Radioimmunoassay of atrial natriuretic peptide in human plasma: application to studies of volume and blood pressure homeostasis

Sensitive radioimmunoassay for determination of immunoreactive atrial natriuretic peptide (ANP) in human plasma was developed and employed for the study of plasma ANP concentrations in healthy controls under basal conditions (2.4 +/- 0.1 pmol/l) and during volume expansion by saline infusion (9.6 +/- 2.0 pmol/l and 14.2 +/- 1.8 pmol/l, respectively). Plasma renin activity and plasma aldosterone concentration exhibited opposite changes during saline infusion. In pathological states associated with extracellular fluid volume (ECFV) expansion, ANP concentration were significantly higher than in the controls (liver cirrhosis 8.6 +/- 0.9; congestive heart failure 33.1 +/- 4.8; chronic renal failure before haemodialysis 72.2 +/- 6.4 pmol/l). Further volume expansion in liver cirrhosis by saline infusion led to the further increase in ANP (13.3 +/- 1.3 and 16.1 +/- 1.5 pmol/l, respectively) and ECFV reduction by ultrafiltration during haemodialysis in chronic renal failure diminished but did not normalize plasma ANP (22.5 +/- 2.9 pmol/l). In patients with arterial hypertension the concentration of ANP exceeded the normal range by 62.5% and reached 8.0 +/- 0.5 pmol/l on the average. Our results support the suggestion that ANP is an important regulatory humoral mechanism participating in the regulation of sodium, volume and blood pressure homeostasis.     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Insulin and glucagon stimulation of (Na+-K+)-ATPase transport activity in isolated rat hepatocytes

The effects of insulin and glucagon on the (Na+-K+)-ATPase transport activity in freshly isolated rat hepatocytes were investigated by measuring the ouabain-sensitive, active uptake of 86Rb+. The active uptake of 86Rb+ was increased by 18% (p less than 0.05) in the presence of 100 nM insulin, and by 28% (p less than 0.005) in the presence of nM glucagon. These effects were detected as early as 2 min after hepatocyte exposure to either hormone. Half-maximal stimulation was observed with about 0.5 nm insulin and 0.3 nM glucagon. The stimulation of 86Rb+ uptake by insulin occurred in direct proportion to the steady state occupancy of a high affinity receptor by the hormone (the predominant insulin-binding species in hepatocytes at 37 degrees C. For glucagon, half-maximal response was obtained with about 5% of the total receptors occupied by the hormone. Amiloride (a specific inhibitor of Na+ influx) abolished the insulin stimulation of 86Rb+ uptake while inhibiting that of glucagon only partially. Accordingly, insulin was found to rapidly enhance the initial rate of 22Na+ uptake, whereas glucagon had no detectable effect on 22Na+ influx. These results indicate that monovalent cation transport is influenced by insulin and glucagon in isolated rat hepatocytes. In contrast to glucagon, which appears to enhance 86Rb+ influx through the (Na+-K+)-ATPase without affecting Na+ influx, insulin stimulates Na+ entry which in turn may increase the pump activity by increasing the availability of Na+ ions to internal Na+ transport sites of the (Na+-K+)-ATPase.     

Characterization of a Na(+)-K(+)-2Cl- cotransport system in oocytes from Xenopus laevis

In order to characterize the transport systems mediating K+ uptake into oocytes, flux studies employing 86Rb were performed on Xenopus oocytes stripped of follicular cells by pretreatment with Ca2(+)-Mg2(+)-free Barth's medium. Total Rb+ uptake consisted of an ouabain-sensitive and an ouabain-insensitive flux. In the presence of 100 mmol/l NaCl and 0.1 mmol/l ouabain the ouabain-insensitive flux amounted to 754.7 +/- 59.9 pmol/oocyte per h (n = 30 cells, i.e., 10 cells each from three different animals). In the absence of Na+ (Na+ substituted by N-methylglucamine) or when Cl- was replaced by NO3- the ouabain-insensitive flux was reduced to 84.4 +/- 42.9 and 79.2 +/- 12.1 pmol/oocyte per h, respectively (n = 50 cells). Furthermore, this Na(+)- and Cl(-)-dependent flux was completely inhibited by 10(-4) mol/l bumetanide, a specific inhibitor of the Na(+)-K(+)-2Cl- cotransport system. These results suggest that K+ uptake via a bumetanide-sensitive Na(+)-K(+)-2Cl- cotransport system represents a major K+ pathway in oocytes.     

