Na+/H+ countertransport and calcium exchange in peripheral blood cells of healthy subjects and patients with chronic heart failure

A comparative study is performed of Na⁺/H⁺ exchange and Ca²⁻ mobilization in erythrocytes and platelets of patients with stage I–II chronic heart failure caused by dilative cardiomyopathy and ischemic heart disease. A significant rise in the Na⁺/H⁺ exchange rate is found in the cells of chronic heart failure patients, which correlates with an elevated erythrocyte and platelet concentration of Ca²⁺ and an increased “calcium” response of platelets to inductors. The findings testify to a certain functional relationship between various cation-transporting cellular systems whose change in properties upon chronic heart failure can play an important pathogenic role. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 572–575, December, 1994     

Short-term and long-term stimulation of Na+−H+ exchange in cortical brush-border membranes during compensatory growth of the rat kidney

The effect of unilateral nephrectomy on Na⁺–H⁺ exchange in rat renal cortical brush-border membrane vesicles (BBMV) was studied by the method of acridine orange fluorescence quenching. The exchanger activity in BBMV from remnant kidney increased rapidly by 70–75% within first 30 min following uninephrectomy. Only a slight further increase was found in later stages of renal growth, i.e. 30 min to 7 days following uninephrectomy. The changes in antiporter activity were restricted toV _max, whereas theK _m for Na⁺ was similar in control and compensatory growing kidney. The increase of Na⁺–H⁺ exchange at 15 min was not affected by actinomycin D in vivo, whereas the increase at 48 h was completely abolished indicating that protein synthesis could be involved in the late, but not in the initial stimulation of renal Na⁺–H⁺ exchange. The late, but not the initial stimulations of Na⁺–H⁺ exchange were associated with elevated activities of cortical (Na⁺+K⁺)-ATPase indicating that changes in antiporter activity precede those in the (Na⁺+K⁺)-pump. The early stimulation of Na⁺–H⁺ exchange in BBMV in one kidney was induced also by the occlusion of blood flow through the contralateral kidney for 15 min, without removing it. Thirty min after the occlusion was removed and the reflow established, the Na⁺–H⁺ exchange in BBMV from the intact kidney decreased to the control values. The observed modulations in renal Na⁺–H⁺ exchanger may be regulated by phosphorylation-dephosphorylation events. In support, the concentration of a well known protein kinase C activator, 1,2-diacylglycerol, in the cortical tissue of the remnant kidney increased up to 100% within 5 min following unilateral nephrectomy and preceded the increase in Na⁺–H⁺ exchange. The early stimulation of Na⁺–H⁺ exchange may be a trigger in initiating the kidney growth.     

Regulation of the cytosolic pH set point for activation of the Na+/H+ antiport in human platelets: the roles of the Na+/Ca2+ exchange,the Na+-K+-2Cl− cotransport and cellular volume

To explore further the mechanisms that regulate the Na⁺/H⁺ antiport in human platelets, we examined the effect of Na⁺ pump inhibition by ouabain and K⁺ removal from the extracellular medium on parameters of this transport system. Treatment with ouabain resulted in increased cytosolic free Ca²⁺ and Na⁺, coupled with an alkaline shift in the cytosolic pH set point for the Na⁺/ H⁺ antiport. Inhibition of the Na⁺ pump by the removal of K⁺ from the medium increased the cytosolic Na⁺ but not the cytosolic Ca²⁺; yet this treatment also produced a substantial alkaline shift in the cytosolic pH set point for the Na⁺/H⁺ antiport. This effect appeared to relate to a decline in cellular volume and it was attenuated by the Na⁺-K⁺-2Cl^– cotransport inhibitor, bumetanide. These findings indicate: (a) a link between the Na⁺ pump and the Na⁺/H⁺ antiport, mediated by the Na⁺/Ca²⁺ exchange and the cytosolic free Ca²⁺, and (b) a link between the Na⁺/H⁺ antiport and the Na⁺-K⁺-2Cl^– cotransport through cellular volume.This work was supported by grants from the National Heart, Lung, and Blood Institute (HL34807, HL42856) and the American Diabetes Association. M. Kimura is a postdoctoral research fellow of the American Heart Association, New Jersey Affiliate     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Abnormal erythrocyte sodium leak in a subset of essential hypertensive patients

