Cyclic nucleotides attenuate thrombin-evoked alterations in parameters of platelet Na/H antiport. The role of cytosolic Ca.

In this work, we explored the role of cyclic nucleotides in modulating parameters of the Na/H antiport in human platelets. Sodium nitroprusside and iloprost, as well as cyclic nucleotide analogues, were used to raise cellular levels of cAMP and cGMP. Cyclic nucleotides reversed the thrombin-evoked alkaline shift in cytosolic pH set point and the activity of the Na/H antiport, concurrently with attenuation of thrombin-induced rise in cytosolic free Ca. No effect of cyclic nucleotides was observed in platelets not treated with thrombin, or platelets subjected to phorbol 12-myristate 13-acetate. cAMP did not reverse ionomycin-induced changes in the parameters of the Na/H antiport. Collectively, these observations indicate that cyclic nucleotides modulate the Na/H antiporter in human platelets through their effect on thrombin-evoked changes in cytosolic free Ca. Presumably, this effect holds for other agonists which stimulate phospholipase C, raise cytosolic-free Ca, and activate the Na/H antiport through protein kinase C dependent and protein kinase C-independent mechanisms.

Race and sex differences in erythrocyte Na+, K+, and Na+-K+-adenosine triphosphatase.

Several reports indicate that erythrocytes (RBCs) from blacks and men have higher sodium concentrations than those from whites and women. One possible mechanism to explain this finding is a difference in the activity of Na+-K+-ATPase. To explore this possibility, we have studied the Na+ and K+ kinetics of RBC Na+-K+-ATPase and RBC Na+ and K+ concentrations in 37 normotensive blacks and whites, both males and females. The maximal initial reaction velocity (Vmax) values for RBC Na+-K+-ATPase were lower in blacks and men as compared with whites and women. Higher RBC Na+ levels were observed in blacks and males vs. whites and females. Significant inverse correlations were noted between the Na+-K+-ATPase activity and RBC Na+ concentrations. These findings indicate that cellular Na+ homeostasis is different in blacks and men as compared with whites and women. Since higher RBC Na+ concentrations have also been observed in patients with essential hypertension as compared with normotensive subjects, the higher intracellular Na+ concentrations in blacks and men may contribute to the greater predisposition of these groups to essential hypertension.

A higher cellular sodium turnover rate in cultured skin fibroblasts from blacks.

Differences in cellular Na+ and K+ regulation may relate to the pathogenesis of essential hypertension and the predisposition of blacks to this disease. To explore these tenets, we examined several aspects of cellular Na+ homeostasis in serially passed, cultured skin fibroblasts from 30 subjects (15 hypertensive blacks and whites and normotensive subjects matched for sex, age, and race.) Fibroblasts from blacks demonstrated higher cellular Na+ turnover rates than did those from whites. This difference was expressed by accelerated Na+-K+ pump activity (ouabain-sensitive Na+ washout rate, 3.46 +/- 0.216 for blacks vs 1.84 +/- 0.283 mEq/L/min for whites; p = 0.0006) and a higher rate of cellular accumulation of Na+ in the presence of ouabain (0.964 +/- 0.0743 vs 0.562 +/- 0.0440 mEq/L/min for blacks and whites, respectively; p = 0.0045). Associated with these findings, fibroblasts from blacks had higher cellular Na+ concentration than did those from whites (9.78 +/- 0.512 vs 7.50 +/- 0.400 mEq/L; p = 0.0170, as measured by atomic absorption, and 7.84 +/- 0.470 vs 5.03 +/- 0.980 mEq/L; p = 0.0141, as derived from the equilibrium distribution ratio of 22Na+). It is concluded that blacks differ from whites with respect to cellular Na+ turnover rate, which is evidenced by an increased Na+ influx and accelerated Na+-K+ pump activity in their fibroblasts. Our findings support the tenet that innate racial differences in cellular Na+ regulation may underlie the predisposition of blacks to hypertension.

Erythrocyte ghost Na+,K+-ATPase and blood pressure.

