Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

Aluminium interactions with K+(86Rb+) and 45Ca2+ fluxes in three cultivars of sugar beet (Beta vulgaris)

Three cultivars of sugar beet (Beta vulgaris L.), which are sensitive to aluminium (Al) in the order Primahill > Monohill > Regina, were grown in water culture for 2 weeks. Nutrients were supplied at 15% increase of amounts daily, corresponding to the nutrient demand for maximal growth. The 2.4-dinitrophenol (DNP)-sensitive (metabolic) and DNP-insensitive (non-metabolic) uptake of aluminium, phosphate. ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in roots were measured as well as transport to shoots of intact plants. All 3 cultivars absorbed more aluminium if DNP was present during the aluminium treatment than in its absence. It is suggested that sugar beets are able to extrude aluminium activity or that they possess an active mechanism to keep Al outside the cell. The presence of Al in the medium during the 1-h experiment affected the metabolic and non-metabolic fluxes of ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) in different ways. In the presence of DNP, the influx of both ⁴⁵Ca²⁺ and K⁺(⁸⁶Rb⁺) and the efflux of ⁴⁵Ca²⁺ were inhibited by Al in a competitive way. At inhibition of ⁴⁵Ca²⁺ influx, 2 Al ions are probably bound per Ca²⁺ uptake site in cv. Regina (Al-tolerant), but in cvs Primahill and Monohill only one Al ion is bound (more Al sensitive). Aluminium competitively inhibited the active efflux of ⁴⁵Ca²⁺ (absence of DNP) in almost the same way in the 3 cultivars. In contrast, aluminium stimulated the influx of K⁺(⁸⁶Rb⁺) in cvs Primahill, Monohill and Regina in the absence of DNP. Thus, the Al effects on active and passive K⁺(⁸⁶Rb⁺) influx are different. The total influx of K⁺(⁸⁶Rb⁺) increased in the presence of Al and might be connected to an active exclusion of Al. Regina is the least Al-sensitive cultivar, probably because Al interferes less with the Ca²⁺ fluxes and because this cultivar actively excludes phosphate in the presence of Al. Thus Al-phosphate precipitation within the plant could be avoided.     

Oxidative stress increases potassium efflux from pancreatic islets by depletion of intracellular calcium stores

Oxidative stress to B-cells is thought to be of relevance in declining B-cell function and in the process of B-cell destruction. In other tissues including heart, brain and liver, oxidative stress has been shown to elevate the intracellular free calcium concentration and to provoke potassium efflux. We studied the effect of oxidative stress on Ca²⁺ and K⁺ (Rb⁺) outflow from pancreatic islets using the thiol oxidants DIP and BuOOH. Both compounds reversibly increased ⁸⁶Rb⁺ efflux in the presence of 3 and 16.7 mmol/l glucose. Stimulation of ⁸⁶Rb⁺ efflux was also evident in the absence of calcium. DIP evoked release of ⁴⁵Ca²⁺ from the pancreatic islets both in the presence or absence of extracellular calcium. Employing inhibitors of the calcium-activated potassium channel (K_Ca) and the high conductance K⁺-channel (BK_Ca), the effect of DIP on ⁸⁶Rb⁺ efflux was slightly diminished. Tolbutamide had no effect on ⁸⁶Rb⁺ efflux in the presence of DIP. On the other hand thapsigargin, a blocker of the Ca²⁺-ATPase of the endoplasmic reticulum, completely suppressed the DIP-mediated ⁸⁶Rb⁺ outflow. The data suggest that thiol oxidant-induced potassium efflux from pancreatic islets is mainly mediated through liberation of intracellular calcium and subsequent stimulation of calcium-activated potassium efflux.     

