86Rb Efflux Research Articles

Leishmania Plasma Membrane Mg2+-ATPase Is a H+/K+-Antiporter Involved in Glucose Symport: STUDIES WITH SEALED GHOSTS AND VESICLES OF OPPOSITE POLARITY

Experiments from other laboratories conducted with Leishmania donovani promastigote cells had earlier indicated that the plasma membrane Mg2+-ATPase of the parasite is an extrusion pump for H+. Taking advantage of the pellicular microtubular structure of the plasma membrane of the organism, we report procedures for obtaining sealed ghost and sealed everted vesicle of defined polarity. Rapid influx of H+ into everted vesicles was found to be dependent on the simultaneous presence of ATP (1 mm) and Mg2+ (1 mm). Excellent correspondence between rate of H+ entry and the enzyme activity clearly demonstrated the Mg2+-ATPase to be a true H+ pump. H+ entry into everted vesicle was strongly inhibited by SCH28080 (IC50 = approximately 40 microm) and by omeprazole (IC50 = approximately 50 microm), both of which are characteristic inhibitors of mammalian gastric H+,K+-ATPase. H+ influx was completely insensitive to ouabain (250 microm), the typical inhibitor of Na+,K+-ATPase. Mg2+-ATPase activity could be partially stimulated with K+ (20 mm) that was inhibitable (>85%) with SCH28080 (50 microm). ATP-dependent rapid efflux of 86Rb+ from preloaded vesicles was completely inhibited by preincubation with omeprazole (150 microm) and by 5,5'-dithiobis-(2-nitrobenzoic acid) (1 mm), an inhibitor of the enzyme. Assuming Rb+ to be a true surrogate for K+, an ATP-dependent, electroneutral stoichiometric exchange of H+ and K+(1:1) was established. Rapid and 10-fold active accumulation of [U-(14)C]2-deoxyglucose in sealed ghosts could be observed when an artificial pH gradient (interior alkaline) was imposed. Rapid efflux of [U-(14)C]d-glucose from preloaded everted vesicles could also be initiated by activating the enzyme, with ATP. Taken together, the plasma membrane Mg2+-ATPase has been identified as an electroneutral H+/K+ antiporter with some properties reminiscent of the gastric H+,K+-ATPase. This enzyme is possibly involved in active accumulation of glucose via a H+-glucose symport system and in K+ accumulation.

Antisecretory Effect of Loperamide in Colon Epithelial Cells by Inhibition of Basolateral K+ Conductance

Background: The mechanism of the antisecretory effect of loperamide was investigated in cultured highly differentiated colon epithelial cells (HT-29/B6). Methods: Chloride secretion was stimulated via cAMP by forskolin (FSK, 10-5 M), via Ca2+ by the muscarinic agonist carbachol (CCh, 10-4 M), and via protein kinase C by the phorbol ester PMA (5·10-9 M). Stimulated Cl- secretion was quantified as short circuit current (ISC) of HT-29/B6 monolayers mounted in Ussing-type chambers. Results: Loperamide (5·10-5 M) inhibited ISC stimulated by FSK, CCh and PMA. The antisecretory action of loperamide was unaffected by preincubation with naloxone (10-5 M). Furthermore, loperamide strongly inhibited basolateral 86Rb efflux. Like loperamide, the calmodulin antagonist trifluoperazine (10-4 M) inhibited ISC induced by FSK, CCh or PMA. The Ca2+ channel blocker verapamil (5·10-5 M), on the other hand, inhibited only PMA-stimulated ISC, but had no effect on FSK or CCh-induced ISC. Conclusions: Loperamide exerts a direct antisecretory action on chloride secretion of colon epithelial cells independently of the respective stimulatory signal transduction pathway. This antisecretory effect is not mediated by opiate receptors and reflects inhibition of basolateral K+ conductance.

