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.     

Intracellular Na+ measurements in smooth muscle using SBFI – changes in [Na+], Ca2+ and force in normal and Na+-loaded ureter

 Our understanding of the control and effects of intracellular [Na⁺] ([Na⁺]_i) in intact smooth muscle is limited by the lack of data concerning [Na⁺]_i. The initial aim of this work was therefore to investigate the suitability of using the Na⁺-sensitive fluorophore SBFI in intact smooth muscle. We find this to be a good method for measuring [Na⁺]_i in ureteric smooth muscle. Resting [Na⁺]_i was found to be around 10 mM and rose to 25 mM when the Na⁺-K⁺-ATPase was inhibited by ouabain. This relatively low [Na⁺]_i in the absence of Na⁺-K⁺-ATPase suggests that other cellular processes, such as Na⁺-Ca²⁺ exchange, play a role in maintaining [Na⁺]_i under these conditions. Simultaneous measurements of [Na⁺]_i or [Ca²⁺] _i and force showed that Na⁺-Ca²⁺ exchange can play a functional role in ureteric smooth muscle. We found that the greater the driving force for Na⁺ exit and hence Ca²⁺ entry, the larger the contraction. In addition the Na⁺-Ca²⁺ exchanger activity under these conditions was found to be pH sensitive: acidification reduced the contraction and concomitant changes in [Ca²⁺] and [Na⁺]_i. We conclude that SBFI is a useful method for monitoring [Na] in smooth muscle and that Na⁺-Ca²⁺ exchange may play a functional role in the ureter. Received: 26 August 1997 / Received after revision: 27 October 1997 / Accepted: 28 October 1997     

Renal sodium/calcium exchange; a vasodilator that is defective in salt‐sensitive hypertension

The Na⁺ : Ca²⁺ exchanger is an important plasma membrane ion transport pathway that plays a major role in controlling [Ca²⁺]_i. In smooth muscle cells, it may function as a Ca²⁺ extrusion pathway and may help lower [Ca²⁺]_i in response to vasoconstrictor‐induced increases in [Ca²⁺]_i. It may also extrude [Ca²⁺]_i and lead to vasodilation in response to vasodilators. Our recent studies have been performed to determine the existence and regulation of the Na⁺ : Ca²⁺ exchanger in renal contractile cells which include afferent and efferent arterioles and mesangial cells. Exchanger activity is present in all three of these contractile elements but is higher in afferent arterioles vs. efferent arterioles. We have also examined the role of altered regulation of the exchanger in the SHR and in salt‐sensitive hypertension. With the establishment of high blood pressure, Na⁺ : Ca²⁺ exchanger activity is reduced in afferent but not in efferent arterioles in both models of hypertension. Other works in cultured mesangial cells and freshly dissected afferent arterioles, have shown that protein kinase C (PKC) up‐regulates the Na⁺ : Ca²⁺ exchanger from Dahl/Rapp salt‐resistant rats while it fails to do so in arterioles and mesangial cells from salt‐sensitive rats. This defect in PKC regulation of Na⁺ : Ca²⁺ exchange is the result of a loss of PKC‐mediated translocation of the exchanger to the plasma membrane in S mesangial cells. Thus, a defect in the PKC‐Na⁺ : Ca²⁺ exchanger‐translocation pathway may cause dysregulation of [Ca²⁺]_i and help explain the dramatic decrease in GFR that occurs in this model of hypertension.     

Mechanisms of calcium transport across the basolateral membrane of the rabbit cortical thick ascending limb of Henle's loop