Presence of a Ca2+-dependent K+ channel in brown adipocytes. Possible role in maintenance ofα1-adrenergic stimulation

We have previously demonstrated mobilization of Ca²⁺ in the efflux of Rb⁺ (K⁺) from isolated hamster brown adipocytes as a consequence of norepinephrine stimulation. We have now investigated the adrenoceptor subtype specificity of these responses and found them both to be of theα₁-subtype. Futher, we have found that the Rb⁺ (K⁺) effux was dependent upon a primary Ca²⁺mobilization event in response to the α₁-adrenergic stimulation, since the Rb⁺ efflux could also be demonstrated by the addition ionophore A23187 to the cells. The norepinephrine- and A23187-stimulated Rb⁺ effluxes were both inhibited by the Ca²⁺-dependent K⁺ -channel blocker apamin. Apamin also significantly attenuated Ca²⁺ mobilization in cells in response to a submaximal concentration of norepinephrine. We conclude that α₁-adrenergic stimulation of brown fat cells leads to a mobilization of intracellular Ca²⁺ which, in itself or via other mechanisms, leads to an increase in cytosolic Ca²⁺ concentration which, in turn, activates a Ca²⁺ -dependent K⁺ channel leading to a K⁺ release from these cells. A possible role for this channel to sustain and augment the response toα₁-adrenergic stimulation is discussed.     

The effect of a hyposmotic shock and purinergic agonists on K(Rb) efflux from cultured human breast cancer cells

The effect of a hyposmotic shock and extracellular ATP on the efflux of K⁺(Rb⁺) from human breast cancer cell lines (MDA-MB-231 and MCF-7) has been examined. A hyposmotic shock increased the fractional efflux of K⁺(Rb⁺) from MDA-MB-231 cells via a pathway which was unaffected by Cl⁻ replacement. Apamin, charybdotoxin or removing extracellular Ca²⁺ had no effect on volume-activated K⁺(Rb⁺) efflux MDA-MB-231 cells. An osmotic shock also stimulated K⁺(Rb⁺) efflux from MCF-7 cells but to a much lesser extent than found with MDA-MB-231 cells. ATP-stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells in a dose-dependent fashion but had little effect on K⁺(Rb⁺) release from MCF-7 cells. ATP-stimulated K⁺(Rb⁺) efflux was only inhibited slightly by replacing Cl⁻ with NO₃⁻. Removal of external Ca²⁺ during treatment with ATP reduced the fractional efflux of K⁺(Rb⁺) in a manner suggesting a role for cellular Ca²⁺ stores. Charybdotoxin, but neither apamin nor iberiotoxin, inhibited ATP-stimulated K⁺(Rb⁺) release from MDA-MB-231 cells. Suramin inhibited the ATP-activated efflux of K⁺(Rb⁺). UTP also stimulated K⁺(Rb⁺) efflux from MDA-MB-231 cells whereas ADP, AMP and adenosine were without effect. A combination of an osmotic shock and ATP increased the fractional efflux of K⁺(Rb⁺) to a level greater than the sum of the individual treatments. It appears that the hyposmotically-activated and ATP-stimulated K⁺ efflux pathways are separate entities. However, there may be a degree of ‘crosstalk’ between the two pathways.     

Rabbit distal colon epithelium: III. Ca2+-activated K+ channels in basolateral plasma membrane vesicles of surface and crypt cells

Summary In the mammalian distal colon, the surface epithelium is responsible for electrolyte absorption, while the crypts are the site of secretion. This study examines the properties of electrical potential-driven⁸⁶Rb⁺ fluxes through K⁺ channels in basolateral membrane vesicles of surface and crypt cells of the rabbit distal colon epithelium. We show that Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels are present in both surface and crypt cell derived vesicles with half-maximal activation at 5×10^–7 m free Ca²⁺. This suggests an important role of cytoplasmic Ca²⁺ in the regulation of the bidirectional ion fluxes in the colon epithelium.The properties of K⁺ channels in the surface cell membrane fraction differ from those of the channels in the crypt cell derived membranes. The peptide toxin apamin inhibits Ca²⁺-activated K⁺ channels exclusively in surface cell vesicles, while charybdotoxin inhibits predominantely in the crypt cell membrane fraction. Titrations with H⁺ and tetraethylammonium show that both high-and low-sensitive⁸⁶Rb⁺ flux components are present in surface cell vesicles, while the high-sensitive component is absent in the crypt cell membrane fraction. The Ba²⁺-sensitive, Ca²⁺-activated K⁺ channels can be solubilized in CHAPS and reconstituted into phospholipid vesicles. This is an essential step for further characterization of channel properties and for identification of the channel proteins in purification procedures.     

