Beneficial Effects of Different Flavonoids on Vascular and Renal Function in L-NAME Hypertensive Rats.

BACKGROUND: we have evaluated the antihypertensive effect of several flavonoid extracts in a rat model of arterial hypertension caused by chronic administration (6 weeks) of the nitric oxide synthesis inhibitor, L-NAME. METHODS: Sprague Dawley rats received L-NAME alone or L-NAME plus flavonoid-rich vegetal extracts (Lemon, Grapefruit + Bitter Orange, and Cocoa) or purified flavonoids (Apigenin and Diosmin) for 6 weeks. RESULTS: L-NAME treatment resulted in a marked elevation of blood pressure, and treatment with Apigenin, Lemon Extract, and Grapefruit + Bitter Orange extracts significantly reduced the elevated blood pressure of these animals. Apigenin and some of these flavonoids also ameliorated nitric oxide-dependent and -independent aortic vasodilation and elevated nitrite urinary excretion. End-organ abnormalities such as cardiac infarcts, hyaline arteriopathy and fibrinoid necrosis in coronary arteries and aorta were improved by these treatments, reducing the end-organ vascular damage. CONCLUSIONS: the flavonoids included in this study, specially apigenin, may be used as functional food ingredients with potential therapeutic benefit in arterial hypertension.

Bile Acids Do Not Contribute to the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state and related to an enhanced intracellular calcium release evoked by thrombin and an increased amount of calcium stored in the intracellular organelles. In the present study we have investigated the role of bile acids in those alterations of the BDL cirrhotic model. Cholic acid (CA) or deoxycholic acid (DCA) did not change P-selectin expression or platelet aggregation in any group but elevated baseline platelet calcium levels. Incubation with both bile acids reduced calcium release after stimulation with thrombin in the absence of extracellular calcium. Pretreatment with CA but not with DCA reduced significantly thrombin-induced calcium entry in all three experimental groups. The capacitative calcium entry was also significantly lower in platelets pretreated with both bile acids. The simultaneous addition of thapsigargin and ionomycin to estimate the total amount of calcium in platelet internal stores was decreased by pretreatment with both CA and DCA, although these changes were significantly different in the control rats only with CA and in the BDL platelets with DCA. These results indicate that CA and DCA reduce calcium movements in platelets of control and BDL animals, thus suggesting that bile acids do not participate in the alterations observed in the BDL cirrotic model.

Role of homocysteine and folic acid on the altered calcium homeostasis of platelets from rats with biliary cirrhosis.

Previously, we have found that intracellular calcium homeostasis is altered in platelets from an experimental model of liver cirrhosis, the bile-duct ligated (BDL) rat; these alterations are compatible with the existence of a hypercoagulable state. Different studies indicate that cholestatic diseases are associated with hyperhomocysteinemia; thus, we hypothetized that it could contribute to those platelet alterations. In the present study, we have investigated the role of homocysteine (HCY) in platelet aggregation and calcium signaling in the BDL model. The effect of chronic folic acid treatment was also analyzed. Acute treatment with HCY increased the aggregation response to ADP and calcium responses to thrombin in platelets of control and BDL rats. Capacitative calcium entry was not altered by HCY. Chronic treatment with folic acid decreased platelet aggregation in control and BDL rats, but this decrease was greater in BDL rats. In folic acid-treated rats, thrombin-induced calcium entry and release were decreased in platelet of control rats but unaltered in BDL rats; however, capacitative calcium entry was decreased in platelets of control and BDL rats treated with folic acid. Reactive oxygen species were produced at higher levels by BDL platelets after stimulation with HCY or thrombin and folic acid normalized these responses. HCY plays a role in the enhanced platelet aggregation response of BDL rats, probably through an enhanced formation of ROS. Folic acid pretreatment normalizes many of the platelet alterations shown by BDL rats.

Altered calcium signalling in platelets from bile-duct-ligated rats.

