It takes two to tango: the structure and function of LIM, RING, PHD and MYND domains.

LIM (Lin-11, Isl-1, Mec-3), RING (Really interesting new gene), PHD (Plant homology domain) and MYND (myeloid, Nervy, DEAF-1) domains are all zinc-binding domains that ligate two zinc ions. Unlike the better known classical zinc fingers, these domains do not bind DNA, but instead mediate interactions with other proteins. LIM-domain containing proteins have diverse functions as regulators of gene expression, cell adhesion and motility and signal transduction. RING finger proteins are generally associated with ubiquitination; the presence of such a domain is the defining feature of a class of E3 ubiquitin protein ligases. PHD proteins have been associated with SUMOylation but most recently have emerged as a chromatin recognition motif that reads the methylation state of histones. The function of the MYND domain is less clear, but MYND domains are also found in proteins that have ubiquitin ligase and/or histone methyltransferase activity. Here we review the structure-function relationships for these domains and discuss strategies to modulate their activity.

Microalbuminuria: do we need a new threshold?

Microalbuminuria (30-300 mg of albumin/24 h) is a well-known independent risk factor for kidney and cardiovascular disease and of mortality in diabetic, hypertensive and in the general population. However, recent studies indicate that increased risk is observed at levels of albuminuria much lower than those currently employed to define microalbuminuria. Such low levels were shown to predict heart disease and death, independent of age, sex, renal function, diabetes, hypertension and lipids, in subjects with cardiovascular disease, hypertension and in the general population; as well as to predict progression to hypertension. Correction of obesity and metabolic derangements lowered levels of albuminuria below 30 mg/24 h to levels not associated with increased risk (5-7 mg/24 h). Despite the lack of outcome studies, there is substantial evidence to indicate that the threshold for defining microalbuminuria (that is, albuminuria associated with increased risk) should be lowered by nearly three to four-fold from the currently defined threshold. It would be advisable that clinical scores and future guidelines would consider including microalbuminuria at the lower rates as an independent risk factor, and as an indication for implementing early intervention. Unfortunately, and despite the abundance of evidence, albuminuria measurements are still underutilized in clinical practice.

Effects of lifestyle changes and metformin on salt sensitivity and nitric oxide metabolism in obese salt-sensitive Hispanics.

Salt sensitivity is associated with obesity, and increased cardiovascular morbidity and mortality. We investigated whether treatment of obesity and its associated metabolic abnormalities corrects salt sensitivity and restores impaired nitric oxide (NO) metabolism characteristic of salt sensitivity. Twenty, otherwise, healthy obese salt-sensitive subjects completed a 12-month program of caloric restriction, aerobic exercise and metformin. Two salt sensitivity tests were performed, that is at baseline and end of program. Lifestyle-metformin treatment decreased weight (9.8+/-0.3 kg), body mass index (3.9+/-0.2 kg/m(2)), waist (11.5+/-0.5 cm), systolic blood pressure (SBP) (8.6+/-0.4 mm Hg), diastolic blood pressure (DBP) (5.5+/-0.4 mm Hg), triglyceride (40+/-5 mg/dl), fasting (8.3+/-1 microIU/ml) and post-load (20+/-4 microIU/ml) insulin levels, and salt sensitivity. Going from a high-sodium ( approximately 300 mmol) to a low-sodium diet ( approximately 30 mmol of sodium/day) lowered SBP/DBP by 14.7+/-1.7/7.4+/-0.9 mm Hg at baseline and by 8.6+/-1.9/3.2+/-1.2 mm Hg after treatment (P<0.001). More importantly, blood pressure (BP) sensitivity to customary levels of dietary salt ( approximately 150 mmol of sodium/day) was abolished by the lifestyle-metformin treatment. Differences in SBP/DBP between usual and low salt averaged 11+/-1/8+/-1 mm Hg before treatment, and 3+/-1/1+/-0.5 mm Hg after treatment (P<0.001). At baseline, NO-metabolite excretion was inhibited during high salt; this impairment was corrected by the lifestyle-metformin treatment. In conclusion, acquired correctable factors play an important role in the pathogenesis of salt sensitivity associated with obesity. Correction of salt sensitivity may account for the BP lowering induced by weight reduction. Restoration of the inability to increase or sustain NO production in response to high salt could account for the correction of salt sensitivity induced by the lifestyle-metformin treatment.