Ba2+-inhibitable86Rb+ fluxes across membranes of vesicles from toad urinary bladder

Summary ⁸⁶Rb⁺ fluxes have been measured in suspensions of vesicles prepared from the epithelium of toad urinary bladder. A readily measurable barium-sensitive, ouabain-insensitive component has been identified; the concentration of external Ba²⁺ required for half-maximal inhibition was 0.6mm. The effects of externally added cations on⁸⁶Rb⁺ influx and efflux have established that this pathway is conductive, with a selectivity for K⁺, Rb⁺ and Cs⁺ over Na⁺ and Li⁺. the Rb⁺ uptake is inversely dependent on external pH, but not significantly affected by internal Ca²⁺ or external amiloride, quinine, quinidine or lidocaine. It is likely, albeit not yet certain, that the conductive Rb⁺ pathway is incorporated in basolateral vesicles oriented right-side-out. It is also not yet clear whether this pathway comprises the principle basolateral K⁺ channel in vivo, and that its properties have been unchanged during the preparative procedures. Subject to these caveats, the data suggest that the inhibition by quinidine of Na⁺ transport across toad bladder does not arise primarily from membrane depolarization produced by a direct blockage of the basolateral channels. It now seems more likely that the quinidine-induced elevation of intracellular Ca²⁺ activity directly blocks apical Na⁺ entry.     

Inhibition of 86Rb+ and 22Na+ efflux of Ehrlich lettré tumor cells by Ca2+.

The mechanism of the protective effect of Ca²⁺ on cellular K⁺ content was studied by examination of the effect of Ca²⁺ on efflux of the K⁺ analog, ⁸⁶Rb⁺, from preloaded cells with the use of compounds which interfere with monovalent cation movements. Ca²⁺ decreased ⁸⁶Rb⁺ efflux to the same extent in the presence and absence of ouabain, suggesting that Ca²⁺ did not alter the activity of the (Na⁺ + K⁺)-adenosine triphosphatase pump. Ca²⁺ exerted a similar protective effect in the presence of furosemide, an inhibitor of K⁺-K⁺ exchange, indicative that Ca²⁺ was not inhibiting this pathway. Since Ca²⁺ did not influence these pathways, it is concluded that Ca²⁺ exerts its primary effect by slowing passive diffusion. In support of this, Ca²⁺ also slowed ²²Na⁺ efflux. In addition, ethanol-induced leakage of ⁸⁶Rb⁺ was reversed by extracellular Ca²⁺, suggestive of a Ca²⁺-membrane phospholipid interaction.     

Ca2+-activated K+ permeability in human erythrocytes: Modulation of single-channel events

Elevated levels of intracellular Ca²⁺ activate a K⁺-selective permeability in the membrane of human erythrocytes. Currents through single channels were analysed in excised inside-out membrane patches. The effects of several ions that are known to inhibit K⁺ fluxes are described with respect to the single-channel events. The results suggest that the blocking ions can partly move into the channels (but cannot penetrate) and interact with other ions inside the pore. The reduction of single-channel conductance by Cs⁺, tetraethylammonium and Ba²⁺ and of single-channel activity by quinine and Ba²⁺ is referred to different rates of access to the channel. The concentration- and voltage-dependent inhibition by ions with measurable permeability (Na⁺ and Rb⁺) can be explained by their lower permeability, with single-file movement and ionic interactions inside the pore.     

The cyclic nucleotide‐gated channel AtCNGC10 transports Ca2+ and Mg2+ in Arabidopsis