In the present study, we have analysed the mechanisms of Ca(2+) entry and release in platelets obtained from BDL (bile-duct-ligated) rats, 11-13 days and 4 weeks after surgery. Platelets were washed and loaded with fura-2, and [Ca(2+)](i) (cytosolic Ca(2+) concentration) was determined in cell suspensions by means of fluorescence spectroscopy. Basal [Ca(2+)](i) was similar in platelets from BDL rats compared with those from their respective controls, both in the absence and presence of extracellular Ca(2+). Platelet stimulation with thrombin in the absence and presence of extracellular Ca(2+) induced a rapid rise in [Ca(2+)](i) that was of greater magnitude in platelets from BDL rats than in controls. Ca(2+) storage was significantly elevated in platelets from BDL rats, as well as the activity of SERCA (sarcoplasmic/endoplasmic-reticulum Ca(2+)-ATPase). Capacitative Ca(2+) entry, as evaluated by inhibition of SERCA with thapsigargin, was also altered in platelets from BDL rats, having lower rates of Ca(2+) entry. In conclusion, chronic BDL alters intracellular Ca(2+) homoeostasis in platelets, such that an enhanced Ca(2+) release is evoked by thrombin, which may be due to an increased amount of Ca(2+) stored in the intracellular organelles and secondary to an enhanced activity of SERCA. These alterations are already evident before cirrhosis has completely developed and occurs during the cholestasis phase.

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.

Reduced capacitative calcium entry in the mesenteric vascular bed of bile duct-ligated rats.

In this work, we analyzed the interaction of nitric oxide (NO) with some of the mechanisms known to regulate intracellular calcium levels in order to gain insight into the mechanisms responsible for the reduced vascular pressor response to vasoconstrictors observed in an experimental model of liver cirrhosis. Specifically, we hypothesized that the entry of calcium through capacitative channels is defective in this model. The experiments were performed with isolated, Krebs-perfused and de-endothelialized mesenteric arterial bed of rats with bile duct ligation (4 weeks) and their controls. Pretreatment with thapsigargin to inhibit calcium uptake into sarcoplasmic reticulum potentiated the pressor responses to methoxamine, but the response of the cirrhotic vessels was significantly lower than that of the controls. Under the same conditions, perfusion of the mesenteries with zero calcium-Krebs resulted in lower pressor responses to methoxamine, especially in the mesenteries of the bile duct-ligated rats. To specifically analyze the entry of calcium through store-operated calcium channels, the pressor response to the addition of calcium was studied in mesenteries perfused with zero calcium-Krebs and in the presence of thapsigargin. Again, the response of the cirrhotic mesenteric beds was significantly lower than that of the control vessels. Under all these experimental conditions, the differences between control and cirrhotic responses were abolished by pretreatment with the NO synthesis inhibitor N(w)-nitro-L-arginine (NNA). These results indicate that, in the mesenteric bed of bile duct-ligated rats, an excess of nitric oxide interferes with the release of calcium from thapsigargin-sensitive internal stores and also reduces the capacitative entry of calcium into vascular muscular cells induced by the depletion of calcium from internal stores. This mechanism may have an important role in the reduced pressor response observed in the mesenteric vascular bed in cirrhosis.

Role of vascular nitric oxide in experimental liver cirrhosis.

One of the most important features of liver cirrhosis is the splanchnic and systemic arterial vasodilation, related to an increase in vascular capacity and an active vasodilation. This arterial vasodilation seems to be the consequence of the excessive generation of vasodilating substances, which also contributes to a lower than normal pressor response to circulating nervous or humoral substances. The following review analyzes the mechanisms responsible for the vascular hyporesponse to vasoconstrictors observed in the experimental models of liver cirrhosis. It has become increasingly clear that, among the great variety of substances studied, nitric oxide (NO) seems to be one of the main contributors to this vascular alteration, since elimination of the endothelium or inhibition of its synthesis corrects it. The mechanism by which NO interferes with the contractile apparatus in smooth muscle cells seems to be related to a direct effect on calcium entry from the extracellular space and release from the internal stores.

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

BACKGROUND: In 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. RESULTS: Basal 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. CONCLUSIONS: It 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.