Effects of 20-HETE on Na+ transport and Na+ -K+ -ATPase activity in the thick ascending loop of Henle

Previous studies have indicated that 20-hydroxyeicosatetraenoic acid (20-HETE) inhibits Na+ transport in the medullary thick ascending loop of Henle (mTALH), but the mechanisms involved remain uncertain. The present study compared the effects of 20-HETE with those of ouabain and furosemide on intracellular Na+ concentration ([Na+]i), Na+ -K+ -ATPase activity, and 86Rb+ uptake, an index of Na+ transport, in mTALH isolated from rats. Ouabain (2 mM) increased, whereas furosemide (100 microM) decreased, [Na+]i in the mTALH of rats. Ouabain and furosemide inhibited 86Rb+ uptake by 91 and 30%, respectively. 20-HETE (1 microM) had a similar effect as ouabain and increased [Na+]i from 19 +/- 1 to 30 +/- 1 mM. 20-HETE reduced Na+ -K+ -ATPase activity by 30% and 86Rb+ uptake by 37%, but it had no effect on 86Rb+ uptake or [Na+]i in the mTALH of rats pretreated with ouabain. 20-HETE inhibited 86Rb+ uptake by 12% and increased [Na+]i by 19 mM in mTALH pretreated with furosemide. These findings indicate that 20-HETE secondarily inhibits Na+ transport in the mTALH of the rat, at least, in part by inhibiting the Na+ -K+ -ATPase activity and raising [Na+]i.     

The mechanism of insulin stimulation of (Na+,K+)-ATPase transport activity in muscle

Since the mechanism underlying the insulin stimulation of (Na+,K+)-ATPase transport activity observed in multiple tissues has remained undetermined, we have examined (Na+,K+)-ATPase transport activity (ouabain-sensitive 86Rb+ uptake) and Na+/H+ exchange transport (amiloride-sensitive 22Na+ influx) in differentiated BC3H-1 cultured myocytes as a model of insulin action in muscle. The active uptake of 86Rb+ was sensitive to physiological insulin concentrations (1 nM), yielding a maximum increase of 60% without any change in 86Rb+ permeability. In order to determine the mechanism of insulin stimulation of (Na+,K+)-ATPase activity, we demonstrated that insulin also stimulates passive 22Na+ influx by Na+/H+ exchange transport (maximal 200% increase) and an 80% increase in intracellular Na+ concentration with an identical time course and dose-response curve as insulin-stimulated (Na+,K+)-ATPase transport activity. Incubation of the cells with high [Na+] (195 mM) significantly potentiated insulin stimulation of ouabain-inhibitable 86Rb+ uptake. The ionophore monensin, which also promotes passive Na+ entry into BC3H-1 cells, mimics the insulin stimulation of ouabain-inhibitable 86Rb+ uptake. In contrast, incubation with amiloride or low [Na+] (10 mM), both of which inhibit Na+/H+ exchange transport, abolished the insulin stimulation of (Na+,K+)-ATPase transport activity. Furthermore, each of these insulin-stimulated transport activities displayed a similar sensitivity to amiloride. These results indicate that insulin stimulates a large increase in Na+/H+ exchange transport and that the resulting Na+ influx increases the intracellular Na+ concentration, thus activating the internal Na+ transport sites of the (Na+,K+)-ATPase. This Na+ influx is, therefore, the mediator of the insulin-induced stimulation of membrane (Na+,K+)-ATPase transport activity classically observed in muscle.     

Ouabain-insensitive Na+-ATPase of proximal tubules is an effector for urodilatin and atrial natriuretic peptide

In the present paper we studied the effect of urodilatin and atrial natriuretic peptide (ANP) on the proximal tubule Na+-ATPase and (Na+K+)ATPase activities. Urodilatin and ANP inhibit the Na+-ATPase activity but not the (Na+K+)ATPase activity. Maximal effect was observed at a concentration of 10(-11) M for both peptides. In this condition, the enzyme activity decreases from 10.8 +/- 1.6 (control) to 5.7 +/- 0.9 or 6.1 +/- 0.7 nmol Pi mg(-1) min(-1) in the presence of urodilatin or ANP, respectively. This effect was completely reversed by 10(-6) M LY83583, a guanylyl cyclase inhibitor, and mimicked by 10 nM cGMP. Furthermore, both ANP and urodilatin increase cGMP production by 33% and 49%, respectively. This is the first demonstration that it was shown that urodilatin and ANP directly modulate primary active sodium transport in the proximal tubule. The data obtained indicate that this effect is mediated by the activation of the NPR-A/guanylate cyclase/cGMP pathway.     

