Fernando González-Fernández

Expression of Constitutive and Inducible Nitric Oxide Synthases in the Vascular Wall of Young and Aging Rats

Two NO synthase (NOS) isoforms have been described in vessels, an endothelial constitutive NOS (eNOS) and an inducible NOS (iNOS). The purpose of the present study was to examine the endothelium-dependent and endothelium-independent hypotensive response in aging rats, analyzing the ability of their vessels to produce NO. The studies were performed in 2 groups of euvolemic, conscious, male Wistar rats: aging rats (n=20, 18 months old) and young rats (n=20, 5 months old). The hypotensive responses to acetylcholine, bradykinin, and sodium nitroprusside were determined. Furthermore, the expression of the NOS isoforms by Western blot and the eNOS and iNOS activities, defined as Ca2+-dependent and Ca2+-independent conversion of [14C]L-arginine into [14C]L-citrulline, respectively, were also determined. In the aging rats, we found an impaired hypotensive response to acetylcholine and bradykinin (2 NO- and endothelium-dependent hypotensive agents) that was accompanied by a preserved hypotensive response to sodium nitroprusside. Aging rats also demonstrated an enhanced sensitivity response to the pressor effect of the L-arginine antagonist L-Nomega-nitro-L-arginine and a reduced vasoconstrictor response to angiotensin II. The inhibition of NO synthesis normalized the pressor effect of angiotensin II in the aging animals. Nitrite plus nitrate plasma levels were increased in aging rats. Furthermore, cGMP content was also higher in the aging vessels. In the aging aortas, the expression of both eNOS and iNOS isoforms was enhanced. However, in aging rats, the activity of the eNOS isoform was markedly reduced, a finding that was accompanied by the presence of iNOS activity. The vessel wall of aging rats showed an enhanced expression of eNOS and iNOS isoforms. However, eNOS activity was reduced in the aging animals. These findings could explain the impaired endothelium-dependent hypotensive response associated with aging.

Estrogen Stimulates Neuronal Nitric Oxide Synthase Protein Expression in Human Neutrophils

Abstract —Recent studies have postulated the contribution of nitric oxide (NO) released by the endothelium to the beneficial effects of estrogen. Despite a neuronal-type NO synthase (nNOS) described in neutrophils, less is known about the effect of estrogen in these cells. The aim of the present study was to analyze the expression of nNOS protein in human neutrophils under different estrogenic conditions. We first analyzed nNOS expression in neutrophils obtained from premenopausal women. During the first 2 days of the follicular phase (low circulating estrogen concentrations), nNOS expression in neutrophils was reduced with respect to that found in neutrophils obtained from the same donors during the ovulatory phase (high circulating estrogen concentrations). Moreover, the expression of nNOS protein in neutrophils obtained from postmenopausal women after transdermal estrogen therapy was markedly enhanced with respect to that observed before the treatment. In vitro incubation of neutrophils derived from men for 6 hours with 17β-estradiol (10−10 to 10−8 mol/L) upregulated the expression of nNOS protein. The 17β-estradiol receptor antagonists, tamoxifen (10−8 mol/L) and ICI 182780 (10−8 mol/L), inhibited the upregulation of nNOS protein induced by 17β-estradiol. The putative functional implication was denoted by a reduced expression of the CD18 antigen on the surface of 17β-estradiol–incubated neutrophils, which was accompanied by a decreased adhesive capacity. Both effects were prevented by an NO antagonist. In conclusion, the in vivo levels of circulating estrogen concentrations seem to be associated with the level of nNOS protein expression in neutrophils from women. Moreover, low doses of 17β-estradiol upregulate nNOS protein expression in neutrophils from men. The increased ability of 17β-estradiol–incubated neutrophils derived from men to produce NO reduced their adhesive properties.

Effect of losartan on human platelet activation.

