Feedback inhibition of sodium uptake in K+-depolarized toad urinary bladders

Ouabain-blocked toad urinary bladders were maintained in Na⁺-free mucosal solutions, and a depolarizing solution of high K⁺ activity containing only 5 mM Na⁺ on the serosal side. Exposure to mucosal sodium (20 mM activity) evoked a transient amiloride-blockable inward current, which decayed to near zero within one hour. The apical sodium conductance increased in the initial phase of the current decay and decreased in the second phase. The conductance decrease required Ca²⁺ to be present on the serosal side and was more rapid when the mucosal Na⁺ activity was higher. At 20 mM mucosal Na⁺ and 3 mM serosal Ca²⁺ the initial (maximal) rate of inhibition amounted to 20% in 10 min. The conductance decrease could be accelerated by raising the serosal Ca²⁺ activity to 10 mM. The inhibition reversed on lowering the serosal Ca²⁺ to 3 μM and, in addition, the mucosal Na⁺ to zero. Exposure of the mucosal surface to the ionophore nystatin abolished the Ca²⁺ sensitivity of the transcellular conductance, showing that the Ca²⁺-sensitive conductance resides in the apical membrane. The data imply that in the K⁺-depolarized epithelia, cellular Ca²⁺, taken up from the serosal medium by means of a Na⁺-Ca²⁺ antiport, cause feedback inhibition by blockage of apical Na⁺ channels. However, the rate of inhibition is small, such that this regulatory mechanism will have little effect at 1 mM serosal Ca²⁺ and less than 20 mM cellular Na⁺.     

Carrier-mediated Potassium Efflux Across the Cell Membrane of Red Beet

The flux ratio (influx/efflux) of K⁺ across the plasmalemma of beet cells at an external potassium concentration of 0.6 mm does not respond to changes of membrane potential in the manner expected for the free diffusion of ions. The K⁺ efflux is affected by the presence of adsorbed Ca²⁺, but is apparently unrelated to the electrical potential or to the net uptake of potassium. The K⁺ efflux is greater than the efflux of the sulfate and organic anions which are accumulated with potassium, and is partially dependent on the presence of external potassium. Thus the loss of ⁴²K from the cell does not appear to be a leakage of freely diffusing K⁺ ions, nor a leakage of ion pairs, but a carrier-mediated transport or exchange of potassium across the cell membrane.     

Activation of Apical K<Superscript>+</Superscript> Conductances by Muscarinic Receptor Stimulation in Rat Distal Colon: Fast and Slow Components

In the epithelium of rat distal colon the acetylcholine analogue carbachol induces a transient increase of short-circuit current (I_sc) via stimulation of cellular K⁺ conductances. Inhibition of the turnover of inositol-1,4,5-trisphosphate (IP₃) by LiCl significantly reduced both the amplitude and the duration of this response. When the apical membrane was permeabilized with nystatin, LiCl nearly abolished the carbachol-induced activation of basolateral K⁺ conductances. In contrast, in epithelia, in which the basolateral membrane was bypassed by a basolateral depolarization, carbachol induced a biphasic increase in the K⁺ current across the apical membrane consisting of an early component carried by charybdotoxin- and tetraethylammonium-sensitive K⁺ channels followed by a sustained plateau carried by channels insensitive against these blockers. Only the latter was sensitive against LiCl or inhibition of protein kinases. In contrast, the stimulation of the early apical K⁺ conductance by carbachol proved to be resistant against inhibition of phospholipase C or protein kinases. However, apical dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, or a Ca²⁺-free mucosal buffer solution significantly reduced the early component of the carbachol-induced apical K⁺ current. The presence of an apically localized Na⁺/Ca²⁺-exchanger was proven immunohistochemically. Taken together these experiments reveal divergent regulatory mechanisms for the stimulation of apical Ca²⁺-dependent K⁺ channels in this secretory epithelium, part of them being activated by an inflow of Ca²⁺ across the apical membrane.