Although net Ca²⁺ absorption takes place in the thick ascending limb of Henle's loop, detailed mechanisms are unknown. Because it has been reported that the Ca²⁺ entry step across the luminal membrane is mediated by Ca²⁺ channels inserted by stimulation with parathyroid hormone, we studied the mechanism of Ca²⁺ transport across the basolateral membrane of rabbit cortical thick ascending limb (CTAL) perfused in vitro by using microscopic fluorometry of cytosolic Ca²⁺ ([Ca²⁺]_i) with fura-2. The resting [Ca²⁺]_i in this segment was 49.8±4.5 nmol/l. Neither Na⁺ removal from the bathing solution nor addition of ouabain (0.1 mmol/l) to the bath increased [Ca²⁺]_i, indicating that a Na⁺/Ca²⁺ exchanger in the basolateral membrane may not contribute in any major way to [Ca²⁺]_i of CTAL. To confirm our technical accuracy, similar protocols were conducted in the connecting tubule, where the existence of a Na⁺/Ca²⁺ exchanger has been reported. In this segment, Na⁺ removal from the bath increased cell Ca²⁺ from 148.6 ±6.4 nmol/l to 647.6±132.0 nmol/l, confirming the documented fact. [Ca²⁺]_i in the CTAL was markedly increased when 1 mmol/l NaCN was added to the bath in the absence of glucose. Calmodulin inhibitors (trifluoperazine or W-7) increased [Ca²⁺]_i. When the bath pH was made alkaline, [Ca²⁺]_i was also increased. This response was abolished when Ca²⁺ was eliminated from the bath, indicating that the Ca²⁺ entry across the basolateral membrane is dependent on bath pH. Increase in [Ca²⁺]_i induced by an alkaline bath was inhibited by increased the bath K⁺ from 5 nmol/l to 50 mmol/l, suggesting that the Ca²⁺ entry system is voltage-dependent. However, the pH-dependent [Ca²⁺]_i increase was unaffected by 0.1–10 mol/l nicardipine in the bath. We conclude that Ca²⁺ transport across the basolateral membrane of CTAL is mediated by a pump-and-leak system of Ca²⁺ rather than a Na⁺/Ca²⁺ exchanger secondarily linked to a Na⁺, K⁺ pump.     

The effects of calcium and calcium channel blockers on sodium pump

The effects of 10 mM Ca²⁺ and Ca²⁺ channel blockers verapamil, diltiazem and flunarizine on the ouabain-sensitive electrogenic Na⁺, K⁺ pump activity of mouse diaphragm muscle fibres enriched with Na⁺ were compared with the changes in cytosolic [Ca²⁺]. The electrogenic Na⁺ pump activity produced by adding K⁺ to muscles previously bathed for 4 h in a K⁺-free, 2-mM [Ca²⁺] solution increased the resting membrane potential by about 18 mV. This hyperpolarization was completely inhibited after 10 min incubation in 10 mM Ca²⁺. Verapamil 10^–5M, 10^–5M diltiazem and 10^–7 M flunarizine effectively prevented the effect of elevated [Ca²⁺]. At these concentrations, these drugs did not affect the K⁺-induced hyperpolarization. In mouse diaphragm, the basal cytosolic [Ca²⁺] measured by the fluorescent indicator 1-[2-(5-carboxyoxazol-2-yl)-6-aminobenzofuran-5-oxy]2-(2-amino 5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (fura-2/AM) was 261±6 nM. After 4 h in a Liley K⁺-free, 2 mM [Ca²⁺] solution, the cytosolic [Ca²⁺] increased to 314±28 nM. Increase in [Ca²⁺] from 2 to 10 mM caused a twofold increase of cytosolic [Ca²⁺] to 637±26 nM. This rise was, like the Ca²⁺-induced inhibition of electrogenic pump, prevented by 10^–5 M verapamil, 10^–5M diltiazem and 10^–7 M flunarizine. The results suggest that substances which block Ca²⁺ entry into the cell prevent the Ca²⁺ induced inhibition of the Na⁺ pump.     

Carboxyeosin decreases the rate of decay of the [Ca2+]i transient in uterine smooth muscle cells isolated from pregnant rats

 In myometrial smooth muscle cells the rate of decline of intracellular calcium ([Ca²⁺]_i) is determined by Ca²⁺ extrusion from the cell and uptake into intracellular stores. The relative quantitative contribution of these processes however, has not been established. We therefore examined the effect of the sarcolemmal Ca²⁺ pump inhibitor, carboxyeosin, on the rate of the [Ca²⁺]_i transient decline in myocytes isolated from pregnant rat uterus. Indo-1 was used in conjunction with the whole-cell patch-clamp technique to measure [Ca²⁺]_i simultaneously with transmembrane calcium current (I _Ca). [Ca²⁺]_i transients were elicited by repetitive membrane depolarization to simulate the natural pattern of uterine electrical activity. The rate of [Ca²⁺]_i removal was calculated from the falling phase of the [Ca²⁺]_i transient. Pre-treatment of the cells with 2 μM carboxyeosin led to a marked decrease in the rate of [Ca²⁺]_i transient decay, suggesting that the sarcolemmal Ca²⁺ pump is involved in the calcium extrusion process. Removal of the extracellular Na also decreased the rate of [Ca²⁺]_i decay, indicating an important role for the Na⁺/Ca²⁺ exchange. When both the sarcolemmal Ca²⁺ pump and Na⁺/Ca²⁺ exchange were inhibited the cell failed to restore [Ca²⁺]_i after the stimulation. Comparison of the rate constants of [Ca²⁺]_i decay in control conditions and after carboxyeosin treatment shows that approximately 30% of [Ca²⁺]_i decay is due to the sarcolemmal calcium pump activity. The remaining 70% can be attributed to the activity of Na⁺/Ca²⁺ exchanger and the intracellular calcium stores. Received: 17 July 1998 / Received after revision: 23 September 1998 / Accepted: 25 September 1998     