The suppression of the cyclic nucleotide‐gated channel (CNGC) AtCNGC10 alters K⁺ transport in Arabidopsis plants. Other CNGCs have been shown to transport Ca²⁺, K⁺, Li⁺, Cs⁺ and Rb⁺ across the plasma membrane when expressed in heterologous systems; however, the ability of the AtCNGC10 channel to transport nutrients other than K⁺ in plants has not been previously tested. The ion fluxes along different zones of the seedling roots, as estimated by the non‐invasive ion‐specific microelectrode technique, were significantly different in two AtCNGC10 antisense lines (A2 and A3) in comparison to the wild type (WT). Most notably, the influxes of H⁺, Ca²⁺ and Mg²⁺ in the meristem and distal elongation zones of the antisense A2 and A3 lines were significantly lower than in the WT. The lower Ca²⁺ influx from the external media corresponded to a lower intracellular Ca²⁺ activity, which was estimated by fluorescence lifetime imaging measurements (FLIM). On the other hand, the intracellular pH values in the meristem zone of the roots of A2 and A3 seedlings were significantly lower (more acidic) than that of the WT, which might indicate a feedback block of H⁺ influx into meristematic cells caused by low intracellular pH. Under the control conditions, mature plants from the A2 and A3 lines contained significantly higher K⁺ and lower Ca²⁺ and Mg²⁺ content in the shoots, indicating disturbed long‐distance ion transport of these cations, possibly because of changes in xylem loading/retrieval and/or phloem loading. Exposing the plants in the flowering stage to various K⁺, Ca²⁺ and Mg²⁺ concentrations in the solution led to altered K⁺, Ca²⁺ and Mg²⁺ content in the shoots of A2 and A3 plants in comparison with the WT, suggesting a primary role of AtCNGC10 in Ca²⁺ (and probably Mg²⁺) transport in plants, which in turn regulates K⁺ transporters' activities.     

Evidence for the Induction of Repetitive Action Potentials in Synaptosomes by K+-Channel Inhibitors: An Analysis of Plasma Membrane Ion Fluxes

Abstract: The effects of four K⁺-channel inhibitors on synaptosomal free Ca²⁺ concentrations and ⁸⁶Rb⁺ fluxes are analysed. 4-Aminopyridine, α-dendrotoxin, charybdotoxin, and tetraethylammonium all increase the free Ca²⁺ concentration, although their potencies differ widely. In each case, the elevation in free Ca²⁺ concentration is reversed by the subsequent addition of tetrodotoxin. The transient ⁸⁶Rb⁺ efflux from preequilibrated synaptosomes induced with high concentrations of veratridine is partially inhibited by 4-aminopyridine and α-dendrotoxin. In contrast, when 4-aminopyridine or α-dendrotoxin is added to polarized synaptosomes, an enhanced⁸⁶Rb⁺ flux is seen, both for uptake and for efflux with no change in the total ⁸⁶Rb⁺/K⁺ content of the synaptosomes and with only a slight time-averaged plasma membrane depolarization (6.4 and 3.3 mV, respectively). The enhancements of flux by 4-aminopyridine or α-dendrotoxin are sensitive to ouabain and/or to tetrodotoxin. Furthermore, these flux changes show the same concentration dependencies as the blocked component of veratridine-stimulated ⁸⁶Rb⁺ efflux, the elevation of free Ca²⁺ concentration, and the facilitation of glutamate exocytosis that are elicited by 4-aminopyridine or α-dendrotoxin. It is concluded that these findings support the proposal of spontaneous, repetitive firing of synaptosomes evoked by K⁺-channel inhibitors and that the enhanced ⁸⁶Rb⁺ flux is a consequence of the activity of 4-aminopyridine- and α-dendrotoxin-insensitive K⁺ channels during these action potentials.     

Interdependence of H+ and K+ fluxes during the Ca2+-pumping activity of sarcoplasmic reticulum vesicles

The release of H⁺ during the oxalate-supported Ca²⁺ uptake in sarcoplasmic reticulum vesicles is kinetically coincident with the initial phase of Ca²⁺ accumulation. The Ca²⁺ uptake is increased and the H⁺ release is decreased in the presence of KCl and other monovalent chloride salts as expected for a H⁺-monovalent cation exchange. The functioning of the Ca²⁺-pump is disturbed by the presence of potassium gluconate and, to a lesser extent, of choline chloride. These salts do not inhibit the ATPase activity of Ca²⁺-permeable vesicles, suggesting a charge imbalance inhibition which is specially relevant in the case of gluconate. Therefore, K⁺, and also Cl^–, appear to be involved in secondary fluxes during the active accumulation of Ca²⁺. The microsomal preparation seems homogeneous with respect to the K⁺-channel, showing an apparent rate constant for K⁺ release of approximately 25 s^–1 measured with the aid of⁸⁶Rb⁺ tracer under equilibrium conditions. A Rb⁺ efflux, sensitive to Ca²⁺-ionophore, can be also detected during the active accumulation of Ca²⁺. The experimental data suggest that both monovalent cations and anions are involved in a charge compensation during the Ca²⁺ uptake and H⁺ release. Fluxes of these highly permeable ions would contribute to cancel the formation of a resting membrane potential through the sarcoplasmic reticulum membrane.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

