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.     

Potassium transport in epithelial cells isolated from small intestine of the chicken

The transport of potassium has been studied in epithelial cells isolated from chicken small intestine using⁸⁶Rb as a tracer for K⁺. (i) The uptake studies revealed that about 60% of the total K⁺ net flux is inhibited by ouabain and therefore mediated by the Na⁺–K⁺-ATPase. About 20% of the ouabain-insensitive K⁺ net influx was inhibited by furosemide, bumetanide and by either Na⁺ or Cl^– removal from the incubation solution, suggesting that a Na⁺/Cl^–/K⁺ cotransport system might be present in chicken enterocytes. (ii) The efflux of K⁺ was measured from cells preloaded with⁸⁶Rb. K⁺ efflux was inhibited by Ba²⁺, quinine and verapamil; it was stimulated by A23187, and it was unaffected by 3,4-diaminopyridine. Apamin, that has no effect on basal rates of K⁺ efflux, abolished the effect of A23187. These findings suggest that K⁺ efflux appears to occur through Ca²⁺-activated K⁺ channels.     

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⁺.     

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.     

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     

Single calcium-activated potassium channel in cultured mammary epithelial cells

The properties of Ca²⁺-activated K⁺ channels in mouse mammary epithelial cells in primary culture were studied by the patch-clamp technique. In cell-attached patches, spontaneous channel openings were sometimes observed; the slope conductance of the currents was about served; the slope conductance of the currents was about 12 pS at negative membrane potentials with a physiological solution (152 mM Na⁺, 5.4 mM K⁺) in the pipette. External application of A23187, a calcium ionophore, activated this channel. In excised inside-out patches, the channel was activated by increasing the internal Ca²⁺ concentration (10^–7 to 10^–6 M). No voltage dependence of the channel activity was observed. Internal Na⁺ blocked the outward K⁺ current in a voltage dependent manner and this block led to the non-linear I–V relationship at positive membrane potentials. The channel was blocked by internal Ba²⁺ (0.1 mM) and tetracthylammonium (TEA⁺, 20–50 mM). Ba²⁺ reduced the open probability but not the single channel conductance, whereas TEA⁺ reduced the single channel conductance. The single channel conductance of this channel, measured from the inward current with a high-K⁺ solution (150 mM K⁺) in the pipette, was large (about 40 pS), and showed inward rectification. These results suggest that this channel is different from the usual small conductance Ca²⁺-activated K⁺ channels observed in many other cells.     

Transcellular sodium transport and basolateral rubidium uptake in the isolated perfused cortical collecting duct

The relation between transcellular Na⁺ absorption, intracellular Na⁺ concentration and Na⁺/K⁺-ATPase activity (the last estimated by the rubidium uptake across the basolateral cell membrane) was examined in the different cell types of the rabbit cortical collecting duct (CCD). Experiments were performed on isolated perfused CCD in which Na⁺ absorption was varied by perfusing the tubule with solutions containing different Na⁺ concentrations (nominally Na⁺-free, 30 mM and 144 mM). Experiments were terminated by shock-freezing the tubules during perfusion. Precisely 30 s before shock-freezing, the K⁺ in the bathing solution was exchanged for Rb⁺. Intracellular element concentrations, including Rb⁺, were determined in freeze-dried cryosections of the tubules using energy-dispersive X-ray analysis. Increasing Na⁺ concentration in the perfusion solution caused significant rises in intracellular Na⁺ concentration and Rb⁺ uptake of principal cells. Principal cell Na⁺ and Rb⁺ concentrations were 7.8±0.9 and 7.0±0.8 mmol/kg wet weight respectively, when the perfusion solution was Na⁺-free, 10.1±0.7 and 11.6±0.6 mmol/kg wet weight with 30 mM Na⁺ in the perfusion solution, and 14.5±1.5 and 14.9 ±0.9 mmol/kg wet weight with 144 mM Na⁺ in the perfusion solution. In contrast, a comparable relationship between lumen Na⁺ concentration, intracellular Na⁺ concentration and basolateral Rb⁺ uptake was not seen in intercalated cells. These results support the notion that principal, but not intercalated, cells are involved in transepithelial Na⁺ absorption. In addition, the data demonstrate that apical Na⁺ entry and basolateral Na⁺/K⁺-AT-Pase activity are closely coupled in principal cells of the rabbit CCD. A rise in lumen Na⁺ concentration leads to increased Na⁺ entry and augmented intracellular Na⁺ concentration, which then secondarily stimulates active basolateral Na⁺/K⁺(Rb⁺) exchange.     