Cytosolic free calcium in single microdissected rat cortical collecting tubules

Cytosolic free Ca²⁺ ([Ca²⁺]_i) was measured in single fragments of rat cortical collecting tubule (CCT) by using fura-2 and a tubule superfusion device. Under basal conditions, i.e. with 1 mM of external Ca²⁺ ([Ca²⁺]_o), the average steady state [Ca²⁺]_i was 179±16 nM (n=44 tubules). Random alterations of [Ca²⁺]_o between 0 mM and 4 mM led to corresponding variations in steady state [Ca²⁺]_i levels, which were linearly correlated with [Ca²⁺]_o (average slope 93±34 nM [Ca²⁺]_i per 1 mM [Ca²⁺]_o for six tubules). In contrast, [Ca²⁺]_i was little affected by decreasing external Na⁺ concentration. Cell membrane depolarization with 100 mM of external K⁺ induced a sustained drop in [Ca²⁺]_i (21% as an average). The data suggest that steady state [Ca²⁺]_i in CCT cells resulted from a non-saturable passive entry of calcium ions across cell membranes balanced with an active extrusion by calcium ATPase (pump and leak mechanism). The passive component cannot be accounted for either by Na⁺/Ca²⁺ exchangers nor by voltage-dependent calcium channels; it is best explained by the presence of voltage-independent calcium channels in cell membranes.     

External calcium sensitivity of low sodium contractures in the control and hypertrophied right ventricle of the ferret

The existence of possible differences of calcium (Ca²⁺) fluxes through the sarcolemmal sodium-calcium (Na⁺/Ca²⁺) exchanger during hypertrophy has been tested by comparing the characteristics of the contracture—as an indicator of the intracellular Ca²⁺ concentration—induced by partial or total withdrawal of external sodium (Na⁺), in the absence of external potassium, in the right ventricular trabeculae of adult ferret hearts. Pressure-overload was induced by pulmonary artery clipping and led to an increase of the right ventricular weight of 60%. At an external Ca²⁺ concentration ([Ca²⁺]₀) of 3 mm, the dependence of the contractures on extracellular sodium concentration ([Na⁺]₀), the rate of tension development, the time course of spontaneous relaxation and the time course for the repriming of the contracture were unchanged by hypertrophy. However, the relationship between [Ca²⁺]₀ and contracture amplitude at various [Na⁺]₀ showed that the apparent affinity of the contracture for [Ca²⁺]₀ was decreased in hypertrophied preparations. Thus, in 0 mM [Na⁺]₀, half-maximal contracture was induced at a [Ca²⁺]₀ of 0.012 ± 0.016 mm and 0.171 ± 0.021 mM in control (n= 11) and hypertrophy (n= 12) respectively (P < 0.001). Although these data may be indicative of a decreased Ca²⁺ influx through the Na⁺/Ca²⁺ exchanger, it cannot be excluded that intracellular buffering mechanism may also be involved in this differential response to [Na⁺]₀ withdrawal.     

Effects of calcium and sodium on contracture tension in the smooth muscle of the rat portal vein

