Role of Nitric Oxide in the Altered Calcium Homeostasis of Platelets from Rats with Biliary Cirrhosis.

INTRODUCTION: Previously, we found that intracellular calcium (Ca2+) homeostasis is altered in platelets from an experimental model of liver cirrhosis, namely the bile-duct-ligated (BDL) rat. These alterations are compatible with the existence of a hypercoagulable state. OBJECTIVE: In the present study, we analyzed the role of nitric oxide in the abnormal calcium signaling responses of an experimental cirrhosis model, the bile duct-ligated rat. METHODS: Chronic treatment with L-NAME was used to inhibit NO production in a group of control and BDL animals, and the responses compared to those obtained in a control and BDL untreated group (n = 6 each). The experiments were conducted on isolated platelets loaded with fura-2, using fluorescence spectrometry. RESULTS: Chronic treatment with L-NAME increased thrombin-induced Ca2+ release from internal stores in both control and BDL rats. However, the effect was significantly greater in the BDL rats (p < 0.05). Thrombin-induced calcium entry from the extracellular space was also elevated but at lower doses and, similarly in both control and BDL platelets, treated with the NO synthesis inhibitor. Capacitative calcium entry was also enhanced in the control platelets but not in platelets from BDL rats treated with L-NAME. Total calcium in intracellular stores was elevated in untreated platelets from BDL rats, and L-NAME pretreatment significantly (p < 0.05) elevated these values both in controls and in BDL but significantly more in the BDL rats (p < 0.05). CONCLUSIONS: Our results suggest that nitric oxide plays a role in the abnormal calcium signaling responses observed in platelets from BDL rats by interfering with the mechanism that releases calcium from the internal stores.

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.

Effects of Flavonoids in Experimental Models of Arterial Hypertension.

Flavonoids are a class of substances of a vegetal origin with many interesting actions from the point of view of human disease. Interest in flavonoids in the diet has increased in recent years due to the publication of basic, clinical and epidemiological studies that have shown a whole array of salutary effects related to intake of flavonols and flavones as well as a lower morbility and mortality of cardiovascular diseases. Since arterial hypertension is the most common modifiable risk factor for cardiovascular diseases, this review will focus mainly on the effects of flavonoids on the cardiovascular system with relation to the elevation of blood pressure. Its antihypertensive effects as well as the many investigations performed in experimental models of arterial hypertension, are reviewed in this mini-review.

Robust In Vitro and In Vivo Immunosuppressive and Anti-inflammatory Properties of Inducible Caspase-9-mediated Apoptotic Mesenchymal Stromal/Stem Cell.

Mesenchymal stromal stem/cells (MSC) therapies are clinically used in a wide range of disorders based on their robust HLA-independent immunosuppressive and anti-inflammatory properties. However, the mechanisms underlying MSC therapeutic activity remain elusive as demonstrated by the unpredictable therapeutic efficacy of MSC infusions reported in multiple clinical trials. A seminal recent study showed that infused MSCs are actively induced to undergo apoptosis by recipient cytotoxic T cells, a mechanism that triggers in vivo recipient-induced immunomodulation by such apoptotic MSCs, and the need for such recipient cytotoxic cell activity could be replaced by the administration of ex vivo-generated apoptotic MSCs. Moreover, the use of MSC-derived extracellular vesicles (MSC-EVs) is being actively explored as a cell-free therapeutic alternative over the parental MSCs. We hypothesized that the introduction of a "suicide gene" switch into MSCs may offer on-demand in vivo apoptosis of transplanted MSCs. Here, we prompted to investigate the utility of the iCasp9/AP1903 suicide gene system in inducing apoptosis of MSCs. iCasp9/AP1903-induced apoptotic MSCs (MSCiCasp9+) were tested in vitro and in in vivo models of acute colitis. Our data show a very similar and robust immunosuppressive and anti-inflammatory properties of both "parental" alive MSCGFP+ cells and apoptotic MSCiCasp9+ cells in vitro and in vivo regardless of whether apoptosis was induced in vivo or in vitro before administering MSCiCasp9+ lysates. This development of an efficient iCasp9 switch may potentiate the safety of MSC-based therapies in the case of an adverse event and, will also circumvent current logistic technical limitations and biological uncertainties associated to MSC-EVs.

Perception of Medical Professionalism among Medical Residents in Spain.