Increased blood pressure reactivity to dietary salt in patients with the metabolic syndrome.

The metabolic syndrome is a predictor of type II diabetes mellitus and cardiovascular disease. The mechanisms of the increased blood pressure (BP) in patients with the metabolic syndrome are poorly understood. We investigated if salt-sensitivity is a characteristic of the metabolic syndrome. A total of 301 subjects (87 male subjects, 214 female subjects) of 41.5+/-0.7 years of age completed a salt sensitivity test, and were evaluated for the presence of metabolic syndrome. BP and 24-h sodium excretion were obtained under usual, high- and low-salt intakes. BP reactivity to salt was markedly increased in subjects with the metabolic syndrome; its magnitude was directly related to the severity of the syndrome. Reducing dietary salt from the average usual intake (8.2 g/day) to nearly 2.3 g/day lowered systolic blood pressure (SBP) by 8.7+/-1.3 mm Hg in subjects with four and five traits, 6.0+/-1.1 in those with three traits and failed to modify the BP of subjects with one or no traits of the syndrome (P < 0.0001). Salt restriction reduced the percentage of subjects with metabolic syndrome that were hypertensive (8.2 g/day of salt) from 23.8 to 8.2% (chi2: 23.6; P<0.0001). BP of non-hypertensive subjects with metabolic syndrome was also significantly reduced by salt restriction (7.1+/-1.5 and 4.2+/-1.1 mm Hg in those with four or five traits and three traits, respectively). In conclusion, the metabolic syndrome is a strong clinical predictor of salt sensitivity. The enhanced BP reactivity to dietary salt observed in subjects with the metabolic syndrome, may determine the increased BP levels commonly associated with the syndrome.

NAD(P)H oxidase p22phox gene C242T polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics.

Mutations in the NAD(P)H oxidase gene may be associated with abnormal superoxide generation, nitric oxide (NO) availability and cardiovascular diseases. We investigated the prevalence of the NAD(P)H oxidase p22phox gene C242T polymorphism, and its possible association with blood pressure, NO production, salt sensitivity and cardiovascular risk factors in Hispanics. Genotype frequencies were as follows: CC, 52.9%; CT, 40.3%; and TT, 6.8%. There were no significant differences in systolic blood pressure, diastolic blood pressure, age, weight, fasting and post-load glucose levels, LDL and HDL cholesterol, triglyceride and urinary albumin levels in subjects with CC, CT or the TT genotypes. Presence of the T allele was associated with increased salt sensitivity in women, but not in men. NO metabolite excretion was markedly decreased both in women and men with the TT genotype (CC: 868+/-79 micromol/day; CT: 839+/-75 micromol/day; TT: 534+/-78 micromol/day; P<0.05). In conclusion, the prevalence of the NAD(P)H oxidase p22phox gene C242T polymorphism in Venezuelans was comparable to that of Caucasians, but different from that of Chinese and Japanese. Although the T allele was not associated with cardiovascular risk factors, hyperinsulinaemia or hypertension, in women, it appeared to be a genetic susceptibility factor for salt sensitivity. Both in women and men, the p22phox gene may play a role in the genetic control of NO levels.

Endothelial nitric oxide synthase polymorphism, nitric oxide production, salt sensitivity and cardiovascular risk factors in Hispanics.