Membrane-permeabilizing activities of cyclic lipodepsipeptides, syringopeptin 22A and syringomycin E from Pseudomonas syringae pv. syringae in human red blood cells and in bilayer lipid membranes

The pore-forming activities of cyclic lipodepsipeptides (CLPs), syringopeptin 22A (SP22A) and syringomycin E (SRE) were compared on the human red blood cell (RBC) membrane and on bilayer lipid membranes (BLMs). SP22A above a concentration of 4×10 5 molecules/cell significantly increased the RBC membrane permeability for 86Rb. With electric current measurements on BLM, it was proved that like SRE, the SP22A formed two types of ion channels in the membrane, small and large, the latter having six times larger conductance and longer dwell time. Both CLPs formed clusters consisting of six small channels, and the channel-forming activity of SP22A is about one order of magnitude higher than that of SRE. A Hill coefficient of 2–3 estimated from the concentration dependence of these CLPs-induced lysis gave a proof of the pore oligomerization on RBCs. Transport kinetic data also confirmed that SP22A pores were oligomers of at least three monomers. While SRE pores were inactivated in time, no pore inactivation was observed with SP22A. The 86Rb efflux through SP22A-treated RBCs approached the tracer equilibrium distribution with a constant rate; a constant integral current was measured on the BLM for as long as 2.5 h as well. The partition coefficient ( K p=2×10 4 l/mol) between the RBC membrane and the extracellular space was estimated for SRE to be at least six times higher than that for SP22A. This finding suggested that the higher ion permeability of the SP22A-treated cells compared to that of SRE was the result of the higher pore-forming activity of SP22A.

Structural determinants of PIP(2) regulation of inward rectifier K(ATP) channels.

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) activates K(ATP) and other inward rectifier (Kir) channels. To determine residues important for PIP(2) regulation, we have systematically mutated each positive charge in the COOH terminus of Kir6.2 to alanine. The effects of these mutations on channel function were examined using (86)Rb efflux assays on intact cells and inside-out patch-clamp methods. Both methods identify essentially the same basic residues in two narrow regions (176-222 and 301-314) in the COOH terminus that are important for the maintenance of channel function and interaction with PIP(2). Only one residue (R201A) simultaneously affected ATP and PIP(2) sensitivity, which is consistent with the notion that these ligands, while functionally competitive, are unlikely to bind to identical sites. Strikingly, none of 13 basic residues in the terminal portion (residues 315-390) of the COOH terminus affected channel function when neutralized. The data help to define the structural requirements for PIP(2) sensitivity of K(ATP) channels. Moreover, the regions and residues defined in this study parallel those uncovered in recent studies of PIP(2) sensitivity in other inward rectifier channels, indicating a common structural basis for PIP(2) regulation.

Regulation of Monovalent Ion Homeostasis and pH by the Ser-Thr Protein Phosphatase SIT4 in Saccharomyces cerevisiae

A gene, SIT4, was identified as corresponding to a serine/threonine protein phosphatase and when overexpressed confers lithium tolerance in galactose medium to the budding yeast Saccharomyces cerevisiae. This gene has been previously identified as a regulator of the cell cycle and involved in nitrogen sensing. It is shown that the transcription levels of SIT4 are induced by low concentrations of Li(+) in a time-dependent manner. Na(+) and K(+) at high concentrations, but not sorbitol, also induce transcription. As a response to Na(+) or Li(+) stress, yeast cells lower the intracellular K(+) content. This effect is enhanced in cells overexpressing SIT4, which also increase (86)Rb efflux after the addition of Na(+) or Li(+) to the extracellular medium. Another feature of SIT4-overexpressing cells is that they maintain a more alkaline pH of 6.64 compared with 6.17 in the wild type cells. It has been proposed that the main pathway of salt tolerance in yeast is mediated by a P-type ATPase, encoded by PMR2A/ENA1. However, our results show that in a sit4 strain, expression of ENA1 is still induced by monovalent cations, and overexpression of SIT4 does not alter the amount of ENA1 transcript. These results show that SIT4 acts in a parallel pathway not involving induction of transcription of ENA1 and suggest a novel function for SIT4 in response to salt stress.

Selective inhibition of glucose-stimulated beta-cell activity by an anion channel inhibitor.

