Calcium-activated potassium channels in human platelets

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

Calcium-activated potassium channels in chondrocytes.

The presence of calcium-activated potassium channels in chondrocytes of growing cartilage was tested. Results obtained with fura-2 on cultured resting chondrocytes indicate that the cells respond to an elevation of extracellular calcium concentration ([Ca2+]o) from 0.1 to 2 mM increasing the intracellular concentration of the ion ([Ca2+]i) from 117 to 187 nM. This increment may be blocked by 3 microM La3+. Patch clamp experiments in cell-attached configuration showed that, when [Ca2+]i rises, the open probability (Po) of the K+ channels increases. Increments in both Po and unitary currents of the K+ channels can be obtained after applying 2.5 microM A23187 with 2 mM [Ca2+]o. Hence, the results demonstrate that, in chondrocytes, a class of Ca(2+)-activated K+ channels is present and their activity is related to an increase of [Ca2+]i.     

Influence of Ca2+ on the plasma membrane potential and electrogenic uptake of glycine by myeloma cells. Involvement of a Ca2+-activated K+ channel

The involvement of Ca2+-activated K+ channels in the regulation of the plasma membrane potential and electrogenic uptake of glycine in SP 2/0-AG14 lymphocytes was investigated using the potentiometric indicator 3,3'-diethylthiodicarbocyanine iodide. The resting membrane potential was estimated to be -57 +/- 6 mV (n = 4), a value similar to that of normal lymphocytes. The magnitude of the membrane potential and the electrogenic uptake of glycine were dependent on the extracellular K+ concentration, [K+]o, and were significantly enhanced by exogenous calcium. The apparent Vmax of Na+-dependent glycine uptake was doubled in the presence of calcium, whereas the K0.5 was not affected. Ouabain had no influence on the membrane potential under the conditions employed. Additional criteria used to demonstrate the presence of Ca2+-activated K+ channels included the following: (1) addition of EGTA to calcium supplemented cells elicited a rapid depolarization of the membrane potential that was dependent on [K+]o; (2) the calmodulin antagonist, trifluoperazine, depolarized the membrane potential in a dose-dependent and saturable manner with an IC50 of 9.4 microM; and (3) cells treated with the Ca2+-activated K+ channel antagonist, quinine, demonstrated an elevated membrane potential and depressed electrogenic glycine uptake. Results from the present study provide evidence for Ca2+-activated K+ channels in SP 2/0-AG14 lymphocytes, and that their involvement regulates the plasma membrane potential and thereby the electrogenic uptake of Na+-dependent amino acids.     

Influence of external K+ on potassium efflux in isolated chicken enterocytes.

1. Efflux of K+ was measured in pre-loaded (86Rb+) chicken enterocytes incubated in buffers with external K+ concentration ([K+]0) between 1 and 40 mM. 2. A decrease in [K+]0 from 6 to 1 mM reduced the rate constant of K+ efflux, whereas it was stimulated by increasing [K+]0 from 6 to 40 mM. 3. The inhibitory effect of low [K+]0 on K+ efflux was: (i) higher than that expected from a change in the electrical driving force, suggesting that membrane K+ permeability has been decreased, and (ii) attenuated by A23187 and Na(+)-free buffers. 4. The effect of A23187 on K(+)-induced K+ efflux was abolished by apamin and that of Na(+)-free buffers by apamin, quinine or verapamil, which suggests that the effect of low K+ on K+ efflux seems to be due to decreased intracellular Ca2+ concentration. 5. The stimulatory effect of 40 mM K0+ on K+ exit can be accounted for by an increase in the electrical driving force. 6. The efflux of K+ at 40 mM K0 appears to occur through Ca2(+)-activated K+ channels (KCa) since it was prevented by 500 microM quinine and unaffected by bumetanide or 3,4-diaminopyridine. 7. In addition, the current results show that an increase in external K+ concentration reduced the ability of quinine to inhibit KCa channels, and even abolished that of Ba2+ and apamin.     

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.     

Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from - 100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.     

Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.     

