Brain nitric oxide production by a proline-rich decapeptide from Bothrops jararaca venom improves baroreflex sensitivity of spontaneously hypertensive rats

Baroreflex sensitivity is disturbed in many people with cardiovascular diseases such as hypertension. Brain deficiency of nitric oxide (NO), which is synthesized by NO synthase (NOS) in the citrulline-NO cycle (with argininosuccinate synthase (ASS) activity being the rate-limiting step), contributes to impaired baroreflex. We recently showed that a decapeptide isolated from Bothrops jararaca snake venom, denoted Bj-PRO-10c, exerts powerful and sustained antihypertensive activity. Bj-PRO-10c promoted vasodilatation dependent on the positive modulation of ASS activity and NO production in the endothelium, and also acted on the central nervous system, inducing the release of GABA and glutamate, two important neurotransmitters in the regulation of autonomic systems. We evaluated baroreflex function using the regression line obtained by the best-fit points of measured heart rate (HR) and mean arterial pressure (MAP) data from spontaneously hypertensive rats (SHRs) treated with Bj-PRO-10c. We also investigated molecular mechanisms involved in this effect, both in vitro and in vivo. Bj-PRO-10c mediated an increase in baroreflex sensitivity and a decrease in MAP and HR. The effects exerted by the peptide include an increase in the gene expression of endothelial NOS and ASS. Bj-PRO-10c-induced NO production depended on intracellular calcium fluxes and the activation of a G(i/o)-protein-coupled metabotropic receptor. Bj-PRO-10c induced NO production and the gene expression of ASS and endothelial NOS in the brains of SHRs, thereby improving baroreflex sensitivity. Bj-PRO-10c may reveal novel approaches for treating diseases with impaired baroreflex function. Hypertension Research (2010) 33, 1283-1288; doi: 10.1038/hr.2010.208

Chronic methionine load-induced hyperhomocysteinemia impairs the relaxation induced by bradykinin in the isolated rat carotid

This study investigates the effects of chronic methionine intake on bradykinin (BK)-relaxation. Vascular reactivity experiments were performed on carotid rings from male Wistar rats. Treatment with methionine (0.1, 1 or 2 g kg(-1) per day) for 8 and 16 weeks, but not for 2 and 4 weeks, reduced the relaxation induced by BK. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, and SQ29548, a selective thromboxane A(2) (TXA(2))/prostaglandin H(2) (PGH(2)) receptor antagonist prevented the reduction in BK-relaxation observed in the carotid from methionine-treated rats. Conversely, AH6809, a selective prostaglandin F(2 alpha) (PGF(2 alpha)) receptor antagonist did not alter BK-relaxation in the carotid from methionine-treated rats. The nitric oxide synthase (NOS) inhibitors L-NAME, L-NNA and 7-nitroindazole reduced the relaxation induced by BK in carotids from control and methionine-treated rats. In summary, we found that chronic methionine intake impairs the endothelium-dependent relaxation induced by BK and this effect is due to an increased production of endothelial vasoconstrictor prostanoids (possibly TXA(2)) that counteracts the relaxant action displayed by the peptide.

Volume-activated chloride channels in mice Leydig cells

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume- activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of similar to 75 mOsm. These currents display the typical biophysical signature of volume- activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(-)>Cl(-)>F(-)). Staurosporine (200 nM) did not block the activation of I(Cl), swell. The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 mu M), SITS (200 mu M), ATP (500 mu M), pyridoxalphosphate-6- azophenyl-2`,4`-disulfonate (PPADS; 100 mu M), and Suramin (10 mu M) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(d)(0) and fractional distance of the binding site (delta) of 334 mu M and 47%, 880 mu M and 35%, 2,100 mu M and 49%, 188 mu M and 27%, and 66.5 mu M and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that ICl, swell in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Genomic and nongenomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments

Leite-Dellova DC, Malnic G, Mello-Aires M. Genomic and non-genomic stimulatory effect of aldosterone on H(+)-ATPase in proximal S3 segments. Am J Physiol Renal Physiol 300: F682-F691, 2011. First published December 29, 2010; doi:10.1152/ajprenal.00172.2010.-The genomic and nongenomic effects of aldosterone on the intracellular pH recovery rate (pHirr) via H(-)(+)ATPase and on cytosolic free calcium concentration ([Ca(2+)](i)) were investigated in isolated proximal S3 segments of rats during superfusion with an Na(+)-free solution, by using the fluorescent probes BCECF-AM and FLUO-4-AM, respectively. The pHirr, after cellular acidification with a NH(4)Cl pulse, was 0.064 +/- 0.003 pH units/min (n = 17/74) and was abolished with concanamycin. Aldosterone (10(-12), 10(-10),10(-8), or 10(-6) M with 1-h or 15- or 2-min preincubation) increased the pHirr. The baseline [Ca(2+)](i) was 103 +/- 2 nM (n = 58). After 1 min of aldosterone preincubation, there was a transient and dose-dependent increase in [Ca(2+)](i) and after 6-min preincubation there was a new increase in [Ca(2+)](i) that persisted after 1 h. Spironolactone [mineralocorticoid (MR) antagonist], actinomycin D, or cycloheximide did not affect the effects of aldosterone (15- or 2-min preincubation) on pHirr and on [Ca(2+)](i) but inhibited the effects of aldosterone (1-h preincubation) on these parameters. RU 486 [glucocorticoid (GR) antagonist] and dimethyl-BAPTA (Ca(2+) chelator) prevented the effect of aldosterone on both parameters. The data indicate a genomic (1 h, via MR) and a nongenomic action (15 or 2 min, probably via GR) on the H(+)-ATPase and on [Ca(2+)](i). The results are compatible with stimulation of the H(+)-ATPase by increases in [Ca(2+)](i) (at 10(-12)-10(-6) M aldosterone) and inhibition of the H(+)-ATPase by decreases in [Ca(2+)](i) (at 10(-12) or 10(-6) M aldosterone plus RU 486).