BACKGROUND: Medical professionalism, defined as commitment to the primacy of patient welfare, is the basis for doctor-patient-society relationships, but previous research with medical students has shown that professionalism and social commitment to medicine may be waning. To determine if this trend also appears in recently qualified practicing doctors, we surveyed 90 newly graduated doctors currently working as medical residents in two university hospitals in Murcia, Spain. A previously validated questionnaire that studies the perception of six categories (responsibility, altruism, service, excellence, honesty and integrity, and respect) defining medical professionalism was used. RESULTS: A good perception of professionalism was found among medical residents, with more than 70% positive responses in all these six categories. There is an increasing trend in the number of negative responses as the residency goes on. Altruism was the category with the greatest percentage of negative answers (22.3%) and Respect was the category with the lowest percentage (12.9%). CONCLUSIONS: The results show a good professionalism perception in medical residents, but also a slight decline in positive answers that began during medical school. A significant trend was found when including both students and residents. Although there were some differences between students and residents, these were not statistically significant. Educational interventions are needed both at the level of medical school and postgraduate medical residency.

Platelet function and microvesicle generation in patients with hemophilia A.

Our results do not support any effect of FVIII on platelet function in patients with severe HA treated under the regime of prophylaxis.

Chronic model of tympanic perforation in rats with mitomycin C and dexamethasone.

A rat model of chronic tympanic membrane perforation was developed to be used in the search of new materials for the sealing of these perforations. A longitudinal study was carried out in rats subjected to incisional myringotomy followed by the application of mitomycin C alone or with dexamethasone. Rats were checked at days 3, 7, 10, 14 and weekly thereafter until perforation closure, for up to 6 months. The addition of dexamethasone is a key component in order to obtain a chronic opening. Myringotomies treated with saline had a mean healing time of 8.5 days. At 8 weeks, between 62.5% and 77.7% of tympanic membranes treated with mitomycin C and dexamethasone remained perforated and at 6 months this number fell to 21.4%. This technique is able to maintain most tympanic membrane perforations patent for at least 8 weeks. This rat model is adequate for its use in preclinical or translational research.

Silk fibroin scaffolds seeded with Wharton's jelly mesenchymal stem cells enhance re-epithelialization and reduce formation of scar tissue after cutaneous wound healing.

BACKGROUND: The treatment of extensive and/or chronic skin wounds is a widespread and costly public health problem. Mesenchymal stem cells (MSCs) have been proposed as a potential cell therapy for inducing wound healing in different clinical settings, alone or in combination with biosynthetic scaffolds. Among them, silk fibroin (SF) seeded with MSCs has been shown to have increased efficacy in skin wound healing experimental models. METHODS: In this report, we investigated the wound healing effects of electrospun SF scaffolds cellularized with human Wharton's jelly MSCs (Wj-MSCs-SF) using a murine excisional wound splinting model. RESULTS: Immunohistopathological examination after transplant confirmed the presence of infiltrated human fibroblast-like CD90-positive cells in the dermis of the Wj-MSCs-SF-treated group, yielding neoangiogenesis, decreased inflammatory infiltrate and myofibroblast proliferation, less collagen matrix production, and complete epidermal regeneration. CONCLUSIONS: These findings indicate that Wj-MSCs transplanted in the wound bed on a silk fibroin scaffold contribute to the generation of a well-organized and vascularized granulation tissue, enhance reepithelization of the wound, and reduce the formation of fibrotic scar tissue, highlighting the potential therapeutic effects of Wj-MSC-based tissue engineering approaches to non-healing wound treatment.

Effects of chronic L-NAME on nitrotyrosine expression and renal vascular reactivity in rats with chronic bile-duct ligation.

In liver cirrhosis, elevated levels of NO and ROS (reactive oxygen species) might greatly favour the generation of peroxynitrite. Peroxynitrite is a highly reactive oxidant and it can potentially alter the vascular reactivity and the function of different organs. In the present study, we evaluated whether peroxynitrite levels are related to the progression of renal vascular and excretory dysfunction during experimental cirrhosis induced by chronic BDL (bile-duct ligation) in rats. Experiments were performed at 7, 15 and 21 days after BDL in rats and in rats 21 days post-BDL chronically treated with L-NAME (N(G)-nitro-L-arginine methyl ester). Sodium balance, BP (blood pressure), basal RPP (renal perfusion pressure) and the renal vascular response to PHE (phenylephrine) and ACh (acetylcholine) in isolated perfused kidneys were measured. NO levels were calculated as 24-h urinary excretion of nitrites, ROS as TBARS (thiobarbituric acid-reacting substances), and peroxynitrite formation as the renal expression of nitrotyrosine. BDL rats had progressive sodium retention, and decreased BP, RPP and renal vascular responses to PHE and ACh in the time following BDL. They also had increasing levels of NO and ROS, and renal nitrotyrosine accumulation,especially in the medulla. All of these changes were either prevented or significantly decreased by chronic L-NAME administration. In conclusion, these results suggest that the increasing levels of peroxynitrite might contribute to the altered renal vascular response and sodium retention in the development of the experimental biliary cirrhosis. Moreover, the beneficial effects of decreasing NO synthesis are, at least in part, mediated by anti-peroxinitrite-related effects.