Calcium-dependent K efflux from rat submandibular gland: The effects of trifluoperazine and quinidine

The Ca²⁺-dependent K⁺ efflux from rat submandibular gland was studied using a K⁺-sensitive electrode. A K⁺ efflux was induced by either adrenalin or by using the divalent cation ionophore A23187 plus added Ca²⁺ to bypass the receptor mechanism. Trifluoperazine, which was used to investigate the role of calmodulin, was found to block the adrenalin-induced K⁺ efflux but not the A23187/Ca²⁺-induced K⁺ efflux. The adrenalin-induced K⁺ efflux was abolished by quinidine and the A23187/Ca²⁺-induced K⁺ efflux was significantly reduced by quinidine. In other experiments, the presence of indomethacin did not inhibit the adrenalin-induced K⁺ efflux, and exogenously added arachidonic acid did not induce a K⁺ efflux. It is concluded that neither prostaglandin synthesis, nor a cytosolic Ca²⁺-calmodulin complex is involved in the agonist-induced K⁺ efflux from rat submandibular gland. A similarity between the Ca²⁺-dependent K⁺ efflux mechanism of erythrocyte ghosts and submandibular tissue is indicated by their common response to quinidine.     

Reactions of corn root tissue to calcium

Washing corn (Zea mays L.) root tissue in water causes loss of about one-third of the exchangeable Ca²⁺ over the first 10 to 15 minutes. Upon transfer to K⁺-containing solutions, the tissue shows a short period of rapid K⁺ influx which subsequently declines. Addition of 0.1 millimolar Ca²⁺ decreases the initial rapid K⁺ influx, but increases the sustained rate of K⁺ and Cl⁻ uptake. It was confirmed (Elzam and Hodges 1967 Plant Physiol 42: 1483-1488) that 0.1 millimolar Ca²⁺ is more effective than higher concentrations for the initial inhibition, and that Mg²⁺ will substitute.

The inhibition arises from a mild shock affect of restoring Ca²⁺. With 0.1 millimolar Ca²⁺ net H⁺ efflux is blocked for 10 to 15 minutes and the cells are depolarized by about 30 millivolts. However, 1 millimolar Ca²⁺ rapidly produces increased K⁺ influx and blocks net H⁺ efflux for only a few minutes; blockage is preceded by a brief net H⁺ influx which may restore and increase ion transport by reactivating the plasmalemma H⁺-ATPase.

Stimulation of electrogenic H⁺-pumping with fusicoccin eliminates the shock responses and minimizes Ca²⁺ effects on K⁺ influx. Fusicoccin also strongly decreases Ca²⁺ influx, but has no effect on Ca²⁺ efflux. Ice temperatures and high pH decreased Ca²⁺ efflux, but uncoupler and chlorpromazine did not.

It is suggested that the inhibitory and promotive actions of Ca²⁺ are manifested through decreases or increases in the protonmotive force.

     

K+ transport in ‘tight’ epithelial monolayers of MDCK cells: Evidence for a calcium-activated K+ channel

Measurements of ⁸⁶Rb efflux across the apical and basal-lateral aspects of intact monolayers of ‘high-resistance’ MDCK cells mounted in Ussing chambers have been made. A transient increase in ⁸⁶Rb efflux across both epithelial borders upon stimulation with adrenalin or ionophore A23187 is observed. The increased ⁸⁶Rb across the basal cell aspects is of greatest quantitative importance. Measurements of total cellular K⁺ contents by flame photometry of tissue extracts indicate a net loss of K⁺ following adrenalin addition. The effects of adrenalin and ionophore A23187 upon ⁸⁶Rb efflux are abolished in ‘Ca²⁺-free’ media. The properties of the Ca²⁺ -dependent increase in ⁸⁶Rb efflux show similarities to Ca²⁺-activated K⁺ conductances in other tissues, notably human red cells, including inhibition by quinine (1 mM), tetraethylammonium (25 mM) and insensitivity to bee venom toxin (apamin) (25 nM). Adrenalin is only effective when applied to the basal bathing solution suggesting that the receptors mediating adrenalin action are located upon the basal-lateral membranes. Half maximal stimulation of ⁸⁶Rb efflux by adrenalin is observed at 9.1·10^?7 M. The action of various adrenergic receptor agonists and antagonists are consistent with adrenalin action being mediated by an α-adrenergic receptor.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