Mutations in the endothelial nitric oxide synthase (eNOS) gene may be associated with abnormal nitric oxide (NO) production and cardiovascular diseases. In this study, we investigated the prevalence of two eNOS polymorphisms, the Glu298Asp variant on exon 7, and the 4a/b variable number of tandem repeats (VNTR) on intron 4, and their association with blood pressure (BP), NO production, salt sensitivity and cardiovascular risk factors in healthy Venezuelans. The prevalence of both polymorphisms in Venezuelans was comparable to that described for Caucasians, but significantly different from that known for African-Americans and Japanese. The 4a/b genotype was associated with reduced levels of NO metabolites (25% decrease), larger BP lowering in response to salt restriction (9.0 vs 4.8 mmHg, P<0.05), greater prevalence of salt sensitivity (39% in 4a/b and 27% in 4b/b; P<0.05) and with higher LDL-cholesterol levels. The Glu298T polymorphism did not affect NO production, nor it was associated with salt sensitivity. Glu298Asp polymorphism was positively associated with higher weight, triglycerides and LDL-cholesterol. Neither polymorphism was associated with changes in fasting or postload serum glucose, BP, obesity and albuminuria. In conclusion, the prevalence of eNOS polymorphisms is strongly determined by ethnic factors. The 4a/b gene polymorphism could be a genetic susceptibility factor for the BP response to salt intake and for the genetic control of NO production. The reduced NO production in subjects with the 4a/b genotype may be responsible for the increased sensitivity of their BP to salt.

Insulin resistance and upper-normal glucose levels in hypertension: a review.

Reduced insulin-mediated glucose disposal, indicative of insulin resistance, has been demonstrated in lean male hypertensives both with the hyperinsulinaemic euglycaemic clamp and the insulin suppression test. In lean hypertensives, insulin resistance was not accompanied by increases in fasting plasma insulin and glucose levels; but with modest hyperglycaemia and hyperinsulinaemia after a glucose load. Population studies (no stratification) reveal that: (1) insulin sensitivities vary widely in normotensives and hypertensives, (2) there are hypertensives and normotensives with similar degrees of insulin resistance, (3) not all hypertensives are insulin resistant, and (4) insulin resistance does not contribute to the blood pressure level of the hypertensive population. In large cross-sectional studies, the clustering of obesity, dyslipidaemia and type 2 diabetes is largely responsible for the observed associations between insulin or insulin resistance and hypertension. Recent studies indicate a role of glucose in blood pressure control. Glucose has been shown to elevate blood pressure in the presence of endothelial dysfunction and glucose values in the upper-normal range have been shown to be associated with increased cardiovascular mortality. Since endothelial dysfunction is present in hypertensives, dyslipidaemic, obese and in glucose intolerant individuals, lowering of high-normal glucose levels becomes a new, additional therapeutic target in the management of these patients. Hyperglycaemia together with endothelial dysfunction may account for the increased incidence of hypertension in obesity and diabetes mellitus. Because of the strong association between insulin resistance, hyperglycaemia and endothelial dysfunction, and the clustering of risk factors in these subjects, we propose the lowering of high normal glucose levels as part of the therapeutic strategy to prevent cardiovascular and metabolic disease.

Differential blood pressure effects of oral glucose and intravenous L-arginine in healthy lean normotensive and obese hypertensive subjects.

We investigated the role of insulin and glucose in the pathophysiology of hypertension associated with obesity. The comparative effects of an oral glucose load and of an L-arginine infusion on plasma glucose, plasma insulin and blood pressures (BP) were assessed in lean normotensive and in obese hypertensive males. Oral glucose (75 g in 1-2 min) induced a small but significant lowering of BP in lean normotensives, but failed to modify BP in obese hypertensives. L-arginine infusion (30 min, 500 mg/kg total dose) reduced BP; significantly greater reductions in systolic and diastolic BP were observed in obese hypertensives than in the control group. Both oral glucose and L-arginine induced greater increases in plasma insulin in obese hypertensives than in lean normotensives. Endothelial dysfunction which accompanies the insulin resistant state of obesity, glucose intolerance and hypertension, may account for the different BP effects induced by glucose and L-arginine in obese hypertensives and lean normotensives.

Clustering of silent cardiovascular risk factors in apparently healthy Hispanics.