4,4'-Dithiocyanatostilbene-2,2'-disulfonic acid (DIDS), an inhibitor of the volume-sensitive anion channel, was used to investigate the role of this channel in the stimulation of rat pancreatic beta-cells by glucose and by tolbutamide. Glucose-stimulated electrical activity in beta-cells was markedly and reversibly inhibited by DIDS. The increase in cytosolic [Ca2+] and stimulated insulin release evoked by glucose were also inhibited by DIDS. In contrast to its inhibitory effect on glucose-induced beta-cell activity, DIDS had no effect on electrical activity, the rise in [Ca2+]i or insulin release induced by tolbutamide. DIDS failed to increase beta-cell input conductance, an index of whole-cell K(ATP) channel activity, or the rate of efflux of 86Rb+ from perifused islets, a measure of net K+ permeability. Furthermore, DIDS had no effect on intracellular pH or on regulatory volume increase following exposure of cells to hypertonic solutions, indicating that the effects of DIDS were not the result of inhibition of Cl- transport systems. It is suggested that the DIDS-induced repolarization is caused by inactivation of the volume-sensitive anion channel. The stimulation of beta-cell electrical and secretory activity by glucose, but not tolbutamide, may therefore involve activation of the anion channel.

A Role for Protein Kinase Cε in the Inhibitory Effect of Epidermal Growth Factor on Calcium-stimulated Chloride Secretion in Human Colonic Epithelial Cells

Epidermal growth factor (EGF) inhibits carbachol-induced chloride secretion in T(84) colonic epithelial cells and has been shown to activate phosphatidylinositol (PI) 3-kinase, leading to inhibition of a basolateral potassium conductance. We asked whether the inhibitory effect of EGF on secretion is due to activation of specific isoforms of protein kinase C (PKC) by PI 3-kinase. Western analysis revealed that PKCalpha, gamma, epsilon, eta, mu, lambda/iota, and zeta were expressed in T(84) cells. Ro318220 (an inhibitor active against PKCepsilon, 10 micrometer) but not Gö6983 (an inhibitor active against PKCzeta, 10 micrometer) reversed the inhibitory effect of EGF (100 ng/ml) on carbachol-stimulated chloride secretion. EGF induced the rapid translocation of PKCepsilon from the cytoplasm to the membrane. Wortmannin (50 micrometer) and LY294002 (20 nm), which are PI 3-kinase inhibitors that by themselves had no effect on PKCepsilon activity, significantly suppressed PKCepsilon translocation activated by EGF. LY294002 also reversed the inhibitory action of EGF on chloride secretion. PI (3,4)P(2) increased membrane-associated PKCepsilon and reduced carbachol-induced (86)Rb(+) efflux. Antisense oligonucleotides against PKCepsilon decreased PKCepsilon mass and prevented the inhibitory effect of EGF on carbachol-induced (86)Rb(+) efflux. Thus, the inhibitory effect of EGF on carbachol-induced chloride secretion involves the activation of PKCepsilon mediated by PI 3-kinase. Our findings contribute to the understanding of the cellular mechanisms that control chloride secretion.

Activation of H(+)-K(+)-ATPase by CO(2) requires a basolateral Ba(2+)-sensitive pathway during K restriction.

We studied the activation of H(+)-K(+)-ATPase by CO(2) in the renal cortical collecting duct (CCD) of K-restricted animals. Exposure of microperfused CCD to 10% CO(2) increased net total CO(2) flux (J(t CO(2))) from 4.9 +/- 2.1 to 14.7 +/- 4 pmol. mm(-1). min(-1) (P < 0. 05), and this effect was blocked by luminal application of the H(+)-K(+)-ATPase inhibitor Sch-28080. In the presence of luminal Ba, a K channel blocker, exposure to CO(2) still stimulated J(t CO(2)) from 6.0 +/- 1.0 to 16.8 +/- 2.8 pmol. mm(-1). min(-1) (P < 0.01), but peritubular application of Ba inhibited the stimulation. CO(2) substantially increased (86)Rb efflux (a K tracer marker) from 93.1 +/- 23.8 to 249 +/- 60.2 nm/s (P < 0.05). These observations suggest that during K restriction 1) the enhanced H(+)-K(+)-ATPase-mediated acidification after exposure to CO(2) is dependent on a basolateral Ba-sensitive mechanism, which is different from the response of rabbits fed a normal-K diet, where activation of the H(+)-K(+)-ATPase by exposure to CO(2) is dependent on an apical Ba-sensitive pathway; and 2) K/Rb absorption via the apical H(+)-K(+)-ATPase exits through a basolateral Ba-sensitive pathway. Together, these data are consistent with the hypothesis of cooperation between H(+)-K(+)-ATPase-mediated acidification and K exit pathways in the CCD that regulate K homeostasis.