Summary The purpose of the present study was to determine the quantitative relationship between membrane potential (or [K⁺]₀) and contracture tension in the smooth muscle of the rat portal vein, and to examine the influence of Ca⁺⁺ and Na⁺ on this relationship. However, electrical all-or-none responses were successfully abolished only in Na⁺-free sucrose-Krebs due to hyperpolarization and in K⁺-high Krebs due to depolarization. It did not seem possible to eliminate spike discharge at intermediate levels of membrane potential without the simultaneous loss of contractility. In the hyperpolarized state the muscle remained relaxed despite the very low levels of [Na⁺]₀ and despite increases in [Ca⁺⁺]₀ up to 20 mM. The depolarized portal vein developed a contracture which was intimately dependent on [Ca⁺⁺]₀, the threshold concentration being of the order of 0.1 to 0.3 mM. Spike-induced, phasic contractions showed a similar Ca⁺⁺-dependence. Variations in [Na⁺]₀ had only a slight and irregular influence on the Ca⁺⁺ dose-response curve of the depolarized muscles.Differences in the effects of Na⁺ on the rate of rise and the rate of fall of the contracture tension, respectively, suggested that Na⁺ is more important for the removal of Ca⁺⁺ from the contractile system than for the supply of Ca⁺⁺ to the system. With regard to the interaction of Ca⁺⁺ and Na⁺ in the excitation-contraction coupling the vascular smooth muscle seemed to differ from both heart muscle and skeletal muscle.The present study was supported by grants from the Swedish Medical Research Council (B 70-14x-28-06 A), from Air Force Office of Scientific Research through the European Office of Aerospace Research, OAR, United States Air Force under Contract F 1052-68-C-0044, from U.S. Public Health Service (HE-05678-08), from AB Hässle, Göteborg, and from the Deutsche Forschungsgemeinschaft (Bi 122/1).     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

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.     

Simultaneous measurements of cytosolic pH and calcium interactions in bovine lactotrophs using optical probes and four-wavelength quantitative video microscopy

The properties of pH and calcium homeostasis have been investigated in single bovine lactotrophs by the use of the fluorescent indicators 2,7-bis(carboxyethyl)-carboxyfluorescein (BCECF) and fura-2 respectively. A method of simultaneous recording from both dyes loaded in the same cell was used. Despite slight crosstalk between the two dyes, physiologically relevant information about the interrelationship between pH and calcium homeostasis was obtained. Three types of interactions were recorded. First, an increase in calcium due to the discharge of intracellular stores by thyrotropin-releasing hormone resulted in no change in cytosolic pH. Secondly, alkalinization by the addition of a weak base, NH₄Cl, induced a large transient (around 1000 nM) and a small (a few tens of nanomoles per liter) sustained increase in cytosolic calcium. The former is partly due to release from agonist-sensitive stores. Thirdly, upon the removal of NH₄Cl the cytoplasm became acidic, which induced a release of calcium from intracellular stores in some cells. In addition we demonstrate that the recovery from acid load is sensitive to extracellular Na⁺, suggesting the presence of Na⁺/H⁺ exchange mechanisms in bovine lactotrophs. Interestingly we have also found that, at rest, removal of Na⁺ from the bathing medium results in a decrease in resting [Ca²⁺]_i, paralleled by a reduction in pH_i. This suggests a role for Na⁺/H⁺ exchange in determining resting [Ca²⁺]_i.     

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.     

Contribution of Na+/Ca2+ exchanger to the regulation of myogenic tone in isolated rat small arteries

The contribution of the Na⁺/Ca²⁺ exchanger to the myogenic vascular tone was examined in rat isolated skeletal muscle small arteries (A_SK) with pronounced myogenic tone and mesenteric small arteries (A_MS) with little myogenic tone. Myogenic tone was assessed by the vascular inner diameter at transmural pressures of 40 and 100 mmHg. To depress the Na⁺/Ca²⁺ exchanger, the extracellular Na⁺ concentration ([Na⁺]o) was lowered from 143 to 1.2 mM by substituting choline‐Cl for NaCl. The A_SK developed significant myogenic tone and constricted further in low [Na⁺]o. Nifedipine (1 μM ) reduced both myogenic tone and low [Na⁺]o‐induced contraction. Because the membrane potential of A_SK was not changed by low [Na⁺]o (–35 ± 2 mV at 143 mM [Na⁺]o, ?37 ± 3 mV at 1.2 mM [Na⁺]o), depolarization‐induced Ca²⁺ influx was not a cause of the low [Na⁺]o‐induced contraction. The A_MS did not develop significant myogenic tone. Although low [Na⁺]o also constricted A_MS, the magnitude of constriction was significantly weaker than that in A_SK (17 ± 4 vs. 47 ± 6%, P < 0.01, at 58 mM Na⁺). With Bay K 8644, A_MS developed myogenic tone, and low [Na⁺]o‐induced constriction was significantly increased. In conclusion, Na⁺/Ca²⁺ exchanger may play an important role in regulating myogenic tone, likely via mediating Ca²⁺‐extrusion.     

