Calcium-activated sodium and chloride fluxes modulate platelet volume: role of Ca2+ stores.

An increase in cytosolic ionized Ca2+ concentration ([Ca2+]i) initiates volume changes in various types of cells. In response to increases in [Ca2+]i most cell types contract by efflux of K+ and Cl-, whereas platelets expand in response to rises in [Ca2+]i. This study examined the importance of the source of Ca2+, the flux of ions responsible for the volume change, and the role of Ca(2+)-dependent protein kinases in regulating these ionic fluxes. The baseline platelet volume was independent of extracellular Ca2+ but when stimulated by the Ca2+ ionophore A-23187 (50 nM) the volume increased in both the presence and absence of extracellular Ca2+ (1.18 +/- 0.08 vs. 0.83 +/- 0.06 fl, respectively). The increased volume was caused by the gain of Na+ and Cl-. Na+ entered through both conductive and nonconductive (Na+/H+ exchange) pathways, whereas the influx of Cl- was conductive and inhibited by the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. The Ca(2+)-induced volume change was blocked by both calmodulin and protein kinase inhibitors. Thus the activation of Ca(2+)-dependent protein kinases promotes platelet swelling by stimulating Na+ and Cl- influx.

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.

Lower Na(+)-H+ antiport activity in vascular smooth muscle cells of Wistar-Kyoto rats than spontaneously hypertensive and Wistar rats.

To determine whether increased Na(+)-H+ antiport activity in vascular smooth muscle cells may relate to the pathogenesis of hypertension in the spontaneously hypertensive rat (SHR), we monitored Na(+)-dependent alkalinization of acidified cells from the hypertensive strain and two normotensive controls, the Wistar-Kyoto rat (WKY) and the Wistar rat. Changes in intracellular pH (pHi) of cultured aortic cells were measured using the fluorescent probe 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF). The initial maximal reaction velocity of Na(+)-dependent alkalinization was significantly higher in SHR and Wistar than WKY cells. Similar results were obtained for the maximal velocity of the proton equivalent efflux: SHR, 7.51 +/- 0.71; Wistar, 9.14 +/- 0.85; WKY, 4.38 +/- 0.55 mmol H+/liter x 10 s. There were no differences in the basal pHi or cellular buffering power among the three rat strains. These findings indicate that the activity of the Na(+)-H+ antiport is higher in SHR vascular smooth muscle cells than in WKY cells. However, by itself, this difference cannot explain the hypertensive process in the SHR, since this transport system is also higher in vascular cells of the Wistar rat.

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)

Na(+)-H+ antiport activity in skin fibroblasts from blacks and whites.

The predisposition of black people to salt (NaCl)-sensitive essential hypertension may relate to racial differences in cellular Na+ metabolism. This tenet was investigated by examining the Na(+)-H+ antiport in serially passed skin fibroblasts from blacks and whites. Na(+)-dependent stimulation of the Na(+)-H+ antiport by cellular acidification resulted in a greater maximal velocity (Vmax) (mean +/- SEM) of this transport system in quiescent fibroblasts from blacks than fibroblasts from whites; the Vmax for recovery from cellular pH (pHi) of 6.6 was 5.84 +/- 0.50 versus 4.39 +/- 0.34 mmol H+/l X 20 seconds for blacks and whites, respectively (p less than 0.05). Although the Na+ concentration producing 50% stimulation of the Na(+)-H+ antiport for blacks (35.1 +/- 5.7 mM) was greater than for whites (24.1 +/- 3.5 mM), this difference was not statistically significant. No racial differences were observed in the Hill coefficient (n, 1.35 +/- 0.21 for blacks and 1.46 +/- 0.28 for whites). Compared with whites, cells from blacks exhibited a greater response to cytoplasmic acidification over the range of pHi values 6.20-6.60, as exhibited by an augmented rate of recovery in the pHi. These differences were not due to different basal pHi values or cellular buffering capacities, which were similar for blacks and whites. Na(+)-H+ antiport activity was not correlated with family history of hypertension. Increased activity of the Na(+)-H+ antiport in fibroblasts from blacks was confirmed without cellular acidification by stimulating quiescent cells with 10% human serum. This study demonstrates innate racial differences in cellular membrane Na(+)-H+ antiport activity.