A potent diazoxide analogue activating ATP-sensitive K+ channels and inhibiting insulin release.

To characterise the effects of BPDZ 73 (7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide), a newly synthesised diazoxide analogue, on insulin secretory cells. Measurements of 86Rb, 45Ca outflow, membrane potential, [Ca2+]i, insulin release in secretory cells as well as measurements of smooth muscle contractile activity and glycaemia were carried out. The analogue BPDZ 73 induced a dose-dependent decrease in insulin output. The IC50 value averaged 0.73 +/- 0.05 mumol/l. The drug increased the rate of 86Rb (42K substitute) outflow from perifused rat pancreatic islets. This effect was inhibited by glibenclamide, a KATP channel blocker. Measurements of DiBAC4(3) fluorescence further indicated that BPDZ 73 hyperpolarised the insulin secreting cells. It also decreased 45Ca outflow from pancreatic islets perifused throughout in the presence of 16.7 mmol/l glucose and extracellular Ca2+. By contrast, the drug did not affect the increase in 45Ca outflow mediated by K+ depolarisation. In single beta cells, BPDZ 73 inhibited the glucose-induced but not the K(+)-induced rise in [Ca2+]i. Moreover, in Wistar rats, i.p. injection of BPDZ 73 provoked a considerable increase in blood glucose concentration whereas diazoxide induced a modest rise in glycaemia. Lastly, the vasorelaxant properties of BPDZ 73 were slightly less pronounced than those of diazoxide. The inhibitory effect of BPDZ 73 on the insulin-releasing process results from the activation of KATP channels with subsequent decrease in Ca2+ inflow and [Ca2+]i. The drug seems to be a KATP channel opener, more potent and more selective than diazoxide for insulin secreting cells.

Protein Kinase C (PKC) Inhibits Fas Receptor-induced Apoptosis through Modulation of the Loss of K+ and Cell Shrinkage: A ROLE FOR PKC UPSTREAM OF CASPASES

Cell shrinkage and loss of intracellular K(+) are early requisite features for the activation of effector caspases and apoptotic nucleases in Fas receptor-mediated apoptosis of Jurkat cells, although the mechanisms responsible for both process remain unclear (Bortner, C. D., Hughes, F. M., Jr., and Cidlowski, J. A. (1997) J. Biol. Chem. 272, 32436-32442). We have now investigated the role of protein kinase C (PKC)-dependent signaling in the regulation of Fas-induced cell shrinkage and loss of K(+) during apoptosis. Anti-Fas induced cell shrinkage was blocked during PKC stimulation by the phorbol ester 12-O-tetradecanoylphorbol-3-acetate (PMA) and by bryostatin-1. Conversely, inhibition of PKC with Gö6976, enhanced the anti-Fas-mediated loss of cell volume. Analyses of mitochondrial membrane potential and DNA fragmentation revealed that the PKC-mediated effect observed in cell volume is propagated to these late features of apoptosis. Flow cytometric analyses and (86)Rb efflux experiments revealed that a primary effect of PKC appears to be on the modulation of Fas-induced K(+) efflux, since both PMA and bryostatin-1 inhibited extrusion of K(+) that occurs during Fas-mediated cell death, and Gö6976 exacerbated the effect of anti-Fas. Interestingly, high extracellular K(+) significantly blocked the effect of anti-Fas alone or anti-Fas combined with Gö6976, suggesting an underlying effect of PKC on K(+) loss. Western blot analyses showed the caspase-dependent proteolysis of PKC isotypes delta, epsilon, and theta in whole cell extracts from anti-Fas treated Jurkat T cells. However, stimulation of PKC by PMA or bryostatin-1 prevented this isotypic-specific PKC cleavage during apoptosis, providing further evidence that PKC itself exerts an upstream signal in apoptosis and controls the caspase-dependent proteolytic degradation of PKC isotypes. Finally, we show that PMA or bryostatin-1 prevents the activation of caspase-3 and caspase-8. Thus, this study shows that the protective effect that PKC stimulation exerts in the Fas-mediated apoptotic pathway occurs at a site upstream of caspases-3 and -8.