K+ and Cl− conductances in the apical membrane from secreting oxyntic cells are concurrently inhibited by divalent cations

Summary This study concerns the properties of rapid K⁺ and Cl transport pathways that are present in the (H⁺+K⁺)-ATPase membrane from stimulated, and secreting, gastric oxyntic cells. Ion permeabilities in the isolated stimulation-associated vesicles were monitored via the rates of H⁺ efflux under conditions of exclusive H⁺/K⁺ counterflux or H⁺–Cl co-efflux, as well as by comparison of equilibration rates for⁸⁶Rb and³⁶Cl under conditions of equilibrium exchange and unidirectional salt flux. These latter studies suggest that Rb⁺ and Cl pathways are conductive and independent. In spite of the functional independence of the ion pathways, several divalent cations inhibit Rb⁺ and Cl isotopic exchange as well as the H⁺ efflux that is dependent on either K⁺ or anion (Cl, SCN, NO₂) fluxes. Zn²⁺ is the more potent inhibitor, reducing by 50% the sensitive component of K⁺, Cl, and NO₂ fluxes at about 20 m; Mn²⁺ has a similar effect at 200 m. Ni²⁺ and Co²⁺ were roughly equipotent to Mn²⁺ while Mg²⁺ and Ca²⁺ had not inhibitory effect. These results suggest that the stimulation-induced permeabilities, while functioning independently, may be physically linked, i.e., residing within a single entity. In similar studies carried out in (H⁺+K⁺)-ATPase vesicles obtained from nonstimulated cells, no vestiges of sensitivity to the inhibitory divalent cations could be detected. The implications of these findings for the physiology of the oxyntic cell in the context of a model for membrane fusion are discussed.     

K+(Rb+) and Ca2+ fluxes in young winter wheat plants exposed to sub-zero temperatures

Ice crystal formation temperature was determined in the region of the crown in one group of 7-day-old intact unhardened high-salt plants of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) with TA (Thermal Analysis) and DTA (Differential Thermal Analysis) methods. After exposure of another group of plants, grown for the first 7 days in the same way as the first group, to various sub-zero temperatures (-1 to 5°C), influx in roots of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) and contents of K⁺ and Ca²⁺ were determined at intervals during 7 days of recovery. Ice crystal formation in the crown tissue was probably extracellular and took place at about -4°C. There was a large loss of K⁺ from the roots after treatment at sub-zero temperatures. This loss increased as the temperature of the sub-zero treatment decreased. During recovery, roots of plants exposed to -1, -2 and -3°C gradually reabsorbed K⁺. Reabsorption of K⁺ in roots of plants exposed to -4°C was greatly impaired. Rb⁺ influx decreased and Ca²⁺ influx increased after sub-zero temperature treatments of the plants. Active Rb⁺ influx mechanisms and active extrusion of Ca²⁺ were impaired or irreversibly damaged by the exposure. While Rb⁺ influx mechanisms were apparently repaired during recovery in plants exposed to temperatures down to -3°C, Ca²⁺ extrusion mechanisms were not. The temperature for ice crystal formation in the region of the crown tissue coincides with the temperature at which the plants lost the ability to reabsorb K⁺ and to repair Rb⁺ influx mechanisms during the recovery period. Plants were lethally damaged at temperatures below ?4°C.     

Changes in Rb+ and Ca2+ influx in winter wheat roots before, during and after exposure to low temperature and short days