Calcium-activated potassium channels in human platelets.

The cationic fluorescent probe, DiSC3(5) was used to measure the membrane potential in human platelets. Hyperpolarization was induced by the addition of Ca2+ to the medium and also by the addition of the Ca2+ ionophore, A23187. In the absence of extracellular Ca2+ ([Ca2+]o) there was no response to A23187. The threshold concentration for [Ca2+]o was 20 microM and for A23187 was 12 nM. The increase polarity induced by [Ca2+]o was not affected by various K+ channel blockers. However, the effect of A23187 was inhibited by quinine and charybdotoxin, while apamin, tetraethylammonium, and the calmodulin inhibitors trifluoperazine and compound R24571 were ineffective. The resting membrane potential was -66 +/- 0.9 mV and was decreased by quinine. There are three conclusions from this study: (i) Ca2+-activated K+ channels exist in human platelets; (ii) they are the type that are apamin insensitive, charybdotoxin sensitive; and (iii) they may contribute to the resting membrane potential.

Differences of Ca2+ regulation between fibroblasts from blacks and whites.

To explore the underlying causes predisposing blacks to essential hypertension, we have studied 45Ca2+ washout and cytosolic free Ca2+ (Cai2+) in quiescent skin fibroblasts from normotensive blacks and whites. Under basal conditions, there were no racial differences in 45Ca2+ washout and Cai2+. However, when stimulated by human serum, fibroblasts from blacks exhibited a higher 45Ca2+ washout associated with higher levels of Cai2+ transients than fibroblasts from whites. We conclude that fibroblasts from blacks demonstrate increased responsiveness to agonists in serum. If the same trend exists in vascular smooth muscle cells, it may predispose blacks to essential hypertension.

Cytosolic Ca2+ regulation attenuates ANP-induced cGMP response in vascular smooth muscle cells of the SHR.

Cultured vascular smooth muscle cells (VSMCs) of the spontaneously hypertensive rat (SHR) show a higher density (Bmax) of ANP receptors and a blunted cyclic (c) GMP response to the hormone. To explore the idea that a higher cytosolic free Ca2+ (Cai2+) may be responsible for a blunted cGMP response to ANP, we examined the effect of raising Cai2+ by the Ca2+-ionophore A23187 or K+ depolarization on the ANP-induced cGMP response. Treatment of VSMCs from the SHR, Wistar-Kyoto rats (WKY), or American Wistar (WIS) rats with A23187 or high K+ resulted in a uniform reduction of the ANP-cGMP response in 1.8 mmol/L Ca2+ medium but not Ca2+-free medium. We conclude that a higher level of Cai2+, probably at the domain adjacent to the membrane, may inhibit ANP-induced activation of the particulate guanylate cyclase, and that this may be the cause of the blunted cGMP response of SHR VSMCs to ANP.

Differences of Ca2+ regulation in skin fibroblasts from blacks and whites.

