Effect of nebivolol on cardiovascular changes associated with a rat model of insulin-resistance.

Nebivolol is a vasodilator that combines beta-adrenergic blocking activity with a relaxant effect on vascular smooth muscle cells (VSMC) mediated by the endothelial nitric oxide (NO) pathway. FFR provide a model of dietary-induced insulin-resistance syndrome, which has been used to study the pathophysiological mechanisms associated with this syndrome. Our main objective was to examine the effect of long-term administration of nebivolol on metabolic and cardiovascular variables in fructose-fed rats (FFR), a model in which an altered bioavailability of NO has been already described. Male Wistar rats were randomly assigned to 4 groups (n = 8 each): I. Control (C); II. Control + nebivolol (C+N): 1 mg/kg(-1) x day(-1) in drinking water during the last 4 weeks. III. FFR: rats receiving fructose in drinking water as a 10% (w/v) solution during 8 weeks, and IV. FFR+N: idem II plus III. During the 8 weeks experimental period, variations in systolic blood pressure (SBP), glucose tolerance test (GTT) and plasma thiobarbituric acid-reactive substances (TBARS) were assessed. At the end of this experimental period, rats were killed and heart and kidneys were excised for calculation of relative heart weight (RHW) and histological evaluation of lumen to media ratio (L/M) in renal arteries. Rats from FFR group increased their SBP and RHW, showed glucose intolerance and an increment in lipid peroxidation. Moreover, FFR showed vascular remodeling in renal arteries evidenced by changes in L/M. Although the metabolic changes were not reverted by the administration of nebivolol, this drug successfully decreased SBP, TBARS levels and reverted structural changes such as cardiac hypertrophy and renal arterial remodeling. Data demonstrate that nebivolol administration could participate in the reversion of cardiovascular structural changes associated with the insulin-resistance syndrome.

Chronic administration of losartan reverses cardiovascular changes in hypertensive fructose-fed rats.

The cluster of risk factors including hyperinsulinemia, insulin resistance, hypertriglyceridemia and hypertension has been called syndrome X. Several evidences link the insulin resistance syndrome with endothelial dysfunction. Since the participation of the renin-angiotensin system (RAS) in this pathology is still unclear, the present study examined the effect of chronic administration of an angiotensin AT1 receptor antagonist, losartan (L), on endothelial nitric oxide synthase (eNOS) activity in aortic endothelium and cardiac tissue, and on the proliferation of primary cultured aortic smooth muscle cells (SMC), obtained from fructose-fed rats (FFR), an experimental model of syndrome X Male Wistar rats were used: Control, FFR and FFR+L (n = 8 in each group). After 8 weeks, tissue samples were obtained and 10% fetal calf serum (FCS) proliferative effect was examined in SMC by 3H-thymidine incorporation and cell counting. The eNOS activity was estimated in aortic endothelial lining and cardiac homogenates by conversion of 3H-arginine into 3H-citrulline. FFR aortic SMC showed a significantly increased 10% FCS-induced 3H-thymidine incorporation and cell number compared to controls. FFR aortic and cardiac eNOS activities were significantly decreased. Chronic treatment with L decreased systolic blood pressure,reverted cardiac hypertrophy, abolished the increased SMC proliferation and restoredeNOS activity. These data confirm that changes in SMC proliferation and endothelial dysfunction at different levels of the cardiovascular system are involved in syndrome "X", and that AT1 receptor blocking can revert those changes, suggesting an important role of the RAS, possibly mediated by AT2 receptors and kinins, in the physiopathological mechanisms of this model.

Proteoglycans production by aortic vascular smooth muscle cells from hypertensive rats

Remodeling of large and small arteries contributes to the development and complications of hypertension. Artery structural changes in chronic sustained hypertension include vascular smooth muscle cells (VSMC) proliferation and extracellular matrix (ECM) modifications. Extracellular constituents such as proteoglycans (PGs), may modulate vascular stiffness and VSMC growth and differentiation. We examined the effect of growth factors on secreted and membrane-bound PGs synthesis by cultured aortic smooth muscle cells (SMC) from 12- to 14- week-old spontaneously hypertensive rats (SHR) and age-matched Wistar rats. After stimulation with platelet-derived growth factor (PDGF-BB), 10% fetal calf serum (FCS) or 0.1% FCS as control, PGs synthesis (dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method using both [3H]-glucosamine and [35S]-sodium sulfate which are incorporated into all complex carbohydrates or only into sulfated dysaccharides, respectively. Data are presented as percent of the control (0.1% FCS). SHR VSMC displayed a significantly greater synthesis of M-ECM [3H]-PGs than Wistar rat cells, with both treatments, but no differences in M-ECM [35S] uptake were found in any case. In the P-ECM, both PDGF-BB and 10% FCS produced a greater effect on [3H]-PGs and sulfated PGs synthesis in VSMC from SHR. An important change seen in SHR cells was a significant decreased sulfation, assessed by [35S]/[3H] ratio, in basal and stimulation conditions. Present results indicate the existence of changes in PGS synthesis and modulation in VSMC from a conduit-artery of SHR and support the pathophysiological role proposed for matrix proteoglycans in the vascular wall changes associated to hypertension and related vascular diseases as atherosclerosis.