To examine the relationship between body mass index, blood pressure, and the Na+,K+-adenosine triphosphatase (ATPase) system, we measured the erythrocyte ghost Na+,K+-ATPase and the erythrocyte Na+ concentration in 120 blacks and 127 whites (136 males and 111 females). Blacks showed a 13.9% higher erythrocyte Na+ (7.63 +/- 0.19 vs 6.70 +/- 0.11 [SEM] mEq/L; p = 0.0001) and a 16.1% lower erythrocyte ghost Na+,K+-ATPase activity (140.3 +/- 4.2 vs 167.3 +/- 4.7 nmol inorganic phosphate/mg protein/hr; p = 0.0002) than whites. Male subjects demonstrated a 6.4% higher erythrocyte Na+ (7.35 +/- 0.17 vs 6.91 +/- 0.14 mEq/L; p = 0.043) and an 11.5% lower Na+,K+-ATPase activity (145.7 +/- 3.7 vs 164.7 +/- 5.5 nmol inorganic phosphate/mg protein/hr; p = 0.0015) than female subjects. Significant (p less than 0.001) negative correlations were identified for the systolic, diastolic, and mean blood pressure levels and the erythrocyte ghost Na+,K+-ATPase. These findings were complemented by positive correlations for the blood pressure levels and erythrocyte Na+ concentrations. The body mass index was negatively correlated with erythrocyte ghost Na+,K+-ATPase and it accounted for 6.7%, 5.6%, and 6.1% of the variabilities in the systolic, diastolic, and mean blood pressure levels, respectively. Variabilities of 1.4% systolic, 12.3% diastolic, and 11.1% in mean arterial pressure were attributable to the erythrocyte ghost Na+,K+-ATPase activity. Provided that findings in erythrocytes also reflect the relative status of the vascular smooth muscle cell Na+,K+-ATPase, the predisposition of black, male, and obese persons to hypertension may relate, among other factors, to a lower activity of this enzyme system, which results in an increased vascular tone.

Erythrocyte sodium/potassium adenosine triphosphatase in thyroid disease and nonthyroidal illness.

Thyroid hormone is known to modulate cell membrane sodium/potassium adenosine triphosphatase (Na/K-ATPase). To determine whether the activity of this enzyme differed in patients with nonthyroidal illness with low levels of circulating thyroid hormones and patients with documented clinical hypothyroidism, we measured Na/K-ATPase activity in red blood cells from patients with hypo- and hyperthyroidism, patients with nonthyroid disease with and without reduced circulating levels of thyroid hormone, and normal subjects. We also assessed whether the activity of this enzyme reflects decreased thyroid hormone action at the cellular level in patients with nonthyroidal illness. Hyperthyroidism was associated with decreased and hypothyroidism with increased erythrocyte Na/K-ATPase activity [142 +/- 24 (+/- SE) and 371 +/- 37 nmol Pi/mg X h; P less than 0.05 and P less than 0.01 compared to normal]. Enzyme activity in cells from patients with nonthyroidal illness and low levels of circulating T3 was significantly higher than that in cells from normal subjects (289 +/- 11 vs. 223 +/- 16 nmol Pi/mg X h; P less than 0.01), but was not significantly different from that in cells from hypothyroid patients. Red cell Na/K-ATPase activity in patients with nonthyroidal illness and normal thyroid function tests (185 +/- 38 nmol Pi/mg X h was indistinguishable from normal values. These data confirm that hyperthyroid patients have decreased red cell Na/K-ATPase activity and provide direct evidence that erythrocyte ATPase activity is increased in hypothyroid patients. The change in enzyme activity in patients with nonthyroidal illness and decreased circulating T3 levels was comparable to that in hypothyroidism. These results suggest that since red cell Na/K-ATPase activity does not distinguish between ill patients with low thyroid function tests and those with hypothyroidism, tissue hypothyroidism may exist in the former group of patients.

[3H]ouabain binding of red blood cells in whites and blacks.