Black people have a higher propensity than caucasians toward essential hypertension. To explore the possibility that this racial difference relates to cellular Ca2+ metabolism, we measured 45Ca2+ washout and uptake and cytosolic free concentration of Ca2+ [Ca2+]i in serially passed skin fibroblasts from normotensive black and white males. Depending on the experimental conditions, 45Ca2+ washout in these cells was described by either two or three exponential functions, whereas 45Ca2+ uptake was described only by a two-exponent function. There were no racial differences in 45Ca2+ uptake and washout of unstimulated fibroblasts. However, stimulation by human serum resulted in an increase in the 45Ca2+ washout that was higher in fibroblasts from blacks than from whites. The racial differences were expressed primarily by higher values of the apparent washout rate constant (k1) of 45Ca2+ from the largest and most rapidly exchangeable cellular pool. The effect of human serum was not related to its origin (blacks vs. whites). In 2 mM Ca2+ medium and 10% serum from blacks, the respective k1 (mean +/- SEM; x 10(-2)/min) values for fibroblasts from blacks and whites were 89.68 +/- 5.23 and 73.29 +/- 4.0; in the presence of 10% serum from whites, the k1 values for cells from blacks and whites were 84.14 +/- 2.80 and 76.36 +/- 3.23 (overall significance of P less than .01). In Ca2+-deficient medium in the presence of 10% human serum, the k1 for fibroblasts from blacks and whites were 115.57 +/- 3.76 and 102.15 +/- 3.30 (P less than .05). Serum substantially increased the 45Ca2+ uptake in fibroblasts from both blacks and whites; however, racial differences were not observed. Basal levels of [Ca2+]i were not different in fibroblasts of blacks vs. whites (46.8 +/- 6.8 and 43.2 +/- 7.1 nM for blacks and whites, respectively). However, the peak response of Cai2+ transients for cell stimulated by 5% human serum was significantly higher in blacks than whites (blacks = 963 +/- 213, whites = 481 +/- 162 nM; P = .0286). We conclude that Ca2+ regulation is different in serum-stimulated fibroblasts from blacks and whites and that, at least in part, this difference may relate to a greater agonist-induced mobilization of Ca2+ in fibroblasts from blacks.

Cytosolic Ca2+ attenuates ANF-induced cyclic GMP response in vascular smooth muscle cells.

Despite a high density of atrial natriuretic factor (ANF) receptors, cultured vascular smooth muscle cells of the spontaneously hypertensive rat (SHR) manifest a blunted cyclic GMP (cGMP) response to ANF. We explored the role of cytosolic free Ca2+ ([Ca2+]i) in the ANF-induced cGMP response of cultured aortic vascular smooth muscle cells from SHR and two normotensive rat strains: Wistar-Kyoto (WKY) and American Wistar. Exposure to 500 nmol/l A23187 in Ca2+-containing but not in Ca2+-deficient medium resulted in a decline in the ANF-induced cGMP response at maximal ANF concentration (500 nmol/l; SHR from 1004 +/- 98 to 423 +/- 67, P less than 0.001; WKY from 1791 +/- 209 to 625 +/- 90, P less than 0.001; American Wistar from 1496 +/- 125 to 559 +/- 96 fmol/10(6) cells/4 min, P less than 0.001). The same phenomenon was observed by depolarization with 50 mmol/l KCl in Ca2+-containing medium. There were no significant differences among the rat strains in basal levels of [Ca2+]i. If Ca2+ plays a role in the blunted cGMP response to ANF in vascular smooth muscle cells of the SHR, this effect may be exerted by a distinct pool of the ion in the submembrane domain which is associated with the particulate guanylate cyclase system.

Restriction of tumor growth in mice by sodium-deficient diet.

Generalized malnutrition results in inhibition of tumorigenesis and tumor growth in experimental animal models. Neither the specific nutrient deficiency nor the mechanism has been definitely elucidated. We have shown previously that dietary sodium deprivation in rapidly growing rats retards protoplasmic growth. This effect was correlated to the extracellular fluid (ECF) volume expansion which is dependent on sodium accumulation. Since solid tumors are composed of a large quantity of ECF (which includes plasma volume) it was postulated that preventing the accumulation of new ECF by means of sodium restriction would influence tumor growth. The present study was designed to determine the effects of salt restriction on tumor growth and to relate these effects to ECF volume. Approximately 10(6) viable B16 melanoma cells were injected into C57BL/6 x DBA/2 F1 and C57 mice. A salt restricted diet (sodium less than 3 microeq/g) was provided ad libitum. The drinking solution was distilled water for the experimental group and 0.45% saline solution for the controls. There was a significant decrease in tumor growth rates during sodium restriction. The total body ECF volume increased when dietary sodium was supplied but did not change during salt restriction. Therefore, the only source for the ECF in the tumor mass was from nontumorous tissue. We conclude that during dietary sodium restriction solid tumor growth is retarded and can proceed only to the extent that ECF is released from cachectic body tissues.

The reliability of calculated bicarbonate in clinical practice.

