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- and time-dependent K+ channel currents in the basolateral membrane of villus enterocytes isolated from guinea pig small intestine

Patch-clamp studies were carried out in villus enterocytes isolated from the guinea pig proximal small intestine. In the whole-cell mode, outward K+ currents were found to be activated by depolarizing command pulses to -45 mV. The activation followed fourth order kinetics. The time constant of K+ current activation was voltage-dependent, decreasing from approximately 3 ms at -10 mV to 1 ms at +50 mV. The K+ current inactivated during maintained depolarizations by a voltage- independent, monoexponential process with a time constant of approximately 470 ms. If the interpulse interval was shorter than 30 s, cumulative inactivation was observed upon repeated stimulations. The steady state inactivation was voltage-dependent over the voltage range from -70 to -30 mV with a half inactivation voltage of -46 mV. The steady state activation was also voltage-dependent with a half- activation voltage of -22 mV. The K+ current profiles were not affected by chelation of cytosolic Ca2+. The K+ current induced by a depolarizing pulse was suppressed by extracellular application of TEA+, Ba2+, 4-aminopyridine or quinine with half-maximal inhibitory concentrations of 8.9 mM, 4.6 mM, 86 microM and 26 microM, respectively. The inactivation time course was accelerated by quinine but decelerated by TEA+, when applied to the extracellular (but not the intracellular) solution. Extracellular (but not intracellular) applications of verapamil and nifedipine also quickened the inactivation time course with 50% effective concentrations of 3 and 17 microM, respectively. Quinine, verapamil and nifedipine shifted the steady state inactivation curve towards more negative potentials. Outward single K+ channel events with a unitary conductance of approximately 8.4 pS were observed in excised inside-out patches of the basolateral membrane, when the patch was depolarized to -40 mV. The ensemble current rapidly activated and thereafter slowly inactivated with similar time constants to those of whole-cell K+ currents. It is concluded that the basolateral membrane of guinea pig villus enterocytes has a voltage-gated, time-dependent, Ca(2+)-insensitive, small-conductance K+ channel. Quinine, verapamil, and nifedipine accelerate the inactivation time course by affecting the inactivation gate from the external side of the cell membrane.     

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 effect of ions on the adhesion and internalization of Chlamydia trachomatis by HeLa cells

The adhesion and internalization of Chlamydia trachomatis by HeLa cells was unaffected by removal of K+, Mg2+, or glucose from the incubation medium, slightly reduced by removal of Na+, and significantly reduced by omission of Ca2+, Sr2+, Mg2+, and Mn2+ could replace Ca2+ in the adhesion but only Sr2+ supported internalization, and La3+, Co2+, Fe3+, Ba2+, and Zn2+ all reduced internalization more than adhesion. During initial infection there was no measurable difference in the uptake or release of 45Ca2+ or 86Rb+ between infected and noninfected HeLa monolayers. Infection was not prevented by pretreatment of the monolayers with the calcium channel blockers, verapamil, D600, and nitrendipine, or the calmodulin inhibitors, TMB-8 or trifluperazine. The results suggest that divalent cations are not essential for chlamydial infection but that the process of internalization is facilitated by the presence of cations, particularly Na+ and Ca2+.     

Modulation of carbachol-stimulated inositol phospholipid breakdown in rat cerebral cortical miniprisms by excitatory amino acids and by BAY K-8644 is dependent upon the assay calcium and potassium concentrations used.