Lymphocyte membrane potential assessed with fluorescent probes

The membrane potential of mouse spleen lymphocytes has been assessed with two fluorescent probes. 3,3'-Dipropylthiadicarbocyanine (diS-C3-(5)) was used for most of the experiments. Solutions with high K+ concentrations depolarised the cells. Valinomycin, an inophore which adds a highly K+-selective permeability membranes, slightly hyperpolarised cells in standard (6 mM K+) solution, and in 145 mM K+ solution produced a slight additional depolarisation. These findings indicate a membrane whose permeability is relatively selective for K+. Very small changes in potential were seen when choline replaced Na+, or gluconate replaced Cl-, supporting the idea of K+ selectivity. The resting potential could be estimated from the K+ concentration gradient at which valinomycin did not change the potential-the "valinomycin null point" - and under the conditions used the resting potential was approx.-60 mV. B cell-enriched suspensions were prepared either from the spleens of nu/nu mice or by selective destruction of T cells in mixed cell populations. The membrane potential of these cells was similar to that estimated for the mixed cells. In solution with no added K+, diS-C3-(5) itself appeared to depolarise the lymphocytes, in a concentration dependent manner. With the 100 nM dye normally used, the membrane potential in K+-free solution was around -45 mV, and 500 nM dye almost completely depolarised the cells. In standard solution quinine depolarised the cells. Valinomycin could still depolarise these cells indicating that depolarisation had not been due to dissipation of the K+ gradient. Since in K+-free solution diS-C3-(5) blocks the Ca2+-activated K+ channels in human red blood cell ghosts and quinine also blocks this K+ channel it is suggested that the resting lymphocyte membrane may have a similar Ca2+-activated K+ permeability channel. Because of the above mentioned effect of diS-C3-(5) and other biological side effects, such as inhibition of B cell capping, a chemically distinct fluorescent probe of membrane potential, bis(1,3-diethylthiobarbiturate)-trimethineoxonol was used to support the diS-C3-(5) data. This new probe proved satisfactory except that it formed complexes with valinomycin, ruling out the use of this ionophore. Results with the oxonol on both mixed lymphocytes and B cell-enriched suspensions gave confirmation of the conclusions from diS-C3-(5) experiments and indicated that despite its biological side effects, diS-C3-(5) could still give valid assessment of membrane potential.     

Inhibition of endothelium-dependent vasorelaxation by extracellular K(+): a novel controlling signal for vascular contractility

The effects of extracellular K+ on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) were examined in mouse aorta, mouse aorta endothelial cells (MAEC), and human umbilical vein endothelial cells (HUVEC). In mouse aortic rings precontracted with prostaglandin F2alpha or norepinephrine, an increase in extracellular K+ concentration ([K+]o) from 6 to 12 mM inhibited EDR concentration dependently. In endothelial cells, an increase in [K+]o inhibited the agonist-induced [Ca2+]i increase concentration dependently. Similar to K+, Cs+ also inhibited EDR and the increase in [Ca2+]i concentration dependently. In current-clamped HUVEC, increasing [K+]o from 6 to 12 mM depolarized membrane potential from -32.8 +/- 2.7 to -8.6 +/- 4.9 mV (n = 8). In voltage-clamped HUVEC, depolarizing the holding potential from -50 to -25 mV decreased [Ca2+]i significantly from 0.95 +/- 0.03 to 0.88 +/- 0.03 microM (n = 11, P < 0.01) and further decreased [Ca2+]i to 0.47 +/- 0.04 microM by depolarizing the holding potential from -25 to 0 mV (n = 11, P < 0.001). Tetraethylammonium (1 mM) inhibited EDR and the ATP-induced [Ca2+]i increase in voltage-clamped MAEC. The intermediate-conductance Ca2+-activated K+ channel openers 1-ethyl-2-benzimidazolinone, chlorozoxazone, and zoxazolamine reversed the K+-induced inhibition of EDR and increase in [Ca2+]i. The K+-induced inhibition of EDR and increase in [Ca2+]i was abolished by the Na+-K+ pump inhibitor ouabain (10 microM). These results indicate that an increase of [K+]o in the physiological range (6-12 mM) inhibits [Ca2+]i increase in endothelial cells and diminishes EDR by depolarizing the membrane potential, decreasing K+ efflux, and activating the Na+-K+ pump, thereby modulating the release of endothelium-derived vasoactive factors from endothelial cells and vasomotor tone.     