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37°C, SRE pore inactivation began 15 min after and at 20°C, 40 min after SRE addition. At 6°C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14°C, but at 23°C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.

Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K(+) channel Kir6.2 combination expressed in HEK-293 cells.

1. The pharmacological properties of K(ATP) channels generated by stable co-expression of the sulphonylurea receptor SUR1 and the inwardly rectifying K(+) channel Kir6.2 were characterized in HEK-293 cells. 2. [(3)H]-Glyburide (glibenclamide) bound to transfected cells with a B(max) value of 18.5 pmol mg(-1) protein and with a K(D) value of 0.7 nM. Specific binding was displaced by a series of sulphonylurea analogues with rank order potencies consistent with those observed in pancreatic RINm5F insulinoma and in the brain. 3. Functional activity of K(ATP) channels was assessed by whole cell patch clamp, cation efflux and membrane potential measurements. Whole cell currents were detected in transfected cells upon depletion of internal ATP or by exposure to 500 microM diazoxide. The currents showed weak inward rectification and were sensitive to inhibition by glyburide (IC(50)=0.92 nM). 4. Metabolic inhibition by 2-deoxyglucose and oligomycin treatment triggered (86)Rb(+) efflux from transfected cells that was sensitive to inhibition by glyburide (IC(50)=3.6 nM). 5. Diazoxide, but not levcromakalim, evoked concentration-dependen decreases in DiBAC(4)(3) fluorescence responses with an EC(50) value of 14.1 microM which were attenuated by the addition of glyburide. Diazoxide-evoked responses were inhibited by various sulphonylurea analogues with rank order potencies that correlated well with their binding affinities. 6. In summary, results from ligand binding and functional assays demonstrate that the pharmacological properties of SUR1 and Kir6.2 channels co-expressed in HEK-293 cells resemble those typical of native K(ATP) channels described in pancreatic and neuronal tissues.

3-Alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1,1-dioxides structurally related to diazoxide and pinacidil as potassium channel openers acting on vascular smooth muscle cells: design, synthesis, and pharmacological evaluation.

A series of 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides structurally related to diazoxide and pinacidil were synthesized and tested as possible K(ATP) channel openers on isolated pancreatic endocrine tissue as well as on isolated vascular, intestinal, and uterine smooth muscle. In contrast to previously described 3-alkylamino-4H-pyrido[4,3-e]-1,2,4-thiadiazine 1, 1-dioxides, most of the new compounds were found to be poorly active on B-cells but exhibited clear vasorelaxant properties. 3-(3, 3-Dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (4d) and 7-chloro-3-(3, 3-dimethyl-2-butylamino)-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxide (5d), two compounds bearing the alkyl side chain of pinacidil, were found to be the most active representatives of their respective series on rat aorta rings. 3-Cycloalkylalkylamino- and 3-aralkylamino-7-chloro-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides also expressed myorelaxant activity on electrically stimulated guinea pig ileum and on oxytocin-induced contractions of the rat uterus. Further biological investigations ((86)Rb efflux measurements, vasodilator potency on 30 and 80 mM KCl-induced contractions in the absence and presence of glibenclamide) revealed that compounds 4d and 5d, but not compound 5f, expressed the pharmacological profile of classical K(ATP) channel openers. In conclusion, by changing the position of the nitrogen atom in the pyridine ring, we now have obtained a family of drugs expressing an opposite tissue selectivity. Taken as a whole, the present findings also suggest that 3-alkylamino-4H-pyrido[2,3-e]-1,2,4-thiadiazine 1, 1-dioxides such as 4c, 4d, 5c, and 5d may be considered as new examples of K(ATP) channel openers expressing a pharmacological profile similar to that of pinacidil and diazoxide.

Factors affecting membrane permeability and ionic homeostasis in the cold-submerged frog.