ObjectivePrevious studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation.MethodsPlatelets were obtained from 15 healthy men, aged 26–40 years. Platelet activati

Aspirin inhibits inducible nitric oxide synthase expression and tumour necrosis factor‐α release by cultured smooth muscle cells

Inflammatory related cardiovascular disease, i.e. cardiac allograft rejection, myocarditis, septic shock, are accompanied by cytokine production, which stimulates the expression of inducible nitric oxide (iNOS).

Expression of Inducible Nitric Oxide Synthase After Endothelial Denudation of the Rat Carotid Artery Role of Platelets

There is functional evidence suggesting that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. In the present study we have analyzed the time course of iNOS protein expression in the arterial wall after in vivo deendothelialization. Endothelial denudation was performed in the left carotid artery of Wistar rats, and the right carotid artery was used as control. Whereas iNOS protein was weakly expressed 6, 24, and 48 hours after endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after vascular damage. Because platelet adhesion and aggregation occur early after endothelial damage, we studied the role of activated platelets in the negative modulation of iNOS protein expression during the first 2 days after endothelial denudation. Early after in vivo endothelial injury, platelet-depleted rats showed a marked iNOS protein expression in the vascular wall. Similar results were obtained by blocking the platelet glycoprotein (GP) IIb/IIIa. Although iNOS protein is present in the arterial wall several days after endothelial denudation, early after arterial wall injury iNOS protein is weakly expressed. Platelets play a crucial role in preventing iNOS protein expression early after endothelial damage, an effect that can be avoided with GP IIb/IIIa blockers. Although iNOS protein was weakly expressed in vivo in the rat carotid artery wall 6, 24, and 48 hours after balloon endothelial denudation, a marked iNOS expression was found 7, 14, and 30 days after arterial damage. iNOS expression could be increased early after endothelial injury by removing circulating platelets and by an antibody against the GP IIb/IIIa. In conclusion, platelets prevent iNOS protein expression early after endothelial balloon damage, an effect that can be avoided with GP IIb/IIIa blocking agents.

Doxazosin modifies Bcl-2 and Bax protein expression in the left ventricle of spontaneously hypertensive rats.

BACKGROUND: Increased apoptosis has recently been reported in the heart of spontaneously hypertensive rats (SHRs). OBJECTIVE: To investigate the molecular basis of apoptosis in the left ventricle of SHRs in terms of the expression of Bcl-2 protein (which protects from apoptosis) and Bax protein (which acts as an apoptotic promoter). In addition, we analysed the involvement of alpha -adrenergic receptors in the left ventricular apoptosis of SHRs. METHODS: The study was performed in untreated SHRs (n=16) and SHRs that were orally treated with doxazosin (10 mg/kg body weight per day, for 15 days), a selective alpha1-receptor blocker (n=16). A group of Wistar-Kyoto (WKY) rats (n=16) was used as the control. RESULTS: The left ventricles of untreated SHRs showed a significant increase in Bcl-2 protein expression and a reduced presence of Bax protein. The ratio of Bcl-2:Bax in SHRs was higher than in WKY rats, suggesting an anti-apoptotic state. Paradoxically, both the number of apoptotic cardiac cells and the cleavage of an 85-kDa fragment of the poly (ADP-ribose) polymerase (PARP), a marker of caspase-3 activity, were higher in the left ventricle of SHRs than in WKY rats, suggesting an apoptotic situation. Bax promotes cell apoptosis when it is bound to Bcl-2. We then determined the abundance of Bax-Bcl-2 complexes in the left ventricle of the two groups of animals. Bax-Bcl-2 complexes were more abundant in SHRs than WKY rats. In a second set of experiments, we analysed the role of alpha1-adrenergic blockade by doxazosin in the above-described mechanisms. Doxazosin treatment reduced the formation of Bax-Bcl-2 complexes in the left ventricle of SHRs, and this was accompanied by a decrease in the levels of 85kDa PARP and a reduction in apoptotic left ventricular cells. CONCLUSIONS: The present work suggests that the presence of Bax-Bcl-2 complexes in the left ventricle could be a more reliable marker of the apoptotic state than the determination of the absolute expression of Bcl-2 and Bax proteins. Moreover, the inhibition of alpha1 -adrenergic receptors by doxazosin decreased the abundance of BaxBcl-2 complexes and promoted a reduction of apoptosis in the left ventricle of SHRs.