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺ (⁴⁵Ca²⁺) in roots of intact winter wheat (Triticum aestivum L. cv. Weibulls Starke II) was determined at intervals before, during and after exposure to cold acclimation conditions (2°C and 8 h light period). The plants were grown in nutrient medium of two ionic strengths. During the initial two weeks of growth at 16°C and 16 h light period, Rb⁺ influx into roots decreased with increasing age, probably as a consequence of a decreasing proportion of metabolically active roots. The presence of 10^?4M 2,4-dinitrophenol (DNP) reduced Rb⁺ influx to a low and constant level, indicating that metabolic influx was the dominant process. In contrast, Ca²⁺ influx in plants grown in full strength nutrient solution was higher in the presence than in the absence of DNP. This effect may have been due to an active extrusion mechanism mediating re-export of absorbed Ca²⁺(⁴⁵Ca²⁺) during the uptake experiment. With the metabolic uncoupler inhibiting such extrusion the Ca²⁺(⁴⁵Ca²⁺) influx mesured would increase. During cold treatment, Rb⁺ influx remained at a low level, and was further decreased when DNP was present in the uptake solution. This effect may have been due to inhibition of residual active influx of Rb⁺ at 2°C by the uncoupler and/or to a decrease in membrane permeability. In contrast to Rb⁺, Ca²⁺ influx increased during cold treatment, which could again be explained as inhibition of re-export. The presence of DNP reduced Ca²⁺ influx at 2°C, indicating decreased membrane permeability by DNP at low temperature. After transfer of plants from cold acclimation conditions to 16°C, Rb⁺ and Ca²⁺ influx increased in plants grown at both ionic strengths. Influx levels were independent of the length of the cold acclimation period (1, 6 and 8 weeks), but the patterns were different for the two ions. After each of the cold acclimation periods, Rb⁺ influx increased during the first week and decreased or remained at the same level during the second week, while Ca²⁺ influx always decreased during the second week of post-cold treatment.     

K+ Secretion in Rat Distal Jejunum

The Ussing chamber technique was used to measure unidirectional Rb⁺ fluxes under short-circuit conditions across tissue sheets from proximal, central, and distal jejunum of rats. Whereas the proximal and central parts of the jejunum did not show any net transport of Rb⁺, there was a net secretion of around 0.2 μmol hr⁻¹ cm⁻² in the distal segment. This secretion could not be influenced significantly by mucosal application of K⁺ channel blockers such as Ba²⁺ (5 mm), tetraethylammonium (20 mm) or quinine (1 mm). Serosal ouabain (1 mm) blocked net secretion by increasing mucoserosal flux. Blockers of H⁺/K⁺ ATPases could not alter net fluxes of Rb⁺. Stimulation of Cl⁻ secretion by forskolin (10 μm) or of Na⁺ absorption by serine (10 mm) failed to influence the observed secretion of Rb⁺. Adrenaline (10 μm) also had no effect on Rb⁺ fluxes. Blocking Na⁺/H⁺ exchange by 5-(N-Ethyl-N-isopropyl)-amilorid (100 μm) blocked net secretion by increasing mucoserosal flux, as did the addition of Na⁺ acetate (30 mm) to the mucosal solution. We conclude that the distal jejunum of the rat secretes K⁺ under short-circuit conditions. This secretion does not seem to occur via K⁺ channels, but through a pH dependent mechanism. Received: 16 February 1999/Revised: 29 June 1999     

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

K⁺ channels, membrane voltage, and intracellular free Ca²⁺ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K⁺ channel and a calcium-activated K⁺ channel. The inwardly rectifying K⁺ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K⁺ channel blockers quinidine, tetraethylammonium chloride and Ba²⁺ and elevated extracellular K⁺ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca²⁺ followed by a long-lasting Ca²⁺ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca²⁺, illustrating a transmembrane flux of Ca²⁺ following its electrochemical gradient. We conclude that K⁺ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca²⁺, a messenger in the mitogenic signal cascade of human melanoma cells. Received: 9 May 1995/Revised: 30 January 1996     

Influence of low temperature on regulation of Rb+ and Ca2+ influx in roots of winter wheat

