Metformin improves putative longevity effectors in peripheral mononuclear cells from subjects with prediabetes. A randomized controlled trial.

BACKGROUND AND AIMS: Prediabetes increases cardiovascular risk and is associated with excess mortality. In preclinical models, metformin has been shown to exert anti-ageing effects. In this study, we sought to assess whether metformin modulates putative effector longevity programs in prediabetic subjects. METHODS AND RESULTS: In a randomized, single-blind, placebo-controlled trial, 38 prediabetic subjects received metformin (1500 mg/day) or placebo for 2 months. At baseline and after treatment, we collected anthropometric and metabolic parameters. Gene and protein levels of SIRT1, mTOR, p53, p66Shc, SIRT1 activity, AMPK activation, telomere length, and SIRT1 promoter chromatin accessibility were determined in peripheral blood mononuclear cells (PBMCs). Plasma N-glycans, non-invasive surrogate markers of ageing, were also analysed. Compared to baseline, metformin significantly improved metabolic parameters and insulin sensitivity, increased SIRT1 gene/protein expression and SIRT1 promoter chromatin accessibility, elevated mTOR gene expression with concomitant reduction in p70S6K phosphorylation in subjects' PBMCs, and modified the plasma N-glycan profile. Compared to placebo, metformin increased SIRT1 protein expression and reduced p70S6K phosphorylation (a proxy of mTOR activity). Plasma N-glycans were also favourably modified by metformin compared to placebo. CONCLUSION: In individuals with prediabetes, metformin ameliorated effector pathways that have been shown to regulate longevity in animal models. ClinicalTrials. gov identifier: NCT01765946 - January 2013.

Phosducin rs12402521 polymorphism predicts development of hypertension in young subjects with overweight or obesity.

BACKGROUND AND AIMS: The G-protein regulator phosducin has been shown to be associated with stress-dependent blood pressure, but whether obesity is a modulator of the relationship between phosducin and risk of hypertension is unknown. We studied the effect of two phosducin polymorphisms on risk of hypertension in 273 overweight or obese (Ov-Ob) young-to-middle-age participants from the HARVEST and 287 normal weight (NW) participants. METHODS AND RESULTS: Genotyping of phosducin SNPs rs12402521 and rs6672836 was performed by real time PCR. For rs12402521, 64.6% of the participants were homozygous for the G allele, 27.9% heterozygous, and 7.5% homozygous for the A allele. During 7.7 years of follow-up, 339 subjects developed hypertension. In a Cox multivariable model, carriers of the A allele had a 1.28 (95% CI,1.00-1.63, p = 0.046) increased risk of hypertension. However, increased incidence of hypertension associated with A allele (AA + AG, 79% and GG, 59%, p = 0.001) was observed only among Ov-Ob individuals with a hazard ratio of 1.60 (95% CI, 1.13-2.21, p = 0.007) whereas in NW subjects the incidence of hypertension did not differ by genotype (56% in both groups). In the whole cohort, there was a significant interaction of phosducin genotype with body mass index on the risk of hypertension (p = 0.012). For SNP rs6672836 no association was found with incident hypertension. No haplotype effect was detected on the risk of hypertension. CONCLUSION: These data suggest that phosducin rs12402521 polymorphism is an important genetic predictor of obesity-related hypertension. In Ov-Ob carriers of the A allele aggressive nonpharmacological measures should be implemented.

MAPKinase and regulation of the sodium-proton exchanger in human red blood cell.

The sodium-proton exchanger is activated by various agonists, including insulin, even in human red blood cell. MAPKinase, a family of ubiquitous serine/threonine kinases, plays an important role in the signal transduction pathways which lead to sodium-proton exchanger activation. The aim of our study was to establish the existence of MAPKinase in human red blood cell and to investigate the effects of its activation by insulin and okadaic acid on the sodium-proton exchanger. Immunoblot with antiMAPK antibody revealed the presence of two isoforms, p44(ERK1) and p42(ERK2). Insulin stimulated MAPKinase activity and increased the phosphorylation of MAPK tyrosine residues, with a peak time between 3 and 5 min. Okadaic acid, an inhibitor of serine/threonine phosphatases, stimulated MAPKinase activity. In the presence of PD98059, an inhibitor of MEK, the upstream activator of MAPKinase, insulin and okadaic acid failed to stimulate MAPKinase. Insulin and okadaic acid increased the activity of the sodium-proton exchanger and this effect was abolished by PD98059. In conclusion, we first describe the presence and activity of MAPKinase in human red blood cell. Furthermore, we demonstrate that in human red blood cell, insulin modulates the sodium-proton exchanger through MAPKinase activation.