Current clinical laboratory methods utilize the Henderson-Hasselbalch equation to calculate plasma bicarbonate from measured pH and pCO2. This practice assumes that the apparent first dissociation constant for carbon dioxide in serum, pK'1, is invariable in clinical situations. This assumption has been questioned recently. Our study examined arterial blood samples from 50 acutely ill patients who were routinely sent for blood gas analysis. The pH and pCO2 were measured on a blood gas analyzer and the total CO2 was determined on the same blood sample using a microgasometer. The calculated pK'1 from these parameters was 6.10 +/- 0.018 (m +/- SD) with a range of 6.06 to 6.15. This amount of variability could be explained by the error in the methods. The correlation for calculated to measured total CO2 was y = 094x + 1.81, r = 0.98, p less than 0.001. These findings indicate that pK'1 is functionally fixed in clinical practice and that the methods used to calculate serum bicarbonate are acceptable.

Blood pressure elevation in young dogs during low-level lead poisoning.

Clinical and experimental studies suggest an association between low-level lead exposure and hypertension. This association was investigated in six 3-month-old dogs who were randomly paired with their littermates. The daily oral dose of lead acetate was 1.0 mg Pb/kg body wt for 5 months; the controls received equimolar sodium acetate. Blood pressure was measured indirectly without anesthesia and was similar in the two groups at the start of the study. The mean blood pressure was higher in the lead-exposed group at every follow-up, from 10 days to 20 weeks. This treatment group difference in profiles was statistically significant (repeated-measures ANOVA, p = 0.0048). The final mean blood pressures were 120 +/- 6.4 (x +/- SE) vs 108 +/- 1.5 mm Hg. At 4 weeks the plasma renin activity was higher in the lead-exposed group: 3.4 +/- 0.25 vs 1.2 +/- 0.15 ng/ml/hr. The difference decreased during the study but the elevated trend persisted (repeated-measures ANOVA, p = 0.014). Lead exposure did not alter renal functions or extracellular fluid volume. This study shows that low-level lead intake in young dogs can cause an early increase in blood pressure which persists during ongoing exposure and which is associated with a small increase in the activity of the renin-angiotensin system.

Angiotensin II effect on 22Na+ transport in vascular smooth muscle cells.

It is well established that angiotensin II (AII) rapidly increases free cytosolic Ca2+ in vascular smooth muscle cells (VSMCs). Several studies have indicated that the hormone also plays a role in Na+-K+ regulation of these cells. In this study, we explored the mechanism of AII effect on 22Na+ transport in cultured rat VSMCs. The 22Na+ washout from these cells was described by three exponents with exponential factors k1 greater than k2 greater than k3. In 1.8 mM Ca2+ medium, AII (10(-9)-10(-6) M) increased (in a dose response manner) the k1 value, and consequently the initial washout rate constant (kei) for the isotope. AII had no effect on kei in Ca2+-deficient medium or in the presence of ouabain. Amiloride (10(-3) M) and verapamil (10(-5) M) abolished the AII induced increase in kei. These findings are consistent with angiotensin II stimulation of an amiloride-sensitive Na+ transport, which is likely to represent the Na+/H+ antiport. In cultured VSMCs, the sustained stimulation by AII of this transport system requires the presence of extracellular Ca2+ and its influx into these cells.

Sodium deprivation growth failure in the rat: alterations in tissue composition and fluid spaces.

Dietary control of sodium intake was utilized in weanling rats to study the relationships among body growth, tissue composition and extracellular fluid volume (ECFV). Forty 3-wk-old rats were divided into groups receiving 30, 150, 300, 600 or 900 mu eq sodium/d for 5 wk. The minimal daily requirement for normal growth was 300 mu eq Na, or about 60 mu eq/g of new growth. Lower doses caused dose-related growth failure associated with a reduced ECFV. Analyses of carcass, muscle and bone composition were carried out. In sodium-deprived animals there was retarded growth of protoplasm, fat and bone; the mineral composition of muscle was not altered, whereas in bone calcium concentration was reduced. Plasma concentrations of sodium, potassium and chloride remained normal. Pair-feeding indicated that sodium-deficiency growth retardation could not be attributed to starvation. Sodium-deficient animals ingested a greater amount of food per gram of weight gain, possibly reflecting an increased energy expenditure. Sodium deprivation initially permitted protoplasmic growth to proceed at a rate disproportionate to that of the ECFV. Subsequently, both continued to grow at a reduced but similar rate, suggesting that ECFV may be a controller of protoplasmic growth.