Proteoglycans production by aortic vascular smooth muscle cells from hypertensive rats

Remodeling of large and small arteries contributes to the development and complications of hypertension. Artery structural changes in chronic sustained hypertension include vascular smooth muscle cells (VSMC) proliferation and extracellular matrix (ECM) modifications. Extracellular constituents such as proteoglycans (PGs), may modulate vascular stiffness and VSMC growth and differentiation. We examined the effect of growth factors on secreted and membrane-bound PGs synthesis by cultured aortic smooth muscle cells (SMC) from 12- to 14- week-old spontaneously hypertensive rats (SHR) and age-matched Wistar rats. After stimulation with platelet-derived growth factor (PDGF-BB), 10% fetal calf serum (FCS) or 0.1% FCS as control, PGs synthesis (dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method using both [3H]-glucosamine and [35S]-sodium sulfate which are incorporated into all complex carbohydrates or only into sulfated dysaccharides, respectively. Data are presented as percent of the control (0.1% FCS). SHR VSMC displayed a significantly greater synthesis of M-ECM [3H]-PGs than Wistar rat cells, with both treatments, but no differences in M-ECM [35S] uptake were found in any case. In the P-ECM, both PDGF-BB and 10% FCS produced a greater effect on [3H]-PGs and sulfated PGs synthesis in VSMC from SHR. An important change seen in SHR cells was a significant decreased sulfation, assessed by [35S]/[3H] ratio, in basal and stimulation conditions. Present results indicate the existence of changes in PGS synthesis and modulation in VSMC from a conduit-artery of SHR and support the pathophysiological role proposed for matrix proteoglycans in the vascular wall changes associated to hypertension and related vascular diseases as atherosclerosis. (AU)

Aortic smooth muscle cell proliferation and endothelial nitric oxide synthase activity in fructose-fed rats.

The aim of this study was to evaluate the proliferative behavior of vascular smooth muscle cells in primary culture (pC-SMC) and the endothelial nitric oxide synthase (eNOS) activity in the endothelial lining of the aorta of fructose-fed rats (FFR). This is an experimental model of syndrome X, a cluster of cardiovascular risk factors including hyperinsulinemia, insulin resistance, and hypertension that has been suggested to be of pathophysiologic importance for the development of atherosclerosis. Male Wistar rats were used: Control (n = 12) and FFR (n = 12). After receiving fructose in drinking water (10% w/v) during 8 weeks, biochemical parameters, systolic blood pressure (SBP) and relative heart weight (RHW) were determined. The proliferative effect of 10% fetal calf serum (FCS) was examined in aortic pC-SMC by [3H]thymidine incorporation and by cell counting. Ca2+/calmodulin-dependent NOS activity was estimated in aortic endothelial lining and in heart tissue homogenates by conversion of [3H]arginine into [3H]citrulline. Fructose-fed rats showed hyperinsulinemia (P = .0263), altered glucose tolerance test (P < .001), higher SBP (P < .0001), and RHW (P = .0145), compared to control rats. These animals also showed an increase of 10% FCS-induced [3H]thymidine incorporation (P < .0001) and cell number of aortic pC-SMC (P = .0049) and decreased eNOS activity in both aortic endothelium (P = .0147) and cardiac tissue (P < .0001). These data support the hypothesis that syndrome X is associated to changes in SMC proliferation and endothelial dysfunction, which could be involved in the onset or progression of the atherogenic process.

Proteoglycan production by vascular smooth muscle cells from resistance arteries of hypertensive rats.