Summary We measured the ouabain- and bumetanide-resistant Na⁺ efflux in Mg²⁺-sucrose medium (passive Na⁺ leak) in erythrocytes from 30 normotensive controls and 72 essential hypertensive patients. The mean values (±SEM) of the rate constant of Na⁺ leak (k_pNa) were not significantly different between normotensives and hypertensives. Nevertheless, using the 95% confidence limits of the k_pNa (in 10^–3.h^–1) in the normotensive group as a cut-off point, 7 (9.7%) essential hypertensives exhibited increased values (58.96±10.12) when compared with the other 65 patients (23.86±0.74). revealing increased passive Na⁺ permeability in the former (leak + hypertensives). Na⁺ fluxes depending on the Na⁺-K⁺ pump, outward Na⁺-K⁺ cotransport, and Na⁺-Li⁺ countertransport were also measured in fresh erythrocytes from the same 72 patients. Three of them (4.2%) exhibited decreased values of ouabain-sensitive Na⁺ efflux and 6 (8.3%) of bumetanide-sensitive Na⁺ efflux, while 8 patients (11.1%) showed increased values of Li⁺-stimulated Na⁺ efflux and, finally, 48 patients (59.7%) did not present any evident abnormality in these Na⁺ transport systems. No differences were observed between leak + hypertensives and the remaining 65 patients when both basal erythrocyte Na⁺ content and clinical parameters of hypertension were compared. However, Na⁺ efflux depending on the outward Na⁺-K⁺ cotransport was significantly higher in the leak + hypertensive subset (299.43±43.18 vs 181.52±10.76 µmol.(l cells.h)^–1;P=0.0078), suggesting a compensatory phenomenon. Enhancement of Na⁺ permeability detected in 3% to 16% of essential hypertensives may be implicated in the pathogenesis of primary hypertension.Abbreviations ATPase adenosine triphosphatase - Dcat difference between the external Na⁺ concentration after incubation at 37° C and at zero time - k_pNa rate constant of passive Na⁺ leak - Leak + hypertensive essential hypertensive patient with abnormally high erythrocyte Na⁺ leak - MOPS 4-morpholinopropanesulfonic acid - OBR ouabain- and bumetanide-resistant - PRA plasma renin activity - sPRA plasma renin activity stimulated after furosemide infusion - SEM standard error of the mean Supported in part by Grant 87/1078 of the Fondo de Investigaciones Sanitarias de la Seguridad Social and Grant PA85/0168 of the Comisión Asesora de Investigación Científica y Técnica     

Copper toxicity in cultured human skeletal muscle cells: the involvement of Na+/K+-ATPase and the Na+/Ca2+-exchanger

Copper (Cu²⁺) intoxication has been shown to induce pathological changes in various tissues. The mechanism underlying Cu²⁺ toxicity is still unclear. It has been suggested that the Na⁺/K⁺-ATPase and/or a change of the membrane permeability may be involved. In this study we examined the effects of Cu²⁺ on the Na⁺ and Ca²⁺ homeostasis of cultured human skeletal muscle cells using the ion-selective fluorescent probes Na⁺-binding benzofuran isophtalate (SBFI) and Fura-2, respectively. In addition, we measured the effect of Cu²⁺ on the Na⁺/K⁺-ATPase activity. Cu²⁺ and ouabain increase the cytoplasmic free Na⁺ concentration ([Na⁺]_i). Subsequent addition of Cu²⁺ after ouabain does not affect the rate of [Na⁺]_i increase. Cu²⁺ inhibits the Na⁺/K⁺-ATPase activity with an IC₅₀ of 51 M. The cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) remains unaffected for more than 10 min after the administration of Cu²⁺. Thereafter, [Ca²⁺]_i increases as a result of the Na⁺/Ca²⁺-exchanger operating in the reversed mode. The effects of Cu²⁺ on the Na⁺ homeostasis are reversed by the reducing and chelating agent dithiothreitol and the heavy metal chelator N,N,N,N,-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN). In conclusion, SBFI is a good tool to examine Na⁺ homeostasis in cultured human skeletal muscle cells. Under the experimental conditions used, Cu²⁺ does not modify the general membrane permeability, but inhibits the Na⁺/K⁺-pump leading to an increase of [Na⁺]_i. As a consequence the operation mode of the Na⁺/Ca²⁺-exchanger reverses and [Ca²⁺]_i rises.The authors thank staff and coworkers of the Department of Neurology of the University Hospital Nijmegen, Nijmegen for their kind cooperation in obtaining muscle biopsies. Mr. Arie Oosterhof is gratefully acknowledged for culturing of the human muscle cells. The Prinses Beatrix Fonds and the Dutch-Chinese scientific exchange program contributed financial support for this study.     