The calcium and potassium ion dependency of the inositol phospholipid breakdown response to stimulatory agents has been investigated in rat cerebral cortical miniprisms. The calcium channel agonist BAY K-8644 (10 microM) potentiated the response to carbachol at 6 mM K+ when Ca2(+)-free, but not when 2.52 mM Ca2+ assay buffer was used. In Ca2(+)-free buffer, verapamil (10 microM) inhibited the response to carbachol at both 6 and 18 mM K+ but higher concentrations (30-300 microM) were needed when 2.52 mM Ca2+ was used. At these higher concentrations, however, verapamil inhibited the binding of 2 nM [3H]pirenzepine to muscarinic recognition sites. N-Methyl-D-Aspartate (NMDA, 100 microM) significantly reduced the basal phosphoinositide breakdown rate at 18 mM K+ at 1.3 mM Ca2+, but was without effect on the basal rate at other K+ and Ca2+ concentrations. In the presence of NMDA (100 microM) or quisqualate (100 microM), the responses to carbachol were reduced, the degree of reduction showing a complex dependency upon the assay K+ and Ca2+ concentrations used. These results indicate that the inositol phospholipid breakdown response to carbachol in cerebral cortical miniprisms can be modulated in a manner dependent upon the extracellular calcium and potassium concentrations used.     

Evidence for a role of Na+,K(+)-ATPase in the hydration of Atlantic croaker and spotted seatrout oocytes during final maturation.

During final maturation the oocytes of many marine teleosts swell four to five times their original size due to uptake of water. The involvement of active inorganic ion transport and Na+,K(+)-ATPase in oocyte hydration in Atlantic croaker (Micropogonias undulatus) and spotted seatrout (Cynoscion nebulosus), marine teleosts which spawn pelagic eggs, was investigated by examining changes in the inorganic ion content of ovarian follicles containing mainly oocytes, by performing in vitro incubations of the follicles with ion channel blockers, and by assaying membrane preparations of ovaries containing hydrating and non-hydrating oocytes for Na+,K(+)-ATPase activity and content. There were marked increases in the contents of K+, Mg++, and Ca++, but not Na+, in oocytes of M. undulatus and C. nebulosus during hydration. Incubation of follicle-enclosed oocytes in K(+)-free medium or with ouabain or amiloride, inhibitors of Na+,K(+)-ATPase and Na+ channels, respectively, blocked gonadotropin-induced oocyte hydration in M. undulatus. In addition, Na+,K(+)-ATPase activity increased threefold and the concentration of the enzyme increased 50% in ovarian tissue during oocyte hydration. These results strongly suggest a major role for active ion regulation by a ouabain-sensitive Na+,K(+)-ATPase system in oocyte hydration in two species of sciaenid fishes.     

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle

Summary A Na⁺-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na⁺ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na⁺ removed. At a 3-O-MG concentration of 2mm, the Na⁺-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na⁺-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na⁺-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na⁺-sensitive component was at a 3-O-MG concentration of about 13mm, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5mm), a potent inhibitor specific for Na⁺-coupled glucose transport, reduced the uptake in a solution containing Na⁺ to the level in Na⁺-free Ringer's solution. Glucose of concentrations higher than 20mm suppressed 3-O-MG uptake to a level slightly lower than that in Na⁺-free Ringer's solution. These observations indicate that there are Na⁺-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.     

Characterization of calcium transport by basal plasma membranes from human placental syncytiotrophoblast.

We have studied the mechanisms involved in calcium (Ca2+) transport through the basal plasma membranes (BPM) of the syncytiotrophoblast cells from full-term human placenta. These purified membranes were enriched 25-fold in Na+/K(+)-adenosine triphosphate (ATPase), 37-fold in [3H] dihydroalprenolol binding sites, and fivefold in alkaline phosphatase activity compared with the placenta homogenates. In the absence of ATP and Mg2+, a basal Ca2+ uptake was observed, which followed Michaelis-Menten kinetics, with a Km Ca2+ of 0.18 +/- 0.05 microM and Vmax of 0.93 +/- 0.11 nmol/mg/min. The addition of Mg2+ to the incubation medium significantly decreased this uptake in a concentration-dependent manner, with a maximal inhibition at 3 mM Mg2+ and above. The Lineweaver-Burk plots of Ca2+ uptake in the absence and in the presence of 1 mM Mg2+ suggest a noncompetitive type of inhibition. Preloading the BPM vesicles with 5 mM Mg2+ had no significant effect on Ca2+ uptake, eliminating the hypothesis of a Ca2+/Mg2+ exchange mechanism. This ATP-independent Ca2+ uptake was not sensitive to 10(-6) M nitrendipine nor to 10(-4) M verapamil. An ATP-dependent Ca2+ transport was also detected in these BPM, whose Km Ca2+ was 0.09 +/- 0.02 microM and Vmax 3.4 +/- 0.2 nmoles/mg/3 min. This Ca2+ transport requires Mg2+, the optimal concentration of Mg2+ being approximately 1 mM. Preincubation of the membrane with 10(-6) M calmodulin strongly enhanced the initial ATP-dependent Ca2+ uptake. Finally, no Na+/Ca2+ exchange process could be demonstrated.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Characterization of tetraethylammonium uptake across the basolateral membrane of the Drosophila Malpighian (renal) tubule