Protein kinase C activity is acutely regulated by plasma glucose concentration in human monocytes in vivo.

Activation of protein kinase C (PKC) by hyperglycemia is implicated in the pathogenesis of long-term diabetic complications. Monocyte activation and transformation into macrophages is a key step in the atherosclerotic process. Therefore, in this study, we sought to determine 1) the effect of hyperglycemia on monocyte PKC activity and on the distribution of Ca2+-dependent and diacylglycerol-sensitive PKC isoforms; and 2) whether the effects on these parameters are determined by hyperglycemia per se, independent of the diabetic state. The studies were performed in 19 type 2 diabetic patients and 14 control subjects. Plasma glucose concentration was higher and insulin sensitivity lower (both P < 0.01) in diabetic patients than in control subjects. Monocytes from diabetic patients showed similar cytosol PKC activity to those from control subjects but higher membrane PKC activity (78+/-6 vs. 50+/-5 pmol x min(-1) x mg(-1) protein; P < 0.01). A direct correlation was observed between fasting plasma glucose and membrane PKC activity (r2 = 0.4008, P = 0.0001). In contrast, a reciprocal correlation was observed between membrane PKC activity and insulin sensitivity index (r2 = 0.28, P < 0.05). Using immunoblotting analysis, we found that membrane beta2, but not alpha, isoform of PKC was more abundant in monocytes from diabetic patients. In diabetic patients, when euglycemia was acutely induced, membrane PKC activity decreased by approximately 42% and beta2 isoform by approximately 15%. In two normal subjects in whom hyperglycemia was induced, membrane PKC increased from 63 and 57 to 92 and 128.6 pmol x min(-1) x mg(-1) protein, respectively. This increase was associated with an increase in the membrane isoform beta2; alpha isoform was unchanged. We conclude that 1) monocytes express the glucose-sensitive beta2 isoform of PKC; 2) the prevailing plasma glucose acutely regulates the activity of the membrane PKC and the content of membrane PKC beta2 isoform; and 3) this effect appears to be a direct effect of glucose per se, since the phenomenon was observed in normal control subjects when hyperglycemia was induced. Monocyte PKC activation may account for the accelerated atherosclerosis of patients with type 2 diabetes.

Sodium-lithium countertransport has low affinity for sodium in hyperinsulinemic hypertensive subjects.

We recently reported that incubation of red blood cells with insulin markedly decreases the affinity for external Na+ and increases the maximal transport rate (Vmax) of Na(+)-Li+ countertransport. The association of hypertension with insulin resistance and its compensatory hyperinsulinemia led us to investigate the relationship between insulin levels in vivo and the Na+ activation kinetics of this antiporter. We studied normotensive (n = 28) and hypertensive (n = 25) subjects after they had fasted overnight and determined their plasma glucose and insulin concentrations. Insulin levels were higher in the hypertensive subjects (11.7 +/- 1.5 microU/mL, mean +/- SEM) than in the normotensive subjects (8.2 +/- 1.2 microU/mL), but glucose levels were similar and within normal limits. Antiporter activity was measured as sodium-stimulated Li+ efflux by a new procedure that uses isosmotic conditions to raise external Na+ to 280 mmol/L. In normotensive subjects, Vmax was reached between 50 and 100 mmol/L Na+, whereas in most hypertensive subjects, Na+ concentrations higher than 150 mmol/L were needed. This different kinetic behavior was because the Na+ concentration for half-maximal activation (Km) was twofold higher in hypertensive subjects (58.9 +/- 5.3 mmol/L) than in normotensive subjects (29.8 +/- 2.6 mmol/L, P < .001). Hypertensive subjects with fasting insulin levels greater than 10 microU/mL (n = 12) had a higher Km for Na+ than subjects with insulin levels less than 10 microU/mL (n = 13) (73.4 +/- 8.7 versus 45.6 +/- 3.9 mmol/L, respectively, P < .01) and similar Vmax (0.57 +/- 0.05 versus 0.41 +/- 0.05 mmol.L-1.h-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperglycemia acutely increases monocyte extracellular signal-regulated kinase activity in vivo in humans.