Diuretic-induced growth failure in rats and its reversal by sodium repletion.

The aim of diuretic therapy is the prevention of excessive sodium accumulation. However, sodium retention is necessary for growth. Inasmuch as many of the clinical conditions for which diuretics are used are associated with growth retardation, we investigated the influence of diuretic therapy on growth in an animal model. In Part I, 32 weanling Sprague-Dawley rats were fed a diet adequate for growth which contained 0.08% sodium and 0.17% potassium. Daily i.p. injections of saline (0.4 ml) containing furosemide in doses of 0, 50, 100 or 200 mg/M2 were given for 9 days. There was a dose-related reduction in weight gain which could not be explained by lower food intake. The highest dose group gained only 58% as much as the control group. Balance studies and muscle, bone and carcass analysis demonstrated that this was accounted for by decreases in protoplasmic, bone, fat and extracellular fluid volume accretion. In Part II, 32 weanling rats, all treated daily with furosemide (100 mg/M2 i.p.) received replacement of NaCl, KCl, both or neither in their drinking water. Sodium replacement resulted in increased growth rates whereas potassium replacement alone had no effect on growth. Sodium replacement also increased the balance of all measured minerals. We conclude that diuretic therapy causes growth retardation by preventing retention of sodium needed for growth.

Angiotensin II effect on cytosolic pH in cultured rat vascular smooth muscle cells.

This study investigated fluctuations of cytosolic pH (pHi) of cultured rat vascular smooth muscle cells (VSMCs) in reaction to metabolic alterations induced by angiotensin II (AII). Serially passed VSMCs from Wistar rat aortae were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. A biphasic reaction was seen after exposure of these cells to AII (1 nM to 1 microM); an initial and relatively brief phase of acidification was followed by sustained alkalinization. The rate of acidification and magnitude of alkalinization were dose-dependent. This biphasic effect of AII was also demonstrated in Ca2+-free medium and was mimicked by subjecting VSMCs to the calcium ionophore A23187 (5 microM) in Ca2+-containing medium but not in Ca2+-free medium. Verapamil (10 microM) almost entirely eliminated the AII-induced acidification, whereas amiloride analogues 5-(N-methyl-N-isobutyl)amiloride and 5-(N-ethyl-N-isopropyl)amiloride (100 microM) as well as Na+-deficient medium abolished the subsequent (alkalinization) phase produced by the hormone. Activation of the Na+/H+ antiport by subjecting VSMCs to phorbol 12-myristate 13-acetate (100 nM) prevented a subsequent effect of AII on the pHi profile. This resistance to a further action of the hormone was not mediated via cytoplasmic alkalinization. AII produced a dramatic redistribution in the cellular compartments of 45Ca2+ associated with accelerated 45Ca2+ washout. These findings suggest that the AII-induced acidification phase may relate to activation of the Ca2+ pump (Ca2+/H+ exchange) and that this process can take place in the presence and absence of extracellular Ca2+. The alkalinization phase is the consequence of stimulation of the Na+/H+ antiport, which in cultured VSMCs can be activated by a rise in cytosolic free Ca2+ as well as other mechanisms.

Results of urinary undiversion in children.

Urinary undiversion was performed in 31 patients. Preoperative investigation and criteria for undiversion are discussed. Renal preservation rate is stable in 28 of 31 patients and bladder function satisfactory in 30 of 31 patients. Although prolongation of life and prevention of renal deterioration are of primary concern, psychologic aspects of undiversion must also be considered. Some children who underwent urinary diversion have poor renal reserve and eventually outgrow their kidneys, thus requiring renal transplantation. Their urinary tracts must be prepared for that eventuality.