Basolateral transport of the prototypical type I organic cation tetraethylammonium (TEA) by the Malpighian tubules of Drosophila melanogaster was studied using measurements of basolateral membrane potential (V(bl)) and uptake of [(14)C]-labeled TEA. TEA uptake was metabolically dependent and saturable (maximal rate of mediated TEA uptake by all potential transport processes, reflecting the total transport capacity of the membrane, 0.87 pmol.tubule(-1).min(-1); concentration of TEA at 0.5 of the maximal rate of TEA uptake value, 24 muM). TEA uptake in Malpighian tubules was inhibited by a number of type I (e.g., cimetidine, quinine, and TEA) and type II (e.g., verapamil) organic cations and was dependent on V(bl). TEA uptake was reduced in response to conditions that depolarized V(bl) (high-K(+) saline, Na(+)-free saline, NaCN) and increased in conditions that hyperpolarized V(bl) (low-K(+) saline). Addition of TEA to the saline bathing Malpighian tubules rapidly depolarized the V(bl), indicating that TEA uptake was electrogenic. Blockade of K(+) channels with Ba(2+) did not block effects of TEA on V(bl) or TEA uptake indicating that TEA uptake does not occur through K(+) channels. This is the first study to provide physiological evidence for an electrogenic carrier-mediated basolateral organic cation transport mechanism in insect Malpighian tubules. Our results also suggest that the mechanism of basolateral TEA uptake by Malpighian tubules is distinct from that found in vertebrate renal tubules.     

Acetyl phosphate can act as a substrate for Na+ transport by (Na+ + K+)-ATPase

Experiments using liposomes with (Na+ + K+)-ATPase incorporated showed that in the presence of extravesicular Mg2+, acetyl phosphate was able to stimulate Na+ uptake when the liposomes contained Na+ or choline and were K+-free; this acetyl phosphate-dependent Na+ transport was similar to the ATP-dependent transport observed with 0.003 mM or 3 mM ATP. When the intravesicular solution contained K+, there was an ATP-dependent Na+ uptake which was large with 3 mM ATP and small (about the size seen in K+-free liposomes) with 0.003 mM ATP; in this case, although acetyl phosphate produced a slight activation of Na+ transport, the effect was not statistically significant. All ATP and acetyl phosphate-stimulated Na+ transport disappeared in the absence of extravesicular Mg2+ or in the presence of ouabain in the intravesicular solution. These results are consistent with the hypothesis that, at the concentration used, acetyl phosphate can replace ATP in the catalytic but not in the regulatory site of the (Na+ + K+)-ATPase and active Na+ transport system. This suggests that as far as the early stages of the pump cycle are concerned the role of ATP is simply to phosphorylate.     

Effect of the membrane potential on the Mg2+,ATP-dependent transport of Ca2+ across smooth muscle sarcolemma