Vitamin E prevents renal dysfunction induced by experimental chronic bile duct ligation.

BACKGROUND: The mechanisms by which prolonged cholestasis alters renal hemodynamics and excretory function are unknown but may be related to increased oxidative stress, with subsequent formation of lipid peroxidation-derived products (e.g., F2-isoprostanes) and endothelin (ET). We investigated whether antioxidant therapy prevents chronic bile duct ligation (CBDL)-induced alterations in systemic and renal hemodynamics, and reduces F2-isoprostane and ET levels. METHODS: Sprague-Dawley rats were placed on either a normal or a high vitamin E diet for 7 days and then underwent either CBDL or sham surgery. They were then maintained on their respective diets for 21 more days, at which time the physiologic studies were performed. RESULTS: Thirty-three percent of the CBDL rats died by day 21. The remaining rats had a lower mean arterial pressure (MAP), renal blood flow (RBF), glomerular filtration rate (GFR), and sodium and water excretion than control rats. CBDL rats had higher portal pressure, renal venous pressure, and renal vascular resistance (RVR). These changes were associated with increased levels of systemic and renal venous F2-isoprostanes and ET. Vitamin E normalized MAP, RBF, GFR, RVR, and sodium and water excretion, and improved the 21-day survival without altering portal or renal venous pressures. Surprisingly, vitamin E did not alter the systemic levels of F2-isoprostanes but markedly reduced their levels in the renal venous circulation. CONCLUSION: Vitamin E improves MAP and renal function in CBDL rats, and selectively decreases renal levels of oxidative stress and ET, suggesting that local redox balance is implicated in CBDL-induced renal dysfunction.

Nitric oxide and renal and cardiac dysfunction in cirrhosis.

Nitric oxide (NO) has diverse physiological and pathophysiological effects. The roles of NO in the renal and cardiac dysfunction found in cirrhosis are reviewed. In the kidneys of experimental animals with cirrhosis, several lines of evidence speak in favour of an enhanced production of NO, through the activation of both endothelial constitutive and inducible isoforms of NO synthase. In contrast with the situation in normal animals, inhibition of NO synthesis in rats with cirrhosis improves sodium and water excretion via blood pressure-dependent and -independent mechanisms, which indicates that the renal sodium and water retention of cirrhosis is related to an excess of NO production. The deleterious effect of excessive NO on the kidney may be mediated by peroxynitrite, a potent oxidant that is readily formed whenever superoxide anions and the *NO radical are produced together. The peroxidation of arachidonic acid by peroxynitrite leads to the formation of F(2a)-isoprostanes, which are powerful renal vasoconstrictors. F(2a)-isoprostane levels are correlated with the severity of liver injury during cirrhosis. However, whether peroxynitrite or F(2a)-isoprostanes are the elusive mediator of the NO-induced renal alterations in cirrhosis remains to be firmly established. NO is also involved in cardiac contractility, probably in the normal heart as well as in disease conditions such as non-cirrhotic and cirrhotic cardiomyopathy. In the latter state, evidence suggests that inducible NO synthase attenuates ventricular contractility, mediated by cGMP. Another gas that transduces its signal through cGMP, carbon monoxide, is also likely to play a role in cirrhotic cardiomyopathy, but the nature of the interaction between NO and carbon monoxide in this syndrome remains unclear.

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 Nw-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.

Relación del pH salival y el grado de higiene bucal / Ratio of the pH. saliva and the degree of oral hygiene

Se realiza un estudio preliminar en 93 estudiantes de segundo año de la Facultad de Estomatología de la ciudad de Santiago de Cuba, a los cuales se les valoró la higiene bucal con los criterios de Greene y Vermillion; se tabularon 16 valores distintos de higiene bucal, tomándoseles tres valores de pH en días consecutivos, a una misma hora del día. Se observó que dentro de los valores 0 y 1 de la higiene bucal, se encontró el 88, 17 por ciento de todas las mediciones, lo que indica que la muestra escogida tenía buena higiene bucal; no se obtuvo relación entre el pH' y los valores del índice de higiene bucal revisado(AU)