H+-dependent efflux of Ca2+ from heart mitochondria

A rapid loss of accumulated Ca²⁺ is produced by addition of H⁺ to isolated heart mitochondria. The H⁺-dependent Ca⁺ efflux requires that either (a) the NAD(P)H pool of the mitochondrion be oxidized, or (b) the endogenous adenine nucleotides be depleted. The loss of Ca²⁺ is accompanied by swelling and loss of endogenous Mg^2–. The rate of H⁺-dependent Ca²⁺ efflux depends on the amount of Ca²⁺ and P_i taken up and the extent of the pH drop imposed. In the absence of ruthenium red the H⁺-induced Ca²⁺-efflux is partially offset by a spontaneous re-accumulation of released Ca²⁺. The H⁺-induced Ca²⁺ efflux is inhibited when the P_i transporter is blocked withN-ethylmaleimide, is strongly opposed by oligomycin and exogenous adenine nucleotides (particularly ADP), and inhibited by nupercaine. The H⁺-dependent Ca²⁺ efflux is decreased markedly when Na⁺ replaces the K⁺ of the suspending medium or when the exogenous K⁺/H⁺ exchanger nigericin is present. These results suggest that the H⁺-dependent loss of accumulated Ca²⁺ results from relatively nonspecific changes in membrane permeability and is not a reflection of a Ca²⁺/H⁺ exchange reaction.     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Exposure of colonic epithelial cells to oxidative and endoplasmic reticulum stress causes rapid potassium efflux and calcium influx

Endoplasmic reticulum (ER) stress and oxidative stress have recently been linked to the pathogenesis of inflammatory bowel diseases. Under physiological conditions, intestinal epithelial cells are exposed to ER and oxidative stress affecting the cellular ionic homeostasis. However, these altered ion flux ‘signatures’ during these stress conditions are poorly characterized. We investigated the kinetics of K⁺, Ca²⁺ and H⁺ ion fluxes during ER and oxidative stress in a colonic epithelial cell line LS174T using a non‐invasive microelectrode ion flux estimation technique. ER and oxidative stress were induced by cell exposure to tunicamycin (TM) and copper ascorbate (CuAsc), respectively, from 1 to 24 h. Dramatic K⁺ efflux was observed following acute ER stress with peak K⁺ efflux being ?30·6 and ?138·7 nmolm^?2 s^?1 for 10 and 50 µg ml^?1, respectively (p < 0·01). TM‐dependent Ca²⁺ uptake was more prolonged with peak values of 0·85 and 2·68 nmol m^?2 s^?1 for 10 and 50 µg ml^?1 TM, respectively (p < 0·02). Ion homeostasis was also affected by the duration of ER stress. Increased duration of TM treatment from 0 to 18 h led to increases in both K⁺ efflux and Ca²⁺ uptake. While K⁺ changes were significantly higher at each time point tested, Ca²⁺ uptake was significantly higher only after prolonged treatment (18 h). CuAsc also led to an increased K⁺ efflux and Ca²⁺ uptake. Functional assays to investigate the effect of inhibiting K⁺ efflux with tetraethylammonium resulted in increased cell viability. We conclude that ER/oxidative stress in colonic epithelial cells cause dramatic K⁺, Ca²⁺ and H⁺ ion flux changes, which may predispose this lineage to poor stress recovery reminiscent of that seen in inflammatory bowel diseases. Copyright © 2012 John Wiley & Sons, Ltd.     

Effects of calcium on potassium and water transport in human erythrocyte ghosts

Bulk water transport in reconstituted ghosts is statistically comparable to that in the parent red cells, and is unaffected by incorporation of Ca²⁺ over the range of 0.01 to 1 mM. Brief exposure of ghosts to p-chloromercuribenzene sulfonate results in a supression of osmotic water flow but leaves K⁺ permeability unchanged. Incorporation of p-chloromercuribenzene sulfonate provokes extremely rapid K⁺ loss which can be counteracted by simultaneous inclusion of Ca²⁺.Erythrocyte ghosts, when prepared with a small amount of Ca²⁺, demonstrate recovery of normal impermeability to choline, sucrose, Na⁺ and inulin and have an improved K⁺ retention over Ca²⁺-free preparations.The rate of passive transport of K⁺ from unwashed erythrocyte ghosts was measured during the initial few minutes of efflux. The initial rates vary in a bimodal fashion with the concentration of Ca²⁺ incorporated at the time of hemolysis. In low concentrations (0.01–0.1 mM), Ca²⁺ protects the K⁺ barrier while at higher concentrations (0.1–1.0 mM) it provokes a K⁺ leakage ranging from 7 to 50 times the normal rate of passive K⁺ loss. The Ca²⁺-induced K⁺ leak is thus a graded response rather than a discrete membrane transport state. The transition from a Ca²⁺-protected to a Ca²⁺-damaged membrane occurs upon an increase in Ca²⁺ concentration of less than 50 μmoles/l.     