Atrial natriuretic hormone secretion in patients with renal failure

To study the effects of volume overload and renal failure on plasma levels of immunoreactive atrial natriuretic hormone (IR-ANH), we measured levels of this hormone in normal subjects, in patients with advanced chronic renal failure (CRF) with and without clinically evident volume overload, and in patients with end-stage renal disease (ESRD) treated with chronic hemodialysis. The levels were 13 +/- 2 pmol/l in normal volunteers, 77 +/- 24 pmol/l in patients with CRF without volume overload, and 219 +/- 50 pmol/l in patients with CRF and clinically evident volume overload (analysis of variance, p less than 0.001, alpha = 0.05 compared to normals). In patients with ESRD, the levels of IR-ANH were 145 +/- 46 pmol/l before dialysis and decreased to 87 +/- 31 after dialysis (p less than 0.025). No correlation was found between the decrease in IR-ANH levels and the decrease in weight during dialysis. A significant positive correlation was found between the IR-ANH levels and blood urea nitrogen in patients with CRF (r = 0.658, p less than 0.01). Volume overload appears to be the most important stimulatory factor for ANH secretion in renal failure patients but other mechanisms, especially a decrease in metabolic clearance, may also contribute to elevated plasma levels. The increased secretion of ANH in patients with renal failure may be an important adaptive response to volume overload and hypertension.     

Hyperthyroidism and cation pumps in human skeletal muscle

Skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormone. We examined the possible relation between energy expenditure (EE), thyroid status, and the contents of Ca2+-ATPase and Na+-K+-ATPasein human skeletal muscle. Eleven hyperthyroid patients with Graves' disease were studied before and after medical treatment with methimazole and compared with eight healthy subjects. Muscle biopsies were taken from the vastus lateralis muscle, and EE was determined by indirect calorimetry. Before treatment, the patients had two- to fivefold elevated total plasma T3 and 41% elevated EE compared with when euthyroidism had been achieved. In hyperthyroidism, the content of Ca2+-ATPase was increased: (mean +/- SD) 6,555 +/- 604 vs. 5,212 +/- 1,580 pmol/g in euthyroidism (P = 0.04) and 4,523 +/- 1,311 pmol/g in healthy controls (P = 0.0005). The content of Na+-K+-ATPase showed 89% increase in hyperthyroidism: 558 +/- 101 vs. 296 +/- 34 pmol/g (P = 0.0001) in euthyroidism and 278 +/- 52 pmol/g in healthy controls (P < 0.0001). In euthyroidism, the contents of both cation pumps did not differ from those of healthy controls. The Ca2+-ATPase content was significantly correlated to plasma T3 and resting EE. This provides the first evidence that, in human skeletal muscle, the capacity for Ca2+ recycling and active Na+-K+ transport are correlated to EE and thyroid status.     

Downregulation of Na+-K+-ATPase pumps in skeletal muscle with training in normobaric hypoxia.

To investigate the effects of training in normoxia vs. training in normobaric hypoxia (fraction of inspired O2 = 20.9 vs. 13.5%, respectively) on the regulation of Na+-K+-ATPase pump concentration in skeletal muscle (vastus lateralis), 9 untrained men, ranging in age from 19 to 25 yr, underwent 8 wk of cycle training. The training consisted of both prolonged and intermittent single leg exercise for both normoxia (N) and hypoxia (H) during a single session (a similar work output for each leg) and was performed 3 times/wk. Na+-K+-ATPase concentration was 326 +/- 17 (SE) pmol/g wet wt before training (Control), increased by 14% with N (371 +/- 18 pmol/g wet wt; P < 0.05), and decreased by 14% with H (282 +/- 20 pmol/g wet wt; P < 0.05). The maximal activity of citrate synthase, selected as a measure of mitochondrial potential, showed greater increases (P < 0.05) with H (1.22 +/- 0.10 mmol x h-1 x g wet wt-1; 70%; P < 0.05) than with N (0.99 +/- 0.10 mmol x h-1 x g wet wt-1; 51%; P < 0.05) compared with pretraining (0.658 +/- 0.09 mmol x h-1 x g wet wt-1). These results demonstrate that normobaric hypoxia induced during exercise training represents a potent stimulus for the upregulation in mitochondrial potential while at the same time promoting a downregulation in Na+-K+-ATPase pump expression. In contrast, normoxic training stimulates increases in both mitochondrial potential and Na+-K+-ATPase concentration.     