Moderate Effect of Flavonoids on Vascular and Renal Function in Spontaneously Hypertensive Rats.

Many studies have shown that flavonoids are effective as antihypertensive drugs in arterial hypertension. In the present work, we have analyzed the effects of some flavonoid extracts in the spontaneous hypertensive rat model (SHR). An important feature of this study is that we have used a low dose, far from those that are usually applied in human therapy or experimental animals, a dose that responded to the criterion of a potential future commercial use in human subjects. Treatments were carried out for 6 and 12 weeks in two groups of SHR rats, which received apigenin, lemon extract, grapefruit + bitter orange (GBO) extracts, and cocoa extract. Captopril was used as a positive control in the SHR group treated for 6 weeks (SHR6) and Diosmin was used as the industry reference in the SHR group treated for 12 weeks (SHR12). Captopril and GBO extracts lowered the high arterial pressure of the SHR6 animals, but none of the extracts were effective in the SHR12 group. Apigenin, lemon extract (LE), GBO, and captopril also improved aortic vascular relaxation and increased plasma and urinary excretion of nitrites, but only in the SHR6 group. Kidney and urinary thiobarbituric acid reactive substances (TBARS) were also significantly reduced by GBO in the SHR6 rats. Apigenin also improved vascular relaxation in the SHR12 group and all the flavonoids studied reduced urinary thiobarbituric acid reactive substances (TBARS) excretion and proteinuria. Vascular abnormalities, such as lumen/wall ratio in heart arteries and thoracic aorta, were moderately improved by these treatments in the SHR6 group. In conclusion, the flavonoid-rich extracts included in this study, especially apigenin, LE and GBO improved vascular vasodilatory function of young adult SHRs but only the GBO-treated rats benefited from a reduction in blood pressure. These extracts may be used as functional food ingredients with a moderate therapeutic benefit, especially in the early phases of arterial hypertension.

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.

Flavonoids in Kidney Health and Disease.

This review summarizes the latest advances in knowledge on the effects of flavonoids on renal function in health and disease. Flavonoids have antihypertensive, antidiabetic, and antiinflammatory effects, among other therapeutic activities. Many of them also exert renoprotective actions that may be of interest in diseases such as glomerulonephritis, diabetic nephropathy, and chemically-induced kidney insufficiency. They affect several renal factors that promote diuresis and natriuresis, which may contribute to their well-known antihypertensive effect. Flavonoids prevent or attenuate the renal injury associated with arterial hypertension, both by decreasing blood pressure and by acting directly on the renal parenchyma. These outcomes derive from their interference with multiple signaling pathways known to produce renal injury and are independent of their blood pressure-lowering effects. Oral administration of flavonoids prevents or ameliorates adverse effects on the kidney of elevated fructose consumption, high fat diet, and types I and 2 diabetes. These compounds attenuate the hyperglycemia-disrupted renal endothelial barrier function, urinary microalbumin excretion, and glomerular hyperfiltration that results from a reduction of podocyte injury, a determinant factor for albuminuria in diabetic nephropathy. Several flavonoids have shown renal protective effects against many nephrotoxic agents that frequently cause acute kidney injury (AKI) or chronic kidney disease (CKD), such as LPS, gentamycin, alcohol, nicotine, lead or cadmium. Flavonoids also improve cisplatin- or methotrexate-induced renal damage, demonstrating important actions in chemotherapy, anticancer and renoprotective effects. A beneficial prophylactic effect of flavonoids has been also observed against AKI induced by surgical procedures such as ischemia/reperfusion (I/R) or cardiopulmonary bypass. In several murine models of CKD, impaired kidney function was significantly improved by the administration of flavonoids from different sources, alone or in combination with stem cells. In humans, cocoa flavanols were found to have vasculoprotective effects in patients on hemodialysis. Moreover, flavonoids develop antitumor activity against renal carcinoma cells with no toxic effects on normal cells, suggesting a potential therapeutic role in patients with renal carcinoma.

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.

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.

Las unidades de educación médica en las facultades de Medicina y de Ciencias de la Salud en España. ¿Son necesarias? / Medical education units in Medical and Health Sciences schools in Spain. Are they needed?