In a previous study, we demonstrated that the red blood cell Na+ concentration and Na+,K+-ATPase activity are sex-dependent and race-dependent: a higher intracellular Na+ concentration in blacks and men was associated with a lower Na+,K+-ATPase activity. To examine whether the low Na+,K+-ATPase activity is due to a decreased number of enzyme units, altered structure of the enzyme, or the presence of an endogenous digoxinlike substance, ouabain binding studies were performed on the same subject group. The measurements included displacement of [3H]ouabain from its specific binding sites by unlabeled ouabain or potassium. The results demonstrate that groups with lower enzyme activity manifest lower numbers of total specific ouabain binding sites on the surface of the red blood cell (mean +/- SD: blacks, 654 +/- 24.4; whites, 806 +/- 18.3; women, 806 +/- 26.9; men, 728 +/- 21.2). Other kinetic parameters of [3H]ouabain displacement appear to be the same among the groups. The respective red blood cell Na+ and K+ concentrations were negatively and positively correlated with the number of ouabain binding sites. Our findings suggest that the lower activity of red blood cell Na+,K+-ATPase in blacks and men is a function of a lower number of Na+-K+ pump units. The results also indicate that sex and race should be considered when red blood cell ouabain binding is examined.

Blood pressure inversely correlates with thrombin-evoked calcium rise in platelets.

Earlier investigations showed a positive correlation between basal cytosolic free calcium in human platelets and blood pressure; however, recent studies have failed to show this relation. We undertook the present work to examine which platelet cytosolic calcium parameters (namely, cytosolic calcium in resting or stimulated states in calcium-containing and calcium-free media) present the least variability and best correlation with blood pressure. We studied 17 healthy white men on three different occasions separated by 1- and 4-week intervals. Their manual and ambulatory automated 24-hour blood pressure measurements were correlated with cytosolic calcium in resting and stimulated (thrombin-treated) fura 2-loaded platelets. The following cytosolic calcium parameters were measured in 1 mmol/L calcium and calcium-free media: basal cytosolic calcium, peak thrombin-evoked cytosolic calcium, and post-transient cytosolic calcium 5 minutes after thrombin treatment. The highest and lowest coefficients of variation were respectively shown by the basal cytosolic calcium (22.8%) and peak thrombin-evoked cytosolic calcium (10.1%) in calcium medium. Basal cytosolic calcium did not correlate with any of the blood pressure parameters. Of the cytosolic calcium parameters, peak thrombin-evoked cytosolic calcium in calcium medium demonstrated consistent (negative) correlations with blood pressure, with better correlations shown with diastolic than systolic blood pressure of both automated and manual blood pressure readings. Peak thrombin-evoked cytosolic calcium in calcium medium showed similar correlations with nighttime and daytime automated blood pressure measurements. There were no correlations between peak thrombin-evoked cytosolic calcium in calcium-free medium and blood pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

Variations in the apparent pH set point for activation of platelet Na-H antiport.

To explore the role of the Na-H antiport in essential hypertension, we studied the kinetics of cytosolic pH and external sodium activation of this transport system in platelets from 65 normotensive and essential hypertensive subjects on and off antihypertensive medications. Subjects included both blacks and whites, as well as men and women. The fluorescent dye 2'7-bis(carboxyethyl)-5,6-carboxyfluorescein was used to monitor the cytosolic pH in these cells. Platelets from black (hypertensive and normotensive) men and hypertensive white men demonstrated a highly significant alkaline shift in the apparent cytosolic pH set point for activation of the Na-H antiport. For the hypertensive subgroups, the cytosolic pH set point values (mean +/- SEM) were: white men, 7.45 +/- 0.052; white women, 7.04 +/- 0.089; black men, 7.66 +/- 0.148; and black women, 7.20 +/- 0.082. For the normotensive subgroups, the cytosolic pH set point values were: white men, 7.13 +/- 0.034; white women, 7.05 +/- 0.036; black men, 7.50 +/- 0.110; and black women, 7.20 +/- 0.176 (p = 0.0016 for race and p = 0.0001 for gender, using a three-way analysis of variance by race, gender, and hypertension). There were no race-, gender-, or blood pressure-related differences among the various cohorts in the kinetics of sodium activation of the Na-H antiport, the cellular buffering power, and basal pH. These results suggest that at basal pH the Na-H antiport is quiescent in platelets from both black and white women and normotensive white men.(ABSTRACT TRUNCATED AT 250 WORDS)

Platelet sodium-hydrogen antiport in obese and diabetic black women.