Density-related changes of potassium (86Rb) uptake by amphibian endothelial cells

Potassium influx has been investigated in XTH-2 cells, a line derived from tadpole heart endothelia. In this line, the density at which the cultures become confluent is clearly separated from the density at which growth arrest takes place. Density-related changes in K+ influx were monitored by determining the uptake of 86Rb into well adhering cells kept in culture medium. The main observations were 1) 86Rb uptake is highest in single cells, and on confluency it reaches a low level, which is kept constant at higher cell density regardless of whether the cultures are stationary or still in logarithmic growth phase; 2) the relative amount of 86Rb taken up via the Na+ -K+ -2Cl- cotransport pathway and via the Na+/K+ pump changes from low cell density to confluent cultures; 86Rb uptake of single cells is nearly insensitive to ouabain, a maximum of ouabain sensitivity is reached around confluency, whereas piretanide-sensitive 86Rb uptake is highest in single cells and seems to reach a minimum at the onset of confluency; 3) the variations in Na+/K+ pumping rate reflect neither differences in the amount of enzyme present nor changes in enzyme repartition between apical and basolateral plasma membranes; they seem to result from either "masking" or "unmasking" of the enzyme; 4) no alterations in K+ uptake occur that would be characteristic of the "stationary growth phase." The only changes that seem to be related to arrest of proliferation are concerned with the Na+/K+-ATPase, which achieves an extraordinary susceptibility to stimulation by monensin and exhibits an increase in PNPPase activity.     

Equimolar doses of atrial and brain natriuretic peptides and urodilatin have differential renal actions in overt experimental heart failure

A hallmark of overt congestive heart failure (CHF) is attenuated cGMP production by endogenous atrial natriuretic peptide (ANP) with renal resistance to ANP. ANP and brain natriuretic peptides (BNP) are of myocardial origin, whereas urodilatin (Uro) is thought to be derived from kidney. All three peptides are agonists to the natriuretic peptide-A receptor. Our objective was to compare the cardiorenal and humoral actions of ANP, BNP, and Uro in experimental overt CHF. We determined cardiorenal and humoral actions of 90 min of intravenous equimolar infusion of ANP, BNP, and Uro (2 and 10 pmol.kg-1.min-1) in three separate groups of anesthetized dogs with rapid ventricular pacing-induced overt CHF (240 beats/min for 10 days). BNP resulted in increases in urinary sodium excretion (U(Na)V) (2.2+/-0.7 to 164+/-76 microeq/min, P<0.05) and glomerular filtration rate (GFR) (27+/-4 to 52+/-11 ml/min, P<0.05) that were greater than those with Uro (P<0.05), whereas ANP did not result in increases in U(Na)V or GFR. Increases in plasma cGMP (25+/-2 to 38+/-2 pmol/ml, P<0.05) and urinary cGMP excretion with BNP (1,618+/-151 to 6,124+/-995 pmol/min, P<0.05) were similar to those with Uro; however, there was no change with ANP. Cardiac filling pressures were reduced in all three groups. These studies also support the conclusion that in experimental overt CHF, renal resistance to natriuretic peptides in increasing rank order is BNP相似文献   

Passive transport of Rb+ by hog gastric (H+,K+)-ATPase

Gastric vesicles enriched in (H+,K+)-ATPase were prepared from hog fundic mucosa and studied for their ability to transport K+ using 86Rb+ as tracer. In the absence of ATP, the vesicles elicited a rapid uptake of 86Rb+ (t 1/2 = 45 +/- 9 s at 30 degrees C) which accounted for both transport and binding. Transport was osmotically sensitive and was the fastest phase. It was not limited by anion permeability (C1- was equivalent to SO2-4) but rather by availability of either H+ or K+ as intravesicular countercation suggesting a Rb+-K+ or a Rb+-H+ exchange. Selectivity was K+ greater than Rb+ greater than Cs+ much greater than Na+,Li+. The capacity of vesicles which catalyzed the fast transport of K+ was 83 +/- 4% of maximal vesicular capacity of the fraction. Addition of ATP decreased both rate and extent of 86Rb+ uptake (by 62 and 43%, respectively with 1 mM ATP) with an apparent Ki of 30 microM. Such an effect was not seen on 22Na+ transport. ATP inhibition of transport did not require the presence of Mg2+, and inhibition was also produced by ADP even in the presence of myokinase inhibitor. On the other hand, 86Rb+ uptake was as strongly inhibited by 200 microM vanadate in the presence of Mg2+. Efflux studies suggested that ATP inhibition was originally due to a decrease of vesicular influx with little or no modification of efflux. Since ATP, ADP, and vanadate are known modulators of the (H+,K+)-ATPase, we propose that, in the absence of ATP, (H+,K+)-ATPase passively exchanges K+ for K+ or H+ and that ATP, ADP, and vanadate regulate this exchange.     