Topography of the respiratory and circulatory responses to acetylcholine and nicotine on the ventral surface of the medulla oblongata

The effects of different [Ca²⁺]₀ in the presence of variable [Na⁺]₀ (100 to 37 mM) on the slow action potentials were studied in isolated rat atria in the presence of 25 mM K⁺ plus 10^–6 M isoproterenol. Two modes of electrical stimulation were used, sustained stimulation at 0.5 Hz (steady-state mode), and stimulation by a single stimulus after rest periods of 2 to 5 min (1st response mode). With the first type of stimulation, and for [Ca²⁺]₀ between 0.5 and 10 mM and [Na⁺]₀ between 100 and 65 mM, the slow action potential overshoot increased linearly with the logarithm of the [Ca²⁺]₀ (28.4 mV per 10-fold change in [Ca²⁺]₀). However, elevation of [Ca²⁺]₀ above 10 mM caused depression of the overshoot. This overshoot depression by high [Ca²⁺]₀ was accentuated if [Na⁺]₀ was decreased to 37 mM. With the 1st response mode of stimulation, the overshoot — log [Ca²⁺]₀ relationship was linear within a wider [Ca²⁺]₀ range (0.5 to 25 mM), and was less sensitive to further decreases in [Na⁺]₀. It is suggested that rat atria slow action potentials are generated by selective influx of Ca²⁺ but not Na⁺, and that the depression of amplitudes observed a high [Ca²⁺]₀ and low [Na⁺]₀ is due to a decrease in the Na²⁺ exchange mechanism which results in a higher [Ca²⁺]_i, and not to a decrease in the inward Na⁺ current. Adenosine produced a parallel downward displacement of the overshoot to log [Ca²⁺]₀ relationship. This adenosine effect was concentration dependent, independent of [Ca²⁺]₀ and the frequency of stimulation. In contrast, the effects of 0.4 mM La³⁺ were dependent on the [Ca²⁺]₀ and on the frequency of stimulation. Adenosine also produced a downward shift of the relationship between maximal rate of rise of the slow action potential and membrane resting potential in such a manner that its effects cannot be attributed to changes in inactivation potential of the slow channels. Hence, adenosine and La³⁺ depress the slow Ca²⁺ action potentials by two different mechanisms. Adenosine may act by 1) decreasing the number of functional slow channels, 2) decreasing the conductance of the individual channels, or 3) altering the kinetic properties of these channels. La³⁺ may act by competing with Ca²⁺ for membrane binding sites. These membrane binding sites appear to be characterized by frequency dependence.Supported by Grants NHLBI 10384 and AHA 74-942.Dr. Luiz Belardinelli is the recipient of a fellowship No. 1112. 1273/75 from Conselho Nacional de Desenvolvimento Cientifico e Technologico (CNPq) and Fundacao Universitaria de Cardiologia do Rio Grande do Sul, Brasil.     

The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes

Glutamate is shown to induce increases in intracellular Ca²⁺ concentrations ([Ca²⁺]_i), increases in⁴⁵Ca²⁺ influx, decreases in the activity of Na⁺, K⁺,-ATPase activity, and activation of the Na⁺/Ca²⁺ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with α-tocopherol, superoxide dismutase, and ganglioside GM₁ normalized [Ca²⁺]_i,⁴⁵Ca²⁺, influx, and Na⁺, K⁺-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM₁ activated the Na⁺/K⁺ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca²⁺ influx into cells, decreases in Na⁺, K⁺-ATPase activity, and significant increases in [Ca²⁺]_i, though this could be prevented by antioxidants and gangliosides. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 85, No. 4, pp. 488–496, April. 1999.     