Glycemic spikes may negatively affect the long-term prognosis of patients with diabetes. Extracellular signal-regulated kinases (ERKs) are intracellular mediators of cell proliferation, and they can be activated in response to high glucose levels. However, the modifications of their activity in response to hyperglycemia have been poorly investigated, in vivo, in humans. Thus, we sought to determine in circulating monocytes: 1) the role of hyperglycemia in ERKs activity and phosphorylation, and 2) whether hyperglycemia affects mitogen-activated protein kinase kinase (MEK) activity and mitogen-activated protein phosphatase-1 (MKP-1) expression. These goals were performed in five normal subjects. Baseline monocyte ERKs activity was 60 +/- 5 pmol/min.mg protein; when exogenous hyperglycemia was induced, both monocyte ERKs activity (81 +/- 11 pmol/min.mg protein; P < 0.05) and phosphorylation significantly increased (P < 0.01). MEK activity was significantly increased by hyperglycemia (1251 +/- 136 vs. 2000 +/- 42 cpm; P = 0.0017), whereas no changes were observed in MKP-1 expression. We conclude that hyperglycemia acutely stimulates ERKs activity and phosphorylation in human monocytes by the MEK pathway in vivo. These findings may be relevant in understanding the negative role of acute hyperglycemia on monocyte pathophysiology.

Improvement of insulin sensitivity by metformin treatment does not lower blood pressure of nonobese insulin-resistant hypertensive patients with normal glucose tolerance.

Nine hypertensive patients with body mass indexes between 24-27 kg/m2 and normal glucose tolerance with at least a postchallenge plasma insulin level greater than 360 pmol/L were recruited for a double blind, cross-over study with metformin (850 mg, twice daily) and placebo. Each treatment lasted 1 month. Before and after each treatment, hormone and substrate concentrations were determined, blood pressure was monitored over 24 h, and insulin sensitivity was measured by a euglycemic (4.7 mmol/L) hyperinsulinemic (450 pmol/L) clamp study. Renal cation excretion and erythrocyte membrane cation heteroexchange were measured. Metformin, compared to placebo, did not affect body weight (70 +/- 7 vs. 70 +/- 7 kg), fasting plasma glucose (4.8 +/- 0.1 vs. 4.8 +/- 0.1 mmol/L), total cholesterol (5.38+/0.33 vs. 5.48 +/- 0.38 mmol/L), or triglycerides (1.73 +/- 0.72 vs. 1.91 0.89 mmol/L). Nevertheless, after metformin treatment, the plasma high density lipoprotein cholesterol concentration increased (1.42 +/- 0.18 vs. 1.34 0.16 mmol/L), and the plasma insulin level dropped (62 +/- 10 vs. 88+/- 12 pmol/L; both P < 0.05). Insulin-mediated glucose disposal was higher after metformin treatment (26.1 +/- 2.4 vs. 19.3 +/- 2.3 micromol/min x kg; P < 0.01), whereas hepatic glucose production was completely suppressed. These positive metformin-induced metabolic effects were not associated with a significant change in mean daily blood pressure levels (141 +/- 6/89 +/- 3 vs. 142 +/- 7/90 +/- 3 mm Hg). Compared to placebo, metformin increased the excretion of sodium, potassium, and lithium by enhancing their glomerular filtration rate. Na+/Li+ countertransport was not affected by metformin. However, the apparent affinity for H+ of Na+/H+ exchange was increased, and the Hill coefficient was decreased. In conclusion, 1 month of metformin administration to patients with essential hypertension and normal glucose tolerance 1) reduces the basal plasma insulin concentration, 2) improves whole body insulin-mediated glucose utilization, and 3) improves plasma high density lipoprotein cholesterol levels. Despite these positive effects, metformin did not reduce arterial blood pressure.

Modulatory effect of insulin on release of calcium from human fibroblasts by angiotensin II.