Involvement of the Ca2+-dependent K+ channel activity in the hyperpolarizing response induced by epidermal growth factor in mammary epithelial cells

Epidermal growth factor (EGF) induces a hyperpolarizing response of 5-20 mV amplitude in mouse mammary epithelial cells in culture. The amplitude of the hyperpolarizing response was reduced by more than 60% within several minutes after addition of blockers of voltage and/or Ca2+-dependent K+ channels such as tetraethylammonium (7 mM) or quinine (0.29 mM). Both nifedipine (0.15 mM), a blocker of the Ca2+ channel, and ruthenium red (2 mM), an inhibitor of the Ca2+-binding site, also reduced the amplitude of the hyperpolarizing response by more than 60%. The Ca2+ ionophore, A23187 (3.8 microM), induced a large hyperpolarization, which was 25-40 mV and lasted about 3 min. These data suggest that activity of the Ca2+-dependent K+ channel was involved in the EGF-induced hyperpolarizing response of the mammary epithelial cells.     

Permeability transition pore regulates both mitochondrial membrane potential and agonist-evoked Ca2+ signals in oligodendrocyte progenitors

In this study, we investigated the importance of mitochondrial permeability transition pore (PTP) in agonist-evoked cytosolic Ca2+ ([Ca2+]c) signals in oligodendrocyte progenitor cells (OP cells). We measured transmembrane potential across the mitochondrial inner membrane (delta psi m) and [Ca2+]c in the immediate vicinity simultaneously using tetramethylrhodamine ethyl ester (TMRE) and calcium green respectively. Stimulation of OP cells with methacholine evoked robust [Ca2+]c signals in approximately 80% of cells which were either oscillatory or showed a peak followed by a plateau. Elevations in [Ca2+]c induced by supramaximal concentrations of the agonist (> 200 microM) were accompanied by changes in delta psi m in 33-42% of the total mitochondria investigated. The mitochondria that responded either depolarized (26-29%), hyperpolarized (7-13%) or showed no change (58-67%). Thus, of the responsive mitochondria, most (70%) depolarized during agonist-evoked [Ca2+]c signals. Blockade of PTP with cyclosporin A (CSA) reduced the number of mitochondria that depolarized with a corresponding increase in the number that hyperpolarized. In addition, CSA or its analogue methyl valine-4- CSA (MeVal-CSA), reduced the frequency of agonist-evoked global [Ca2+]c oscillations. In resting cells, CSA (63%) and MeVal-CSA (77%) hyperpolarized a majority of the mitochondria suggesting that PTP is constitutively active and may show flickering openings. Such hyperpolarizations were not mimicked by either cyclosporine H or verapamil and were inhibited by Ru360, which blocks the mitochondrial uniporter. This observation suggested that in resting cells, Ca2+ ions might redistribute between cytosol and mitochondrial matrix through the uniporter and the PTP. Taken together, these data suggest that PTP may play an important role in regulating delta psi m and local [Ca2+]c signals during agonist stimulation in OP cells.     

Measurement of ionized calcium in blood platelets with the photoprotein aequorin. Comparison with Quin 2