Ischaemic heart disease is one of the leading causes of cardiovascular morbidity and mortality. Because most factors leading to cardiovascular disease have a silent course, early screening is needed for prevention and for halting disease progression. In our centre, a programme was implemented in apparently healthy subjects for the early diagnosis and treatment of factors known to increment the risk of developing cardiovascular and metabolic disease. We present data from the first 153 individuals evaluated. The incidence of modifiable risk factors in our healthy population was as follows: overweight 33% (BMI: 25-30 kg/m(2)), obesity 45% (BMI >30 kg/m(2)), sedentarism 84%, arterial hypertension 15% (>140/90 mm Hg), hyperinsulinaemia 50%, glucose intolerance 14% (>160 mg/dl 120 min after 75 g glucose load), type 2 diabetes mellitus 5%, hypercholesterolaemia 50%, hypertriglyceridaemia 28%, and salt sensitivity 25%. Clustering of three or more cardiovascular risk factors was observed in 59% of the apparently healthy subjects. Obesity was associated with greater clustering of risk factors. The cardiovascular dysmetabolic syndrome was present in 72% of the obese individuals. These findings revealed a very high prevalence of cardiovascular risk factors in apparently healthy Hispanics. Even though these individuals were clinically asymptomatic, they are at increased risk for developing cardiovascular disease and type 2 diabetes mellitus. Mechanisms for the early detection and correction of modifiable risk factors in the healthy population must be implemented. Only through prevention will a reduction in the incidence of cardiovascular atherosclerotic disease and of type 2 diabetes mellitus be achieved.

Hiperinsulinemia y sensibilidad a la sal / Hyperinsulinaemia and salt sensitivity

La enfermedad coronaria es una de las primeras causas de morbilidad y mortalidad cardiovascular. Para su prevención es necesario diagnosticar y corregir con medidas farmacológicas y no farmacológicas los factores de riesgo cardivascular modificables. Nosotros hemos desarrollado un programa de diagnóstico precoz y tratamiento de factores que incrementan a desarrollar enfermedades cardiovasculares y metabólicas, en sujetos "sanos", asintomáticos. Hasta el presente hemos evaluado 153 sujetos. En este trabajo evaluamos la asociación a la sensibilidad a la sal con otros factores de riesgo cardiovascular silente e investigamos los posibles factores predictores de sensibilidad a la sal. La comparación de los sujetos sensibles a la sal (SS), con los sujetos sal resistentes (SR) demostró que los sujetos SS tienen mayor edad, peso corporal, cifras tensionales, niveles elevados de triglicéridos, niveles bajos de HDL-colesterol y de actividad físico. Adicionalmente estudiamos el papel de la hiperinsulinemia y de la hiperglicemia en la sensibilidad a la sal, a través de la medición de la incidencia (porcentaje de individuos SS y SR) y severidad de la sensibilidad a la sal en sujetos hiperinsulinémicos e intolerantes glucosados y/o diabéticos comparándolos con su respectivos controles. No hubo diferencias en la distribución de sensibilidad a la sal entre los diferentes grupos (hiper y normo insulinémicos (insulina en ayunas < 15uUL/ml), glucotolerantes (glicemia en ayunas <110;2 horas post carga < 140 mg/d) y en los intolerantes glucosados y/o diabéticos. La severidad de la sensibilidad a la sal también fue similar entre grupos, de hecho, la reducción de la ingesta de sal de 316ñ13 a 26ñ3 mmol/día, produjo cambios similares en la presión arterial en sujetos hiperinsulinémicos o en sujetos con niveles normoinsulinémicos y en sujetos tolerantes e intolerantes glucosados y/o diabéticos. Adicionalmente no se encontró correlación entre la magnitud de los cambios de la presión arterial inducidos por la sal y los niveles de insulina y glucosa en ayunas, 2 horas después de la carga oral de glucosa o el área bajo la curva de insulina. En conclusión, nuestros resultados sugieren los niveles de glicemia y de insulina del paciente, no determinan su reactividad vascular a los cambios en ingesta de sal

Urinary albumin excretion in lean, overweight and obese glucose tolerant individuals: its relationship with dyslipidaemia, hyperinsulinaemia and blood pressure.