Potassium fluxes across the blood brain barrier of the cockroach, Periplaneta americana

Potassium fluxes across the blood-brain barrier of the cockroach Periplaneta americana were measured using the scanning ion-selective microelectrode technique. In salines containing 15 mM or 25 mM K⁺, an efflux of K⁺ from the ganglia of isolated nerve cords was counterbalanced by an influx across the connectives. Metabolic inhibition with CN⁻ resulted in an increase in K⁺ efflux across both the ganglia and the connectives. Depletion of K⁺ by chilling the nerve cords in K⁺-free saline was associated with subsequent K⁺ influx across the connectives in K⁺-replete saline at room temperature. There were dramatic increases in K⁺ efflux across both ganglia and connectives when the nerve cords were exposed to the pore-forming antibiotic amphotericin B. K⁺ fluxes across the ventral nerve cord were also altered when paracellular leakage was augmented by transient exposure to 3 M urea. K⁺ efflux was reduced by the K⁺ channel blockers Ba²⁺ and tetraethylammonium or by exposure to Ca²⁺-free saline and K⁺ efflux from the ganglia was increased by addition of ouabain to the bathing saline. The results provide direct support for a model proposing that K⁺ is cycled through a current loop between the ganglia and the connectives and that both the Na⁺/K⁺-ATPase and K⁺ channels are implicated in extracellular K⁺ homeostasis within the central nervous system.     

The calcium requirement for stimulation of rubidium efflux from isolated rat parotid acinar cells by carbachol.

Stimulation of ⁸⁶Rb⁺ efflux from isolated parotid acinar cells by carbchol was biphasic. The phases of stimulated ⁸⁶Rb⁺ efflux were separated on the basis of their relative requirements for extracellular Ca²⁺. If the isolated cells were incubated in Ca²⁺ free buffer containing 1.0 mM ethylene glycol bis (β-aminoethyl ether) N, N¹ - tetra acetic acid (EGTA) for 30 min. before adding carbachol an initial phase of ⁸⁶Rb⁺ efflux was observed. A second phase of ⁸⁶Rb⁺ efflux was obtained upon addition of 2.0 mM Ca²⁺. However when cells were incubated for 60 min. in Ca²⁺ free buffer containing 1.0 mM EGTA the initial phase of release caused by carbachol was inhibited by 95 percent. If the EGTA was titrated with Ca²⁺ to give 1.0 mM Ca²⁺, following the 60 min. depletion regimen, the second phase was observed. Although 60 min. of Ca²⁺-depletion in EGTA buffer was required for complete inhibition of the effect of carbachol on the initial phase of ⁸⁶Rb⁺ efflux, the response was fully restored within 4 min. after the readdition of Ca²⁺.     

Red Blood Cells of a Transgenic Mouse Expressing High Levels of Human Hemoglobin S Exhibit Deoxy-stimulated Cation Flux