The Ca2+-sensitive photoprotein aequorin (Mr = 20,000) was introduced into human blood platelets by incubation with 10 mM EGTA and 5 mM ATP. Platelet cytoplasmic and granule contents were retained during the loading procedure, and platelet morphology, aggregation, and secretion in response to agonists were normal after aequorin loading. Luminescence indicated an apparent resting cytoplasmic ionized calcium concentration [( Cai2+]) of 2-4 microM in media containing 1 mM Ca2+ and of 0.8-2 microM in 2-4 mM EGTA. The Ca2+ ionophore A23187 and the enzyme thrombin produced dose-related luminescent signals in both Ca2+-containing and EGTA-containing media. Peak [Cai2+] after A23187 or thrombin stimulation of aequorin-loaded platelets was 2-10 microM, while peak [Cai2+] determined using Quin 2 as the [Cai2+] indicator was at least 1 log unit lower. In platelets loaded with both aequorin and Quin 2, the aequorin signal was delayed but not reduced in amplitude. Aequorin loading of Quin 2-loaded cells had no effect on the Quin 2 signal. Ca2+ buffering by Quin 2 (intracellular concentration greater than 1 mM) is also supported by a reciprocal relationship between [Quin 2] and peak [Cai2+] stimulated by A23187 in the presence of EGTA. Parallel experiments with Quin 2 and aequorin may identify inhomogeneous [Cai2+] in platelets and give a more complete picture of platelet Ca2+ homeostasis than either indicator alone.     

Voltage gating of Ca2(+)-activated potassium channels in human lymphocytes

The effect of membrane potential on Ca2+ activated K+ channels was studied on human peripheral lymphocytes. Membrane potential was monitored using bisoxonol and flow cytometry. 1 mM Ca2+ in the presence of 2 microM ionomycin depolarized the control cell population, while 100 microM Ca2+ caused hyperpolarization. However 1 mM Ca2+ had a hyperpolarizing effect on previously partially depolarized cells. Potassium channel blockers did not influence the depolarization, while they inhibited the hyperpolarization. Based on the experimental evidence a voltage gating of Ca2+ activated K+ channels is suggested.     

Chromaffin cell calcium channel kinetics measured isotopically through fast calcium, strontium, and barium fluxes

Fast Ca2+ uptake into K+-depolarized cultured bovine adrenal chromaffin cells has been isotopically measured in a time scale of 1-10 s. Depolarized cells retained as much as 80-fold 45Ca2+ taken up by resting cells; Ca2+ was not taken up by fibroblasts or endothelial-like cells. Because Ca2+ entry was inhibited by inorganic (La3+, Co2+, Mg2+) and organic (nifedipine) Ca2+ channel antagonists and enhanced by the Ca2+ channel activator Bay-K-8644, it seems clear that Ca2+ gains access to the chromaffin cell cytosol mainly through specific voltage-dependent Ca2+ channels. Ca2+ uptake evoked by 59 mM K+ was linear during the first 5 s of stimulation and continued to rise at a much slower rate up to 60 s. The rate of Ca2+ entry became steeper as the external [Ca2+] increased; initial rates of Ca2+ uptake varied from 0.06 fmol/cells . s at 0.125 mM Ca2+ to 2.85 fmol/cell . s at 7.5 mM Ca2+. The early 90Sr2+ uptake was linear but faster than Ca2+ uptake and later on was also saturated; 133Ba2+ was taken up still at a much faster rate and was linear for the entire depolarization period (2-60 s). Increased [K+] gradually depolarized chromaffin cells; Ca2+ and Sr2+ uptakes were not apparent below 30 mM K+ but were linear for 30 to 60 mM K+. In contrast, substantial Ba2+ uptake was seen even in K+-free solutions; and in 5.9 mM K+, Ba2+ uptake was as high as Ca2+ uptake obtained in 60 mM K+. Five to ten-second pulses of 45Ca2+, 90Sr2+, or 133Ba2+ given at different times after pre-depolarization of chromaffin cells served to analyze the kinetics of inactivation of the rates of entry of each divalent cation. Inactivation of Ca2+ uptake was faster than Sr2+, and Ba2+ uptake inactivated very little. Neither voltage changes nor Ca2+ ions passing through the channels seems to cause their inactivation; however, experiments aimed to manipulate the levels of internal Ca2+ using the cell-permeable chelator Quin-2 or the ionophore A23187 strongly suggest that intracellular Ca2+ levels determine the rates of inactivation of these channels.     