The presence of microalbuminuria has become an important tool for therapeutic intervention. In this study we investigated whether the dysmetabolic syndrome of obesity was associated with or could occur in the absence of microalbuminuria. The study was conducted in 71 clinically healthy, glucose tolerant Hispanics (age: 43 +/- 1.4 years, body mass index (BMI): 28.7 +/- 0.6 kg/m(2), systolic blood pressure (SBP): 117 +/- 2 mm Hg, diastolic blood pressure (DBP): 77 +/- 1.3 mm Hg, urinary albumin excretion: 10.2 +/- 0.6 mg/24 h). Subjects were classified as lean (BMI <25), overweight (BMI >25 <30) and obese (BMI >30 kg/m(2)). Greater BMI was associated with higher body weight, waist-to-hip ratio (WHR), BP, fasting insulin, triglyceride, post glucose load insulin and glucose, and lower high-density lipoprotein (HDL) cholesterol levels. However, no significant differences in the urinary albumin excretion (mg/24 h) were found between lean (9.0 +/- 0.9; median: 9.1), overweight (11.3 +/- 1.2; median: 10.5) and obese (11.1 +/- 1.2; median: 9.7) subjects. In addition, microalbuminuria (urinary albumin excretion >30 mg/24 h) was not found in any of the study subjects. For all subjects combined, as well as for each of the groups separately, the urinary albumin excretion was unrelated to the BMI, WHR, body weight, triglyceride, cholesterol (total, LDL or HDL), fasting or post-load glucose and insulin plasma concentrations. Neither in females nor in males, abdominal fat accumulation was associated with an increase in the urinary albumin excretion. However, in the obese groups, urinary albumin excretion was strongly related to the level of SBP (r(2): 0.67; P < 0.0001) and DBP (r(2): 0.55; P < 0.0001). In summary, obesity, hyperinsulinaemia and dyslipidaemia per se are not determinants of increased albumin excretion. However, in the obese subjects, the BP, particularly the SBP, was a strong determinant of the level of albumin in the urine. Microalbuminuria may occur later in the course of the dysmetabolic syndrome, due to worsening of hypertension and development of hyperglycaemia.

Human endothelial cells grow poorly on vitronectin: role of PAI-1.

The cell adhesive protein vitronectin is a common component of interstitial extracellular matrix and circulates in plasma. It competes effectively with other plasma proteins to adsorb to certain biomaterial surfaces, and is likely to represent an important cell adhesion mediator on the luminal surface of vascular grafts. It is also found associated with certain vascular pathologies. We have shown previously that human endothelial cells grow poorly on a vitronectin surface compared with other extracellular matrix molecules. In this paper we show that endothelial cells seeded on vitronectin and fibronectin produced substantially different profiles of extracellular matrix molecules. The most outstanding difference was in the amount of matrix-localised plasminogen activator-inhibitor-1 which was high on vitronectin and negligible on fibronectin. This was correlated with a small but significant inhibition of cell adhesion to vitronectin compared with fibronectin, and very significant interference with dissociation of cell: extracellular matrix contacts, resulting either from direct inhibition of the proteolytic activity of urokinase, or from interference with urokinase-receptor signaling and consequent focal adhesion turnover. Such interference would inhibit cell proliferation by disabling the cells from loosening their matrix contacts in order to proceed through mitosis. This would seriously compromise endothelial recovery in cases of damage to the vascular wall and placement of stents or grafts, where the presence of surface-adsorbed vitronectin is likely to modulate the tissue response.

Nitric oxide synthases and cyclophosphamide-induced cystitis in rats.

The role of inducible (iNOS) and neuronal nitric oxide (nNOS) synthases and of tachykinin NK1 receptors on the pathogenesis of cyclophosphamide (CYP)-induced cystitis was investigated, in rats. CYP-induced cystitis was characterized by large increases in bladder-protein plasma extravasation (PPE), increases in the urinary excretion of nitric oxide (NO) metabolites and histological evidences of urothelial damage, edema, extensive white blood cell infiltrates and vascular congestion of the bladder. The specific iNOS inhibitor, S-methylthiourea (MITU), produced marked inhibition (>90%) of CYP-induced increases in PPE associated with amelioration of tissue inflammatory changes. Treatment with 7-nitroindazole (7-NI; 20, 40 and 80 mg/kg), a selective nNOS inhibitor, did not significantly reduce CYP-induced increases in PPE and failed to produce histological improvement. In addition, treatment with MITU, but not with 7-NI, inhibited the increases in the urinary excretion of NO metabolites induced by CYP treatment. WIN 51,708 (17-beta-hydroxy-17-alpha-ethynyl-androstano[3,2-b]pyrimido[1,2-a]benzimidazole; WIN), a selective NK1-receptor antagonist, reduced the increases in EPP and ameliorated the inflammatory changes in the bladder induced by CYP. However, the maximal degree of protection achieved with WIN was significantly less than that produced by MITU. Combined treatment with the iNOS inhibitor and the NK1 antagonist produced no greater effect than that produced by the iNOS inhibitor alone. Our results suggest that NO plays a fundamental role in the production of the cystitis associated with CYP treatment. The iNOS, and not nNOS, seems responsible for the inflammatory changes. Part of the increases in NO may due to activation of NK1 receptors by neuropeptides such as substance P possibly released from primary afferent fibers.