En una mayoría de países, la educación médica es una especialidad médica más y preside la vida académica en el grado, el posgrado y en la formación continuada. Pero la situación en España es muy mejorable. Aunque existe un creciente interés en la educación médica como disciplina o especialidad, la mayor parte de las facultades de Medicina españolas no disponen de una unidad o departamento de educación médica que se encargue del avance de la disciplina. Algunas facultades han dispuesto una unidad, cátedra, departamento o centro de estudios, adscrita o independiente a la dirección del centro, a veces sin relación orgánica alguna con el proceso de formación. En este artículo describiremos por qué creemos que estas estructuras son necesarias, su utilidad, así como sus funciones y el alcance de sus actividades. Analizaremos la situación actual en España con el ánimo de promocionar la creación de estas estructuras en todas las facultades de Medicina. Igualmente, repasaremos los mecanismos de los que se ha dotado a la formación especializada en el posgrado para dar respuestas a sus necesidades de formación

In a majority of countries, medical education is one more medical specialty and presides over academic life in undergraduate, graduate and continuing education. But, the situation in Spain is very improvable. Although there is a growing interest in medical education as a discipline or specialty, most of the Spanish faculties of Medicine do not have a unit or department of medical education, which is in charge of advancing the discipline. Some faculties have arranged a unit, chair, department or study center, attached or independent to the management of the center, sometimes without any organic relationship to the training process. In this article we will describe why we believe these structures are necessary, their usefulness, as well as their functions and the scope of their activities. We will analyze the current situation in Spain with the aim of promoting the creation of these structures in all the faculties of Medicine. We will also review the mechanisms that specialized postgraduate training has been equipped to provide answers to their training needs

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.

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.

Interaction of nitric oxide with calcium in the mesenteric bed of bile duct-ligated rats.

We have analysed the interaction of NO with calcium in order to study the molecular mechanisms responsible for the vascular hyporesponse of liver cirrhosis. The experiments have been performed in the isolated and perfused mesenteric arterial bed of rats with bile duct ligation (BDL) and their controls. While perfusing the vessels with normal Krebs, methoxamine (MTX) or KCl produced a lower pressor response in the BDL mesenteries. The NO synthesis inhibitor N(w)-nitro-L-arginine (NNA) potentiated those responses and abolished the differences between groups. The administration of MTX under perfusion with zero calcium-Krebs, to analyse the intracellular release of calcium, also induced a lower response in the BDL mesenteries and NNA potentiated and normalized the response. To investigate calcium entry, the vessels were perfused with zero-calcium Krebs containing high potassium to open voltage-dependent calcium channels. Then, the addition of calcium (10(-1) - 3 x 10(-3) M) produced a lower pressor response in the BDL vessels, that was corrected by NNA. To study calcium entry through receptor-operated channels, the vessels were perfused with zero-calcium Krebs containing MTX. The addition of calcium elevated the perfusion pressure less in the BDL mesenteries than in the control and NNA potentiated the responses and eliminated the between groups differences. When calcium entry through both voltage- and receptor-operated channels was simultaneously analysed, similar results were obtained. In the mesenteric bed of bile duct ligated rats, an excess of nitric oxide affects vascular calcium regulation through an interaction with both calcium entry and intracellular calcium release.

Effects of omapatrilat on blood pressure and renal injury in L-NAME and L-NAME plus DOCA-treated rats.

BACKGROUND: This study investigates the effects of chronic administration of omapatrilat (OMA) on blood pressure (BP), renal injury, and other variables in N(omega)-nitro-L-arginine methyl ester (L-NAME) hypertension and in the low-renin model produced by the simultaneous administration of L-NAME and deoxycorticosterone acetate (DOCA). METHODS: The control, DOCA, L-NAME, L-NAME + DOCA, L-NAME + OMA, and L-NAME + DOCA + OMA groups were used. Tail systolic BP was measured twice a week. After 4 weeks of treatment, mean arterial pressure (MAP), and metabolic, morphologic, and renal variables were measured. RESULTS: The final values of MAP were 109 +/- 5.1 mm Hg for the control group, 113 +/- 3.0 mm Hg for DOCA, 175 +/- 3.7 mm Hg for L-NAME, 193 +/- 3.8 mm Hg for L-NAME + DOCA, 117 +/- 3.9 mm Hg for L-NAME + OMA, and 158 +/- 3.0 mm Hg for L-NAME + DOCA + OMA. The rats treated with L-NAME showed mild and scarce renal lesions, which were prevented by OMA treatment and the L-NAME + DOCA group showed proteinuria and hyaline arteriopathy, which were markedly attenuated in the L-NAME + DOCA + OMA group. Plasma urea and creatinine were significantly increased in the L-NAME + DOCA group, whereas these variables were not significantly greater in the L-NAME + DOCA + OMA group versus controls. The L-NAME + DOCA group showed relative renal and cardiac hypertrophy that was not observed in the L-NAME + DOCA + OMA group. CONCLUSIONS: The simultaneous blockade of neutral endopeptidase (NEP) and angiotensin converting enzyme (ACE) completely prevents L-NAME hypertension. Our results also show that OMA attenuates the increased BP and the renal injury in L-NAME hypertensive rats treated with DOCA. Assuming that this is a low-renin model of hypertension, the protective effect of OMA may be due to an increase in vasodilator peptides produced by both ACE and NEP inhibition.

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.