Efecto del triflusal sobre la agregación y secreción de las plaquetas humanas: papel del óxido nítrico

INTRODUCTION AND AIMS: The thrombotic process is a multicellular phenomenon in which not only platelets are involved but also neutrophils are involved. Recent in vitro studies performed in our laboratory have demonstrated that triflusal reduced platelet aggregation by stimulating nitric oxide (NO) production by neutrophils. The aim of the present study was to evaluate whether the in vivo treatment with triflusal could also modify the ability of neutrophils to produce NO. Furthermore, the role of NO released by neutrophils on platelet aggregation and secretion was also tested. METHODS: The study was performed in 12 healthy volunteers of 32 +/- 6 years of age. The volunteers were treated with triflusal (600 mg/day) for 5 days and platelets and neutrophils were isolated before and after treatment. The ability of neutrophils to produce NO and the capacity of inhibiting platelet aggregation and secretion of transforming growth factor-beta (TGF-beta) were assessed. RESULTS: After the treatment with triflusal we obtained the following results: a) an increase in NO production by neutrophils; b) potentiation of the inhibition of platelet aggregation by neutrophils, an effect that was reverted by incubating neutrophils with an L-arginine antagonist, L-NAME, and c) the presence of neutrophils reduced the release of TGF-beta by platelets measured as index of platelet secretion by a NO-independent mechanism. CONCLUSIONS: Triflusal (600 mg/day/5 days) stimulated NO production by neutrophils. After the treatment with triflusal, neutrophils inhibited both platelet aggregation and secretion. The antiaggregating effect of neutrophils was an NO-dependent mechanism while the inhibition of platelet secretion mediated by neutrophils after the treatment with triflusal was an NO-independent mechanism.

Expresión de la óxido nítrico sintasa inducible en carótida de rata tras denudación endotelial: efecto de las plaquetas y del tratamiento con abciximab

BACKGROUND: Functional evidence suggests that endothelial denudation stimulates inducible nitric oxide synthase (iNOS) activity in the vascular wall. In vitro studies done in our laboratory have shown that iNOS expression in smooth muscle cells is reduced by endothelial cells. The object of this study was to analyze the iNOS protein expression in the arterial wall after in vivo deendothelialization, and the role of platelet activation abciximab in the expression of this protein. MATERIALS AND METHODS: Endothelial denudation was performed in the left carotid artery of Wistar rats. The right carotid artery was used as control. RESULTS: iNOS protein was only weakly expressed at 6, 24 and 48 hours after endothelial denudation. Since platelet adhesion and aggregation occur early after endothelial damage, we have analyzed the role of activated platelets in iNOS protein expression during the first two days after angioplasty. Early after in vivo endothelial injury, thrombocytopenic rats showed a marked iNOS protein expression. Similar results were obtained by blocking the platelet glycoprotein IIb/IIIa in rats treated with abciximab (Reopro). CONCLUSIONS: iNOS protein is weakly expressed in the arterial wall after endothelial denudation. Platelets play a crucial role preventing iNOS protein expression early after endothelial damage through a mechanism that depends on GP IIb/IIIa, an effect that can be avoided with glycoprotein IIb/IIIa, blockers, such as abciximab.