Expression of inducible nitric oxide synthase in primary culture of rat bladder smooth muscle cells by plasma from cyclophosphamide-treated rats.

Intraperitoneal administration of cyclophosphamide (50-150 mg/kg) for 6 or 12 h induced edema and hemorrhagic changes in rat bladder, which were both dose and time-dependent. Pretreatment with nitric oxide synthase (NOS) inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME, 40 mg/kg) or with s-methylisothiourea (40 mg/kg) ameliorated the cyclophosphamide-induced cystitis. Cyclophosphamide administration also produced increases in NO-metabolite levels (nitrate+nitrite) in the urine and plasma of rats. Greater increases in NO metabolites were observed with 150 than with 50 mg/kg of cyclophosphamide, and at 12 than at 6 h after cyclophosphamide injection. Pretreatment with L-NAME and s-methylisothiourea significantly reduced cyclophosphamide-induced increases in urine and plasma NO-metabolite levels. To explore the mechanism by which cyclophosphamide increases the expression of inducible NOS (iNOS), primary cultures of rat bladder smooth muscle were developed. Exposure to tumor necrosis factor alpha (TNF-alpha) plus interferon gamma, produced a marked increase in the expression of iNOS and in NO production in the culture medium. However, exposure to cyclophosphamide or to its metabolite acrolein (10(-6)-10(-4) M for 24 h) did not increase iNOS or NO-metabolite levels. On the other hand, incubation of primary cell cultures with plasma from rats treated with cyclophosphamide (150 mg/kg, 12 h) produced a marked increase in iNOS expression and NO production. Taken together, our results indicate that NO plays an important role in the pathogenesis of cyclophosphamide-induced cystitis in rats, and some factors may be released in cyclophosphamide-treated rat plasma which stimulate iNOS expression in primary culture of rat bladder smooth muscle cells.

Nitric oxide and NK(1)-tachykinin receptors in cyclophosphamide-induced cystitis, in rats.

The present study was conducted to investigate the role of NK(1) receptors and of nitric oxide (NO) on the pathogenesis of cyclophosphamide-induced cystitis, in rats. This bladder toxicity was characterized by marked increases in protein plasma extravasation, urothelial damage, edema, white blood cell infiltrates, and vascular congestion. These changes were associated with appearance of Ca(2+)-independent NO-synthase (NOS) activity [characteristic of inducible NOS (iNOS)] in the bladder and with increases in urinary NO metabolites. GR205171, a selective NK(1) antagonist (10-20 mg/kg, i.p.) reduced cyclophosphamide-induced increases in protein plasma extravasation and in the urinary excretion of NO metabolites. N(G)-Nitro-L-arginine (L-NNA) (10 mg/kg, i.p.), a NOS inhibitor, reduced basal and cyclophosphamide-induced increases in NO metabolites and protected against cyclophosphamide-induced protein plasma extravasation. GR205171 had no effect, whereas L-NNA reduced basal NO metabolite excretion. Combined treatment with the NK(1) antagonist and the NO-synthesis inhibitor produced comparable reduction in protein plasma extravasation than that achieved with each drug given separately. Combined drug treatment ameliorated cyclophosphamideinduced urothelial damage, and the extent of edema, vascular congestion, and white blood cell infiltrates in the bladder. In summary, NK(1) receptors and iNOS play a role in NO formation and on cyclophosphamide-induced cystitis. Activation of NK(1) receptors mainly acts through the formation of NO. It is proposed that cyclophosphamide and/or its metabolites would stimulate primary afferent capsaicin-sensitive fibers in the bladder, releasing neuropeptides, which would activate NK(1) receptors. However, additional mechanisms are involved, because neither the NK(1) receptor antagonist nor the NO synthesis inhibitor, either alone or in combination, were able to completely prevent the toxicity.