Epidermal growth factor stimulates Ca2+ uptake of human erythrocytes

To examine the functional significance of epidermal growth factor (EGF) binding sites present on the human erythrocyte membrane [Engelmann et al. (1992) Am J Hematol 39:239–241], the effect of EGF on ⁴⁵Ca²⁺ uptake and on ²²Na⁺ efflux from these cells has been studied. In all cases media contained 1.25 mM Ca²⁺, whereas Na⁺ and K⁺ were varied. In 140 mM Na⁺/5 mM K⁺ medium EGF (250 ng/ml) stimulated ⁴⁵Ca²⁺ uptake by 50%–90% in quin-2-loaded cells, and by up to threefold in untreated cells. Increasing extracellular K⁺ up to 75 mM at the expense of extracellular Na²⁺ stimulated the EGF-induced ⁴⁵Ca²⁺ uptake by about twofold compared to 145 mM Na⁺ medium both in quin-2-loaded and in untreated cells. In 145 mM K⁺ medium, however, no EGF-induced ⁴⁵Ca²⁺ uptake was detectable in quin-2-loaded cells, while in untreated cells Ca²⁺ entry was stimulated twofold by EGF. After increasing intracellular Na⁺ from 6 mmol/l cells to 18 mmol/l cells in untreated cells suspended in 145 mM K⁺ medium, ⁴⁵Ca²⁺ uptake induced by EGF gradually increased. In contrast, in 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, ⁴⁵Ca²⁺ uptake accelerated by EGF was largely unaffected by a modified red cell Na⁺ content. When ²²Na-loaded untreated red cells were suspended in 145 mM K⁺ medium EGF stimulated red cell ²²Na⁺ efflux by more than threefold. In 140 mM Na⁺/5 mM K⁺ as well as in 70 mM Na⁺/75 mM K⁺ medium, no ²²Na⁺ efflux induced by the growth factor was evident. The results are consistent with the idea that EGF stimulates (at least) two components of ⁴⁵Ca²⁺ uptake in human erythrocytes. One of the two is unmasked in 145 mM K⁺ medium, inhibited by quin-2 loading, accelerated by intracellular Na⁺ and appears to involve reversed Na⁺/Ca²⁺ exchange.     

Blood pressure in essential hypertension correlates with the concentration of a circulating inhibitor of the sodium pump

Summary The influence of serum from patients with essential hypertension on the sodium efflux rate constants of human lymphocytes and on the activity of isolated (Na⁺+K⁺)-ATPase was investigated. The ouabain-sensitive sodium efflux rate constant was significantly decreased (p<0.001) in the sera of 19 hypertensives (1.92±0.11 h^–1) compared with the sera of 30 normotensives (2.44±0.07 h^–1). The ouabain-insensitive sodium efflux was unaffected. These results corresponded with a significant difference (p<0.005) of (Na⁺+K⁺)-ATPase activity (1.03±0.04 mU/ml and 0.079±0.06 mU/ml), when an isolated (Na⁺+K⁺)-ATPase was incubated with the sera of 22 normotensives or 18 hypertensives. Both the rate constant of ouabain-sensitive sodium efflux and the (Na⁺+K⁺)-ATPase activity correlated significantly with the diastolic and systolic blood pressure (p<0.001). These data, therefore, demonstrated the close relationship between essential hypertension and the concentration of a circulating inhibitor of the sodium pump.Abbreviations ATP Adenosine triphosphate - EGTA Ethyleneglycol bis(2-aminoethyl)-N,N,N,N-tetraacetic acid This paper contains an essential part of the thesis of K.M. presented to the Fachbereich Veterinärmedizin, GiessenThis work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg (Scho 139/16-2) and by the Fonds der Chemischen Industrie, Frankfurt/Main     

