Noemí M. Atucha

Role of endothelium in the abnormal response of mesenteric vessels in rats with portal hypertension and liver cirrhosis

BACKGROUND & AIMS Previous studies have shown that nitric oxide synthesis inhibition corrects the hyporesponsiveness to vasoconstrictors present in the mesenteric vascular bed of portal-hypertensive rats. The origin of this elevated NO production, whether endothelial or muscular, is unknown. The aim of this study was to evaluate the role of vascular endothelium in the hyporesponsiveness to methoxamine (MTX) in the mesenteric vascular bed of portal vein-ligated (PVL) and cirrhotic rats. METHODS Endothelial denudation was achieved using a combined treatment of cholic acid and distilled water. RESULTS Compared with the respective control groups, PVL rats showed a reduced vascular response to MTX. Similar results were obtained in cirrhotic animals. The presence of ascites was associated with a more severe reduction in the response to MTX. Removal of the endothelium completely corrected the vascular hyporesponsiveness of PVL, cirrhotic nonascitic, and ascitic animals. In these experiments, acetylcholine-mediated vasodilation was practically absent whereas that of sodium nitroprusside was potentiated, which indicates a successful elimination of the endothelium and the preservation of smooth muscle function. Immunostaining for NO synthase isoforms revealed the presence of endothelial NO synthase protein in healthy and PVL rats exclusively in the endothelium. CONCLUSIONS The mesenteric vascular hyporesponsiveness to MTX present in these models of liver diseases and portal hypertension is solely due to endothelium-dependent factors.

Role of AT1 receptors in the renal papillary effects of acute and chronic nitric oxide inhibition

Nitric oxide (NO) is a vasodilator substance controlling renal papillary blood flow (PBF) in the rat. In this study we have evaluated the role of AT1 angiotensin II receptors as modulators of the whole kidney and papillary vasoconstrictor effects induced by the acute or chronic inhibition of NO synthesis. Experiments have been performed in anesthetized, euvolemic Munich-Wistar rats prepared for the study of renal blood flow (RBF) and PBF. In normal rats, acute administration of the NO synthesis inhibitor N ω-nitro-l-arginine methyl ester (l-NAME) increased mean arterial pressure (MAP) and decreased RBF and PBF. Either acute or chronic treatment with the AT1 receptor blocker losartan did not modify the decreases in RBF or PBF secondary to l-NAME. In animals made hypertensive by chronic inhibition of NO, basal MAP was higher, whereas RBF and PBF were lower than in the controls. In these animals, acute or chronic administration of losartan decreased MAP and increased both RBF and PBF significantly. These results indicate that, under normal conditions, the decreases in RBF or PBF induced by the acute inhibition of NO synthesis are not modulated by AT1-receptor stimulation. However, the arterial hypertension, renal vasoconstriction, and reduced PBF present in chronic NO-deficient hypertensive rats is partially due to the effects of angiotensin II, via stimulation of AT1-receptors.Nitric oxide (NO) is a vasodilator substance controlling renal papillary blood flow (PBF) in the rat. In this study we have evaluated the role of AT1 angiotensin II receptors as modulators of the whole kidney and papillary vasoconstrictor effects induced by the acute or chronic inhibition of NO synthesis. Experiments have been performed in anesthetized, euvolemic Munich-Wistar rats prepared for the study of renal blood flow (RBF) and PBF. In normal rats, acute administration of the NO synthesis inhibitor N omega-nitro-L-arginine methyl ester (L-NAME) increased mean arterial pressure (MAP) and decreased RBF and PBF. Either acute or chronic treatment with the AT1 receptor blocker losartan did not modify the decreases in RBF or PBF secondary to L-NAME. In animals made hypertensive by chronic inhibition of NO, basal MAP was higher, whereas RBF and PBF were lower than in the controls. In these animals, acute or chronic administration of losartan decreased MAP and increased both RBF and PBF significantly. These results indicate that, under normal conditions, the decreases in RBF or PBF induced by the acute inhibition of NO synthesis are not modulated by AT1-receptor stimulation. However, the arterial hypertension, renal vasoconstriction, and reduced PBF present in chronic NO-deficient hypertensive rats is partially due to the effects of angiotensin II, via stimulation of AT1-receptors.