+ and Na⁺ transport in RBCs from control mice (C57Bl/6J) and a transgenic (α^Hβ^S[β^MDD]) mouse line that expresses high levels of human α^H and β^S-chains and has a small percent dense cells but does not exhibit anemia. In transgenic mouse RBCs (n= 5) under oxygenated conditions, K⁺ efflux was 0.22 ± 0.01 mmol/L cell × min and Na⁺ influx was 0.17 ± 0.02 mmol/L cell × min. Both fluxes were stimulated by 10 min deoxygenation in transgenic but not in control mice. The deoxy-stimulated K⁺ efflux from transgenic mouse RBCs was about 55% inhibited by 5 nm charybdotoxin (CTX), a blocker of the calcium activated K⁺-channel. To compare the fluxes between human and mouse RBCs, we measured the area of mouse RBCs and normalized values to area per liter of cells. The deoxy-simulated CTX-sensitive K⁺ efflux was larger than the CTX-sensitive K⁺ efflux observed in RBCs from SS patients. These results suggest that in transgenic mice, deoxygenation increases cytosolic Ca²⁺ to levels which open Ca²⁺-activated K⁺ channels. The presence of these channels was confirmed in both control and transgenic mice by clamping intracellular Ca²⁺ at 10 μm with the ionophore A23187 and measuring Ca²⁺-activated K⁺ efflux. Both types of mouse had similar maximal rates of CTX-sensitive, Ca²⁺-activated K⁺ efflux that were similar to those in human SS cells. The capacity of the mouse red cell membrane to regulate cytosolic Ca²⁺ levels was examined by measurements of the maximal rate of calmodulin activated Ca²⁺-ATPase activity. This activity was 3-fold greater than that observed in human RBCs thus indicating that mouse RBC membranes have more capacity to regulate cytosolic Ca²⁺ levels. In summary, transgenic mouse RBCs exhibit larger values of deoxy-stimulated K⁺ efflux and Na⁺ influx when compared to human SS cells. They have a similar Ca²⁺-activated K⁺ channel activity to human SS cells while expressing a very high Ca²⁺ pump activity. These properties may contribute to the smaller percent of very dense cells and to the lack of adult anemia in this animal model. Received: 23 October/Revised: 15 May 1997     

Cation-dependent uptake of zinc in human fibroblasts

The influence of K⁺ and Ca²⁺ on Zn²⁺ transport into cultured human fibroblasts was investigated. Zn²⁺ uptake was markedly reduced in the presence of both valinomycin and nigericin (electrogenic and electroneutral K ⁺ ionophores, respectively), and by reduction in the transmembrane K⁺ gradient produced by replacement of extracellular K⁺ with Na⁺, suggesting that Zn²⁺ may be driven by a Zn²⁺/K⁺ counter-transport system. To test the counter-transport hypothesis, we used ⁸⁶Rb as an analog of K ⁺ for efflux studies. The rate of Rb⁺ efflux was 3760 times that of Zn²⁺ uptake, thus the component of K⁺ involved in the Zn²⁺ counter-transport system was only a small proportion of the total K⁺ efflux. In investigating the effect of Ca²⁺ on Zn²⁺ uptake, we identified two components: (1) a basal Zn²⁺ uptake pathway, independent of hormonal or growth factors which does not require extracellular Ca²⁺ and (2) a Ca²⁺-dependent mechanism. The absence of Ca²⁺ decreased Zn²⁺ uptake, while increasing extracellular C+a²⁺ stimulated Zn²⁺ uptake. The effect was mediated by Ca²⁺ influx as the ionophores A23187 and ionomycin also stimulated Zn²⁺ uptake. We could not ascribe the Ca²⁺ effect to known Ca²⁺ influx pathways. We conclude that Zn²⁺ uptake occurs by a K⁺-dependent process, possibly by Zn²⁺/K⁺ counter-transport and that a component of this is also Ca²⁺-dependent.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

In Vivo Perturbation of Membrane-Associated Calcium by Freeze-Thaw Stress in Onion Bulb Cells : Simulation of This Perturbation in Extracellular KCl and Alleviation by Calcium

Incipient freeze-thaw stress in onion bulb scale tissue is known to cause enhanced efflux of K⁺, along with small but significant loss of cellular Ca²⁺. During the post-thaw period, irreversibly injured cells undergo a cytological aberration, namely, `protoplasmic swelling.' This cellular symptom is thought to be caused by replacement of Ca²⁺ from membrane by extracellular K⁺ and subsequent perturbation of K⁺ transport properties of plasma membrane. In the present study, onion (Allium cepa L. cv Sweet Sandwich) bulbs were slowly frozen to either −8.5°C or −11.5°C and thawed over ice. Inner epidermal peels from bulb scales were treated with fluorescein diacetate for assessing viability. In these cells, membrane-associated calcium was determined using chlorotetracycline fluorescence microscopy combined with image analysis. Increased freezing stress and tissue infiltration (visual water-soaking) were paralleled by increased ion leakage. Freezing injury (−11.5°C; irreversible) caused a specific and substantial loss of membrane-associated Ca²⁺ compared to control. Loss of membrane-associated Ca²⁺ caused by moderate stress (−8.5°C; reversible) was much less relative to −11.5°C treatment. Ion efflux and Ca²⁺-chlorotetracycline fluorescence showed a negative relationship. Extracellular KCl treatment simulated freeze-thaw stress by causing a similar loss of membrane-associated calcium. This loss was dramatically reduced by presence of extracellular CaCl₂. Our results suggest that the loss of membrane-associated Ca²⁺, in part, plays a role in initiation and progression of freezing injury.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L. IV. Comparative ion flux characteristics of the turion and the vegetative frond and the effect of ABA during early turion development