The effect of the membrane potential (K(+)-valinomycin system) on the Mg2+, ATP-dependent transport of Ca2+ in inside-out vesicles of myometrium sarcolemma has been studied. The membrane potential was identified by using a cyanine potential-sensitive probe, diS-C3-(5). In the presence of valinomycin (5.10(-8) M) the inside-out directed K+ gradient (delta psi = -86 mV, with a negative charge inside) stimulated the initial rate of the energy-dependent accumulation of Ca2+ transfer whereas the oppositely directed K+ gradient (delta psi = +72 mV, with a positive charge inside) had no effect on this process. The K+ gradient was formed by isotonic substitution of K+ in intra- or extravesicular space for choline +. At the same time, in the absence of K+ gradient the Mg2+, ATP-dependent accumulation of Ca2+ in membrane vesicles did not depend on the chemical nature of the cations (K+ or choline+) used for isotonicity. The decrease of delta psi from 0 to -86 mV affects the initial rate of Ca2+ accumulation but not the maximal content of the accumulated cation. Preliminary dissipation of the membrane potential (delta psi = -86 mV) in Mg2(+)-free isotonic (with respect of K+ and choline+) media containing ATP and Ca2+ resulted in the inhibition of Mg2+, ATP-dependent Ca2+ transport induced by subsequent addition of Mg2+. These results indicate that the negative (intravesicular) electrical potential activates the Ca-pump of smooth muscle sarcolemma. This activation is based on the increase in the turnover number of the Ca2+ transporting system but not on its affinity for the transfer substrate. The use of the absolute reaction rates theory made it possible to establish that the Ca-pump effectuates the transport of a single positive charge in inside-out vesicles of smooth muscle plasma membranes, i.e., the energy-dependent transport of Ca2+ occurs either as a symport (with an anion (Cl-) or an antiport with a monovalent cation (K+) or a proton. It is assumed that the potential dependence of the Ca-pump in the smooth muscle plasma membrane plays a role in the realization of effects of mediators and physiologically active substances that are manifested as stimulation of the contractile response and depolarization of the sarcolemma. In is quite probable that the delta psi-dependent Ca-pump is also responsible for the maintenance of intracellular homeostasis of monovalent cations (K+, H+, Cl-) in smooth muscle tissues.     

Cyclic AMP and Ca2+-activated K+ transport in a human colonic epithelial cell line

Addition of either vasoactive intestinal peptide (VIP) or the Ca2+ ionophore, A23187, to confluent monolayers of the T84 epithelial cell line derived from a human colon carcinoma increased the rate of 86Rb+ or 42K+ efflux from preloaded cells. Stimulation of the rate of efflux by VIP and A23187 still occurred in the presence of ouabain and bumetanide, inhibitors of the Na+,K+-ATPase and Na+,K+,Cl- cotransport, respectively. The effect of A23187 required extracellular Ca2+, while that of VIP correlated with its known effect on cyclic AMP production. Other agents which increased cyclic AMP production or mimicked its effect also increased 86Rb+ efflux. VIP- or A23187-stimulated efflux was inhibited by 5 mM Ba2+ or 1 mM quinidine, but not by 20 mM tetraethylammonium, 4 mM 4-aminopyridine, or 1 microM apamin. Under appropriate conditions, VIP and A23187 also increased the rate of 86Rb+ or 42K+ uptake. Stimulation of the initial rate of uptake by either agent required high intracellular K+ and was not markedly affected by the imposition of transcellular pH gradients. The effect of A23187, but not VIP or dibutyryl cyclic AMP, was refractory to depletion of cellular energy stores. A23187-stimulated uptake was not significantly affected by anion substitution, however, stimulation of uptake by VIP required the presence of a permeant anion. This result may be due to the simultaneous activation of a cyclic AMP-dependent Cl- transport system. The kinetics of both VIP- and A23187-stimulated uptake and efflux were consistent with a channel-rather than a carrier-mediated K+ transport mechanism. The results also suggest that cyclic AMP and Ca2+ may activate two different kinds of K+ transport systems. Finally, both transport systems have been localized to the basolateral membrane of T84 monolayers, a result compatible with their possible regulatory role in hormone-activated electrogenic Cl- secretion.     