Rapid stimulation of Na+/H+ exchange by 1,25-dihydroxyvitamin D3; interaction with parathyroid-hormone-dependent inhibition

We have examined the rapid effect of 1,25-dihydroxyvitamin-D₃ [1,25(OH)₂D₃] on apical Na⁺/H⁺ exchange activity in opossum kidney (OK) cells and in MCT cells (a culture of simian-virus-40-immortalized mouse cortical tubule cells) grown on filter support. Addition of 1,25(OH)₂D₃ (10 nM) for 1 min increased apical Na⁺/H⁺ exchange activity [recovery from an acid load; measured by 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein] in OK cells (by 56%) and in MCT cells (by 36%). The cellular mechanisms involved in 1,25(OH)₂D₃-dependent stimulation of Na⁺/H⁺ exchange were analysed in OK cells; stimulation of Na⁺/ H⁺ exchange by 1,25(OH)₂D₃ was not prevented by actinomycin D. Applying parathyroid hormone (PTH) reduced Na⁺/H⁺ exchange activity in OK cells (by 34% at 10 nM, 5 min); 1,25(OH)₂D₃ reversed PTH-induced inhibition, either when PTH was added prior to 1,25(OH)₂D₃ or when the two agonists were applied together. 1,25(OH)₂D₃ had no effect on basal OK cell cAMP content or on [Ca²⁺]_i (fura-2). 1,25(OH)₂D₃ attenuated PTH-induced cAMP accumulation and had no effect on the PTH-dependent increase in [Ca²⁺]_i. These data suggest a regulatory control (stimulation) of proximal tubular brush-border Na⁺/H⁺ exchange by 1,25(OH)₂D₃. This effect is non-genomic and might in part be explained by a release from cAMP-dependent control of transport activity.     

The leading role of membrane Ca(2+)-ATPase in recovery of Ca(2+) homeostasis after glutamate shock

Combined blockade of Na⁺/Ca²⁺ exchange, Ca²⁺ uptake by mitochondria and endoplasmic reticulum usually does not prevent recovery of the basal level of intracellular Ca²⁺ after 1-min action of glutamate (100 M) or K⁺ (50 mM). However, replacement of Ca²⁺ with Ba²⁺, which cannot be transported by Ca²⁺-ATPase, considerably delayed the decrease in intracellular Ba²⁺ after its rise caused by glutamate or potassium application in all examined cells, which attest to an important role of Ca²⁺-ATPase in Ca²⁺ extrusion after the action of glutamate or K⁺.     

Erythrocyte cations and Na+,K+-ATPase pump activity in athletes and sedentary subjects

Summary The chronic effect of training on intraerythrocyte cationic concentrations and on red cell Na⁺,K⁺-ATPase pump activity was studied by comparing well-trained athletes with sedentary subjects at rest. Also the acute effect of a 50-min cross-country run on these erythrocyte measurements was studied in the athletes. At rest the intraerythrocyte potassium concentration was increased (P<0.01) in the athletes compared to that of the control subjects. The intraerythrocyte concentrations of sodium and magnesium and red cell Na⁺, K⁺-ATPase pump activity were, however, similar in the trained and the untrained subjects.As compared with the resting condition, the intraerythrocyte potassium concentration was decreased (P<0.05) after exercise in the athletes, and this was accompanied by a minor increase in the intraerythrocyte sodium concentration. Red cell Na⁺,K⁺-ATPase pump activity was slightly increased (P<0.05) after exercise.     