In this investigation we correlated platelet Na-H antiport parameters with blood pressure and serum lipids in a sample population of non-insulin-dependent diabetic obese, nondiabetic obese, and nondiabetic nonobese black women. Parameters of the Na-H antiport were examined in aspirin-treated platelets. These parameters were not altered in resting or in thrombin-stimulated platelets of diabetic patients. The activity index of platelet Na-H antiport after thrombin stimulation was positively correlated with the blood pressure (systolic blood pressure, r = 0.5320 and p = 0.0001; diastolic blood pressure, r = 0.5123 and p = 0.0017). Lower high density lipoprotein cholesterol levels were associated with an alkaline shift in the cytosolic pH set point for activation of the Na-H antiport. Highly significant correlations were also observed between the total cholesterol/high density lipoprotein cholesterol ratio and the cytosolic pH set point for activation of the Na-H antiport. These correlations were independent of diabetes or the body mass index. Together, these observations indicate that parameters of platelet Na-H antiport are altered with an increase in blood pressure and a decrease in serum high density lipoprotein cholesterol.

Malignant lymphoma with "myeloma kidney" acute renal failure.

The first cases of malignant lymphoma with a "myeloma kidney" type of acute renal failure are presented. With appropriate therapy, both patients regained partial renal function; the first after three months of dialysis. It is suggested that the term paraproteinemic nephropathy is preferable to "myeloma kidney."

Differences in platelet calcium regulation between African Americans and Caucasians: implications for the predisposition of African Americans to essential hypertension.

OBJECTIVE: To study differences in cytosolic free calcium regulation between African Americans and Caucasians, using platelets as a model for studying cellular physiology. DESIGN: Platelet calcium regulation in apparently healthy African American and Caucasian males was examined. METHODS: Using fura-2, calcium influx and cytosolic calcium extrusion were monitored after treatment with thapsigargin, an inhibitor of the Ca-ATPase in the dense tubular membrane system. RESULTS: Compared with Caucasians, platelets from African Americans demonstrated a higher calcium influx into the cytosol (from the external medium and dense tubular membrane system), as well as enhanced calcium extrusion from the cytosol. CONCLUSIONS: Such findings indicate that platelets from African Americans have increased intracellular calcium stores or increased cellular calcium turnover, or both. If these racial differences involve other cells, such as vascular smooth muscle cells, they could underlie the predisposition of African Americans to essential hypertension.

Effects of essential hypertension and antihypertensive medications on sweat formation.

OBJECTIVE: Sweat volume and ionic composition depend to a large extent upon the cytosolic free calcium level in secretory sweat cells and sodium and potassium transport in the reabsorptive sweat duct. Since essential hypertension and its treatment with antihypertensive drugs is likely to be associated with altered cellular ionic regulation, the objective of this research was to explore sweat formation and sweat parameters in hypertensive and normotensive subjects. DESIGN: Black and white hypertensive and normotensive subjects of both genders were studied. Essential hypertensives were on or off antihypertensive medication. METHODS: Pilocarpine iontophoresis was used to induce sweat in a 5-cm2 area of the middle forearm. Sweat was analyzed for volume, sodium and potassium concentrations. RESULTS: Females demonstrated lower sweat volumes after pilocarpine stimulation than males. Untreated hypertensive white males exhibited a higher pilocarpine-induced sweat volume and sweat sodium excretion than normotensive white males, whilst hypertensive white males on antihypertensive medication showed a lower sweat volume and sweat sodium excretion than both normotensive white males and untreated essential hypertensive white males. Although untreated hypertensive white females did not show significant alterations in sweat parameters, treated hypertensive white females exhibited lower sweat volume and sweat sodium excretion than both the normotensive and untreated essential hypertensive white females. These hypertension and drug related alterations were not present in hypertensive black males and females. CONCLUSIONS: The results are consistent with the heterogeneous nature of essential hypertension and the diversity of the response to antihypertensive therapy. They suggest that the effect of antihypertensive medication on sweat formation is mediated through cytosolic free calcium.