In the squid axon Na+/Ca2+ exchanger the state of the Cai-regulatory site influences the affinities of the intra- and extracellular transport sites for Na+ and Ca2+

In squid axons, intracellular Mg²⁺ reduces the activity of the Na⁺/Ca²⁺ exchanger by competing with for its regulatory site. The state of the Ca_i-regulatory site (active–inactive) also alters the apparent affinity of intra- and extracellular transport sites. Conditions that hinder the binding of (low pH_i, low [Ca²⁺]_i, high [Mg²⁺]_i) diminish the apparent affinity of intracellular transport sites, in particular for Na_i due to its synergism with H⁺ inhibition, but less noticeably for because of its antagonism towards () and inhibitions. These are kinetic effects unrelated to the true affinity of the sites. With the Ca_i-regulatory site saturated, the intracellular transporting sites are insensitive to [H⁺]_i and to ATP. Likewise, the state of the Ca_i-regulatory site (activated or inactivated) influences the affinity of the extracellular Ca_o and Na_o-transport sites (trans effects). In this case, the effects are opposite to those predicted by any of the transport schemes proposed for the Na⁺/Ca²⁺exchanger; i.e. its mechanism remains unexplained. In addition to their intrinsic importance for a full understanding of the properties of the Na⁺/Ca²⁺ exchanger, these findings show a new way by which the state of the Ca_i-regulatory site may determine net movements of Ca²⁺ through this system.     

Dynamics of Ca2+ i and pHi in Ehrlich ascites tumor cells after Ca2+-mobilizing agonists or exposure to hypertonic solution

 Intracellular free calcium concentration ([Ca²⁺]_i) and intracellular pH (pH_i) were monitored in Ehrlich ascites tumor cells using Fura-2 or 2′,7′,-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), or both probes in combination. An increase in [Ca²⁺]_i induced by thrombin or bradykinin, agonists known to elicit transient cell shrinkage in these cells, evoked a transient intracellular acidification, followed by an alkalinization. The latter was due to activation of a Na⁺/H⁺ exchanger and was inhibited under conditions preventing agonist-induced cell shrinkage without preventing the increase in [Ca²⁺]_i. In contrast, a smaller, slower increase in [Ca²⁺]_i elicited by thapsigargin did not cause cell shrinkage, and did not activate the Na⁺/H⁺ exchanger. Exposure to hypertonic solution was not associated with an increase in [Ca²⁺]_i, but elicited an intracellular alkalinization similar to that induced by thrombin or bradykinin, via activation of the Na⁺/H⁺ exchanger. Thus, activation of the exchanger by the Ca²⁺-mobilizing agonists is suggested to be secondary to the cell shrinkage induced by these compounds. NH₄Cl-induced intracellular alkalinization resulted in an increase in [Ca²⁺]_i, apparently via stimulation of Ca²⁺ influx, whereas shrinkage-induced intracellular alkalinization did not stimulate Ca²⁺ influx. Thus, cell shrinkage appears to inhibit the Ca²⁺ influx otherwise resulting from alkalosis. In agreement with that notion, thapsigargin-induced Ca²⁺ influx was inhibited by cell shrinkage. Received: 6 January 1998 / Received after revision: 10 March 1998 / Accepted: 11 March 1998     

Fluorescence and Laser Photon Counting: Measurements of Epithelial [Ca2+]i or [Na+]i with Ciliary Beat Frequency

We describe a system we developed that enabled simultaneous measurements of either epithelial calcium ion concentration ([Ca²⁺]i) or sodium ion concentration [Na⁺]i with the ciliary beat frequency (CBF) in native ciliated epithelia using either Fura-2 (AM) or SBFI (AM) ratiometric fluorescence photon counting along with nonstationary laser light scattering. Studies were performed using native epithelial tissues obtained from ovine tracheae. The dynamic range of the laser light-scattering system was determined by a simulated light beating experiment. The nonstationary CBF was demonstrated by the time-frequency analysis of the raw photon count sequences of backscattered heterodyne photons from cultured and native epithelia. Calibrations of calcium and sodium ion concentrations were performed using the respective Fura-2 and SBFI impermanent salts as well as in native epithelia. The cumulative responses of 10^–6, 10^–5, and 10^–4} M nifedipine on [Ca²⁺]i together with the CBF as well as the cumulative responses of 10^–5, 10^–4, and 10^–3 M amiloride on [Na⁺]i together with the CBF were also determined. Nifedipine decreased [Ca²⁺]i but had no effect on CBF. Amiloride decreased [Na⁺]i and CBF. Stimulation of CBF corresponded with either an increase of [Na⁺]i or an increase of [Ca²⁺]i. Decreases of [Na⁺]i or substantial decreases of [Ca²⁺]i were associated with decreases in the CBF. These data demonstrate the utility of this system for investigating the regulatory mechanisms of intracellular ions dynamics and the CBF in native epithelia. © 1998 Biomedical Engineering Society. PAC98: 8780+s, 8722-q, 4262Be     

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.