M. Clara Ortiz

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.

Pressor and renal effects of cross-linked hemoglobin in anesthetized cirrhotic rats

BACKGROUND/AIMS Cross-linked hemoglobin (XL-Hb), a hemoglobin-based oxygen carrier, is currently under investigation as a blood substitute. In the present study we have evaluated its pressor and renal effects in a rat model of liver cirrhosis by bile duct ligation. METHODS Experiments were performed 3 weeks after surgery in anesthetized rats In the first protocol, the ability of XL-Hb to recover blood pressure after a hypotensive hemorrhage (0.5 ml/min, 10 min) was analyzed. In the second protocol, the pressor and renal effects produced by the administration of XL-Hb were evaluated during a period of 3 h. RESULTS After a hypotensive hemorrhage (0.5 ml/min, 10 min), resuscitation with XL-Hb resulted in greater and faster recovery of blood pressure than with the administration of blood. In non-hemorrhaged rats, administration of XL-Hb (5% of blood volume) reversibly increased blood pressure in bile duct ligation and in control rats, but this effect was of longer duration in the control animals. XL-Hb also induced brisk increases in water and sodium excretion in both groups of animals, but the response of the control animals was more intense and sustained than that of the bile duct ligation rats. Glomerular filtration rate and renal blood flow showed slight decreases, but they were well maintained around the baseline levels. All the parameters studied were normalized 3 h later. In additional experiments, the effect of a bolus of L-NAME (10 mg/kg), an inhibitor of nitric oxide synthase, 1 h after the administration of XL-Hb was partially reduced, suggesting that the effect of XL-Hb may be secondary to the disappearance of circulating nitric oxide. CONCLUSIONS XL-Hb seems to be effective as a resuscitative solution in case of hemorrhage in cirrhotic rats Moreover, this blood substitute only moderately and reversibly elevates blood pressure and does not adversely affects renal function.

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.

Omapatrilat normalizes renal function curve in spontaneously hypertensive rats

BackgroundThe present study was designed to analyze the chronic renal response to omapatrilat, a new vasopeptidase inhibitor, in spontaneously hypertensive rats (SHR). To that end, the renal and blood pressure response to a 4-day salt loading protocol was analyzed and the respective chronic renal curves constructed.ResultsIn non treated animals, and under normal sodium intake (around 2 mEq/day), mean arterial pressure (MAP), was significantly higher in the SHR as compared with the controls (WKY). After increasing salt intake (8 times normal), MAP did not change significantly in any group and the animals reached a normal sodium balance in four days. In a second group of animals, omapatrilat was given orally for 15 days at the dose of 40 mg/kg/day in the drinking water. In these omapatrilat-treated animals, and under normal sodium intake, MAP was significantly lower in both groups, although the antihypertensive effect was much greater in the SHR, so that the MAP of the SHR group was completely normalized and similar to the WKY-treated group. The subsequent elevation of sodium intake did not significantly elevate MAP in any group and the animals could manage the sodium excess as well as the non treated groups.ConclusionsThese results indicate that chronic treatment with omapatrilat normalizes blood pressure in SHR without affecting adversely the renal ability to eliminate a sodium load. Chronic treatment with omapatrilat resets the chronic pressure natriuresis relationship of the SHR to a normal level, thus without altering the normal salt-independence of this arterial hypertension model.

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.