Molecular origin of Na/Li exchanger: Evidence against the involvement of major cloned erythrocyte transporters

Numerous studies have demonstrated heightened Na⁺/Li⁺ countertransport (NLCT) activity in erythrocytes of patients with essential hypertension or diabetic nephropathy. The same carrier also contributes to the therapeutic action of lithium salt, widely used in the treatment of psychiatric disorders. However, the molecular origin of NLCT remains unknown. This study examined the role of major ion transporters in NLCT by comparative analysis of its activity and that of ion transporters providing inwardly directed ⁸⁶Rb, ²²Na and ³²P fluxes. NLCT was below the detection limit in rat erythrocytes and ∼50-fold higher in rabbits compared to humans. Unlike NLCT, the activities of Na⁺,K⁺-ATPase, Na⁺,K⁺,2Cl⁻ cotransporter and anion exchanger were somewhat similar in the erythrocytes of these species, whereas Na⁺,P_i cotransport was in 1:2:6 proportion in rats, humans and rabbits, respectively. Loading of erythrocytes with Li⁺ for NLCT measurement did not affect the activity of Na⁺,P_i cotransporter. Keeping in mind that NLCT is much higher in rabbits vs humans and rats, we compared the set of membrane proteins in these species using 2-dimensional gel electrophoresis. This approach revealed 174 common spots, whereas 132 proteins were detected only in human and rabbit erythrocyte membranes. Among these proteins, we found 17 spots whose expression was higher by more than 5-fold in rabbit compared to human erythrocytes. Thus, our results argue against the involvement of major ion transporters in NLCT. They also show that comparative proteomics is a potent tool to identify the molecular origin of this carrier.     

Role of Na+/Ca2+ exchange in transcellular Ca2+ transport across primary cultures of rabbit kidney collecting system

Cells from connecting tubule and cortical collecting duct of rabbit kidney were isolated by immunodissection with mAb R2G9 and cultured on permeable filters. Confluent monolayers developed an amiloride-sensitive transepithelial potential difference of –50±1 mV (lumen negative) and a transepithelial resistance of 507±18 cm². Transepithelial Ca²⁺ transport increased dose-dependently with apical [Ca²⁺] and, in solutions containing 1 mM Ca²⁺, the active transcellular Ca²⁺ transport rate was 92±2 nmol h^–1 cm^–2. Transcellular Ca²⁺ transport was dependent on basolateral Na⁺ (Na _b ⁺ ). Isoosmotic substitution of Na _b ⁺ for N-methylglucamine resulted in a concentration-dependent decrease in Ca²⁺ absorption, with maximal inhibition of 67±5%. A Hill plot of the Na⁺-dependence yielded a coefficient of 1.9±0.4, indicating more than one Na⁺ site on a Na⁺-dependent Ca²⁺ transport system. In addition, the absence of Ca _b ²⁺ resulted in a significant increase in Ca²⁺ transport both in the presence and absence of Na _b ⁺ . Added basolaterally, ouabain (0.1 mM) inhibited Ca²⁺ transport to the same extent as did Na⁺-free solutions, while bepridil (0.1 mM), an inhibitor of Na⁺/Ca²⁺ exchange, reduced Ca²⁺ transport by 32±6%. Methoxyverapamil, felodipine, flunarizine and diltiazem (10 M) were without effect. Depolarisation of the basolateral membrane, by raising [K⁺]_b to 60 mM, significantly decreased transcellular Ca²⁺ transport, which is indicative of electrogenic Na⁺/Ca²⁺ exchange. In conclusion, active Ca²⁺ transport in the collecting system of rabbit kidney is largely driven by basolateral Na⁺/Ca²⁺ exchange. However, a residual Ca²⁺ absorption of about 30% was always observed, suggesting that other Ca²⁺ transport mechanisms, presumably a Ca²⁺-ATPase, participate as well.     

