Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR

Crajoinas RO, Lessa LMA, Carraro-Lacroix LR, Davel APC, Pacheco BPM, Rossoni LV, Malnic G, Girardi ACC. Posttranslational mechanisms associated with reduced NHE3 activity in adult vs. young prehypertensive SHR. Am J Physiol Renal Physiol 299:F872-F881, 2010. First published July 14, 2010; doi:10.1152/ajprenal.00654.2009.-Abnormalities in renal proximal tubular (PT) sodium transport play an important role in the pathophysiology of essential hypertension. the Na(+)/H(+) exchanger isoform 3 (NHE3) represents the major route for sodium entry across the apical membrane of renal PT cells. We therefore aimed to assess in vivo NHE3 transport activity and to define the molecular mechanisms underlying NHE3 regulation before and after development of hypertension in the spontaneously hypertensive rat (SHR). NHE3 function was measured as the rate of bicarbonate reabsorption by means of in vivo stationary microperfusion in PT from young prehypertensive SHR (Y-SHR; 5-wk-old), adult SHR (A-SHR; 14-wk-old), and age-matched Wistar Kyoto (WKY) rats. We found that NHE3-mediated PT bicarbonate reabsorption was reduced with age in the SHR (1.08 +/- 0.10 vs. 0.41 +/- 0.04 nmol/cm(2)xs), while it was increased in the transition from youth to adulthood in the WKY rat (0.59 +/- 0.05 vs. 1.26 +/- 0.11 nmol/cm(2)xs). Higher NHE3 activity in the Y-SHR compared with A-SHR was associated with a predominant microvilli confinement and a lower ratio of phosphorylated NHE3 at serine-552 to total NHE3 (P-NHE3/total). After development of hypertension, P-NHE3/total increased and NHE3 was retracted out of the microvillar microdomain along with the regulator dipeptidyl peptidase IV (DPPIV). Collectively, our data suggest that the PT is playing a role in adapting to the hypertension in the SHR. the molecular mechanisms of this adaptation possibly include an increase of P-NHE3/total and a redistribution of the NHE3-DPPIV complex from the body to the base of the PT microvilli, both predicted to decrease sodium reabsorption.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Univ São Paulo, Sch Med, Heart Inst InCor, BR-05403900 São Paulo, BrazilUniv São Paulo, Inst Biomed Sci, Dept Physiol & Biophys, BR-05403900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Physiol, São Paulo, BrazilWeb of Scienc

The antiapoptotic effect of granulocyte colony-stimulating factor reduces infarct size and prevents heart failure development in rats

Background/Aim. Granulocyte colony-stimulating factor (G-CSF) reduces myocardial injury and improves cardiac function after myocardial infarction (MI). We investigated the early alterations provided by G-CSF and the chronic repercussions in infarcted rats. Methods. Male Wistar rats (200-250g) received vehicle (MI) or G-CSF (MI-GCSF) (50 mu g/kg, sc) at 7, 3 and 1 days before MI surgery. Afterwards MI was produced and infarct size was measured 1 and 15 days after surgery. Expression of anti-and proapoptotic proteins was evaluated immediately before surgery. 24 hours after surgery, apoptotic nuclei were evaluated. Two weeks after MI, left ventricular (LV) function was evaluated, followed by in situ LV diastolic pressure-volume evaluation. Results. Infarct size was decreased by 1 day pretreatment before occlusion (36 +/- 2.8 vs. 44 +/- 2.1% in MI; P<0.05) and remained reduced at 15 days after infarction (28 +/- 2.2 vs. 36 +/- 1.4% in MI; P<0.05). G-CSF pretreatment increased Bcl-2 and Bcl-xL protein expression, but did not alter Bax in LV. Apoptotic nuclei were reduced by treatment (Sham: 0.46 +/- 0.42, MI: 15.5 +/- 2.43, MI-GCSF: 5.34 +/- 3.34%; P<0.05). Fifteen days after MI, cardiac function remained preserved in G-CSF pretreated rats. The LV dilation was reduced in MI-G-CSF group as compared to MI rats, being closely associated with infarct size. Conclusion. The early beneficial effects of G-CSF were essentials to preserve cardiac function at a chronic stage of myocardial infarction2813340CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESPsem informaçã

L-NAME treatment enhances exercise-induced content of myocardial heat shock protein 72 (Hsp72) in rats