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.     

Fast Na+ current in circular smooth muscle cells of the large intestine

Whole-cell voltage clamp was carried out on freshly dispersed single smooth muscle cells from adult rat and human colons to investigate the regulation of the Ca²⁺ channels. In this study, we unexpectedly discovered the existence of a fast Na⁺ channel current. With normal physiological salt solution (PSS) plus 4-amino-pyridine (3 mM) in the bath and high-Cs⁺ solution in the pipette to inhibit outward K⁺ currents, an inward current possessing fast and slow components was observed when the cell membrane was depolarized to a value more positive than –20 mV from a holding potential of –100 mV. When Ca²⁺ ions were removed from the PSS, or when nifedipine (10 M) and Ni²⁺ (30 M) were simultaneously applied, the slow component disappeared and the fast component remained. The fast current component became almost completely inactivated within 10 ms. This fast component was dependent on extracellular Na⁺ concentration and was inhibited by tetrodotoxin (TTX) dose dependently (IC₅₀ of 130 nM in rat and 14 nM in human). These results suggest that the slow component of inward current was a Ca²⁺ channel current, whereas the fast component was a TTX-sensitive fast Na⁺ channel current. The threshold voltage, the voltage for peak current, and the reversal potential for the fast Na⁺ current were, respectively, about –50, –20, and + 50 mV in rats, and –40, 0, and + 60 mV in humans. The incidence of cells possessing fast Na⁺ currents depended on the region of the colon. In rat proximal colon, the incidence was 64% (14 out of 22 cells tested); in distal colon, it was 10% (2 out of 21 cells tested). In humans, the incidence in the ascending colon was 73% (16 out of 22 cells tested), and in the descending colon was 22% (7 out of 32 cells tested). The densities of fast Na⁺ and Ca²⁺ currents were 3.2 and 4.5 pA/pF in rats and 1.0 and 1.4 pA/pF in humans, respectively. The ratio of both current densities (Na⁺ vs Ca²⁺) was 0.71, in both rats and humans. We conclude that the major ion channels associated with the generation of inward currents in the circular smooth muscle cells of rat and human colon are voltage-dependent Ca²⁺ channels and TTX-sensitive Na⁺ channels. The fast Na⁺ current may facilitate propagation of excitation.     

Na/Ca exchangers in collecting cells of rat kidney

Single pieces of fura-2-loaded cortical collecting tubule (CCT) isolated either from normal or adrenalectomized (ADX) rats were superfused in vitro, and the cytosolic calcium concentration ([Ca²⁺]_i) was calculated from fluorescence recordings. The effects of altering the sodium gradient across cell membranes were investigated. Switching external sodium from 164 mM to 27 mM (low [Na⁺]_o) had little effect on [Ca²⁺]_i in normal tubules (106±9 versus 101±9 nM, n=15) whereas it resulted in a large peak of [Ca²⁺]_i in CCT from ADX-rats (270±32 versus 135±11 nM, n=21). Since CCT from ADX rats are known to have a reduced Na-pump activity, the effect of ouabain treatment on CCT from normal rats was also tested. When CCT from normal rats were exposed to 1 mM of ouabain in the presence of 164 mM of [Na⁺]_o, [Ca²⁺]_i increased only moderately (123±15 versus 111±11 nM, n=13); when the low [Na⁺]_o solution was applied to these ouabain-treated tubules, a large and transient increase in [Ca²⁺]_i was obtained (287±38 versus 123±15 nM, n=13). This response was absent with [Ca²⁺]_o=0. The data suggest the presence of 3 Na⁺/1 Ca²⁺ exchangers in cell membranes of rat CCT. The calcium flux equation derived by Läuger for the exchanger indicates a non-linear relationship between net calcium flux and driving force which could account for the difference observed here between the poor effect of applying either low [Na⁺]_o or ouabain alone and the large peak of [Ca²⁺]_i induced by combining these two conditions.     