Sodium-potassium exchange in sea urchin egg. II. Ionic events stimulating the Na+-K+ pump activity at fertilization

The preceding paper (Ciapa et al., 1984) provided biochemical and kinetic characterization of the Na+-K+ exchange in Paracentrotus lividus eggs. The present work is a study of the ionic events involved in the stimulation of the Na+-K+ transporter after fertilization. Fertilization in low Na+-external medium containing amiloride (0.1 mM) suppresses the stimulation of the net efflux of H+ and 86Rb uptake. Activation of eggs with the ionophore A23187 leads to stimulation of both Na+-H+ exchange and ouabain-sensitive 86Rb influx. When eggs were activated with A23187 in artificial seawater, 86Rb uptake and 24Na influx showed similar saturable kinetics with respect to the external Na+. A23187 treatment of eggs in Na+-free artificial seawater did not stimulate the Na+-K+ exchange until 10 mEq Na+ was added. Activation of eggs by NH4Cl (5 mM) stimulated 86Rb influx and Na+ exit; both fluxes were ouabain sensitive. Monensin increased cell Na+ of unfertilized eggs without any significant increase in intracellular pH: a condition in which 86Rb influx was not markedly stimulated. Addition of 10 mEq Na+ to unfertilized eggs in Na+-free artificial seawater stimulated 86Rb uptake but to a lower extent that did 10 mEq Na+ plus sperm. It is concluded that (1) the stimulation of the Na+-K+ pump at fertilization has an absolute requirement for the Na+-H+ exchange; (2) the alkalinization of eggs resulting from the acid efflux is a prerequisite for the enhancement of the Na+-K+ pump; (3) the amount of Na+ entering eggs at fertilization determines the intensity of the Na+-K+ exchange; (4) early events of fertilization such as exocytosis and calcium release which may be involved in the stimulation of the Na+-K+ pump must necessarily be coupled to cell alkalinization.     

Atrial natriuretic peptide in acute mountain sickness

To test the hypothesis that elevated atrial natriuretic peptide (ANP) may be involved in altered fluid homeostasis at high altitude, we examined 25 mountaineers at an altitude of 550 m and 6, 18, and 42 h after arrival at an altitude of 4,559 m, which was climbed in 24 h starting from 3,220 m. In 14 subjects, symptoms of acute mountain sickness (AMS) were absent or mild (group A), whereas 11 subjects had severe AMS (group B). Fluid intake was similar in both groups. In group B, urine flow decreased from 61 +/- 8 (base line) to 36 +/- 3 (SE) ml/h (maximal decrease) (P less than 0.05) and sodium excretion from 7.9 +/- 0.9 to 4.6 +/- 0.7) mmol.l-1.h-1 (P less than 0.05); ANP increased from 31 +/- 4 to 87 +/- 26 pmol/l (P less than 0.001), plasma aldosterone from 191 +/- 27 to 283 +/- 55 pmol/l (P less than 0.01 compared with group A), and antidiuretic hormone (ADH) from 1.0 +/- 0.1 to 2.9 +/- 1.2 pmol/l (P = 0.08 compared with group A). These variables did not change significantly in group A, with the exception of a decrease in plasma aldosterone from 189 +/- 19 to 111 +/- 17 pmol/l (P less than 0.01). There were no measurable effects of elevated ANP on natriuresis, cortisol, or blood pressure. The reduced diuresis in AMS may be explained by increased plasma aldosterone and ADH overriding the expected renal action of ANP. The significance of elevated ANP in AMS remains to be established.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relationships between membrane lipids and ion transport in red blood cells of Dahl rats