BACKGROUND: Angiotensin II stimulates synthesis and deposition of collagen and might contribute to the vascular and cardiac dysfunction associated with arterial hypertension. Insulin attenuates angiotensin II-induced responses of intracellular Ca2+ concentration ([Ca2+]) in many cell types but this effect is less in insulin-resistant states. The mechanisms of the interaction between insulin and angiotensin II are still not known. OBJECTIVE: To characterize the effects of angiotensin II on intracellular [Ca2+] and the effects of insulin on the angiotensin II-induced response of intracellular [Ca2+] in human skin fibroblasts. METHODS: Spectrofluorophotometric measurements of intracellular [Ca2+] in monolayers of cultured human skin fibroblasts from 15 normotensive patients were performed using Fura-2 at 510 nm emission with excitation wavelengths of 340 and 380 nm. RESULTS: Basal intracellular [Ca2+] in quiescent (24 h serum-deprived) human fibroblasts was 75 +/- 3 nmol/l (n = 20). Administration of angiotensin II elevated intracellular [Ca2+] dose-dependently with a concentration for half-maximal effect of 20 nmol/l. Administration of 100 nmol/l angiotensin II stimulated a rapid and transient increase in intracellular [Ca2+] (from 75 +/- 3 to 130 +/- 2 nmol/l, n = 20). Removal of extracellular calcium did not change peak intracellular [Ca2+], but it did reduce the time to recovery of [Ca2+] (from 64 +/- 4 to 48 +/- 2 s, n = 10, P < 0.01), suggesting that an angiotensin II-induced transmembrane calcium influx had occurred. This hypothesis was confirmed by quenching studies with manganese. The angiotensin II-induced changes in intracellular [Ca2+] were completely blocked by administration of 100 nmol/l of the angiotensin II type 1 receptor inhibitor losartan but not by administration of 100 nmol/l of the angiotensin II type 2 receptor blocker CGP42112A. Acute (20 min) exposure to 100 nmol/l insulin did not alter basal intracellular [Ca2+] in quiescent fibroblasts, but significantly blunted angiotensin II-stimulated peak of [Ca2+] (to 101 +/- 3 nmol/l, P < 0.01, n = 18) and delayed recovery of [Ca2+] (to 99 +/- 5 s, P < 0.01). The inhibitory effect of insulin was observed both with and without extracellular Ca2+. CONCLUSIONS: Our results demonstrate that administration of angiotensin II increases intracellular [Ca2+] in human skin fibroblasts by release of Ca2+ from intracellular Ca2+ stores and by influx of Ca2+ and that administration of insulin attenuates the response of [Ca2+] to angiotensin II but prolongs the time to recovery of [Ca2+].

Inhibition of furosemide-sensitive cation transport and activation of sodium-lithium exchange by endogenous circulating factor(s) in Bartter's and Gitelman's syndromes.

BACKGROUND: The nature of the cellular abnormality causing hypokalemia, hypotension, and hypovolemia in Bartter's and Gitelman's syndromes is still being debated. In fact, despite the recent descriptions of an array of nonconservative missense or point mutations in some ion transporters and in K+ channel, the lack of detectable defects in some patients suggests that other abnormalities of cell ion homeostasis may be involved in the pathophysiology of these syndromes. The study of the activity of cell ion transporters in patients with these syndromes using red blood cells (RBC) as a cellular model never investigated the role of plasma factor(s) affecting ion transport. OBJECTIVE: To evaluate the effect of plasma from patients with these syndromes on furosemide-sensitive lithium efflux (FSLE) from lithium (Li+)-loaded RBC of healthy subjects in vitro. METHODS: RBC of healthy controls were loaded with Li+ in the presence of nystatin and FSLE was evaluated in the presence of various concentrations of plasma from controls and patients with the two syndromes. RESULTS: Plasma from controls did not affect FSLE (0.08 +/- 0.02 mmol/l cells per h with 1:4 vol:vol and 0.07 +/- 0.02 mmol/l cells per h with 1:2 vol:vol plasma dilution). In contrast, doubling concentrations of plasma from patients with either syndrome in the efflux solution halved FSLE (from 0.10 +/- 0.0 mmol/l cells per h with 1:4 vol:vol to 0.05 +/- 0.01 mmol/l cells per h with 1:2 vol:vol plasma dilution, P < 0.05). Na+/Li+ exchange was significantly greater for RBC from patients with either syndrome than it was for RBC from controls (0.373 +/- 0.06 versus 0.257 +/- 0.01 mmol/l cells per h, P < 0.01), but the kinetic properties of furosemide-sensitive Na+-K+-2Cl- cotransport were similar. CONCLUSION: These data provide evidence for the hypothesis that plasma factor(s) affect ion transport in patients with these two syndromes. Since FSLE estimates Na+-K+-2Cl- cotransport the data suggest that plasma factor(s) contribute(s) to K+ wasting, hypokalemia, and hypotension by inhibiting cotransport in patients with these syndromes. The increase of Na+/Li+ exchange is most likely a secondary phenomenon associated with the hypermineralocorticoid state.