Properties of receptors for the Ca2+-channel blocker verapamil in transverse-tubule membranes of skeletal muscle. Stereospecificity, effect of Ca2+ and other inorganic cations, evidence for two categories of sites and effect of nucleoside triphosphates

The verapamil receptor associated with the voltage-dependent calcium channel of rabbit skeletal muscle transverse tubule membranes has the following properties. (i) This receptor is stereospecific and discriminates between the different stereoisomers of verapamil, gallopamil and diltiazem. (ii) Inorganic divalent cations inhibit the binding of [3H]verapamil to its receptor in an apparently non-competitive fashion. The rank order of potency is: Ca2+ = Mn2+ greater than Mg2+ greater than Sr2+ greater than Ba2+ much greater than Co2+ much greater than Ni2+. Ca2+ and Mn2+ have inhibition constants of 0.3 mM. Binding of [3H]verapamil is also sensitive to monovalent cations such as Cs+, K+, Li+ and Na+. The most active of these cations (Cs+ and K+) have inhibition constants in the range of 30 mM. (iii) Binding of [3H]verapamil is pH-dependent and reveals the presence on the verapamil receptor of an essential ionizable group with a pKa of 6.5. (iv) A low-affinity binding site for verapamil and for some other Ca2+ channel blockers is detected by studies of dissociation kinetics of the [3H]verapamil receptor in the presence of high concentrations of verapamil, gallopamil, bepridil and diltiazem. (v) GTP and nucleoside analogs change the properties of [3H]verapamil binding to verapamil binding sites. High-affinity binding sites seem to be transferred into low-affinity sites. Dissociation constants obtained from inhibition studies of [3H]verapamil binding are in the range of 0.1-0.3 mM for GTP, ATP and Gpp(NH)p.     

Regulation of 3-O-methyl-D-glucose uptake in isolated bovine adrenal chromaffin cells

We showed earlier that insulin stimulated sugar transport in adrenal chromaffin cells (Bigornia, L. and Bihler, I. Biochim. Biophys. Acta 885, 335-344). Transport regulation and its Ca2+ -dependence was further investigated in isolated bovine adrenal chromaffin cells, serving as a model of a homogeneous neuronal cell population. Uptake of the nonmetabolizable glucose analogue, 3-O-methyl-D-glucose was stimulated by hyperosmolar medium, and this effect was abolished in the absence of external Ca2+, or depressed in the presence of La3+ or the slow Ca2+ channel blocker methoxyverapamil. Basal transport was also stimulated by factors (acetylcholine, carbamylcholine, low-Na+ medium), which cause Ca2+ -dependent catecholamine release, and these effects were abolished in Ca2+ -free medium. In addition insulin, acetylcholine, hyperosmolar and low-Na+ medium significantly increased 45Ca uptake. Thus, glucose transport in adrenal chromaffin cells was stimulated by insulin and hyperosmolarity in a Ca2+ -dependent manner, as in muscle. Sensitivity to secretory stimuli, a regulatory feature perhaps characteristic of this cell type, was also demonstrated. In contrast to muscle, sugar transport was not affected by Na+ -pump inhibition, metabolic inhibitors or the Na+ ionophore monensin, suggesting that Ca2+ influx by Na+/Ca2+ exchange does not play a significant role in the activation of sugar transport in chromaffin cells.     

Regulation of K+ transport in tomato roots by the TSS1 locus. Implications in salt tolerance

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4(+)-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4(+)-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4(+)-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.     

The role of calcium in stimulation of sugar transport in muscle by lithium

We have investigated the relation between the stimulation of sugar transport by Li+ and Li+-induced changes in cellular Ca2+ distribution. The fluxes of 3-O-[14C]methyl-D-glucose and 45Ca were measured in hemidiaphragm, soleus, and cardiac muscles of the rat, and cellular levels of Ca2+, Na+ and K+ were determined. Li+ increased in parallel the fluxes of 3-O-[14C]methyl-D-glucose and 45Ca in rat hemidiaphragm and soleus muscles. Sugar transport and Ca2+ efflux were also stimulated by Li+ in Ca2+-free medium, suggesting that in addition to increasing sarcolemmal Ca2+ influx, Li+ may also cause the release of Ca2+ from intracellular storage sites, presumably the mitochondria. Mitochondria were isolated from preparations of rat ventricular muscle exposed to Li+, and their Ca2+ content was determined. In rat cardiac muscle, Li+ stimulation of sugar transport was associated with decreased mitochondrial Ca2+ levels (indicating mitochondrial Ca2+ release) only under conditions of deteriorating mitochondrial function. Thus, Li+-induced changes in cellular Ca2+ distribution, which would increase cytosolic Ca2+ levels, were associated with stimulation of sugar transport. These observations support the hypothesis that the increased availability of cytosolic Ca2+ regulates the activity of the sugar transport system in muscle.     