Mechanisms of the increased pressor response to vasopressors in the mesenteric bed of nitric oxide-deficient hypertensive rats.

In the present study we analyzed mesenteric vascular reactivity of chronic nitric oxide (NO)-deficient hypertensive rats (NW-nitro-L-Arginine Methyl Ester, L-NAME, 50 mg/kg/day, oral, 3 weeks). Perfusion pressure changes in response to cumulative additions of methoxamine and KCl were significantly increased in the mesenteric vessels of the L-NAME-treated as compared with vessels of the controls. Verapamil reduced the responses to methoxamine, but those of the hypertensive rats were still enhanced. In contrast, responses to KCl were almost completely abolished by verapamil. In mesenteric vessels perfused with zero calcium and high-potassium Krebs, pressor responses to the re-addition of calcium were also significantly enhanced in the hypertensive rats compared to the controls. Vasodilator responses to acetylcholine in KCl-preconstricted vessels, while still significant, were reduced in the L-NAME-treated rats. In this case, acute inhibition of NO blocked the vasodilator responses to acetylcholine and abolished the differences between the two groups. In methoxamine-preconstricted vessels and in the presence of acute inhibition of NO and prostaglandins, vasodilator responses to acetylcholine were significantly greater in the hypertensive vessels than in controls. In conclusion, the mesenteric vessels of L-NAME hypertensive rats show an enhanced response to vasopressors which is related to calcium entry. These data also reveal the existence of an enhanced role of a NO and prostaglandin-independent vasodilator factor, probably endothelium-derived hyperpolarizing factor that may play a compensatory role in the deficiency of NO.

Importance of Nitric Oxide and Prostaglandins in the Control of Rat Renal Papillary Blood Flow

The role of nitric oxide and prostaglandins in the control of rat renal papillary blood flow has been studied in anesthetized Munich-Wistar rats by use of laser Doppler flowmeter. Acute administration of N omega-nitro-L-arginine methyl ester (L-NAME) 10 mg/kg IV (n=8) increased mean arterial pressure by 27.8 +/- 3.6%, decreased papillary blood flow by 39.4 +/- 3.8%, and decreased renal blood flow by 47.4 +/- 1.9%. The subsequent administration of indomethacin (7.5 mg/kg IV) further decreased papillary blood flow (35.2 +/- 2.5%) without significant changes in mean arterial pressure or renal blood flow. In a second group (n = 6), administration of indomethacin before L-NAME decreased papillary blood flow by 39.6 +/- 2.1% without significantly altering mean arterial ressure or renal blood flow. The subsequent injection of L-NAME further decreased papillary blood flow (32.9 +/- 1.8%) and renal blood flow (49.8 +/- 6.6%) while increasing mean arterial pressure to a level not significantly different from that found in the first group. Autoregulation studies showed that L-NAME but not indomethacin reduced the renal perfusion pressure-renal blood flow relationship without altering autoregulation. However, both nitric oxide and prostaglandins importantly affected the renal perfusion pressure-papillary blood flow relationship because L-NAME and indomethacin significantly decreased this relationship in an additive fashion. Although both drugs reduced the sensitivity of the pressure-papillary flow relationship, only L-NAME affected autoregulation so that papillary blood flow was autoregulated at higher renal perfusion pressures. Thus, the present results indicate that both nitric oxide and prostaglandins control a similar percentage of rat renal papillary blood flow, but nitric oxide seems to be more important than prostaglandins as a mediator of the pressure-blood flow relationship. In contrast, only nitric oxide modifies the renal blood flow level, although it does not disturb whole-kidney blood flow autoregulation.

Role of protein kinase C in mesenteric pressor responses of rats with portal hypertension.