Kinetic properties of erythrocyte Na+-Li+ and Na+-H+ exchange in hypertensive patients.

OBJECTIVE: To ascertain the relationships between the kinetic properties of erythrocyte Na+-H+ exchange [maximum kinetic energy (Vmax), Michaelis constant (Km) for internal H+ and Hill's coefficient], Na+-Li+ (Vmax and Km for external Na+), and metabolic parameters in normotensive controls and hypertensive subjects. MATERIALS AND METHODS: Na+-H+ exchange was measured as the Na+ influx driven by intracellular H+, and Na+-Li+ exchange as the Li+ efflux driven by extracellular Na+, in erythrocytes from normotensive (n = 59) and hypertensive (n = 93) subjects. RESULTS: In comparison with normotensives, the hypertensives had a higher Vmax for Na+-Li+ and Na+-H+ exchange, a higher Km for external Na+ for Na+-Li+ exchange and a lower reduced Hill's number for Na+-H+ exchange. Vmax values for Na+-Li+ and Na+-H+ exchange were significantly correlated, as were Km values for internal H+ for Na+-H+ exchange and Km for external Na+ for Na+-Li+ exchange. Insulin resistance and beta-cell function indices were higher in the hypertensives than the normotensives. Upon stepwise multiple regression analysis, Vmax for Na+-Li+ exchange was correlated significantly and independently with Km for external Na+ and with the insulin resistance index, while Km for external Na+ was correlated with Km for internal H+, Vmax for Na+-H+ exchange and mean blood pressure. Vmax and Hill's coefficient for Na+-H+ exchange were correlated only with mean blood pressure. CONCLUSIONS: The demonstration of functional correlations between the kinetic properties of Na+-H+ and Na+-Li+ exchange provides further evidence that erythrocyte Na+-Li+ exchange is a functioning mode of Na+-H+ exchange, which is affected by insulin resistance.

Adrenomedullin stimulates DNA synthesis of rat adrenal zona glomerulosa cells through activation of the mitogen-activated protein kinase-dependent cascade.

BACKGROUND: Adrenal zona glomerulosa cells are provided with adrenomedullin receptors. Adrenomedullin has recently been found to enhance proliferation of cultured rat vascular smooth muscle cells and zona glomerulosa cells. OBJECTIVE: To investigate whether adrenomedullin affects rat zona glomerulosa proliferative activity through the tyrosine kinase and extracellular signal regulated kinases (ERKs) pathways. METHODS: Dispersed rat zona glomerulosa cells were cultured in vitro for 24 h and then exposed to adrenomedullin (10(-7) mol/l), alone or in the presence of tyrphostin-23 (10(-5) mol/l) or PD-98059 (10(-4) mol/l), for 24 or 48 h. To assess the rate of DNA synthesis, 5-bromo-2'-deoxyuridine (BrdU, 20 mg/ml) was also added to the medium and BrdU-positive cells were detected by immunocytochemistry. The expression of ERKs and the effect of adrenomedullin on ERKs phosphorylation and activity were assayed in dispersed zona glomerulosa cells. RESULTS: Adrenomedullin significantly increased the percentage of BrdU-positive (phase-S) zona glomerulosa cells; this effect was blocked by either the tyrosine kinase inhibitor, tyrphostin-23, or the mitogen-activated protein kinase kinase (MEK-1) inhibitor, PD-98059. Both zona glomerulosa and zona fasciculata/reticularis express ERK-1 (44 kDa) and ERK-2 (42 kDa) isoforms. However, adrenomedullin phosphorylated ERK-1 and ERK-2 only in the zona glomerulosa; this effect was blunted by the MEK-1 inhibitor, PD98059, and by the calcitonin gene-related peptide type 1 (CGRP-1) receptor antagonist, CGRP8-37, but not by the adrenomedullin C-terminal fragment, ADM22-52. CONCLUSION: Adrenomedullin stimulates the growth of rat zona glomerulosa cells through activation of CGRP-1 receptor, linked to the tyrosine kinase-MEK-1-ERKs signalling pathway. These results confirm the complex role played by this peptide in the regulation of zona glomerulosa cell physiology.