Cadmium as a tool for studying calcium-dependent cation permeability of the human red blood cell membrane.

Three Ca(2+)-dependent procedures known to increase cation permeability of red blood cell membranes were tested with Cd2+ ions which equal Ca2+ ions both in their charge and the crystal radius, 1. Increase of non-selective permeability for monovalent cations by incubating the red cells in a Ca(2+)-free sucrose medium. Addition of Cd2+ to the suspension of leaky cells failed to restore the initial impermeability of the red cell membrane while a repairing effect of Ca2+ was evident both in the presence and absence of Cd2+. Thus, in low electrolyte medium, Cd2+ could neither mimic Ca2+, nor prevent the latter from interacting with membrane structures which control cation permeability. 2. Increase of the K(+)-selective permeability by propranolol plus Ca2+. Cd2+ added to a Ca(2+)-free Ringer type medium containing propranolol enhanced K+ permeability similar to that obtained with Ca2+. No changes of membrane permeability could be detected in the presence of 0.5 mmol/l Cd2+ in absence of propranolol. The Cd(2+)-stimulated K+ channels were different from those induced by Ca2+. They proved to be insensitive to quinine, exhibited a low K+/Na+ selectivity, and showed no tendency to self-inactivation. 3. Stimulation of K+ permeability by electron donors plus Ca2+. Substitution of Ca2+ by Cd2+ yielded results similar to those obtained with propranolol. The ability of Cd2+ to overtake the role of Ca2+ appears to depend on the system studied. It supplies information allowing to distinguish between the diverse Ca(2+)-dependent systems in cell membranes.     

Augmentation of forskolin-induced cAMP production by ouabain in rat renal papillary collecting tubule cells in culture

The effect of extracellular calcium (Ca2+) on the cellular action of forskolin was studied using a Na+, K(+)-ATPase inhibitor ouabain in rat renal papillary collecting tubule cells in culture. Forskolin-induced cAMP production was enhanced by the pretreatment of cells with ouabain, providing that a dose-dependent curve with forskolin shifted to the left. The enhancement by ouabain of cellular cAMP production in response to forskolin was totally blunted by cotreatment with cobalt, verapamil, or Ca2(+)-free medium containing 1 mM EGTA. In addition, two dissimilar antagonists of calmodulin, namely trifluoperazine and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W - 7), attenuated the ouabain's effect on cAMP production in response to forskolin. These results therefore indicate that ouabain enhances the activation of adenylate cyclase by forskolin, mediated through cellular free Ca2+, in renal papillary collecting tubule cells, and that extracellular Ca2+ is an important source for cellular Ca2+ mobilization by ouabain.     

Ca2+-dependent K+ transport in the Ehrlich ascites tumor cell

The possible presence and properties of the Ca2+-dependent K+ channel have been investigated in the Ehrlich ascites tumor cell. The treatment with ionophore A23187 + CA2+, propranolol or the electron donor system ascorbate-phenazine methosulphate, all of which activate that transport system in the human erythrocyte, produces in the Ehrlich cell a net loss of K+ (balanced by the uptake of Na+) and a stimulation of both the influx and the efflux of 86Rb. These effects were antagonized by quinine, a known inhibitor of the Ca2+-dependent K+ channel in other cell systems, and by the addition of EGTA to the incubation medium. Ouabain did not have an inhibitory effect. These results suggests that the Ehrlich cell possesses a Ca2+-dependent K+ channel whose characteristics are similar to those described in other cell systems.