Sodium-activated potassium current in sensory neurons: a comparison of cell-attached and cell-free single-channel activities

Single-channel currents from Na⁺-dependent K⁺ channels (K_Na) were recorded from cell-attached and inside-out membrane patches of cultured avian trigeminal ganglion neurons by means of the patchclamp technique. Single-channel properties, such as the high elementary conductance and the occurrence of subconductance levels, were unchanged after the patches had been excised from the cells, indicating that they are not under the control of soluble cytoplasmic factors. In cellattached recordings at the cell resting potential the degree of K_Na activity, measured as the probability of the channel being open, P _o, was low in most cases (around 0.01) and similar to that observed in the inside-out configuration when the bath solution contained concentrations of Na⁺ around 30 mM and of K⁺ close to the physiological intracellular levels. However, in some cell-attached patches P _o was high (around 0.2) and comparable to the values measured in cell-free recordings with high Na⁺ concentrations in the bath (100 mM). The excision of a highactivity patch in the presence of 30 mM Na⁺ resulted in a fall of P _o in about 20 s, which is consistent with the wash-out of a soluble cytoplasmic molecule. After the excision all K_Na displayed a similar Na⁺ sensitivity, irrespective of the degree of activation observed in the cellattached mode. In inside-out patches the P _o values observed in the presence of either low or high concentrations of Na⁺ in bath solutions were not modified by internal Ca²⁺ (0.8–8.5 M). The variable degree of K_Na activation observed in cell-attached recordings suggests that either internal Na⁺ concentrations reach very high levels close to the membrane, or soluble factor(s) are involved in the modulation of K_Na activity: under such conditions, the Na⁺-activated K⁺ current may contribute to the maintenance of the resting membrane potential and to control neuronal membrane excitability.     

The stimulus-secretion coupling of glucose-induced insulin release

The effect of glucose upon the handling of²²Na⁺ by pancreatic islets was investigated. Using a triple-isotope technique, the apparent concentration of Na⁺ in islet cells was estimated at 50–75 mM. The pattern of²²Na⁺ efflux from perifused islets indicates that this intracellular Na⁺ load is compartmentalized among a small, possibly organelle-bound pool characterized by a low fractional turnover rate (5%/min) and a large, presumably cystosolic pool displaying a much higher fractional turnover rate (20–34%/min). Glucose provokes a rapid, pronounced and sustained increase in the fractional outflow rate of Na⁺ across the plasma membrane and, under steady-state conditions, moderately reduces the concentration of Na⁺ inside the islet cells. The glucose-induced increase in Na⁺ outflow rate, which is also observed in response to glyceraldehyde and does not require the presence of extracellular Ca²⁺, might be mediated, in part at least, by an ouabain-resistant ionophoretic system. The experimental data suggest that glucose also increases the inward transport of Na⁺ in islet cells by a veratridine-sensitive channel.     

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.     

Cell-volume-dependent vascular smooth muscle contraction: role of Na+, K+, 2Cl− cotransport, intracellular Cl− and L-type Ca2+ channels

This study elucidates the role of cell volume in contractions of endothelium-denuded vascular smooth muscle rings (VSMR) from the rat aorta. We observed that hyposmotic swelling as well as hyper- and isosmotic shrinkage led to VSMR contractions. Swelling-induced contractions were accompanied by activation of Ca²⁺ influx and were abolished by nifedipine and verapamil. In contrast, contractions of shrunken cells were insensitive to the presence of L-type channel inhibitors and occurred in the absence of Ca²⁺_o. Thirty minutes preincubation with bumetanide, a potent Na⁺,K⁺,Cl^– cotransport (NKCC) inhibitor, decreased Cl^–_i content, nifedipine-sensitive ⁴⁵Ca uptake and contractions triggered by modest depolarization ([K⁺]_o=36 mM). Elevation of [K⁺]_o to 66 mM completely abolished the effect of bumetanide on these parameters. Bumetanide almost completely abrogated phenylephrine-induced contraction, partially suppressed contractions triggered by hyperosmotic shrinkage, but potentiated contractions of isosmotically shrunken VSMR. Our results suggest that bumetanide suppresses contraction of modestly depolarized cells via NKCC inhibition and Cl^–_i-mediated membrane hyperpolarization, whereas augmented contraction of isosmotically shrunken VSMR by bumetanide is a consequence of suppression of NKCC-mediated regulatory volume increase. The mechanism of bumetanide inhibition of contraction of phenylephrine-treated and hyperosmotically shrunken VSMR should be examined further.     