Regulation of Na+/H+ exchange in opossum kidney cells by parathyroid hormone,cyclic AMP and phorbol esters

Parathyroid hormone (PTH) controls two proximal tubular brush border membrane transport systems, Na⁺/phosphate co-transport and Na⁺/H⁺ exchange. In OK cells, a cell line with proximal tubular transport characteristics, PTH acts via kinase C and kinase A activation to inhibit Na⁺/phosphate co-transport [6, 8, 9, 19, 22]. In the present study, we show that PTH inhibits Na⁺/H⁺ exchange and that this effect can be mimicked by pharmacological activation of kinase A and kinase C. Ionomycin-dependent increases in cytoplasmic Ca²⁺ concentration do not induce inhibition of Na⁺/H⁺ exchange; PTH-dependent inhibition of Na⁺/H⁺ exchange is not prevented by ionomycin or by the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (Ca²⁺ clamping). Detailed dose-response curves for the different agonists, given either alone or in combination, suggest that the two regulatory cascades (kinase A and kinase C) are operating independent of each other and reach a common final target, resulting in 40–50% inhibition of Na⁺/H⁺ exchange. An analysis of intracellular pH sensitivity of Na⁺/H⁺ exchange suggests that inhibition is not related to a shift in set point, but is rather explained by a reduced V _max of Na⁺/H⁺ exchange and/or reduced affinity for protons at the internal membrane surface. It is suggested that kinase A as well as kinase C can mediate PTH inhibition of renal proximal tubular Na⁺/H⁺ exchange and that the relative importance of a particular regulatory cascade is determined by the PTH-concentration-dependent rates in the liberation of diacylglycerol (phospholipase C/kinase C) and cAMP (adenylate cyclase/kinase A).Abbreviations HEPES 4-(2-hydroxyethyl)-1-piperazineethane-sulphonic acid - EDTA ethylenediaminetetraacetic acid - FCS fetal calf serum - PTH parathyroid hormone - Tris 2-amino-2-hydroxyme-thylpropane-1,3-diol - BCECF 2,7-bis(2-carboxyethyl)-5,6-carboxyfluorescein - P_i inorganic phosphate - pH_i intracellular pH - Ca_i cytosolic free Ca²⁺ - BAPTA 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid - EGTA ethylene glycol-bis(-amino-ethylether)-N,N,N,N-tetraacetic acid - IP₃ inositol 1,4,5-trisphos-phate - DAG 1,2-diacylglycerol     

Mechanismus und Bedeutung der arteriolären Media-Hypertrophie/Hyperplasie bei der arteriellen Hypertonie

Summary The most common haemodynamic abnormality in human essential hypertension is an increase in systemic vascular resistance. Morphologic substrate for increased flow resistance is a narrowing of the lumen of arteriolar resistance vessels. During the course of essential hypertension, this is associated with an increase in wall (mainly media) thickness due to hypertrophy and hyperplasia of vascular smooth muscle cells. In contrast to concepts interpreting media thickening strictly as structural adaptation to increased perfusion pressure, various lines of evidence also point to pressure independent factors. In this context, extracellular factors such as growth factors as well as alterations in the activity of intracellular messenger systems must be considered. Recent studies suggest that substances generally known to act as vasoconstrictors such as angiotensin II, noradrenaline and arginine-vasopressin may also stimulate vascular smooth muscle cell growth and proliferation. Intracellular messenger systems with possible significance in the response to trophins and/or mitogens of vascular smooth muscle cells are phospholipase C, protein kinase C and the Na⁺/H⁺-antiport. These systems have been demonstrated to be altered in hypertension supporting the concept that one endogenous factor in human essential hypertension with pathophysiological significance, at least in a subgroup of patients, may be an enhanced reactivity of vascular smooth muscle cells to trophic and mitogenic stimuli. In this context, intracellular messenger systems such as phospholipase C, protein kinase C and/or the Na⁺/H⁺-antiport may play an important pathophysiological role.