Extracellular matrix (ECM) modifications in the vascular wall contribute to the narrowing of arteries in hypertension. Because direct evidence for the role of proteoglycans (PGs) in the pathological process of resistance-sized arteries has not already been demonstrated, we examined the effect of growth factors on secreted and membrane-bound PG synthesis by cultured mesenteric vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and Wistar rats. After 48 hours of stimulation with angiotensin II (Ang II), platelet-derived growth factor (PDGF-BB), and 10% fetal calf serum (FCS) or 0.1% FCS as control, PG synthesis (in dpm/ng DNA) was evaluated in the medium (M-ECM) and in the cell layer (P-ECM) by a double-isotopic label method with both [(3)H]-glucosamine and [(35)S]-sodium sulfate, which are incorporated into all complex carbohydrates or only into sulfated disaccharides, respectively. VSMC from SHR displayed a significantly lower level of synthesis of M-ECM [(3)H]-PGs than those of Wistar rats in all the experimental groups, including the control group (0. 1% FCS), but no differences in M-ECM [(35)S] uptake were found in any case. In the P-ECM, Ang II was the only factor that produced a lesser effect on [(3)H]-glucosamine and a greater effect on [(35)S]-sodium sulfate uptakes in VSMC from SHR than from Wistar rats. The most prominent change seen in VSMC from SHR was an increased sulfation, assessed by [(35)S]/[(3)H] ratio, in nonstimulated cells and in response to 10% FCS and Ang II but not to PDGF-BB compared with VSMC from Wistar rats. These data indicate the existence of changes in PG modulation in the resistance vessels of SHR, which suggests that PGs may contribute to the development of structural and functional modifications in hypertensive states.

[Accumulation of actin and adhesion to HEp-2 cells of strains of Escherichia coli isolated from children with diarrhea in Mendoza, Argentina]. / Acumulación de actina y adherencia a células HEp-2 de cepas de Escherichia coli aisladas de niños con diarrea en Mendoza, Argentina.

E. coli strains are the major bacterial cause of diarrhea among children under 2 years of age residing in Mendoza, Argentina. Detection of diarrheogenic E. coli is made after coproculture, by agglutination tests using O-group antisera including most enteropathogenic E. coli (EPEC) serogroups and others often isolated in diarrhea. Although there are many O serogroups and H serotypes of E. coli strongly associated with infantile diarrhea, a number of studies have shown differences in the rate of isolation of EPEC between cases and controls using DNA probes. We compared the diagnosis of EPEC infections by traditional serogrouping tests with other detection methods using cell culture, involving the screening of isolates for adherence patterns to HEp-2 cells. A total of 140 isolates from children less than 24 months old with acute, persistent and chronic diarrhea and 40 isolates from controls were recovered. Three distinct patterns of adherence, termed localised (LA), diffuse (DA) and aggregative (EAgg) adherence were found. The fluorescence actin staining assay was used as indicative of the ability of some EPEC strains that produce attaching-effacing (A/E) lesions. Positive serogrouping strains were strongly associated with adherence (P = 0.0001). LA adherence pattern occurred in 11% of cases with acute diarrhea associated with these serogroups (P = 0.001) and children under 12 months (P = 0.0001). The FAS test was positive in 80% of them. EAgg adherence was found only in patients (20% P = 0.0001) and DA occurred both in cases (29%) and controls (2.5% P = 0.0001). Diagnosis of EPEC infections has traditionally been performed by identifying organisms belonging to a number of serogroups or serotypes epidemiologically linked to diarrhea. Evidence is presented in this paper to show that pathogenicity is not restricted to serogroups. Isolation of many adherent strains not belonging to traditional EPEC O serogroups, shows the need for alternative methods to be used to detect and identify E. coli.

Proliferative effect of insulin on cultured smooth muscle cells from rat mesenteric resistance vessels.

The present study evaluates the growth promoting effect of insulin on the proliferative activity of cells from rat mesenteric arteries in culture in order to test the hypothesis that insulin may play a pathogenetic role in the hypertrophy of resistance vessels. The proliferative effect of insulin was studied in cultured vascular smooth muscle cells (SMC) obtained from two functionally different rat vessels: mesenteric arteries and aorta. Growth characteristics (cell number and growth rate) of mesenteric and aortic cells were determined after a quiescent period and followed-up for 24, 72, and 120 h after the addition of insulin. At all studied time intervals, aortic SMC exhibited a significatively higher cell number and specific growth rate than did mesenteric SMC. Aortic SMC also displayed a greater proliferation than did mesenteric SMC in the presence of 10% fetal calf serum (FCS). At a physiological concentration of 100 microU/mL, the proliferative effect of insulin, after a quiescent period, was seen only in aortic SMC, at 72 and 120 h. Higher insulin concentration (500 microU/mL) increased significantly the cell number in SMC of both arteries. The proliferative effect was significant at all studied periods for aortic SMC; however, in mesenteric SMC, insulin increased the cell number only at 72 and 120 h. The proliferative effect of insulin was observed on SMC obtained from functionally different arteries such as aorta and mesenteric, being greater in the former. The different behavior of these SMC in the presence not only of insulin, but also of 10% FCS, provides further evidence for the existence of intervascular heterogeneity. The mild stimulatory effect of insulin in vitro may contribute in this way to the vascular hypertrophy of pathological entities exhibiting hyperinsulinemia.