Influx of Rb⁺(⁸⁶Rb⁺) and Ca²⁺(⁴⁵Ca²⁺) was determined in roots of winter wheat (Triticum aestivum L. cv. Weibulls Starke II) after 14 days at 16°C/16 h light, after 1 and 8 weeks of cold acclimation (2°C/8 h light) and at intervals after deacclimation (16°C/16 h light) for up to 14 days. The plants were cultivated at 3 ionic strengths: 100, 10 and 1% of a full strength nutrient solution, containing 3.0 mM K⁺ and 1.0 mM Ca²⁺. K⁺ concentrations in roots and shoots increased during cold treatment, while Ca²⁺ in the roots decreased. In the shoots Ca²⁺ concentrations remained the same. Influx of Rb⁺ as a function of average K⁺ concentration in the roots of 14-day-old, non-cold-treated plants was high at a certain K⁺ level in the root and decreased at higher root K⁺ levels (negative feedback). The pattern for Ca²⁺ influx versus average concentration of Ca²⁺ in the root was the reverse. Independent of duration of treatment (1–8 weeks), cold acclimation partly changed the regulation of Rb⁺ influx, so that it became less dependent upon negative feedback and more dependent on the ionic strength of the cultivation solution. After exposure to 2°C, Ca²⁺ influx increased at high Ca²⁺ concentrations in the root as compared with influx in roots of 14-day-old non-cold-treated plants. Under deacclimation, Ca²⁺ influx gradually decreased again, and reached the level observed before cold treatment within 7–14 days at 16°C; the number of days depending on the exposure time at 2°C. It is suggested that Rb⁺(K⁺) influx became adjusted to low temperature and that abscisic acid (ABA) may be involved in this mechanism. It is also suggested that extrusion of Ca²⁺ was impaired and/or Ca²⁺ channels were activated at 2°C in roots of plants grown in the full-strength solution and that extrusion was gradually restored and/or Ca²⁺ channels were closed under deacclimation conditions.     

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

Rises in cytosolic Ca²⁺ concentration ([Ca²⁺]_cyt) are central in platelet activation, yet many aspects of the underlying mechanisms are poorly understood. Most studies examine how experimental manipulations affect agonist-evoked rises in [Ca²⁺]_cyt, but these only monitor the net effect of manipulations on the processes controlling [Ca²⁺]_cyt (Ca²⁺ buffering, sequestration, release, entry and removal), and cannot resolve the source of the Ca²⁺ or the transporters or channels affected. To investigate the effects of protein kinase C (PKC) on platelet Ca²⁺ signalling, we here monitor Ca²⁺ flux around the platelet by measuring net Ca²⁺ fluxes to or from the extracellular space and the intracellular Ca²⁺ stores, which act as the major sources and sinks for Ca²⁺ influx into and efflux from the cytosol, as well as monitoring the cytosolic Na⁺ concentration ([Na⁺]_cyt), which influences platelet Ca²⁺ fluxes via Na⁺/Ca²⁺ exchange. The intracellular store Ca²⁺ concentration ([Ca²⁺]_st) was monitored using Fluo-5N, the extracellular Ca²⁺ concentration ([Ca²⁺]_ext) was monitored using Fluo-4 whilst [Ca²⁺]_cyt and [Na⁺]_cyt were monitored using Fura-2 and SFBI, respectively. PKC inhibition using Ro-31-8220 or bisindolylmaleimide I potentiated ADP- and thrombin-evoked rises in [Ca²⁺]_cyt in the absence of extracellular Ca²⁺. PKC inhibition potentiated ADP-evoked but reduced thrombin-evoked intracellular Ca²⁺ release and Ca²⁺ removal into the extracellular medium. SERCA inhibition using thapsigargin and 2,5-di(tert-butyl) l,4-benzohydroquinone abolished the effect of PKC inhibitors on ADP-evoked changes in [Ca²⁺]_cyt but only reduced the effect on thrombin-evoked responses. Thrombin evokes substantial rises in [Na⁺]_cyt which would be expected to reduce Ca²⁺ removal via the Na⁺/Ca²⁺ exchanger (NCX). Thrombin-evoked rises in [Na⁺]_cyt were potentiated by PKC inhibition, an effect which was not due to altered changes in non-selective cation permeability of the plasma membrane as assessed by Mn²⁺ quench of Fura-2 fluorescence. PKC inhibition was without effect on thrombin-evoked rises in [Ca²⁺]_cyt following SERCA inhibition and either removal of extracellular Na⁺ or inhibition of Na⁺/K⁺-ATPase activity by removal of extracellular K⁺ or treatment with digoxin. These data suggest that PKC limits ADP-evoked rises in [Ca²⁺]_cyt by acceleration of SERCA activity, whilst rises in [Ca²⁺]_cyt evoked by the stronger platelet activator thrombin are limited by PKC through acceleration of both SERCA and Na⁺/K⁺-ATPase activity, with the latter limiting the effect of thrombin on rises in [Na⁺]_cyt and so forward mode NCX activity. The use of selective PKC inhibitors indicated that conventional and not novel PKC isoforms are responsible for the inhibition of agonist-evoked Ca²⁺ signalling.