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Abkürzungsverzeichnis G-Protein Guanin-Nukleotid-bindendes Protein - PLC Phospholipase C - PIP₂ Phosphatidylinosit-4,5-Diphosphat - PI Phosphatidylinosit - IP₃ Inosit-1,4,5-Triphosphat - DG Diacylglycerin - Kinase C Proteinkinase C - Na⁺ Natrium - K⁺ Kalium - H⁺ Wasserstoff - Ca⁺⁺ Calcium - Mg⁺⁺ Magnesium - Li⁺ Lithium     

Na+, K+-ATPase activity in neurons and glial cells of the olfactory cortex of the rat brain during the development of long-term potentiation

Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities were studied in neurons and glial cells of the olfactory cortex of the rat by quantitative cytophotometry in conditions of long-term potentiation (LTP), and significant changes in direction and extent were found. Na⁺, K⁺-ATPase activity decreased in neurons in the first 15 min after LTP, with subsequent elevation by 30 min. Mg²⁺-ATPase activity remained unchanged in these conditions. Glial cells showed significant increases in Na⁺, K⁺-ATPase activity in the initial period after LTP, with return to control by 30 min. Again, there were no significant changes in Mg²⁺-ATPase activity. The formation and persistence of LTP in neurons and glial cells was accompanied by significant changes in Na⁺, K⁺-ATPase activity, which were reciprocal in nature. Functional Neurochemistry Laboratory (Director N. A. Emel'yanov), I. P. Pavlov Institute of Physiology, Russian Academy of Sciences, St. Petersburg. Translated from Fiziologicheskii Zhurnal im. I. M. Sechenova, Vol. 81, No. 3, pp. 16–20, March, 1995.     

Aldosterone actions on basolateral Na+/H+ exchange in Madin-Darby canine kidney cells

In recent studies, there has been a re-evaluation of the polarity of Na⁺/H⁺ exchange in Madin-Darby canine kidney (MDCK) cells. This study was designed to examine aldosterone actions on basolaterally located Na⁺/H⁺ exchange of MDCK cell monolayers grown on permeant filter supports; pH_i was analysed in the absence of bicarbonate by using the pH-sensitive fluorescent probe 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein. Pre-exposure of MDCK cells to aldosterone led within 10–20 min to an alkalization of pH_i ( 0.3 pH unit); this effect is prevented by an addition of dimethylamiloride to the basolateral superfusate. Addition of aldosterone led to stimulation of the basolaterally located Na⁺/H⁺ exchange activity (Na⁺-dependent recovery from an acid load); this effect required preincubation (more then 3 min) and was observed at 0.1 nM aldosterone. Preexposure (15 min) of MDCK monolayers to phorbol 12-myristate 13-acetate also led to an activation of Na⁺/H⁺ exchange; pre-exposure to 8-bromo-cAMP led to inhibition of Na⁺/H⁺ exchange activity. An inhibitory effect of aldosterone was observed if Na⁺/H⁺ exchange activity was analysed in the presence of aldosterone; the highest inhibitory effects (20%–30%) occurred at concentrations of 5 nM and higher. Aldosterone-dependent inhibition does not require preincubation and is fully reversible; it was only observed at low (20 mM) but not at high Na⁺ concentrations (130 mM). The data suggest that aldosterone has an instantaneous inhibitory effect on basolaterally located Na⁺/H⁺ exchange activity under conditions of low Na⁺, but stimulates the rate of transport activity upon preincubation under conditions of physiological Na⁺ concentrations.     

Na+ entry and modulation of Na+/Ca2+ exchange as a key mechanism of TRPC signaling

Ion channels formed by canonical transient receptor potential (TRPC) proteins are considered to be key players in cellular Ca²⁺ homeostasis. As permeation of Ca²⁺ through TRPC homo- and/or heteromeric channels has been repeatedly demonstrated, analysis of the physiological role of TRPC proteins was so far based on the concept that these proteins form regulated Ca²⁺ entry channels. The well-recognized lack of cation selectivity of TRPC channels and the ability to generate substantial monovalent conductances that govern membrane potential and cation gradients were barely appreciated as a physiologically relevant issue. Nonetheless, recent studies suggest monovalent, specifically Na⁺ permeation through TRPC cation channels as an important event in TRPC signaling. TRPC-mediated Na⁺ entry may be converted into a distinct pattern of cellular Ca²⁺ signals by interaction with Na⁺/Ca²⁺ exchanger proteins. This review discusses current concepts regarding the link between Na⁺ entry through TRPC channels and cellular Ca²⁺ signaling.     