Kinetic properties of Na+/H+ exchange in cultured bovine pigmented ciliary epithelial cells

Uptake studies with²²Na were performed in cultured bovine pigmented ciliary epithelial cells, in order to characterize mechanisms of Na⁺ transport. A large part of Na⁺ uptake was sensitive to amiloride, quinidine and harmaline. Na⁺ uptake was stimulated by intracellular acidification (using the NH ₄ ⁺ prepulse technique), and was inhibited with increasing extracellular proton concentration. Decreasing extracellular pH from 7.5 to 7.0 increased the apparentK _M for Na⁺ from 38 to 86 mM without considerable changes inV _max. In the presence of 5 mM Na⁺ half maximal inhibition of amiloride sensitive Na⁺ uptake by extracellular protons was observed at a hydrogen concentration of 50 nM. In the presence of 50 mM Na⁺ the proton concentration necessary for 50% inhibition was 139 nM. Thus, the mode of inhibition of extracellular H⁺ seemed to be competitive with aK _i of 20–40 nM. 10 M amiloride increased the apparentK _M for Na⁺ from 33 mM to 107 mM, whileV _max remained nearly unchanged. IC₅₀ for amiloride was 6 M at 5 mM Na⁺ and 36 M in the presence of 150 mM Na⁺. Thus, amiloride behaves as a competitive inhibitor with aK _i of about 5 M. The affinities of Na⁺ to the transport site (K _M16 mM), to the inhibitory site for protons (K _M21 mM), and to the inhibitory site for amiloride (K _M26 mM) were in the same order of magnitude.In summary, we have presented evidence for the presence of a Na⁺/H⁺ exchanger in cultured bovine pigmented ciliary epithelial cells. The kinetic data suggest the presence of only one common extracellular binding site for Na⁺, H⁺ and amiloride.     

Das Problem des zellulären Herzglykosidrezeptors

Zusammenfassung Diese Übersicht beschreibt die Eigenschaften der Na⁺, K⁺-ATPase bzw. der Na⁺, K⁺-Pumpe in der intakten Membran und die hochspezifische Hemmung dieses Transportenzyms durch Herzglykoside.Es wird auf die Wechselwirkung zwischen Glykosid und Enzym eingegangen und da bei insbesondere auf die Steuerung der Glykosidbindung durch ATP, K⁺, Na⁺, Mg²⁺ und Ca²⁺.Die Bedeutung des Enzyms als wahrscheinlicher pharmakologischer Rezeptor für Herzglykoside wird herausgestellt. Zunächst erfolgt eine Beschreibung der Schwierigkeiten und Fortschritte bei der Herstellung eines Zusammenhanges zwischen positiv inotropem Effekt von Herzglykosiden und deren Bindung an die Na⁺, K⁺-Pumpen des Herzmuskels bzw. der Hemmung des Transportsystems durch Herzglykoside.Es werden neuere Erkenntnisse über die Verknüpfung von der Hemmung der Na⁺, K⁺-Pumpe, die zu einer Erhöhung der intrazellulären Na⁺-Konzentration führt, und der indirekten Erhöhung der intrazellulären Ca²⁺-Aktivität referiert.Mitteilungen über den Nachweis einer herzglykosidartigen endogenen Aktivität entsprechen der hohen Rezeptorspezifität für Herzglykoside.     

Effects of Na+, K+ and Mg2+ on45Ca2+ uptake by pancreatic islets

Microdissected pancreatic islets of noninbredob/ob-mice were used to study ionic effects on the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake by islet cells. Omission of Mg²⁺ from the incubation medium had no effect, but the⁴⁵Ca²⁺ uptake was increased by omission of Na⁺ and decreased by omission of K⁺. Excess Mg²⁺ (1.2–15 mM) inhibited and excess K⁺ (4.7–25 mM) stimulated the⁴⁵Ca²⁺ uptake in a concentration-dependent manner. Stimulation of⁴⁵Ca²⁺ uptake in Na⁺-deficient islets was associated with an enhancement of the basal insulin release. Total abolishment of glucose-stimulated⁴⁵Ca²⁺ uptake in K⁺-deficient islets did not preclude a significant secretory response to glucose. It is concluded that the lanthanum-nondisplaceable⁴⁵Ca²⁺ uptake shows a partial correlation to insulin release.     