Effects of canrenoate on red cell sodium transport and calf flow in essential hypertension.

The effects of potassium canrenoate (100 mg/day, orally for 1 month) on blood pressure, calf blood flow at rest and after ouabain (0.5 mg intravenous bolus), and red cell Na homeostasis were investigated in 15 patients (7 men, 8 women, aged 18 to 63) with essential hypertension and without peripheral vascular diseases. On placebo, acute intravenous ouabain administration significantly and transiently reduced calf flow and increased calf vascular resistance without affecting blood pressure. Canrenoate significantly decreased blood pressure (from 159 +/- 21/105 +/- 9 mm Hg to 141 +/- 14/94 +/- 10, P < .05) and the rise of calf resistance after intravenous ouabain bolus. The latter effect was variable, since it was inhibited almost completely in 8 patients and unaffected in the others. In the patients in whom exogenously administered ouabain was antagonized, canrenoate diminished blood pressure through vasodilation and heightened the red cell Na/K pump. None of these parameters changed significantly in the other patients. Thus these data suggest that the fall in vascular resistance induced by canrenoate is mediated, in part, by the antagonism of endogenous ouabain-like factors.

Red blood cell Na+/H+ and Li+/Na+ exchange in patients with essential hypertension.

The increased red blood cell Li+/Na+ exchange found in a subgroup of patients with essential hypertension (EH) may reflect an increased activity of the Na+/H+ exchange. The maximal velocity of the red cells' Na+/H+ (Na+ influx promoted by an outward H+ gradient) and Li+/Na+ (Li+ efflux promoted by external Na+) exchange were therefore measured in 41 EH and in 21 normotensive controls (NT). Both transporters were significantly higher in EH than in NT (74 +/- 39 mmol/L cell x h v 43 +/- 27 for the former, P less than .03, and 0.35 +/- 0.16 v 0.26 +/- 0.10 for the latter, P less than .05). Even though more than 100 times faster, Na+/H+ exchange was weakly but significantly correlated to Li+/Na+ exchange (r = 0.29, P less than .05). Proximal tubule Na+ reabsorption (fractional renal Li+ reabsorption) was significantly greater in EH than in NT (0.78 +/- 0.07, n = 32, v 0.73 +/- 0.06, n = 10, P less than .05) but it was not correlated to either the red cells' Na+/H+ or Li+/Na+ exchanges. Therefore, hyperactivity of Na+/H+ exchange in EH may play a role in blood pressure elevation through mechanisms other than stimulation of renal Na+ reabsorption.

Effect of protein kinase C and insulin on Na+/H+ exchange in red blood cells of essential hypertensives.

The kinetic properties of sodium-proton exchange are abnormal in human red blood cells of hypertensive patients and it has been demonstrated that the transport protein undergoes post-translational modifications able to affect its kinetic properties. Protein kinase C (PKC) activation decreases the affinity constant for intracellular protons while insulin increases the maximal rate of proton translocation. The present study therefore aimed to examine the relationships among PKC activity, fasting insulin levels and the kinetic behaviour of sodium-proton exchange in red blood cells from 20 normotensives and 36 hypertensives. In comparison with normotensive subjects, hypertensive patients had higher body mass index (26.2 +/- 0.7 vs 23.6 +/- 0.6 kg/m2, P < 0.05), higher fasting insulin levels (93.2 +/- 10.8 vs 38.6 +/- 2.9 pmol/L), increased maximal velocity of proton translocation (37.9 +/- 2.7 vs 27.6 +/- 1.9 mmol/L per cell x h, P < 0.05), and reduced Hill's coefficient (1.6 +/- 0.1 vs 2.0 +/- 0.1, P < 0.01) of sodium-proton exchange. Basal PKC activity of the cytosol and membrane was similar in the study groups. However, after treatment with 1 micromol/L phorbol 12-myristate 13-acetate (PMA) for 10 min, membrane PKC activity was stimulated to a larger extent in hypertensives (to 181 +/- 8 pmol/min/mg protein) than in normotensives (to 136 +/- 6 pmol/min/mg protein, P < 0.01). The PMA stimulated PKC activity was positively correlated to fasting insulin levels (r = 0.59, P < 0.01). Stimulation of membrane PKC by PMA corrected the low Hill's coefficient for H(i)+ activation of sodium-proton exchange in the hypertensives, while the constant for half maximal activation for intracellular protons (ie, the affinity for intracellular protons) decreased to a similar extent in both groups. The maximal transport rate was unaffected by PMA. These results indicate that the abnormal proton activation of red blood cell sodium-proton exchange in hypertensives reflects an abnormal regulation of PKC translocation to the cell membrane, associated to hyperinsulinaemia and probably insulin resistance. Therefore, post-translational modifications of the transport protein(s) account for the altered kinetic behaviour of sodium-proton exchange in hypertensives.