Abstract Using the method of compartmental analysis, the ion fluxes and compartment concentrations of Ca²⁺, K⁺ and Cl^- have been compared in the untreated vegetative frond and the abscisic acid (ABA) induced turion of Spirodela polyrrhiza. The ABA-induced turion is characterized by reduced Ca²⁺ exchange across the tonoplast and low vacuolar Ca²⁺ concentration relative to the vegetative frond. In addition the turion exhibits a higher plasmalemma flux with a correspondingly high Ca²⁺ concentration in the cytoplasm. The concentration of K⁺ and Cl^- is much lower in the cytoplasm of the ABA-induced turion than in the vegetative frond with the influx/efflux ratio at both the plasmalemma and the tonoplast being less than 1, a finding exhibited also in dormant storage tissue. Treatment of vegetative fronds with ABA for 18 h resulted in a reduced K⁺ plasmalemma efflux relative to untreated vegetative fronds and a concomitant increase in the cytoplasmic concentration. There was no rapid effect of ABA on Ca²⁺, K⁺ or Cl^- fluxes through either membrane. These results are consistent with the notion that drastic changes in ion fluxes and concentrations in the turion are a secondary consequence of ABA-induced development, possibly due to prior regulation by ABA of enzymes inherent to processes involved in membrane transport.     

Role of external potassium in the calcium-induced potassium efflux from human red blood cell ghosts

Summary The exposure of red cell ghosts to external Ca⁺⁺ and K⁺ leads to a rapid net K⁺ efflux. Preincubation of the ghosts for various lengths of time in the absence of K⁺ in the external medium prior to a challenge with maximally effective concentrations of Ca⁺⁺ and K⁺ renders the ghosts unresponsive to that challenge with a half-time of about 7–10 min. Preincubation at a range of K⁺ concentrations for a fixed length of time (60 min) prior to the challenge revealed that K⁺ concentrations of about 500 m or more suffice to maintain the K⁺ channel in a maximally responsive state for at least 60 min. These K⁺ concentrations are considerably lower than the K⁺ concentrations required to make the responsive channel respond with a maximal rate of K⁺ efflux. Thus, external K⁺ is not only necessary to induce the permeability change but also to maintain the transport system in a functional state.The presence of Mg⁺⁺ or ethylenediamine-tetraacetic acid (EDTA) in the K⁺-free preincubation media preserves the responsiveness to a challenge with Ca⁺⁺ plus K⁺. In contrast to external K⁺, the presence of external Ca⁺⁺ does not reduce but rather enhances the loss of responsiveness. An excess of EDTA prevents the effects of Ca⁺⁺ while washes with EDTA after exposure to Ca⁺⁺ do not reverse them.In red cell ghosts that contain Ca⁺⁺ buffers, the transition from a responsive to a nonresponsive state incubation in the absence of external K⁺ is enhanced. The effects of incubation in the presence of Ca⁺⁺ in K⁺-free media are reversed; external Ca⁺⁺ now reduces the rate at which the responsiveness is lost. The loss of responsiveness after incubation in K⁺-free media prior to a challenge with external K⁺ and internal Ca⁺⁺ does also take place when K⁺-efflux from red cell ghosts is measured by means of⁴²K⁺ into media that have the same K⁺ concentrations as the ghost interior. This confirms that the effects of K⁺-free incubation are due to the modification of the K⁺-selective channel rather than to an inhibition of diffusive Cl^–-efflux.Abbreviation used in text TRIS Tris (hydroxymethyl) aminomethan This paper is dedicated to the memory of Walther Wilbrandt.