Adenosine modulates alpha2-adrenergic receptors through a phospholipase C pathway in brainstem cell culture of rats

Adenosine acts in the nucleus tractus solitarii (NTS), one of the main brain sites related to cardiovascular control. In the present study we show that A(1) adenosine receptor (A(1R)) activation promotes an increase on alpha(2)-adrenoceptor (Alpha(2R)) binding in brainstem cell culture from newborn rats. We investigated the intracellular cascade involved in such modulatory process using different intracellular signaling molecule inhibitors as well as calcium chelators. Phospholipase C, protein kinase Ca(2+)-dependent, IP(3) receptor and intracellular calcium were shown to participate in A(1R)/Alpha(2R) interaction. In conclusion, this result might be important to understand the role of adenosine within the NTS regarding autonomic cardiovascular control. (C) 2009 Elsevier B.V. All rights reserved.

Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule

Pergher PS, Leite-Dellova D, de Mello-Aires M. Direct action of aldosterone on bicarbonate reabsorption in in vivo cortical proximal tubule. Am J Physiol Renal Physiol 296: F1185-F1193, 2009. First published February 18, 2009; doi:10.1152/ajprenal.90217.2008.-The direct action of aldosterone (10(-12) M) on net bicarbonate reabsorption (J(HCO3)(-)) was evaluated by stationary microperfusion of an in vivo middle proximal tubule (S2) of rat kidney, using H ion-sensitive microelectrodes. Aldosterone in luminally perfused tubules caused a significant increase in J(HCO3)(-) from a mean control value of 2.84 +/- 0.08 [49/19 (n degrees of measurements/n degrees of tubules)] to 4.20 +/- 0.15 nmol.cm(-2).s(-1) (58/10). Aldosterone perfused into peritubular capillaries also increased J(HCO3)(-), compared with basal levels during intact capillary perfusion with blood. In addition, in isolated perfused tubules aldosterone causes a transient increase of cytosolic free calcium ([Ca(2+)](i)), monitored fluorometrically. In the presence of ethanol ( in similar concentration used to prepare the hormonal solution), spironolactone (10(-6) M, a mineralocorticoid receptor antagonist), actinomycin D (10(-6) M, an inhibitor of gene transcription), or cycloheximide (40 mM, an inhibitor of protein synthesis), the J(HCO3)(-) and the [Ca(2+)](i) were not different from the control value; these drugs also did not prevent the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). However, in the presence of RU 486 alone [10(-6) M, a classic glucocorticoid receptor (GR) antagonist], a significant decrease on J(HCO3)(-) and on [Ca(2+)](i) was observed; this antagonist also inhibited the stimulatory effect of aldosterone on J(HCO3)(-) and on [Ca(2+)](i). These studies indicate that luminal or peritubular aldosterone (10(-12) M) has a direct nongenomic stimulatory effect on J(HCO3)(-) and on [Ca(2+)](i) in proximal tubule and that probably GR participates in this process. The data also indicate that endogenous aldosterone stimulates J(HCO3)(-) in middle proximal tubule.

Association of NAD(P)H Oxidase with Glucose-Induced Insulin Secretion by Pancreatic beta-Cells

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H] oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C] glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown. (Endocrinology 150: 2197-2201, 2009)

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

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.

