Effects of L-alanine on membrane potential, potassium (86Rb) permeability and cell volume in hepatocytes from Raja erinacea

Isolated hepatocytes from the elasmobranch Raja erinacea were examined for their regulatory responses to a solute load following electrogenic uptake of L-alanine. The transmembrane potential (Vm) was measured with glass microelectrodes filled with 0.5 M KCl (75 to 208 M omega in elasmobranch Ringer's solution) and averaged -61 +/- 16 mV (S.D.; n = 68). L-Alanine decreased (depolarized) Vm by 7 +/- 3 and 18 +/- 2 mV at concentrations of 1 and 10 mM, respectively. Vm did not repolarize to control values during the 5-10 min impalements, unless the amino acid was washed away from the hepatocytes. The depolarizing effect of L-alanine was dependent on external Na+, and was specific for the L-isomer of alanine, as D- and beta-alanine had no effect. Hepatocyte Vm also depolarized on addition of KCN or ouabain, or when external K+ was increased. Rates of 86Rb+ uptake and efflux were measured to assess the effects of L-alanine on Na+/K+-ATPase activity and K+ permeability, respectively. Greater than 80% of the 86Rb+ uptake was inhibited by 2 mM ouabain, or by substitution of choline+ for Na+ in the incubation media. L-Alanine (10 mM) increased 86Rb+ uptake by 18-49%, consistent with an increase in Na+/K+ pump activity, but had no effect on rubidium efflux. L-Alanine, at concentrations up to 20 mM, also had no measurable effect on cell volume as determined by 3H2O and [14C]inulin distribution. These results indicate that Na+-coupled uptake of L-alanine by skate hepatocytes is rheogenic, as previously observed in other cell systems. However, in contrast to mammalian hepatocytes, Vm does not repolarize for at least 10 min after the administration of L-alanine, and changes in cell volume and potassium permeability are also not observed.     

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

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

Na+, Cl− cotransport in Ehrlich ascites tumor cells activated during volume regulation (regulatory volume increase)

Ehrlich ascites cells were preincubated in hypotonic medium with subsequent restoration of tonicity. After the initial osmotic shrinkage the cells recovered their volume within 5 min with an associated KCl uptake. The volume recovery was inhibited when NO-3 was substituted for Cl-, and when Na+ was replaced by K+, or by choline (at 5 mM external K+). The volume recovery was strongly inhibited by furosemide and bumetanide, but essentially unaffected by DIDS. The net uptake of Cl- was much larger than the value predicted from the conductive Cl- permeability. The undirectional 36Cl flux, which was insensitive to bumetanide under steady-state conditions, was substantially increased during regulatory volume increase, and showed a large bumetanide-sensitive component. During volume recovery the Cl- flux ratio (influx/efflux) for the bumetanide-sensitive component was estimated at 1.85, compatible with a coupled uptake of Na+ and Cl-, or with an uptake via a K+,Na+,2Cl- cotransport system. The latter possibility is unlikely, however, because a net uptake of KCl was found even at low external K+, and because no K+ uptake was found in ouabain-poisoned cells. In the presence of ouabain a bumetanide-sensitive uptake during volume recovery of Na+ and Cl- in nearly equimolar amounts was demonstrated. It is proposed that the primary process during the regulatory volume increase is an activation of an otherwise quiescent, bumetanide-sensitive Na+,Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump, stimulated by the Na+ influx through the Na+,Cl- cotransport system.     