1 Hyporesponsiveness to vasoconstrictors is a characteristic abnormality of liver diseases of uncertain origin. In the present study, we have evaluated the involvement of protein kinase C (PKC) in the reduced pressor response to methoxamine (MTX) of a rat model of portal hypertension induced by partial portal vein ligation (PVL). Experiments were performed in the isolated and perfused mesentery. 2 The pressor response to MTX was reduced in PVL compared to that of control animals (Sham) and pretreatment with NG‐nitro‐L‐arginine (L‐NOARG, 10−4 m) or removal of the endothelium potentiated the response of both groups. However, only removal of the endothelium completely eliminated the reduced pressor response to MTX of the PVL vessels. 3 Pretreatment of the mesentric vessels with calphostin C (10−6 m), a PKC inhibitor, reduced the response to MTX of Sham to a level similar to that of untreated PVL vessels, but did not change that of PVL animals. 4 Mesenteric pressor responses to a PKC activator, phorbol 12,13‐dibutyrate (PDBu), were similar in vessels from both PVL and Sham rats and pretreatment with L‐NOARG or removal of the endothelium enhanced those responses while indomethacin (10−5 m) decreased them. In all cases, the responses to PDBU were similar in PVL vessels compared to Sham. 5 These results indicate that the reduced pressor response to MTX of the mesenteric vascular bed of PVL rats is due to an endothelial alteration, compatible with an enhanced production of nitric oxide. The lack of response to calphostin C in PVL vessels suggests an impairment in agonist‐induced PKC activation. Since direct activation of PKC induces a normal pressor response, it is concluded that the endothelial alteration interacts with the mechanism producing PKC activation, which results in a lower pressor response of the PVL mesenteric vaculature.

Effects of angiotensin-converting-enzyme inhibitors in combination with diuretics on blood pressure and renal injury in nitric- oxide-deficiency-induced hypertension in rats

The present study investigates the effects of chronic administration of ACEIs (angiotensin-converting-enzyme inhibitors; either zofenopril or enalapril) in combination with a diruetic (hydrochlorothiazide) on BP (blood pressure) increase and renal injury induced by L-NAME (NG-nitro-L-arginine methyl ester), an inhibitor of NO (nitric oxide) synthesis. Rats were untreated or received L-NAME alone, L-NAME+zofenopril+hydrochlorothiazide or L-NAME+enalapril+hydrochlorothiazide for 8 weeks. L-NAME treatment resulted in marked elevation in BP and mortality. Treatment with either ACEI and diuretic prevented the increase in BP induced by L-NAME, reduced the death rate and improved excretory parameters. Renal injury in the L-NAME group was severe, but, in the groups treated with either ACEI and diuretic, glomerular and tubulointerstitial lesions were not observed and the intensity, number and size of vessels affected was reduced. However, the efficacy of zofenopril+diuretic was superior to that of enalapril+diuretic in reducing vascular alterations. Oxidative stress indices and the expression of NO synthase and nitrotyrosine were normalized by the treatments. In conclusion, the combined treatment of zofenopril or enalapril with hydrochlorothiazide completely prevented the development of arterial hypertension induced by L-NAME. Renal morphological and functional alterations in the hypertensive animals were also almost completely normalized, but the treatment with zofenopril+diuretic produced a more complete organ protection. The protective effect is related to an activation of endothelial NO synthase expression and to a normalization of the oxidative stress parameters due to the inhibition of angiotensin II.

Altered calcium signaling in platelets from nitric oxide-deficient hypertensive rats

BackgroundIn the present study we have analyzed the mechanisms of calcium entry and mobilization in platelets obtained from rats chronically treated with the nitric oxide synthesis inhibitor, N-nitro L-arginine methyl ester [L-NAME, 40 mg/kg/day, 5 days). The platelets were obtained the day of the experiment, washed and loaded with fura-2. The intracellular calcium levels were determined in suspension of cells by means of fluorescence spectroscopy.ResultsBasal calcium levels were always elevated in the platelets of the L-NAME-treated rats, both in the presence and in the absence of extracellular calcium. The administration of thrombin in the absence and in the presence of extracellular calcium induced important elevations in calcium levels that were always of greater magnitude in the platelets of the L-NAME-treated rats than in those of the controls. The addition of calcium to thapsigargin-treated platelets produced a massive elevation in calcium levels in both groups that was significantly greater in the platelets obtained from the hypertensive rats than in those of the controls.ConclusionsIt is concluded that the arterial hypertension induced by the reduction of nitric oxide alters the regulation of platelet calcium levels so that elevated baseline levels and calcium entry and mobilization are enhanced. This could be the result of direct or indirect effects of the lack of nitric oxide synthesis in platelets or in other tissues.