Importance of aliphatic amines in uremia.

Five main aspects were addressed: 1)The demonstration that creatinine is an endogenous precursor of dimethylamine (DMA) in chronic renal failure. 2) The size of the body amine pool measured in transplant patients suggests sequestration in some intracellular compartment. This illustrates the possible error in directly relating serum concentrations to neurological toxicity. 3) Bacterial overgrowth and increased generation of duodenal DMA in the small intestine becomes apparent at a serum creatinine above 8 mg/dl. Two cases show that bacterial overgrowth preceded the increased duodenal DMA. 4)Clinical toxicity is demonstrated by i) correlation of abnormal neurobehavioral parameters with serum amine levels, and ii) by improvement with administration of nonabsorbable broad spectrum antibiotics. Results with adsorption agents are inconclusive. 5) Preliminary tests of behavior modification in a rat model by direct instillation of amines into the brain are positive for TMA but negative for DMA, but no DMA entry into brain cells is demonstrated in the latter. The generation of aliphatic amines represents only one part of a spectrum of alteration induced by proximal intestinal bacterial enzyme action that occurs in renal failure. It is possible that some bacterial activity is beneficial and that the net clinical result is a balance between the "good" and the "evil" bacterial effects.

Biotin supplementation improves glucose and insulin tolerances in genetically diabetic KK mice.

Because biotin treatment may lower blood glucose in insulin-dependent diabetes, we chose to study such an effect in non-insulin dependent diabetes. Twenty-six diabetic KK mice, moderately hyperglycemic and insulin resistant, were treated for 10 weeks: 9 animals with 2 mg of biotin/Kg, 8 with 4 mg of biotin/Kg, and 9 with saline (controls). Blood glucose levels, oral glucose tolerance, insulin response to oral glucose, and blood glucose decrease in response to insulin were quantitated. Compared to controls, biotin treatment lowered post-prandial glucose levels, and improved tolerance to glucose and insulin resistance. Serum immunoreactive insulin levels in biotin-treated mice were like the controls.

Cytosolic Ca profile of resting and thrombin-stimulated platelets from black women with NIDDM.

In this study, human platelets were used as a cellular model for exploring cytosolic free Ca (Cai) regulation in non-insulin-dependent diabetes mellitus (NIDDM). Cai levels were monitored in resting and thrombin-stimulated platelets from obese females with NIDDM; obese, nondiabetic women, and nonobese, nondiabetic women. All subjects were black. Significant and marked elevation of basal Cai levels was observed in platelets from the diabetic subjects when no aspirin was used during platelet isolation. However, no significant differences were observed in Cai between aspirin-treated platelets from women with NIDDM and platelets from nondiabetic women. The rate of the Cai return to basal level after thrombin stimulation was significantly lower in platelets from the diabetic subjects, suggesting an abnormality in platelet Ca extrusion or sequestration in NIDDM. Platelet Cai levels positively correlated with low-density lipoprotein cholesterol/high-density lipoprotein cholesterol ratio (LDL/HDL) and fasting blood glucose. These findings suggest abnormalities in platelet Cai homeostasis in NIDDM that are influenced by the serum lipid profile and perhaps glucose.

Platelet associated IgG in uremia.