Frogs (Rana temporaria) were submerged at 3 degrees C in either normoxic (P(O2)=155 mmHg, P(O2)=20 kPa) or hypoxic (P(O2)=60 mmHg; P(O2)=8 kPa) water for up to 16 weeks, and denied air access, to mimic the conditions of an ice-covered pond during the winter. The activity of the skeletal muscle Na(+)/K(+) pump over the first 2 months of hibernation, measured by ouabain-inhibitable (22)Na(+) efflux, was reduced by 30 % during normoxia and by up to 50 % during hypoxia. The reduction in Na(+)/K(+) pump activity was accompanied by reductions in passive (22)Na(+) influx and (86)Rb(+) efflux (effectively K(+) efflux) across the sarcolemma. This may be due to a decreased Na(+) permeability of the sarcolemma and a 75 % reduction in K(+) leak mediated by ATP-sensitive K(+) channels ('K(ATP)' channels). The lowered rates of (22)Na(+) and (86)Rb(+) flux are coincident with lowered transmembrane ion gradients for [Na(+)] and [K(+)], which may also lower Na(+)/K(+) pump activity. The dilution of extracellular [Na(+)] and intracellular [K(+)] may be partially explained by increased water retention by the whole animal, although measurements of skeletal muscle fluid compartments using (3)H-labelled inulin suggested that the reduced ion gradients represented a new steady state for skeletal muscle. Conversely, intracellular ion homeostasis within ventricular muscle was maintained at pre-submergence levels, despite a significant increase in tissue water content, with the exception of the hypoxic frogs following 4 months of submergence. Both ventricular muscles and skeletal muscles maintained resting membrane potential at pre-submergence levels throughout the entire period of hibernation. The ability of the skeletal muscle to maintain its resting membrane potential, coincident with decreased Na(+)/K(+) pump activity and lowered membrane permeability, provided evidence of functional channel arrest as an energy-sparing strategy during hibernation in the cold-submerged frog.

Efflux of osmolyte amino acids during isovolumic regulation in hippocampal slices.

The efflux of potassium (K(+)) and amino acids from hippocampal slices was measured after sudden exposure to 10% (270 mOsm), 25% (225 mOsm) or 50% (150 mOsm) hyposmotic solutions or after gradual decrease (-2.5 mOsm/min) in external osmolarity. In slices suddenly exposed to 50% hyposmotic solutions, swelling was followed by partial (74%) cell volume recovery, suggesting regulatory volume decrease (RVD). With gradual hyposmotic changes, no increase in cell water content was observed even when the solution at the end of the experiment was 50% hyposmotic, showing the occurrence of isovolumic regulation (IVR). The gradual decrease in osmolarity elicited the efflux of (3)H-taurine with a threshold at -5 mOsm and D-[(3)H]aspartate (as marker for glutamate) and at -20 mOsm for [(3)H]GABA. The efflux rate of [(3)H]taurine was always notably higher than those of [(3)H]GABA and D-[(3)H]aspartate, with a maximal increase over the isosmotic efflux of about 7-fold for [(3)H]taurine and 3- and 2-fold for [(3)H]GABA and D-[(3)H]aspartate, respectively. The amino acid content in slices exposed to 50% hyposmotic solutions (abrupt change) during 20 min decreased by 50. 6% and 62.6% (gradual change). Taurine and glutamate showed the largest decrease. An enhancement in (86)Rb efflux and a corresponding decrease in K(+) tissue content was seen in association with RVD but not with IVR. These results demonstrate the contribution of amino acids to IVR and indicate their involvement in this mechanism of cell volume control.

Inhibition of Na+/K(+)-ATPase may be one mechanism contributing to potassium efflux and cell shrinkage in CD95-induced apoptosis.

To investigate the involvement of K+ efflux in apoptotic cell shrinkage, we monitored efflux of the K+ congener, 86Rb+, and cell volume during CD95-mediated apoptosis in Jurkat cells. An anti-CD95 antibody caused apoptosis associated with intracellular GSH depletion, a significant increase in 86Rb+ efflux, and a decrease in cell volume compared with control cells. Preincubating Jurkat cells with Val-Ala-Asp-chloromethylketone (VAD-cmk), an inhibitor of caspase proteases, prevented the observed 86Rb+ efflux and cell shrinkage induced by the anti-CD95 antibody. A wide range of inhibitors against most types of K+ channels could not inhibit CD95-mediated efflux of 86Rb+, however, the uptake of 86Rb+ by Jurkat cells was severely compromised when treated with anti-CD95 antibody. Uptake of 86Rb+ in Jurkat cells was sensitive to ouabain (a specific Na+/K(+)-ATPase inhibitor), demonstrating Na+/K(+)-ATPase dependent K+ uptake. Ouabain induced significant 86Rb+ efflux in untreated cells, as well as it seemed to compete with 86Rb+ efflux induced by the anti-CD95 antibody, supporting a role for Na+/K(+)-ATPase in the CD95-mediated 86Rb+ efflux. Ouabain treatment of Jurkat cells did not cause a reduction in cell volume, although together with the anti-CD95 antibody, ouabain potentiated CD95-mediated cell shrinkage. This suggests that the observed inhibition of Na+/K(+)-ATPase during apoptosis may also facilitate apoptotic cell shrinkage.