Nebivolol ameliorates nitric oxide deficient hypertension.

Nebivolol is a new selective beta 1-adrenoceptor antagonist with nitric oxide (NO)–releasing properties. In the present study we have analyzed whether nebivolol affects the development of the arterial hypertension that follows the chronic inhibition of nitric oxide synthesis. Nebivolol (1 mg/kg/day, 14 days) was given concurrently with the NO synthesis inhibitor N-nitro-L-arginine methyl ester (L-NAME, 0.1, 1, and 10 mg/kg/day, 14 days) to several groups of rats. Blood pressure, renal function, plasma renin activity (PRA), and NO activity and metabolites were measured at the end of the treatment period. L-NAME treatment alone increased mean arterial pressure dose dependently (103.5 ± 2.4, 110.9 ± 2.0, and 125.8 ± 2.2 mmHg, respectively). Nebivolol completely prevented the development of arterial hypertension in the groups treated with L-NAME at the doses of 0.1 and 1 mg/kg/day and reduced the increase achieved with the L-NAME dose of 10 mg/kg/day (110.3 ± 2.7). There were no differences in glomerular filtration rate or natriuresis between nebivolol-treated and -untreated rats. Plasma nitrates+nitrites and calcium-dependent NO synthase activity in the kidney also decreased dose dependently with L-NAME treatment and nebivolol did not significantly modify it. However, PRA was lower in all groups treated with nebivolol and L-NAME as compared to the rats receiving only L-NAME. These data indicate that nebivolol prevents the development of the arterial hypertension associated with chronic NO deficit and this effect seems to be dependent on the inhibition of renin-angiotensin system.

Altered calcium regulation in freshly isolated aortic smooth muscle cells from bile duct-ligated rats: role of nitric oxide

In the present study we have analyzed the mechanisms of calcium entry and mobilization in smooth muscle cells (SMCs) freshly isolated from the abdominal aorta of rats with bile duct ligation (BDL). The SMCs were obtained in the day of the experiment after collagenase digestion and loaded with fura-2. The intracellular calcium levels ([Ca](i)) were determined in individual cells by fluorescence microscopy. Baseline [Ca](i) was slightly but significantly lower in SMCs from BDL rats (70.14+/-2.02 nM, n=51) than in controls (80.77+/-3.52, n=44). The application of the purinergic agonists ATP and UTP induced a fast calcium peak and a slow return to baseline. But the calcium responses were significantly smaller in the cells from the BDL rats. Also, the area under the curve (AUC) of the calcium responses elicited by the agonists was always lower in the SMCs from BDL rats as compared to the controls. Similar results were obtained with UTP, but the calcium response of the SMCs from the BDL rats was even lower than that observed with ATP. In experiments performed in the absence of extracellular calcium, both agonists also elevated [Ca](i), although the responses were much smaller than those obtained in the presence of calcium. Again, the peak and AUC responses of the SMCs from BDL rats were significantly lower than those of the controls. Incubation with NNA, a non-specific nitric oxide synthase (NOS) inhibitor, or with NIL, an inducible NOS inhibitor (iNOS), potentiated and normalized the calcium responses of the SMCs obtained from BDL rats. These data indicate that, in SMCs from bile duct-ligated rats, both the entry of calcium and the mobilization from internal stores is defective in response to purinergic agonists. NO, of an inducible origin, is involved in this altered calcium regulation.

Corticotropin-releasing factor (CRF) receptor-1 is involved in cardiac noradrenergic activity observed during naloxone-precipitated morphine withdrawal

The negative affective states of withdrawal involve the recruitment of brain and peripheral stress circuitry [noradrenergic activity, induction of the hypothalamic–pituitary–adrenocortical (HPA) axis and activation of heat shock proteins (Hsps)]. Corticotropin‐releasing factor (CRF) pathways are important mediators in the negative symptoms of opioid withdrawal. We performed a series of experiments to characterize the role of the CRF1 receptor in the response of stress systems to morphine withdrawal and its effect in the heart using genetically engineered mice lacking functional CRF1 receptors.