Distinct changes of membrane lipid content could contribute to the abnormalities of ion transport that take part in the development of salt hypertension in Dahl rats. The relationships between lipid content and particular ion transport systems were studied in red blood cells (RBC) of Dahl rats kept on low- and high-salt diets for 5 weeks since weaning. Dahl salt-sensitive (SS/Jr) rats on high-salt diet had increased blood pressure, levels of plasma triacylglycerols and total plasma cholesterol compared to salt-resistant (SR/Jr) rats. Furthermore, RBC of SS/Jr rats differed from SR/Jr ones by increased content of total membrane phospholipids, but membrane cholesterol was not changed significantly. SS/Jr rats had higher RBC intracellular Na+ (Na(i)+) content and enhanced bumetanide-sensitive Rb+ uptake. RBC membrane content of cholesterol and phospholipids correlated positively with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and also with Rb+ leak. The content of phosphatidylserines plus phosphatidylinositols was positively associated with RBC Na(i)+ content, with the activity of Na+-K+ pump and Na+-K+-2Cl- cotransport and with Rb+ leak. The content of sphingomyelins was positively related to Na+-K+-2Cl- cotransport activity and negatively to ouabain-sensitive Rb+-K+ exchange. We can conclude that observed relationships between ion transport and the membrane content of cholesterol and/or sphingomyelins, which are known to regulate membrane fluidity, might participate in the pathogenesis of salt hypertension in Dahl rats.     

Characterisation of a Na+/K+/Cl- cotransporter in alkylating agent-sensitive L1210 murine leukemia cells

The mode of influx of 86Rb+, a K+ congener, to exponentially proliferating L1210 murine leukemia cells, incubated in a Krebs-Ringer buffer, has been characterised. The influx was composed of a ouabain-sensitive fraction (approx. 40%), a loop diuretic-sensitive fraction (approx. 40%) and a fraction which was insensitive to both types of inhibitor (approx. 15%). The fraction of ouabain-insensitive 86Rb+ influx, which was fully inhibited by furosemide (1 mM) or bumetanide (100 microM), was completely inhibited when Cl- was completely substituted by nitrate or gluconate ions, but was slightly (29 +/- 12%) stimulated if the Cl- was substituted by Br-. The substitution of Na+ by Li+, choline or tetramethylammonium ions inhibited the loop diuretic-sensitive fraction of 86Rb+ uptake. These results suggested that a component of 86Rb+ influx to L1210 cells was mediated via a Na+/K+/Cl- cotransporter. 86Rb+ efflux from L1210 cells which had been equilibrated with 86Rb+ and incubated in the presence or absence of 1 mM ouabain, was insensitive to the loop diuretics. Additionally, efflux rates were found to be independent of the external concentration of K+, suggesting that efflux was not mediated by K+-K+ exchange. The initial rate of 86Rb+ influx to L1210 cells in the plateau phase of growth was reduced to 44% of that of exponentially dividing cells, the reduction being accounted for by significant decreases in both ouabain- and loop diuretic-sensitive influx; these cells were reduced in volume compared to cells in the exponential phase of cell growth. In cells which had been deprived of serum for 18 h, and which showed an increase of the proportion of cells in the G1 phase of the cell cycle, the addition of serum stimulated an immediate increase in the furosemide-sensitive component of 86Rb+ influx. Diuretic-sensitive 86Rb+ influx was not altered by the incubation of the cells with 100 microM dibutyryl cyclic AMP, but was inhibited by 10 microM of the cross-linking agent nitrogen mustard (bis(2-chloro-ethyl)methylamine, HN2).     

Regulation of atrial natriuretic peptide release in normal humans.

Atrial volume, pressure, and heart rate are considered the most important modulators of atrial natriuretic peptide (ANP) release, although their relative role is unknown. Continuous positive-pressure breathing in normal humans may cause atrial pressure and atrial volume to go in opposite directions (increase and decrease, respectively). We utilized this maneuver to differentially manipulate atrial volume and atrial pressure and evaluate the effect on ANP release. Effective filling pressure (atrial pressure minus pericardial pressure) was also monitored, because this variable has been proposed as another modulator of ANP secretion. We measured right atrial (RA) pressure, RA area, esophageal pressure (reflection of pericardial pressure), and RA and peripheral venous ANP in seven healthy adult males at rest and during continuous positive-pressure breathing (19 mmHg for 15 min). Continuous positive-pressure breathing decreased RA area (mean +/- SE, *P less than 0.05) 13.6 +/- 1.1 to 10.5 +/- 0.8* cm2, increased RA pressure 4 +/- 1 to 16 +/- 1* mmHg, increased esophageal pressure 2 +/- 1 to 12 +/- 1* mmHg, and increased effective filling pressure 2 +/- 0 to 4 +/- 1* mmHg. RA ANP increased from 67 +/- 17 to 91 +/- 18* pmol/l and peripheral venous ANP from 43 +/- 4 to 58 +/- 6* pmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)     