Relation of exocytotic release of γ-aminobutyric acid to Ca2+ entry through Ca2+ channels or by reversal of the Na+/Ca2+ exchanger in synaptosomes

The specific inhibitor of the -aminobutyric acid (GABA) carrier, NNC-711, {1-[(2-diphenylmethylene) amino]oxyethyl}-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride, blocks the Ca²⁺-independent release of [³H]GABA from rat brain synaptosomes induced by 50 mM K⁺ depolarization. Thus, in the presence of this inhibitor, it was possible to study the Ca²⁺-dependent release of [³H]GABA in the total absence of carrier-mediated release. Reversal of the Na⁺/Ca²⁺ exchanger was used to increase the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) to test whether an increase in [Ca²⁺]_i alone is sufficient to induce exocytosis in the absence of depolarization. We found that the [Ca²⁺]_i may rise to values above 400 nM, as a result of Na⁺/Ca²⁺ exchange, without inducing release of [³H]GABA, but subsequent K⁺ depolarization immediately induced [³H]GABA release. Thus, a rise of only a few nanomolar Ca²⁺ in the cytoplasm induced by 50 mM K⁺ depolarization, after loading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange, induced exocytotic [³H]GABA release, whereas the rise in cytoplasmic [Ca²⁺] caused by reversal of the Na⁺/Ca²⁺ exchanger was insufficient to induce exocytosis, although the value for [Ca²⁺]_i attained was higher than that required for exocytosis induced by K⁺ depolarization. The voltage-dependent Ca²⁺ entry due to K⁺ depolarization, after maximal Ca²⁺ loading of the synaptosomes by Na⁺/Ca²⁺ exchange, and the consequent [³H]GABA release could be blocked by 50 M verapamil. Although preloading the synaptosomes with Ca²⁺ by Na⁺/Ca²⁺ exchange did not cause [³H]GABA release under any conditions studied, the rise in cytoplasmic [Ca²⁺] due to Na⁺/Ca²⁺ exchange increased the sensitivity to external Ca²⁺ of the exocytotic release of [³H]GABA induced by subsequent K⁺ depolarization. Thus, our results show that the vesicular release of [³H]GABA is rather insensitive to bulk cytoplasmic [Ca²⁺] and are compatible with the view that GABA exocytosis is triggered very effectively by Ca²⁺ entry through Ca²⁺ channels near the active zones.     

Is there a Na+/Ca2+ exchanger in macrophages and in lymphocytes?

In two blood cell types, peritoneal murine macrophages and Jurkat cells (a human T cell line), we have examined whether a Na⁺/Ca²⁺ exchange was present and what could be its functional importance. In nonstimulated macrophages, the intracellular Ca²⁺ concentration, [Ca²⁺]_i, was unchanged when Li⁺ was substituted for external Na⁺. However, after stimulation by platelet-activating factor (PAF), the Ca²⁺ response was larger when the extracellular solution contained Li⁺ rather than Na⁺ ions. In stimulated macrophages, the rate of Ca²⁺ extrusion was smaller in a Li⁺-than in a Na⁺-containing medium. The net electrochemical gradient for ionic movements through the Na⁺/Ca²⁺ exchanger, during the course of the response of macrophages to PAF, was determined by combining the measurements of membrane potential (in patch-clamp), of [Ca²⁺]_i (with fura-2), and of the intracellular Na⁺ concentration (with sodium-binding benzofuran isophthalate). These results show that macrophages possess a Na⁺/Ca²⁺ exchange that only functions as a Ca²⁺ extruder, and this only when [Ca²⁺]_i has been increased, for instance following PAF stimulation. In T lymphocytes, before or after stimulation by an anti-CD3 antibody, no Na⁺/Ca²⁺ activity could be detected by measuring either [Ca²⁺]_i, or the rate of Ca²⁺ extrusion. Even if a Na⁺/ Ca²⁺ exchanger was present in these cells, its equilibrium potential would be such that it would not allow Ca²⁺ influx but only Ca²⁺ extrusion.