Characterization of low potassium-resistant mutants of the Madin-Darby canine kidney cell line with defects in NaCl/KCl symport

Three independent mutants of the Madin-Darby canine kidney cell line (MDCK) have been isolated which were capable of growth in media containing low concentrations of potassium. All three mutants were deficient to varying extents in furosemide- and bumetanide-sensitive 22Na+, 86+b+, and 36Cl- uptake. The two mutants most resistant to low K+ media had lost essentially all of the 22Na+, 86Rb+, and 36Cl- uptake activities of this system. The third mutant was partially resistant to low K+ media and had reduced levels of bumetanide-sensitive uptake for all three ions. Extrapolated initial uptake rates for 22Na+, 86Rb+, and 36Cl- revealed that the partial mutant exhibited approximately 50% of the parental uptake rates for all three ions. The stoichiometries of bumetanide-sensitive uptake in both the parental cell line and the partial mutant approximated 1 Rb+:1 Na+:2 Cl-. The results of this study provide genetic evidence for a single tightly-coupled NaCl/KCl symporter in MDCK cells. The correlation between the ability to grow in low K+ media and decreased activity of the bumetanide-sensitive co-transport system suggests that the bumetanide-sensitive transport system catalyzes net K+ efflux from cells in low K+ media. The results of 86Rb+ efflux studies conducted on ouabain-pretreated mutant and parental cells are consistent with this interpretation. Cell volume measurements made on cells at different densities in media containing normal K+ concentrations showed that none of the mutants differed significantly in volume from the parental strain at a similar cell density. Furthermore, all three mutants were able to readjust their volume after suspension in hypotonic media. These results suggest that in the MDCK cell line, the bumetanide-sensitive NaCl/KCl symport system does not function in the regulation of cell volume under the conditions employed.     

Na+ and K+ transport at basolateral membranes of epithelial cells. II. K+ efflux and stoichiometry of the Na,K-ATPase

Changes of 42K efflux (J23K) caused by ouabain and/or furosemide were measured in isolated epithelia of frog skin. From the kinetics of 42K influx (J32K) studied first over 8-9 h, K+ appeared to be distributed into readily and poorly exchangeable cellular pools of K+. The readily exchangeable pool of K+ was increased by amiloride and decreased by ouabain and/or K+-free extracellular Ringer solution. 42K efflux studies were carried out with tissues shortcircuited in chambers. Ouabain caused an immediate (less than 1 min) increase of the 42K efflux to approximately 174% of control in tissues incubated either in SO4-Ringer solution or in Cl-Ringer solution containing furosemide. Whereas furosemide had no effect on J23K in control tissues bathed in Cl-rich or Cl-free solutions, ouabain induced a furosemide-inhibitable and time-dependent increase of a neutral Cl-dependent component of the J23K. Electroconductive K+ transport occurred via a single-filing K+ channel with an n' of 2.9 K+ efflux before ouabain, normalized to post-ouabain (+/- furosemide) values of short-circuit current, averaged 8-10 microA/cm2. In agreement with the conclusions of the preceding article, the macroscopic stoichiometry of ouabain-inhibitable Na+/K+ exchange by the pump was variable, ranging between 1.7 and 7.2. With increasing rates of transepithelial Na+ transport, pump-mediated K+ influx saturated, whereas Na+ efflux continued to increase with increases of pump current. In the usual range of transepithelial Na+ transport, regulation of Na+ transport occurs via changes of pump-mediated Na+ efflux, with no obligatory coupling to pump-mediated K+ influx.     

Exodus of 42K+ and 86Rb+ from rat thymic and human blood lymphocytes exposed to phytohemagglutinin.

We have found that PHA produces an alteration in the lymphocyte membrane which allows 86Rb+ or 42K+ in prelabeled lymphocytes to exchange for cations present in washing solutions. These observations suggested that PHA might induce an increase in the exodus of intracellular potassium during incubation in physiologic media. We, therefore, examined 86Rb+ and 42K+ efflux from rat and human lymphocytes during incubation in tissue culture medium. The rate constant for efflux, Ke, was significantly increased by PHA. 86Rb+ efflux was increased by 27% in rat thymic lymphocytes and by 78% in human blood lymphocytes following PHA treatment.     