Nitric oxide and salt sensitivity.

Studies in laboratory animals suggest that altered nitric oxide (NO) production may be associated with salt sensitivity. In this investigation we determined whether the endogenous NO production was altered in salt-sensitive human subjects when salt intake was changed. Salt sensitivity was assessed from the magnitude of the blood pressure (BP) lowering obtained when the salt intake was reduced from high to a low intake. The combined urinary excretion of nitrites and nitrates, the major metabolites of NO, was employed as an index of endogenous NO production. Salt-sensitive subjects (n = 23) were older, heavier, and had greater waist-to-hip ratios and higher baseline BP than salt-resistant individuals (n = 25). In salt-sensitive subjects, mean blood pressure (MBP) decreased 11.8+/-0.7 mm Hg, and NO metabolite excretion increased from 823+/-102 to 1530+/-148 mmol/24 h, when salt intake was reduced from 316 to 28 micromol/day. NO metabolite excretion was 45% lower during high salt (0.66+/-0.1 micromol/mg creatinine) than during low salt intake (1.12+/-0.1 micromol/mg creatinine) (P < .001). In contrast, when salt intake was reduced, salt-resistant subjects exhibited no significant mean changes in BP or NO metabolite excretion. During low salt intake, NO metabolite excretion (micromol/ day) was significantly higher in salt-sensitive individuals. The magnitude of decrease of systolic blood pressure, diastolic blood pressure, or MBP induced by reducing salt intake was not related to the increase in urinary excretion of NO metabolite levels (r2 = 0.009; P = .66). In summary, to the extent that urinary NO metabolite levels reflect the activity of the endogenous NO system, our results support the view that salt sensitivity may in part be determined by an inability to increase or to sustain NO production in response to high salt. Insufficient NO production during high salt may in turn lead to altered pressure-natriuresis relationships and to an increase in BP. The possibility that the increase in BP induced by high salt intake in salt-sensitive individuals could be the key factor in reducing NO metabolite levels can not be ruled out.

Dictyostelium discoideum as expression host: isotopic labeling of a recombinant glycoprotein for NMR studies.

The advantages of the organism Dictyostelium discoideum as an expression host for recombinant glycoproteins have been exploited for the production of an isotopically labeled cell surface protein for NMR structure studies. Growth medium containing [(15)N]NH(4)Cl and [(13)C]glycerol was used to generate isotopically labeled Escherichia coli, which was subsequently introduced to D. discoideum cells in simple Mes buffer. A variety of growth conditions were screened to establish minimal amounts of nitrogen and carbon metabolites for a cost-effective protocol. Following single-step purification by anion-exchange chromatography, 8 mg of uniformly (13)C,(15)N-labeled protein secreted by approximately 10(10) D. discoideum cells was isolated from 3.3 liters of supernatant. Mass spectrometry showed the recombinant protein of 16 kDa to have incorporated greater than 99.9% isotopic label. The two-dimensional (1)H-(13)C HSQC spectrum confirms (13)C labeling of both glycan and amino acid residues of the glycoprotein. All heteronuclear NMR spectra showed a good dispersion of cross-peaks essential for high-quality structure determination.

Effects of short- and long-term exercise on urinary cGMP excretion in healthy subjects and in patients with coronary artery disease.