Acumulación de actina y adherencia a células HEp-2 de cepas de Escherichia coli aisladas de niños con diarrea en Mendoza, Argentina / Actin accumulation and HEp-2 cell adherence assay for Escherichia coli isolated from children in Mendoza, Argentina

Entre los niños menores de 2 años atendidos en el Hospital Infantil H. Notti de Mendoza, Argentina, es frecuente la diarrea causada por E. coli. La identificación se realiza por medio de marcadores bioquímicos y seroagrupamiento con antisueros que incluyen la mayoría de los serogrupos tradicionales de E. coli enteropatógeno (EPEC). Aunque se ha comprobado que existen serogrupos O y serotipos H de E. coli comúnmente asociados a diarrea infantil, en algunos estudios existe discrepancia tanto en relación a su presencia en controles sanos, en cuanto a su clasificación como EPEC en estudios genotípicos con sondas de ADN. En este trabajo se comparó la información proporcionada por la metodología estándar con los patrones de adherencia a células HEp-2, de cepas obtenidas de coprocultivos de 140 niños menores de 2 años con diarrea aguda, prolongada y crónica. Se hizo un grupo control de 40 casos sin diarrea. Las cepas se clasificaron según serología positiva (+) y negativa (-) frente a antisueros polivalentes. Se identificaron 3 tipos de adherencia: localizada (LA), difusa (DA) y EAagg (agregativa). Además, se realizó la prueba denominada "Fluorescence Actin Staining" (FAS) que pone en evidencia la lesión característica de EPEC, denominada "attaching-effacing" (A/E) producida por la formación de un pedestal de filamentos de actina en el sitio de adhesión bacteriana, en tejido intestinal y células en cultivo. En diarreas agudas, la adherencia fue relacionada con la serología + (P=0,001). La presencia de LA en diarreas fue del 11 por ciento con predominio significativo en niños menores de 12 meses (P=0,0001) y serología + (P=0,0001). El 80 por ciento de estas cepas dieron positiva la prueba FAS. El patrón DA apareció en diarreas (29 por ciento P=0,0001) y controles (2,5 por ciento); EAgg sólo en diarreas (20 por ciento P=0,0001). Este trabajo postula que la patogenicidad no estaría restringida a serogrupos. Debido a la frecuencia del aislamiento de cepas enteroadherentes de las que no se conoce bien la patogenia y que no son detectadas por las pruebas fenotípicas estándares, sería importante complementar al laboratorio clínico con técnicas que evidencien factores de virulencia

Acumulación de actina y adherencia a células HEp-2 de cepas de Escherichia coli aisladas de niños con diarrea en Mendoza, Argentina / Actin accumulation and HEp-2 cell adherence assay for Escherichia coli isolated from children in Mendoza, Argentina

Entre los niños menores de 2 años atendidos en el Hospital Infantil H. Notti de Mendoza, Argentina, es frecuente la diarrea causada por E. coli. La identificación se realiza por medio de marcadores bioquímicos y seroagrupamiento con antisueros que incluyen la mayoría de los serogrupos tradicionales de E. coli enteropatógeno (EPEC). Aunque se ha comprobado que existen serogrupos O y serotipos H de E. coli comúnmente asociados a diarrea infantil, en algunos estudios existe discrepancia tanto en relación a su presencia en controles sanos, en cuanto a su clasificación como EPEC en estudios genotípicos con sondas de ADN. En este trabajo se comparó la información proporcionada por la metodología estándar con los patrones de adherencia a células HEp-2, de cepas obtenidas de coprocultivos de 140 niños menores de 2 años con diarrea aguda, prolongada y crónica. Se hizo un grupo control de 40 casos sin diarrea. Las cepas se clasificaron según serología positiva (+) y negativa (-) frente a antisueros polivalentes. Se identificaron 3 tipos de adherencia: localizada (LA), difusa (DA) y EAagg (agregativa). Además, se realizó la prueba denominada "Fluorescence Actin Staining" (FAS) que pone en evidencia la lesión característica de EPEC, denominada "attaching-effacing" (A/E) producida por la formación de un pedestal de filamentos de actina en el sitio de adhesión bacteriana, en tejido intestinal y células en cultivo. En diarreas agudas, la adherencia fue relacionada con la serología + (P=0,001). La presencia de LA en diarreas fue del 11 por ciento con predominio significativo en niños menores de 12 meses (P=0,0001) y serología + (P=0,0001). El 80 por ciento de estas cepas dieron positiva la prueba FAS. El patrón DA apareció en diarreas (29 por ciento P=0,0001) y controles (2,5 por ciento); EAgg sólo en diarreas (20 por ciento P=0,0001). Este trabajo postula que la patogenicidad no estaría restringida a serogrupos. Debido a la frecuencia del aislamiento de cepas enteroadherentes de las que no se conoce bien la patogenia y que no son detectadas por las pruebas fenotípicas estándares, sería importante complementar al laboratorio clínico con técnicas que evidencien factores de virulencia (AU)