Increases in intracellular pH and Ca(2+) are essential for K(+) channel activation after modest 'physiological' swelling in villus epithelial cells.

We studied the relationship between changes in intracellular pH (pH(i)), intracellular Ca(2+)([Ca(2+)](i)) and charybdotoxin sensitive (CTX) maxi-K(+) channels occurring after modest 'physiological' swelling in guinea pig jejunal villus enterocytes. Villus cell volume was assessed by electronic cell sizing, and pH(i) and [Ca(2+)](i) by fluorescence spectroscopy with 2,7, biscarboxyethyl-5-6-carboxyfluorescein and Indo-1, respectively. In a slightly (0.93 x isotonic) hypotonic medium, villus cells swelled to the same size they would reach during d-glucose or l-alanine absorption; the subsequent Regulatory Volume Decrease (RVD) was prevented by CTX. After the large volume increase in a more hypotonic (0.80 x isotonic) medium, RVD was unaffected by CTX. After modest swelling associated with 0.93 x isotonic dilution, the pH(i) alkalinized but N-5-methyl-isobutyl amiloride (MIA) prevented this DeltapH(i) and the subsequent RVD. Even in the presence of MIA, alkalinization with added NH(4)Cl permitted complete RVD which could be inhibited by CTX. The rate of (86)Rb efflux which also increased after this 0.93 x isotonic dilution was inhibited an equivalent amount by CTX, MIA or Na(+)-free medium. Modest swelling transiently increased [Ca(2+)](i) and Ca(2+)-free medium or blocking alkalinization by MIA or Na(+)-free medium diminished this transient increase an equivalent amount. RVD after modest swelling was prevented in Ca(2+)-free medium but alkalinization still occurred. After large volume increases, alkalinization of cells increased [Ca(2+)](i) and volume changes became sensitive to CTX. We conclude that both alkalinization of pH(i) and increased [Ca(2+)](i) observed with 'physiological' volume increase are essential for the activation of CTX-sensitive maxi-K(+) channels required for RVD.

Influence of CA2+ on K+ efflux during regulatory volume decrease in cultured astrocytes

The calcium (Ca2+) dependence of potassium (K+) efflux activated by hyposmolarity in cultured cerebellar astrocytes was investigated, measuring in parallel experiments (86)Rb release and changes in cytosolic Ca2+ ([Ca2+]i). Hyposmotic (50%) medium increased [Ca2+]i from 117 to 386 nM, with contributions of extracellular Ca2+ and Ca2+ from the endoplasmic reticulum. Hyposmotic medium increased (86)Rb efflux rate from 0.015 min(-1) to a maximal of 0. 049 min(-1) and a net release of 30%. This osmosensitive efflux was inhibited by Ba(2+) (0.028 min(-1)), quinidine (0.024 min(-1)), and charybdotoxin (0.040 min(-1)), but was unaffected by TEA, 4-AP, or apamin. Removal of external Ca2+ from the hyposmotic medium increased (86)Rb efflux to a maximal rate constant of 0.056 min(-1) and a net release of 38% and caused a delay of inactivation. These changes were due to the overlaping of an efflux activated by Ca2+ removal in isosmotic medium. This isosmotic 86Rb efflux was unaffected by TEA or 4-AP, reduced by verapamil, and abolished by Ba2+, nitrendipine, and Mg2+. With the swelling-induced [Ca2+]i rise suppressed by ethyleneglycoltetraacetic acid-acetoxy-methyl ester (EGTA-AM), hyposmotic (86)Rb was 30% reduced. The Ca2+ entry blockers Cd2+, Ni2+, La3+, and Gd3+ did not affect (86)Rb efflux. A 40% decrease observed with verapamil and nitrendipine was found unrelated to Ca2+, because these agents did not affect the [Ca2+]i rise and the inhibition persisted in the absence of external Ca2+. The phospholipase C blocker U-73122 did not affect [Ca2+]i nor (86)Rb efflux. Blockers of Ca2+/calmodulin W7 and KN-93 decreased (86)Rb efflux to the same extent as EGTA-AM. Ionomycin markedly potentiated (86)Rb release in hyposmotic conditions only when [Ca2+]i was raised to about 1 microM, suggesting the implication of maxi-K+ channels at this [Ca2+]i threshold, which nonetheless, was not attained during hyposmotic swelling. It is concluded that (86)Rb efflux in cerebellar astrocytes is largely (70%) Ca2+-independent and the Ca2+-dependent fraction is sustained essentially by Ca2+ released from the endoplasmic reticulum and mediated by a mechanism involving Ca2+/calmodulin.