Platelet-associated IgG (PAIgG) levels, a useful adjunct for the evaluation of patients with immunologic mediated thrombocytopenia, were obtained in 36 patients with end stage renal disease undergoing maintenance hemodialysis. Twenty-five of the 36 had elevated PAIgG. Nine of the 36 were thrombocytopenic and only 3 of the 9 had elevated PAIgG. Fifteen patients in the group admitted to recent substance abuse. All but one had elevated PAIgG and only one patient was thrombocytopenic. In contrast to patients with idiopathic immune thrombocytopenic purpura (ITP), a direct relationship was not found between PAIgG and total platelet protein (TPP) in the uremic group. It can be concluded that PAIgG values in patients with uremia are difficult to interpret since elevated PAIgG values are found in the majority of uremic patients with normal platelets counts.

Use of hemodialysis and forced diuresis in the treatment of poisoning.

This paper is a brief commentary of the role of forced diuresis and hemodialysis in the treatment of acute poisoning. The importance of informed conservative management is stressed. Rationale and methods of forced diuresis and urinary alkalinization are presented. Dialysis is reserved for special situations where shortening the poison-patient contact time is indicated.

Clinical decision making. Conference on therapeutic and diagnostic options.

Clinical decision-making conference will be a new quarterly feature. Suggestions or contributions should be addressed to the authors. The authors discuss a 67-year-old male with coronary heart disease and renal failure.

Defects in membrane transport of ions as possible pathogenic factors in hypertension.

Although abnormalities in cellular ion transport have been shown in a variety of cells of essential hypertensives, the mechanistic link between these abnormalities and elevated blood pressure is poorly understood. Reduced sodium-potassium ATPase activity, with and without elevated levels of a circulating inhibitor of this transport system, has been reported by a number of studies. The recent characterization of the endogenous ouabain or its isomer will facilitate the testing of the hypothesis that salt-sensitive essential hypertension relates to higher levels of this factor. The erythrocyte sodium-lithium countertransport may serve as a marker for a subpopulation of essential hypertensives. However, this transport system has no physiologic counterpart and thus does not provide insight into mechanisms associated with altered cellular ionic homeostasis in essential hypertension. Increased activity of the sodium-hydrogen antiport in essential hypertension relates to an alkaline shift in the cytosolic pH set-point for activation of this transport system. This process may reflect increased cytosolic free calcium concentration with or without augmented protein kinase C activity.

Thapsigargin-evoked changes in human platelet Ca2+, Na+, pH and membrane potential.

1. In this work we explored the effect of thapsigarin on the intracellular Ca2+, pH, Na+ and membrane potential in human platelets. These parameters were monitored using the fluorescent probes fura-2, 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein, sodium-binding benzofuran isophthalate, and 3,3'-dipropylthiadicarbocyanine iodide. 2. Thapsigargin caused an increase in the cytosolic Ca2+, coupled with cytosolic alkalinization. Thapsigargin-induced alkalinization was Na(+)-dependent, indicating that thapsigargin stimulated the Na(+)-H+ exchange. 3. Using Mn2+ as a Ca2+ surrogate, we showed that thapsigargin activated Ca2+ channels at relatively low levels of cytosolic Ca2+, suggesting that a rise in cytosolic free Ca2+ is not the signal for the activation of these channels. 4. Thapsigargin-induced increase in the cytosolic free Ca2+ was greater in Na(+)-containing medium than in Na(+)-free medium, suggesting that Na(+)-dependent mechanisms participate in the regulation of platelet cytosolic Ca2+. 5. Thapsigargin not only increased the cytosolic Ca2+, but also elevated the cytosolic free Na+. The latter effect was more pronounced in Ca(2+)-free medium, a finding that may indicate that some of the Na+ enters through Ca2+ entry pathways. 6. Finally, thapsigargin evoked sustained platelet hyperpolarization which was attenuated by charybdotoxin, indicating thapsigargin-induced stimulation of Ca(2+)-sensitive K+ channels. 7. Together these observations demonstrate a multifactorial effect of thapsigargin on platelets that can be utilized to further understand platelet ionic homeostasis.