Na~+-K~+-2Cl~-共转运蛋白在L6骨骼肌细胞调节性体积增加中的作用(英文)

在许多类型的哺乳动物细胞中,细胞体积对介导胰岛素发挥其生理功能起关键作用.细胞体积调节机制在胰岛素的主要靶组织骨骼肌中尤为重要.为了解该组织中细胞体积调节的机制,对Na+K+2Cl-共转运蛋白在大鼠L6骨骼肌细胞中的表达及其在调节性体积增加中的作用进行了研究.应用免疫印迹法,在L6肌管细胞中检测到分子量为170kD的钠钾氯共转运蛋白.K+(86Rb+)输入实验结果表明,54%的86Rb+是通过钠钾氯共转运蛋白输入细胞的,而其余86Rb+的输入是通过钠钾三磷酸腺苷酶和其他未知转运蛋白实现的.当L6细胞被置于高渗透压溶液(420mosmolL)中,导致细胞体积减少40%,同时钠钾氯共转运蛋白的活性迅速增加(高达2倍).细胞体积在60min内恢复至正常,但在钠钾氯共转运蛋白抑制剂bumetanide存在时,这种调节性体积增加的过程被阻断.这些结果表明,L6肌管细胞表达具有生理活性的钠钾氯共转运蛋白;此转运蛋白被高渗透压诱导造成的细胞体积缩小激活的现象表明,它是细胞调节性体积增加(RVI)机制中的关键元素,在L6骨骼肌细胞体积的调节中起重要作用.     

Sodium influx rate and ouabain-sensitive rubidium uptake in isolated guinea pig atria.

1. Ouabain-sensitive 86Rb+ uptake by tissue preparations has been used as an estimate of Na+ pump activity. This uptake, however, may be a measure of the Na+ influx rate, rather than capacity of the Na+ pump, since intracellular Na+ concentration is a determinant of the active Na+/Rb+ exchange reaction under certain conditions. This aspect was examined by studying the effect of altered Na+ influx rate on ouabain-sensitive 86Rb+ uptake in atrial preparations of guinea pig hearts. 2. Electrical stimulation markedly enhanced ouabain-sensitive 86Rb+ uptake without affecting nonspecific, ouabain-insensitive uptake. Paired-pulse stimulation studies indicate that the stimulation-induced enhancement of 86Rb+ uptake is due to membrane depolarizations, and hence related to the rate of Na+ influx. 3. Alterations in the extracellular Ca2+ concentration failed to affect the 86Rb+ uptake indicating that the force of contraction does not influence 86Rb+ uptake. 4. Reduced Na+ influx by low extracellular Na+ concentration decreased 86Rb+ uptake, and an increased Na+ influx by a Na+-specific ionophore, monensin, enhanced 86Rb+ uptake in quiescent atria. 5. Grayanotoxins, agents that increase transmembrane Na+ influx, and high concentrations of monensin appear to have inhibitory effects on ouabain-sensitive 86Rb+ uptake in electrically stimulated and in quiescent atria. 6. Electrical stimulation or monensin enhanced ouabain binding to (Na+ + K+)-ATPase and also increased the potency of ouabain to inhibit 86Rb+ uptake indicating that the intracellular Na+ available to the Na+ pump is increased under these conditions. 7. The ouabain-sensitive 86Rb+ uptake in electrically stimulated atria was less sensitive to alterations in the extracellular Na+ concentration, temperature and monensin than that in quiescent atria. 8. These results indicate that the rate of Na+ influx is the primary determinant of ouabain-sensitive 86Rb+ uptake in isolated atria. Electrical stimulation most effectively increases the Na+ available to the Na+ pump system. The ouabain-sensitive 86Rb+ uptake by atrial preparations under electrical stimulation at a relatively high frequency seems to represent the maximal capacity of the Na+ pump in this tissue.