PH regulation of K(+) efflux from myocytes in isolated rat hearts: (87)Rb, (7)Li, and (31)P NMR studies.

This study investigates the effects of intracellular (pH(i)) and extracellular pH (pH(e)) on the efflux of Rb(+) and Li(+) in isolated rat hearts. (87)Rb and (7)Li NMR were used to measure Rb(+) and Li(+) content, respectively, of hearts, and (31)P NMR was used to monitor pH(i), pH(e), and phosphate levels. After 30-min equilibration with Rb(+) or Li(+), effluxes were initiated by switching perfusion to a Rb(+)- or Li(+)-free, high-K(+) (20.7 mM) Krebs-Henseleit buffer with 15 microM bumetanide. Monensin (2 microM) increased pH(i) from 7.10 +/- 0.05 to 7.32 +/- 0.07 and resulted in activation of Rb(+) efflux; the first-order rate constant (k x 10(3), in min(-1)) increased from 42 +/- 2 to 116 +/- 16. Glibenclamide (4 microM) did not inhibit monensin-activated Rb(+) efflux (k = 110 +/- 17), whereas quinine (0.2 mM) slightly inhibited it by 19 +/- 9%. Infusion of 15 mM NH(4)Cl during Rb(+) washout increased k for Rb(+) efflux by 93% (81 +/- 8), which was glibenclamide and quinine insensitive, and caused a transient increase in pH(i) to 7.25 +/- 0.08. Intracellular Li(+) inhibited NH(4)Cl-stimulated Rb(+) efflux by 55%. Monensin and NH(4)Cl stimulated Li(+) efflux by 40%, increasing k from 29 +/- 3 to 43 +/- 7 and 41 +/- 3, respectively. The stimulation was not sensitive to 10 microM dimethylamiloride. Intracellular acidosis that resulted from the washout of NH(4)Cl (pH 6.86 +/- 0.2) slightly inhibited Rb(+) efflux (k = 36 +/- 5), whereas NH(4)Cl itself in the absence of pH(i) changes did not markedly affect Rb(+) efflux. A moderate increase in pH(i) (7.17 +/- 0.06) produced by washout of 15 mM 2, 2-dimethylpropionate (DMP)-Tris from hearts preequilibrated with DMP did not markedly affect Rb(+) efflux. Neither global alkalosis (pH(i) 7.4, pH(e) 7.55) nor acidosis (pH(i) approximately pH(e) 6.8) produced by 3 mM Tris base or 5 mM MES, respectively, affected Rb(+) efflux. We suggest that intracellular alkalosis stimulates Rb(+) (K(+)) and Li(+) effluxes by activating a nonselective sarcolemmal K(+) (Li(+))/cation exchanger or a K(+) (Li(+))-anion symporter.

Regional cerebral blood flow difference between dementia with Lewy bodies and AD.

The authors studied 14 patients with dementia with Lewy bodies (DLB), 14 patients with AD, and 14 healthy control subjects with N-isopropyl-p-[123I]iodoamphetamine SPECT. Comparison with the statistical parametric mappings revealed that relative cerebral blood flow was lower in the occipital lobes and higher in the right medial temporal lobe in the DLB group than in the AD group. Decreased occipital perfusion and relatively well preserved medial temporal perfusion are features that distinguish DLB from AD.