Magnesium: effects on reperfusion arrhythmias and membrane potential in isolated rat hearts.

The effects of Mg2+ concentration (Mg2+o, 0, 1.2, 2.4, and 4.8 mM) on the incidence of reperfusion arrhythmias and on the cellular electrical activity were studied in spontaneously beating rat hearts. The surface electrogram and the membrane potential were recorded in control conditions, during 10 min of regional ischemia (ligature of the left anterior descending coronary artery), and on reflow. Changes in Mg2+o did not alter action potential morphology but the depolarization induced by ischemia decreased with increasing Mg2+o. In hearts perfused with Mg2+ free solution or 1.2 mM subthreshold delayed afterdepolarizations (DADs) were often detected during ischemia. Moreover, DADs could be identified as initial events in the production of extrabeats or tachycardia appearing on reperfusion under these conditions. Chaotic electrical activity during fibrillation precluded the observation of DADs. The overall incidence (100%) and severity of ventricular tachyarrhythmias (80% tachycardia and fibrillation) was similar in both groups. At high Mg2+o, subthreshold DADs were occasionally observed during ischemia and often on reperfusion where they did not lead to the development of overt arrhythmias. Consequently, the incidence, severity, and duration of arrhythmic episodes on reflow was markedly reduced. Raising Mg2+ only on reperfusion did not prevent the development of arrhythmias, whose morphology in the intracellular recordings was similar to that found in hearts perfused without Mg2+ or with 1.2 mM. The recovery of sinus rhythm after 10 min of reperfusion was linearly related to Mg2+o. Our data strengthen the view that reperfusion arrhythmias belong to the Ca2+ mediated non reentrant type and suggest that Mg2+ counteracts these arrhythmias by depressing cytosolic Ca2+ oscillations. Besides, it appears that raising Mg2+o reduces ischemic K+o accumulation. The resulting changes in resting potential could contribute to lower DADs amplitude and thus decrease the arrhythmogenic potential of the Ca2+i oscillations induced by reperfusion.

Effect of beta adrenoreceptor blocking drugs on the responses of isolated rat mesenteric arteries.

The effect of several beta-blockers on the vasoactive effect of several substances was studied on the perfused isolated rat mesenteric vessels. The agonists were tested while the beta-blocker was infused; the effects were compared in the same experiment with those observed in a control period in which the agonists were injected without simultaneous infusion of beta-blocker. l-propranolol, infused at three different doses, did not modify the vascular effect of either angiotensin II (AII) or vasopressin (VP). On the other hand, the constrictor effect of epinephrine (E) and norepinephrine (NE) was significantly reduced. Timolol, but not sotalol, showed the same results. d-propranolol produced the same effect as l-propranolol. A high dose of l-propranolol did not protect alpha adrenoceptors of blockade induced by phentolamine.

Effect of beta adrenoreceptor blocking drugs on the responses of isolated rat mesenteric arteries.

The effect of several beta-blockers on the vasoactive effect of several substances was studied on the perfused isolated rat mesenteric vessels. The agonists were tested while the beta-blocker was infused; the effects were compared in the same experiment with those observed in a control period in which the agonists were injected without simultaneous infusion of beta-blocker. l-propranolol, infused at three different doses, did not modify the vascular effect of either angiotensin II (AII) or vasopressin (VP). On the other hand, the constrictor effect of epinephrine (E) and norepinephrine (NE) was significantly reduced. Timolol, but not sotalol, showed the same results. d-propranolol produced the same effect as l-propranolol. A high dose of l-propranolol did not protect alpha adrenoceptors of blockade induced by phentolamine.