The effects of ionophore A23187 and concanavalin A on the membrane potential of human peripheral blood lymphocytes and rat thymocytes

Effects of the Ca2+-ionophore A23187 and concanavalin A on the membrane potential of human lymphocytes and rat thymocytes have been studied using the fluorescent potential probe diS-C3-(5). At concentrations of 10(-8) to 10(-6) M A23187 changes the membrane potential, inducing both hyper- and depolarization. Depending on concentrations of A23187 and the external Ca2+, and on the type of lymphocytes, one of these effects predominates. The hyperpolarization induced by A23187 is caused by activation of Ca2+-dependent K+ channels. It is blocked by quinine and high concentrations of extracellular K+. The dependence of Ca2+-activated K+ transport on extracellular Ca2+ and its sensitivity to calmodulin antagonists is different for human lymphocytes and for thymocytes. As distinct from lymphocytes, in thymocytes calmodulin is not involved in activation of Ca2+-dependent K+ transport. The depolarization induced in lymphocytes by A23187 is caused by an increase in Na+ permeability of the lymphocyte plasma membrane: it is eliminated in a low-Na+ medium. At mitogenic concentrations concanavalin A does not change the membrane potential of the lymphocytes. The results obtained permit elucidation of the relationship between two early events in lymphocyte activation, namely the increase in intracellular Ca2+ concentration and the increase in lymphocyte plasma membrane permeabilities to monovalent cations.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

Stimulation of leucine transport by a mitogen through intracellular Ca2+ increase in human peripheral lymphocytes

The effect of concanavalin A and ionophore A23187 on leucine uptake by human peripheral lymphocytes has been examined. Preincubation of the cells with 32 micrograms/ml concanavalin A or 0.1 microM A23187 increased leucine uptake by 67% and 100%, respectively. Both concanavalin A and A23187 could, within 2 min, induce a more than 2-fold increase in the cytoplasmic free Ca2+ concentration ([Ca2+]i). This increase by concanavalin A was completely blocked by the addition of 0.1 mM 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8) to incubation medium; TMB-8 partially blocked the action of A23187. The stimulation of leucine uptake by concanavalin A and A23187 was strongly inhibited by the presence of TMB-8 in the medium, whereas the basal uptake was not affected by this intracellular Ca2+ antagonist. Amiloride did not inhibit the stimulation of leucine uptake by concanavalin A. The concanavalin A- and A23187-induced elevation of [Ca2+]i was accompanied by membrane hyperpolarization. Concanavalin A-stimulated leucine uptake was greatly inhibited by the presence of an excess of 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid. These results indicate that the increase in [Ca2+]i may function as a signal of the stimulation by mitogen of leucine uptake mediated by system L, finally inducing membrane hyperpolarization in human lymphocyte.     

Effect of cyclosporin A on the membrane potential and Ca2+ level of human lymphoid cell lines and mouse thymocytes

The effect of the immunosuppressive cyclosporin A (CsA) on the cytosolic free Ca2+ concentration ([Ca2+]i) and membrane potential of human B and T lymphoblastoid cells and mouse thymocytes was studied in order to reveal some features of the early stage of drug-cell interaction. Cytosolic free Ca2+ concentration of the cells was measured by spectrofluorimetry using indo-1 and quin2 fluorescent calcium indicators. Membrane potential was monitored in a flow cytometer with oxonol dye. CsA applied at 2-20 micrograms/ml final concentrations caused a dose-dependent, rapid, transient rise of [Ca2+]i in all cell types. This effect could be blocked by chelating the extracellular Ca2+ with EGTA but was not sensitive to Ca2+ channel blockers verapamil and nifedipine or K+ channel blocker 4-aminopyridine. A possible explanation for the calcium mobilizing effect of CsA is an ionophore-like mode of action at the cell membrane level. Besides directly interfering with mitogenic signals, the elevation of [Ca2+]i could be responsible for an initial hyperpolarization observed in CsA-treated T lymphocytes. This hyperpolarization, however, was not detectable in B lymphoblastoid cells. A further difference between B and T cells was the diverse pattern of depolarization following CsA treatment. This variance in the behaviour of T and B lymphocytes and the diversity of membrane transport systems in its background could account for the different final outcome of the drug-cell interaction.     