Effect of losartan on human platelet activation

OBJECTIVE Previous studies have demonstrated that losartan can block the thromboxane A2 receptor on the vascular wall. The aim of the present study was to assess the effect of losartan on human platelet activation. METHODS Platelets were obtained from 15 healthy men, aged 26-40 years. Platelet activation was measured by changes in the light transmission of platelet-rich plasma stimulated by the thromboxane A2 analog U46619 (5 x 10(-6) mol/l) or ADP (10(-5) mol/l). RESULTS U46619-stimulated platelet aggregation was significantly inhibited by losartan in a dose-dependent manner. Only a high dose of EXP 3174 (5 x 10(-5) mol/l), the in vivo active metabolite of losartan, was able to attenuate U46619-induced platelet activation. Captopril, an angiotensin I converting inhibitor, failed to modify U46619-induced platelet aggregation. Furthermore, the binding of [3H]-U46619 to platelets was competitively inhibited by losartan, whereas only a high dose of EXP 3174 reduced the binding of [3H]-U46619. Captopril failed to modify the binding of [3H]-U46619 to platelets. Losartan also reduced the platelet activation induced by ADP (10(-5) mol/l), a platelet agonist partially dependent on thromboxane A2. In addition, when thromboxane A2 generation was blocked by aspirin, ADP-induced platelet aggregation was inhibited to a similar degree to the inhibition induced by losartan. Exogenous angiotensin II did not elicit any modification of either U46619- or ADP-stimulated platelet aggregation. CONCLUSIONS Losartan decreased platelet aggregation by a thromboxane A2-dependent mechanism. EXP 3174 was less potent than losartan in reducing thromboxane A2-dependent platelet activation. Captopril and exogenous angiotensin II had no effect on human platelet activation. These results suggest that losartan reduced thromboxane A2-dependent platelet activation independently of its effect on angiotensin II.

Doxazosin modifies Bcl-2 and Bax protein expression in the left ventricle of spontaneously hypertensive rats

Background Increased apoptosis has recently been reported in the heart of spontaneously hypertensive rats (SHRs). Objective To investigate the molecular basis of apoptosis in the left ventricle of SHRs in terms of the expression of Bcl-2 protein (which protects from apoptosis) and Bax protein (which acts as an apoptotic promoter). In addition, we analysed the involvement of α1-adrenergic receptors in the left ventricular apoptosis of SHRs. Methods The study was performed in untreated SHRs (n = 16) and SHRs that were orally treated with doxazosin (10 mg/kg body weight per day, for 15 days), a selective α1-receptor blocker (n = 16). A group of Wistar–Kyoto (WKY) rats (n = 16) was used as the control. Results The left ventricles of untreated SHRs showed a significant increase in Bcl-2 protein expression and a reduced presence of Bax protein. The ratio of Bcl-2:Bax in SHRs was higher than in WKY rats, suggesting an anti-apoptotic state. Paradoxically, both the number of apoptotic cardiac cells and the cleavage of an 85-kDa fragment of the poly (ADP-ribose) polymerase (PARP), a marker of caspase-3 activity, were higher in the left ventricle of SHRs than in WKY rats, suggesting an apoptotic situation. Bax promotes cell apoptosis when it is bound to Bcl-2. We then determined the abundance of Bax–Bcl-2 complexes in the left ventricle of the two groups of animals. Bax–Bcl-2 complexes were more abundant in SHRs than WKY rats. In a second set of experiments, we analysed the role of α1-adrenergic blockade by doxazosin in the above-described mechanisms. Doxazosin treatment reduced the formation of Bax–Bcl-2 complexes in the left ventricle of SHRs, and this was accompanied by a decrease in the levels of 85-kDa PARP and a reduction in apoptotic left ventricular cells. Conclusions The present work suggests that the presence of Bax–Bcl-2 complexes in the left ventricle could be a more reliable marker of the apoptotic state than the determination of the absolute expression of Bcl-2 and Bax proteins. Moreover, the inhibition of α1-adrenergic receptors by doxazosin decreased the abundance of Bax–Bcl-2 complexes and promoted a reduction of apoptosis in the left ventricle of SHRs.