Increased red cell sodium-lithium countertransport and lymphocyte cytosolic calcium are separate phenotypes in patients with essential hypertension.

Increased red blood cell sodium-lithium countertransport (SLC) activity and elevated intracellular calcium have been observed in hypertensive patients. The association of these ion transport abnormalities with each other and with another phenotype, insulin resistance, has been suggested. We investigated whether elevated SLC activity and increased lymphocyte cytosolic calcium (Ca(cyt)) occur in the same individuals and whether either is associated with hyperinsulinaemia. We measured SLC activity, lymphocyte Ca(cyt)and fasting insulin levels in hypertensive patients and normal subjects. Consistent with prior studies, SLC activity was significantly and positively correlated with fasting insulin levels (r = 0.45, P < 0.01). However, SLC activity and lymphocyte Ca(cyt) were significantly but inversely correlated (r = -0.42, P < 0.01) and lymphocyte Ca(cyt) was also inversely correlated with fasting insulin (r = -0.55, P < 0.001). When the study participants were instead separated into two groups based on fasting insulin levels, those above the median (15 microU/ml) had significantly higher SLC activity and significantly lower Ca(cyt). When separated by lymphocyte Ca(cyt) levels (above or below 120 nM) those patients with low lymphocyte Ca(cyt) had significantly higher SLC activity and significantly higher insulin levels. Multiple linear regression showed that fasting insulin was significantly predictive of SLC activity (P = 0.05) and Ca(cyt) (P < 0.01). Thus, elevated SLC activity and increased lymphocyte Ca(cyt) are separate and distinct ion transport phenotypes in hypertensive patients, linked through a relationship to hyperinsulinaemia that is direct with SLC activity and inverse with lymphocyte Ca(cyt).

Ouabain-inhibiting activity of aldosterone antagonists.

It has been suggested that endogenous substances (known as ouabain-like factors, OLF), secreted from the central nervous system in response to salt and water retention, inhibit the cell membrane Na+/K+ pump in the renal tubules and reduce sodium reabsorption. However, by also acting upon vascular smooth muscle cells, they may induce cell Na+ and Ca++ accumulation, vasoconstriction and systemic hypertension. Recently, an endogenous Na+/K+ pump inhibitor was isolated from human plasma; this inhibitor is indistinguishable from the cardiac glycoside ouabain based on biochemical and immunological criteria. Its plasma concentration is close to the therapeutic range for ouabain (around 0.4 nmol/L). Since plant ouabain promotes natriuresis, vasoconstriction, and hypertension; endogenous ouabain may therefore control extracellular fluid volume and blood pressure. The highest plasma concentrations of endogenous ouabain and OLF were found in congestive heart failure, aldosterone producing adenoma, human and animal models of volume expanded hypertension (reduced renal mass and DOCA-salt hypertension), and in Milan hypertensive rats (MHS). Aldosterone antagonists (canrenone and canrenoate) exert both agonist and antagonist effects on the digitalis receptor site of the Na+/K+ pump. They are effective antihypertensive agents in animal models of hypertension sustained by OLF (reduced renal mass-Na+ and DOCA-salt hypertension in rats). Moreover, in a subgroup of essential hypertensives, 4 weeks of canrenoate administration reduced blood pressure, heightened red blood cell Na+/K+ pump activity, and antagonized ouabain-induced vasoconstriction. None of these effects was seen in the other hypertensives. These data suggest that aldosterone antagonists stimulate the Na+/K+ pump inhibited by endogenous ouabain and exert their antihypertensive action at least in part through this mechanism.