The possibility that systemic formation of cyclic guanosine monophosphate (cGMP) could reflect the level of cardiovascular fitness was investigated. The relations between physical activity and systemic formation of cGMP were evaluated in healthy volunteers and in patients with coronary artery disease (CAD). No significant differences were observed in the basal urinary excretion of cGMP in highly trained runners, sedentary subjects, and in patients with CAD, despite the large differences in aerobic exercise training between groups. In addition, the basal levels of cGMP in CAD patients failed to increase after a 12-week cardiac rehabilitation program. Short-term exercise, on the other hand, was associated with significant increases in urinary cGMP excretion. A 42-km marathon increased urinary cGMP excretion by 272%. The 15-km race increased urinary cGMP excretion by 330%. In CAD patients, 30 min of supervised exercise on a treadmill, at 80% of patient's maximal heart rate, induced a 60% increase in urinary cGMP, which returned to preexercise levels 90 min after termination of the exercise. Completion of the 12-week cardiac rehabilitation program improved exercise capacity and the magnitude of increase in cGMP levels induced by short-term treadmill exercise. Our findings suggest that cGMP increases during and shortly after short-term exercise and that the magnitude of the increase seems dependent on the intensity of the exercise and on physical fitness. Exercise training in healthy subjects and in CAD patients enhanced the amount of cGMP produced during short-term exercise, which might be responsible for some of the protective cardiovascular actions of exercise. The short half-life of cGMP may explain why the basal resting levels of cGMP are not appropriate predictors of a subject's physical fitness.

Insulin and blood pressure responses to changes in salt intake.

In this study we evaluated the role of insulin in hypertension and on salt sensitivity. The study was conducted in 47 consecutive patients attending the Center for the Detection and Treatment of Cardiovascular and Metabolic Risk factors. The relationships between fasting and post-glucose load insulin levels and the blood pressure (BP) responses to changes in salt intake, were investigated. No correlation was observed between fasting or 2-h post-load insulin levels and mean BP (MBP), systolic BP (SBP) or diastolic BP (DBP). The plasma concentrations of insulin were not significantly related to body mass index (BMI) (r2 = 0.05; P = 0.135). Neither fasting nor 2-h post-load insulin predicted the BP response to changes in salt intake. A reduction in salt intake from 316 +/- 13 to 26 PM 3 mmoles/day, produced similar BP lowering in subjects with fasting insulin >15 microU/ml and in subjects with normal fasting insulin levels (<15 microU/ml). In addition, no relationship was observed between the magnitude of the BP responses to salt and the levels of insulin, either fasting (r2 = 0.007; P = 0.86) or 2-h after a glucose load (r2 = 0.01; P = 0.213). A very strong association was found between body weight or BMI and MBP (r2 = 0.443; P< 0.0001). In conclusion, our results are against the view of a cause-effect relationship between insulin and BP levels. In addition, the insulin status of a patient does not predict (nor determines) his (her) vascular reactivity to changes in salt intake. Finally, our findings further support the existence of a strong and direct association between body weight and hypertension, and speak against a major role of insulin in the pathogenesis of hypertension associated with obesity.

Role of NK1 receptors on cisplatin-induced nephrotoxicity in the rat.

The role of NK1 receptors on the nephrotoxicity associated with cisplatin treatment was evaluated. Adult Sprague-Dawley male rats (300-400 g) were treated with saline (0.1 ml/100 g, i.p., every 8 h for 72 h) or the selective NK1 receptor antagonist (GR205171; 2 mg/kg, i.p., every 8 h for 72 h). Treatments were started 5 min before cisplatin (7.5 mg/kg, i.p., single dose). All evaluations were made from 72 h to 96 h after cisplatin. An oral load of 10 ml/kg of water was given at time 0 (72 h after cisplatin). Cisplatin reduced the urinary volume (-45%), creatinine clearance (>90%), lithium clearance (-76%) and urinary potassium excretion (-54%). Protein and sodium excretion was not affected by cisplatin. GR205171 prevented the reduction in urine volume induced by cisplatin. In addition, the decreases in creatinine and lithium clearances induced by cisplatin were also attenuated by the NK1 receptor antagonist. The clearance of creatinine averaged 0.57+/-0.2 ml/min in controls, 0.004+/-0.01 ml/min after cisplatin, and 0.09+/-0.02 ml/min after cisplatin + GR205171. The lithium clearance was 0.09+/-0.04 ml/min in controls, 0.02+/-0.01 ml/min after cisplatin and 0.06+/-0.01 ml/min after cisplatin + GR205171. Cisplatin induced marked necrosis, vacuolation and edema of proximal renal tubules; these changes were considerably reduced in GR205171-treated animals. These results indicate that treatment with a selective NK1 receptor antagonist ameliorated cisplatin-induced renal toxicity in rats, as evidenced by improvements in renal function and histology.