Patch-clamp study of rubidium and potassium conductances in single cation channels from mammalian exocrine acini

Single-channel current recordings were carried out on excised inside-out patches of baso-lateral plasma membrane from exocrine acinar cells. The mouse pancreas and submandibular gland as well as the pig pancreas were investigated.In the mouse pancreas the voltage-insensitive Ca²⁺-activated cation channel was studied. Single-channel current-voltage (i/v) relationships were studied in symmetrical Rb⁺-rich solutions and in asymmetrical Rb⁺/Na⁺ and Na⁺/Rb⁺ solutions. In all cases the i/v relations were linear and had the same slope representing a single-channel conductance of about 33 pS which is identical to that previously obtained with symmetrical Na⁺ solutions or asymmetrical Na⁺/K⁺ solutions.In the mouse submandibular gland and the pig pancreas the voltage and Ca²⁺-activated K⁺ channel was studied. The outward currents observed after depolarization in the presence of quasi-physiological Na⁺/K⁺ gradients were immediately abolished when all the K⁺ in the bath fluid was replaced by Rb⁺ (bath fluid in contact with inside of plasma membrane). This effect was immediately and fully reversible upon return to the high K⁺ solution.The voltage and Ca²⁺-activated K⁺ channel was also studied in asymmetrical K⁺/Rb⁺ and Rb⁺/K⁺ solutions. In the first case inward (K⁺) currents could be observed but not outward (Rb⁺) currents, while in the other case inward (Rb⁺) currents could not be seen whereas outward (K⁺) currents were measured. The current-voltage relationships were approximately linear and the null potential was close to 0 mV in both situations. In contrast the null potential for current through the K⁺ channel in the presence of asymmetrical Na⁺/K⁺ or Li⁺/K⁺ solutions was about –70 mV and with reversed gradients about +60 mV.Outward K⁺ currents of reduced size (through the voltage and Ca²⁺-activated K⁺ channel) could be observed when the bath fluid contained 75 mM K⁺ and 75 mM Rb⁺, but not (in the same membrane patches) when 150 mM Rb⁺ and no K⁺ was present.It is concluded that the large voltage- and Ca²⁺-activated K⁺ channel has an extremely low Rb⁺ conductance. It is possible, however, that the permeability for Rb⁺ may be about the same as for K⁺. The voltage-insensitive Ca²⁺-activated cation channel does not discriminate between K⁺ and Rb⁺.     

Cyanide inhibits the Na+/Ca2+ exchanger in isolated cardiac pacemaker cells of the cane toad

The effects of the metabolic inhibition on the activity of the Na⁺/Ca²⁺ exchanger (NCX) were studied in single isolated pacemaker cells from the cane toad. Ca²⁺ influx on NCX (reverse mode) was estimated by measuring the increase in intracellular calcium concentration ([Ca²⁺]_i) in response to extracellular Na⁺-free solution. After application of 2 mM sodium cyanide for 3–5 min, the peak [Ca²⁺]_i in Na⁺-free solution was significantly decreased from 377±42 nM to 260±46 nM, suggesting inhibition of NCX. To study Ca²⁺ efflux on NCX (forward mode), we recorded the tail currents on repolarization which were abolished by Ni²⁺ and by Na⁺-free solution. Cyanide decreased the amplitude of tail currents by 36±3%. To investigate the intrinsic properties of NCX during the metabolic inhibition, we used rapid application of caffeine to trigger sarcoplasmic reticulum Ca²⁺ release, which then stimulates NCX current (I_NCX ). Both the caffeine-induced peak [Ca²⁺]_i and the peak I_NCX were reduced by cyanide exposure. When I_NCX was plotted against [Ca²⁺], the slope of the decay phase was decreased in the presence of CN^– to 44±8% of control, indicating that for a given [Ca²⁺]_i there was less I_NCX produced. These results show that cyanide (CN^–) inhibits NCX activity at least partly through changes in the intrinsic properties of NCX. The inhibition of NCX probably contributes to the slower firing rate of pacemaker cells in CN^–.