Interactions between insulin and sodium homeostasis in essential hypertension.

It has been proposed, therefore, that hyperinsulinemia may favor the development of hypertension through sodium retention, sympathetic nervous system activation, and vascular hypertrophy. In insulin-resistant hypertensive subjects, insulin infusion during euglycemic clamp promotes a transient sodium retention by stimulating proximal tubular Na+ reabsorption, but chronic hypertension usually is not associated with extracellular fluid and plasma volume expansion. In essential hypertensive subjects, intracellular potassium is decreased and intracellular sodium increased, which is consistent with insulin resistance. The latter is also associated with high red blood cell Li+/Na+ exchange, and chronic insulin treatment in insulin-dependent diabetics induces a slight increase in Li+/Na+ CT. This is a functioning mode of the Na+/H+ exchange, and its increase may reflect either an increased number of transport units or abnormal kinetic properties. Experiments in vitro and in vivo suggested that any change in insulin concentration and insulin sensitivity may affect Li+/Na+ and Na+/H+ counter-transport. High Li+/Na+ and Na+/H+ CT are associated with a significant cardiac and vascular remodeling in essential hypertension, insulin-dependent diabetes, and familiar hypertrophic cardiomyopathy. Reduced insulin sensitivity is associated with salt-sensitive hypertension. Finally, insulin potentiates the effects of other agonists (eg, thromboxane A2, angiotensin II) on vascular contraction and cell growth. These data indicate that insulin may play a role in the pathogenesis of hypertension and its major complications by amplifying the effects of sodium, vasoconstrictors, and growth factors.

Red blood cell membrane cation transport in normotensive psoriatics.

Psoriasis might be a widespread membrane disorder. To verify whether under the influence of psoriasis the red cell membrane cation metabolism is altered, we compared 46 psoriatics with 23 normotensive controls. A significant increase was observed in intracellular K+ content, in the maximal velocity of the Na(+)-K+ ATPase, of the Na(+)-K(+)-Cl- outward co-transport, of the Na+/H+ exchange, as well as in the outward passive permeability for Na+. No difference was seen between the two groups in Na+ content, Li+/Na+ exchange or in the passive permeability to K+. In 8 psoriatics treated with etretinate (10-75 mg/day for 1-36 months), Na(+)-K+ ATPase, Na(+)-K(+)-Cl- co-transport and the passive permeability for Na+ were not significantly different from controls. These results suggest that the primary abnormality might be an increased Na+ influx, in part through the Na+/H+ exchange, which is compensated by increased activity of outward transports, and confirm that the red cells are a useful model for the study of membrane transport in this disease. Our results indicate also that these membrane transport abnormalities can be corrected by etretinate treatment.

Red blood cell Li+/Na+ exchange in patients with diabetic nephropathy and essential hypertension: therapeutic implications.

Patients who develop diabetic nephropathy, one of the leading causes of end-stage renal diseases in Western communities, have an increased red cell Li+/Na+ countertransport (CT). Li+/Na+ CT is a membrane function which exchanges intracellular Li for extracellular Na in vitro. High Li+/Na+ CT reflects abnormal kinetic properties of red cell membrane Na/H exchange. A widespread abnormality of Na/H exchange could play a major role in the pathogenesis of diabetic nephropathy as well as of cardiovascular diseases since Na/H exchange is involved in the regulation of cell pH and cell volume; in the cellular response to hormones, mitogens, and growth factors; and in the renal reabsorption of Na and bicarbonate. Li+/Na+ CT is under genetic control and raised in a subgroup of patients with essential hypertension. Among these patients, high Li+/Na+ CT is associated with increased glomerular filtration rate, filtration fraction, proximal fractional Na reabsorption, microalbuminuria, plasma renin activity, and kidney and cardiac volume. Increased Li+/Na+ CT is often associated with hyperlipidemia, hyperuricemia, reduced insulin sensitivity, and obesity. The whole of these observations may explain why patients with diabetes or essential hypertension and increased Li/Na CT are at risk of early renal and cardiac impairment.