Na+-K+ pump activation inhibits endothelium-dependent relaxation by activating the forward mode of Na+/Ca2+ exchanger in mouse aorta

The effect of Na+-K+ pump activation on endothelium-dependent relaxation (EDR) and on intracellular Ca2+ concentration ([Ca2+]i) was examined in mouse aorta and mouse aortic endothelial cells (MAECs). The Na+-K+ pump was activated by increasing extracellular K+ concentration ([K+]o) from 6 to 12 mM. In aortic rings, the Na+ ionophore monensin evoked EDR, and this EDR was inhibited by the Na+/Ca2+ exchanger (NCX; reverse mode) inhibitor KB-R7943. Monensin-induced Na+ loading or extracellular Na+ depletion (Na+ replaced by Li+) increased [Ca2+]i in MAECs, and this increase was inhibited by KB-R7943. Na+-K+ pump activation inhibited EDR and [Ca2+]i increase (K+-induced inhibition of EDR and [Ca2+]i increase). The Na+-K+ pump inhibitor ouabain inhibited K+-induced inhibition of EDR. Monensin (>0.1 microM) and the NCX (forward and reverse mode) inhibitors 2'4'-dichlorobenzamil (>10 microM) or Ni2+ (>100 microM) inhibited K+-induced inhibition of EDR and [Ca2+]i increase. KB-R7943 did not inhibit K+-induced inhibition at up to 10 microM but did at 30 microM. In current-clamped MAECs, an increase in [K+]o from 6 to 12 mM depolarized the membrane potential, which was inhibited by ouabain, Ni2+, or KB-R7943. In aortic rings, the concentration of cGMP was significantly increased by acetylcholine and decreased on increasing [K+]o from 6 to 12 mM. This decrease in cGMP was significantly inhibited by pretreating with ouabain (100 microM), Ni2+ (300 microM), or KB-R7943 (30 microM). These results suggest that activation of the forward mode of NCX after Na+-K+ pump activation inhibits Ca2+ mobilization in endothelial cells, thereby modulating vasomotor tone.     

Inositol 1,4,5-trisphosphate increases myoplasmic [Ca2+] in isolated muscle fibers. Depolarization enhances its effects

Inositol 1,4,5-trisphosphate (InsP3) has been proposed as an intracellular messenger which mobilizes calcium from the sarcoplasmic reticulum, during excitation-contraction coupling in skeletal muscle. We have measured the myoplasmic free calcium concentration ([Ca2+]i) by means of calcium selective microelectrodes in intact fibers isolated from Leptodactylus insularis microinjected with InsP3. In muscle fibers bathed in normal Ringer, the mean resting [Ca2+]i was 0.11 +/- 0.01 microM (M +/- SEM, n = 30). The microinjection of 0.3, 0.5 and 1 microM InsP3 induced transient increments in the [Ca2+]i to 0.35 +/- 0.02 microM (n = 9), to 0.53 +/- 0.03 microM (n = 11) and 0.94 +/- 0.06 microM (n = 10) respectively. Microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers incubated in low Ca2+ solution induced increments in [Ca2+]i similar to those observed in fibers bathed with normal Ringer. The microinjection of 0.3, 0.5 and 1 microM InsP3 in muscle fibers partially depolarized with 10 mM [K+]o induced transient enhancements of the resting [Ca2+]i that were greater than the transients observed in the normally polarized muscle. In partially depolarized fibers microinjected with 0.3, 0.5 and 1 microM InsP3, the [Ca2+]i was changed to 1.45 +/- 0.14 microM (n = 20), to 3.37 +/- 0.34 microM (n = 7) and to 7.43 +/- 0.70 microM (n = 6) respectively. In all partially depolarized fibers these increments in [Ca2+]i were associated with local contraction.(ABSTRACT TRUNCATED AT 250 WORDS)