic rats is prone to damage induced by ethanol, a phenomenon associated with reduced gastric hyperemic response to acid-back diffusion. The aim of this study was to examine the impact of ablation of capsaicin-sensitive neurons and the tachykinin NK(1) receptor antagonist A5330 on the susceptibility of the portal hypertensive gastric mucosa, to ethanol-induced injury and its effects on gastric cyclooxygenase (COX) and nitric oxide synthase (NOS) mRNA expression. Capsaicin was administered to neonatal, male, Wistar rats and the animals were allowed to grow. Cirrhosis was then induced by bile duct ligation in adult rats while controls had sham operation. Ethanol-induced gastric damage was assessed using ex vivo gastric chamber experiments. Gastric blood flow was measured as well as COX/NOS mRNA expression. Topical application of ethanol produced significant gastric damage in cirrhotic rats compared to controls, which was reversed in capsaicin- and A5330-treated animals. Mean arterial and portal pressure was normalized in capsaicin-treated cirrhotic rats. Capsaicin and A5330 administration restored gastric blood flow responses to topical application of ethanol followed by acid in cirrhotic rats. Differential COX and NOS mRNA expression was noted in bile duct ligated rats relative to controls. Capsaicin treatment significantly modified gastric eNOS/iNOS/COX-2 mRNA expression in cirrhotic rats. Capsaicin-sensitive neurons modulate the susceptibility of the portal hypertensive gastric mucosa to injury induced by ethanol via tachykinin NK(1) receptors and signalling of prostaglandin and NO production/release. (c) 2008 Elsevier B.V. All rights reserved.

Small bowel injury associated to allergy is triggered by platelet-activating factor, mast cells, neutrophils and protected by nitric oxide

Allergy to components of the diet is followed by gut inflammation which in children, sometimes progress to mucosal lesions and anaphylaxis. In newborns suffering of cow`s milk allergy, bloody stools, rectal. bleeding and ulcerations are found. The rat systemic anaphylaxis is a suitable model to study the intestinal lesions associated to allergy. In the present study we used this model to investigate some mechanisms involved. We found that 15 min after antigen challenge of sensitized rats, hemorrhagic lesions develop in the small intestine. The lesions were more severe in jejunum and ileum compared to duodenum. Pretreatment of the rats with a platelet-activating factor-receptor antagonist (WEB-2170) reduced the lesions whereas inhibition of endogenous nitric oxide by L-NAME, greatly increased the hemorrhagic lesions and mortality. Both, lesions and mortality were reversed by L-arginine. The hemorrhagic lesions were also significantly reduced by the mast cell stabilizers, disodium cromoglycate and ketotifen as well as by neutrophils depletion (with anti-PMN antibodies) or inhibition of selectin binding (by treatment with fucoidan). Thus, the intestinal hemorrhagic lesions in this model are dependent on ptatelet-activating factor, mast cell granule-derived mediators and neutrophils. Endogenous nitric oxide and supplementation with L-arginine has a protective role, reducing the lesions and preventing mortality. These results contributed to elucidate mechanisms involved in intestinal lesions which could be of relevance to human small bowel injury associated to allergy. (c) 2007 Elsevier B.V. All rights reserved.

Pivotal Role for Platelet-Activating Factor Receptor in CD36 Expression and oxLDL Uptake by Human Monocytes/Macrophages

The uptake of oxLDL by CD36 is not regulated by intracellular levels of cholesterol, leading to macrophage differentiation into foam cells which play a major role in atherosclerosis. Furthermore, oxLDL competes with PAF in macrophages for binding to PAF receptors (PAFR). Here we investigated the involvement of PAFR in CD36 expression and uptake of oxLDL by human monocytes/macrophages. Adherent peripheral blood mononuclear cells were treated with PAFR-antagonists (WEB2170, CV3988); inhibitors of ERK1/2 (PD98059), p38 (SB203580), JNK (SP600125) or diluents, before stimulation with oxLDL or PAF. After 24 h, uptake of FITC oxLDL and expression of CD36 was determined by flow cytometry and phosphorylation of MAP-kinases by Western blot. It was shown that the uptake of oxLDL was reduced by PAFR antagonists. CD36 expression was up-regulated by oxLDL, an effect reversed by PAFR antagonists. The up-regulation of CD36 and oxLDL uptake both required MAP-kinases activation. The oxLDL induced ERK1/2 and JNK but not p38 phosphorylation was reversed by PAFR-antagonists suggesting that oxLDL signalling involves PAFR dependent and independent pathways. In macrophages from PAFR(-/-) mice, oxLDL was unable to up-regulate CD36 expression and the oxLDL uptake was reduced compared to wild type. These results suggest that oxLDL interacts with PAFR in macrophages to increase CD36 expression and oxLDL uptake. Whereas pharmacological intervention at the level of PAFR would be beneficial in atherosclerosis remains to be determined. Copyright (C) 2011 S. Karger AG, Basel