Background/Aim: Nitric oxide (NO) modulates the expression of the chaperone Hsp72 in the heart, and exercise stimulates both NO production and myocardial Hsp72 expression. The main purpose of the study was to investigate whether NO interferes with an exercise-induced myocardial Hsp72 expression. Methods: Male Wistar rats (70-100 days) were divided into control (C, n= 12), L-NAME-treated (L, n= 12), exercise (E, n= 13) and exercise plus L-NAME-treated (EL, n= 20) groups. L-NAME was given in drinking water (700 mg. L-1) and the exercise was performed on a treadmill (15-25 m.min(-1), 40-60 min. day(-1)) for seven days. Left ventricle (LV) protein Hsp content, NOS and phosphorylated-NOS (p-NOS) isoforms were measured using Western blotting. The activity of NOS was assayed in LV homogenates by the conversion of [H-3] L-arginine to [H-3] L-citrulline. Results: Hsp72 content was increased significantly (223%; p < 0.05) in the E group compared to the C group, but exercise alone did not alter the NOS content, p-NOS isoforms or NOS activity. Contrary to our expectation, L-NAME enhanced (p < 0.05) the exercise-induced Hsp72 content (EL vs. C, L and E groups = 1019%, 548% and 457%, respectively). Although the EL group had increased stimulatory p-eNOS(Ser1177) (over 200%) and decreased inhibitory p-nNOS(Ser852) (similar to 50%) compared to both the E and L groups (p < 0.05), NOS activity was similar in all groups. Conclusions: Our results suggest that exercise-induced cardiac Hsp72 expression does not depend on NO. Conversely, the in vivo L-NAME treatment enhances exercise-induced Hsp72 production. This effect may be due to an increase in cardiac stress. Copyright (C) 2011 S. Karger AG, Basel275479486CONSELHO NACIONAL DE DESENVOLVIMENTO CIENTÍFICO E TECNOLÓGICO - CNPQFUNDAÇÃO DE AMPARO À PESQUISA E INOVAÇÃO DO ESPÍRITO SANTO - FAPESsem informaçã

Inhibition of Giα protein or ERK1/2 activation reversed hypercontractility to phenylephrine induced by β-AR overactivation in aorta of wild-type, but not in β₂KO mice.

<p>Effect of pertussis toxin (PTx, 4 μM) and PD98,059 (1 μM) on the concentration-response curves to phenylephrine in aortic rings of wild-type (WT) (A, D) and β₂KO (B, E) mice treated for 7 days with vehicle or isoproterenol (ISO). The contraction response is expressed as a % of the contraction to KCl (125 mM). Bar graphs show differences in the area under the concentration-response curve (AUC) in the presence or absence of PTx (C) or PD98,059 (F) in WT and β₂KO mice treated or not with ISO. Values are presented as the mean ± SEM. The number of animals used in each group is indicated in parenthesis. Significance was assessed using a 2-way ANOVA: ⁺p<0.05 <i>vs.</i> WT ISO; *p<0.05 <i>vs.</i> WT.</p

Aortic β-AR subtypes expression.

<p>Protein expression of β₁- β₂- and β₃-adrenoceptors (AR) evaluated in the membrane fraction of aortas from WT, β₁KO and β₂KO mice treated for 7 days with vehicle or isoproterenol (ISO). (A) Representative Western-blot autoradiographs for each β-AR subtype in membrane preparations of aorta and positive controls (+C: heart for β₁-AR; skeletal muscle for β₂-AR; adipose tissue for β₃-AR). Densitometric quantification was evaluated for β₁- (B) and β₂-AR (C) but not for β₃-AR, as this subtype was not expressed (n.e.) in the mouse aorta. The number of samples analyzed (pool of 3 aortas in each sample) is indicated in the bar for each group. Values (mean ± SEM) are expressed the fold-change in β-AR expression compared to the WT. Significance was assessed using a 2-way ANOVA.</p

Isoproterenol treatment induces β₂-AR-Gi-ERK1/2 pathway activation and eNOS uncoupling.

<p>Protein expression of Giα-1,2 (A), Giα-3 (B), ERK 1/2 phosphorylated at Thr²⁰² and Tyr²⁰⁴ (C), p38 MAPK phosphorylated at Thr¹⁸⁰ and Tyr¹⁸² (D) and eNOS protein dimerization (E) in aortas from control and 7-day isoproterenol-treated (ISO) wild-type (WT) and β₂KO mice. The top panels in each graph represent typical Western-blot autoradiographs. Giα protein expression was normalized to the α-actin content in each sample, and phosphorylated ERK 1/2 and p38 MAPK were normalized to the total content of ERK 1/2 and p38 MAPK, respectively, and these results were expressed as the fold-change compared to WT aorta. eNOS dimerization was expressed as ratio of dimer:monomer band intensity. The number of animals used in each group is indicated in the bars. Values are presented as the mean ± SEM. Significance was assessed using a 2-way ANOVA: *p<0.05 <i>vs.</i> WT.</p