Testosterone suppresses oxidative stress in human neutrophils

The in vitro effect of testosterone on human neutrophil function was investigated. Blood neutrophils from healthy male subjects were isolated and treated with 10 nM, 0.1 and 10 mu M testosterone for 24 h. As compared with untreated cells, the testosterone treatment produced a significant decrease of superoxide production as indicated by the measurement of extra- and intracellular superoxide content. An increment in the production of nitric oxide was observed at 0.1 and 10 mu M testosterone concentrations, whereas no effect was found for 10 nM. Intracellular calcium mobilization was significantly increased at 10 nM, whereas it was reduced at 10 mu M testosterone. There was an increase in phagocytic capacity at 10 nM and a decrease of microbicidal activity in neutrophils treated with testosterone at 10 mu M. Glutathione reductase activity was increased by testosterone treatment, whereas no effect was observed in other antioxidant enzyme activities. An increase in the content of thiol groups was observed at all testosterone concentrations. Lipid peroxidation in neutrophils evaluated by levels of TBARS was decreased at 10 nM and 0.1 mu M testosterone. These results indicate the antioxidant properties of testosterone in neutrophils as suggested by reduction of superoxide anion production, and lipid peroxidation, and by the increase in nitric oxide production, glutathione reductase activity and the content of thiol groups. Therefore, the plasma levels of testosterone are important regulators of neutrophil function and so of the inflammatory response. Copyright (C) 2010 John Wiley & Sons, Ltd.

Progression of Diet-Induced Diabetes in C57BL6J Mice Involves Functional Dissociation of Ca(2+) Channels From Secretory Vesicles

OBJECTIVE The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS-C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes. Diabetes 59:1192-1201, 2010

Novel endogenous peptide agonists of cannabinoid receptors

Hemopressin (Hp), a 9-residue alpha-hemoglobin-derived peptide, was previously reported to function as a CB(1) cannabinoid receptor antagonist (1). In this study, we report that mass spectrometry (MS) data from peptidomics analyses of mouse brain extracts identified N-terminally extended forms of Hp containing either three (RVD-Hp alpha) or two (VD-Hp alpha) additional amino acids, as well as a beta-hemoglobinderived peptide with sequence similarity to that of hemopressin (VD-Hp beta). Characterization of the alpha-hemoglobin-derived peptides using binding and functional assays shows that in contrast to Hp, which functions as a CB(1) cannabinoid receptor antagonist, both RVD-Hp alpha and VD-Hp alpha function as agonists. Studies examining the increase in the phosphorylation of ERK1/2 levels or release of intracellular Ca(2+) indicate that these peptides activate a signal transduction pathway distinct from that activated by the endo-cannabinoid, 2-arachidonoylglycerol, or the classic CB(1) agonist, Hu-210. This finding suggests an additional mode of regulation of endogenous cannabinoid receptor activity. Taken together, these results suggest that the CB(1) receptor is involved in the integration of signals from both lipid-and peptide-derived signaling molecules.-Gomes, I., Grushko, J. S., Golebiewska, U., Hoogendoorn, S., Gupta, A., Heimann, A. S., Ferro, E. S., Scarlata, S., Fricker, L. D., Devi, L. A. Novel endogenous peptide agonists of cannabinoid receptors. FASEB J. 23, 3020-3029 (2009). www.fasebj.org

Peptidomic Analysis of Human Cell Lines

Peptides have been proposed to function in intracellular signaling within the cytosol. Although cytosolic peptides are considered to be highly unstable, a large number of peptides have been detected in mouse brain and other biological samples. In the present study, we evaluated the peptidome of three diverse cell lines: SH-SY5Y, MCF7, and HEIC293 cells. A comparison of the peptidomes revealed considerable overlap in the identity of the peptides found in each cell line. The majority of the observed peptides are not derived from the most abundant or least stable proteins in the cell, and approximately half of the cellular peptides correspond to the N- or C- termini of the precursor proteins. Cleavage site analysis revealed a preference for hydrophobic residues in the PI position. Quantitative peptidomic analysis indicated that the levels of most cellular peptides are not altered in response to elevated intracellular calcium, suggesting that calpain is not responsible for their production. The similarity of the peptidomes of the three cell lines and the lack of correlation with the predicted cellular degradome implies the selective formation or retention of these peptides, consistent with the hypothesis that they are functional in the cells.

The Central Nervous System as Target for Antihypertensive Actions of a Proline-Rich Peptide from Bothrops jararaca Venom

Pyroglutamyl proline-rich oligopeptides, present in the venom of the pit viper Bothrops jararaca (Bj-PROs), are the first described naturally occurring inhibitors of the angiotensin I-converting enzyme (ACE). The inhibition of ACE by the decapeptide Bj-PRO-10c (intracellular calcium concentration ([Ca(2+)](i)) in primary culture from postnatal rat brain. The N-terminal sequence of the peptide was not essential for induction of calcium fluxes, while any changes of C-terminal Pro or Ile residues affected Bj-PRO-10c`s activity. Using calcium imaging by confocal microscopy and fluorescence imaging plate reader analysis, we have characterized Bj-PRO-10c-induced [Ca(2+)](i) transients in rat brain cells as being independent from bradykinin-mediated effects and ACE inhibition. Bj-PRO-10c induced pertussis toxin-sensitive G(i/o)-protein activity mediated through a yet unknown receptor, influx and liberation of calcium from intracellular stores, as well as reduction of intracellular cAMP levels. Bj-PRO-10c promoted glutamate and GABA release that may be responsible for its antihypertensive activity and its effect on HR. (C) 2010 International Society for Advancement of Cytometry

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)