Volume-sensitive K transport in human erythrocytes

Studies have been carried out on human erythrocytes to examine the alterations of K transport induced by swelling or shrinking the cells by osmotic and isosmotic methods. Hypotonic swelling of erythrocytes (relative cell volume, 1.20) resulted in a striking, four- to fivefold augmentation in the ouabain-resistant K influx over the value obtained at a normal cell volume. Shrinking the cells in hypertonic media resulted in a small but statistically significant reduction in K influx. Three different methods of varying cell volume gave similar results. These include the addition of sucrose and of NaCl to hypotonic media and the isosmotic (nystatin) method. The major fraction of the K influx in swollen cells is specific in its requirement for Cl or Br and is not supported by thiocyanate, iodide, nitrate, methylsulfate, or acetate. Bumetanide (0.1 mM), MK-196 (0.2 mM), and piretanide (1 mM) are poorly effective in suppressing K uptake in swollen cells, but at higher concentrations, bumetanide (1 mM) inhibits 80% of the Cl-dependent K influx in swollen cells. The bumetanide concentration required to inhibit 50% of the Cl-dependent K influx is 0.17 mM. The volume-sensitive K influx is independent of both extracellular and intracellular Na, so that the (Na + K + 2Cl) cotransport pathway is not a likely mediator of the volume-sensitive K transport. A variety of inhibitors of the Ca-activated K channel are ineffective in suppressing swelling-induced K influx. Like K uptake, the efflux of K is also enhanced by cell swelling. Swelling-activated K efflux is Cl dependent, is independent of extracellular and intracellular Na, and is observed with both hypotonic and isosmotic methods of cell swelling. The activation of K efflux by cell swelling is observed in K-free media, which suggests that the volume-sensitive K transport pathway is capable of net K efflux. The addition of external K to hypotonic media resulted in an increase in K efflux compared with the efflux in K-free media, and this increase was probably due to K/K exchange. Thus, hypotonic or isosmotic swelling of human erythrocytes results in the activation of a ouabain-resistant, Cl-dependent, Na-independent transport pathway that is capable of mediating both net K efflux and K/K exchange.     

Effect of extracellular volume expansion on erythrocyte sodium transport in rats.

The effects of extracellular volume expansion (EVE) on the major sodium transport systems and sodium and potassium contents in rat erythrocytes have been examined in the present study. Study has been performed in anesthetized Wistar rat weighing about 300 g. Acute extracellular volume expansion (EVE) was induced by a constant intravenous saline infusion (3% body wt, 3 hours). Rats anaesthetized and catheterized but not expanded were used as controls. Arterial blood samples from control and expanded rats were obtained at the same time, and assayed immediately. Intracellular sodium and potassium concentration and ouabain sensitive (Na(+)-K(+)-pump) and bumetanide sensitive (Na(+)-K(+)-cotransport system) outward Na+ fluxes in erythrocytes were measured. The effect of plasma on erythrocyte transport was also analyzed by measuring 86Rb uptake. Neither of two plasma cations (Na+ and K+) were modified by the EVE. Also intracellular Na+ and K+ levels remained unvariable. Total Na+ efflux was not modified by EVE, but pump-mediated Na+ efflux was smaller after than before EVE. The ouabain-inhibible Na+ efflux rate constant decreased after EVE (from 687 +/- 81 to 525 +/- 29 h-1 x 10(-3); P less than 0.05). Both Na(+)-K(+)cotransport-mediated Na+ efflux and passive permeability increased significantly after EVE. The incubation with plasma from saline-infused animals induced a significant decrease in Rb uptake rate constant, that was not observed after incubation with plasma from non-expanded rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of arginine modification on kidney brush-border-membrane transport activity.

The effect of phenylglyoxylation on brush-border-membrane functions was studied with membrane vesicles from rat kidney cortex. Na+-gradient-dependent uptake of phosphate, glucose and alanine was inhibited by 65, 88 and 70% by pre-incubation of vesicles with 50 mM-phenylglyoxal for 2 min. The inhibition showed a dependency for alkaline pH. Borate co-operativity in butanedione inactivation was used to prove that inhibition was caused by arginine modification. Intravesicular volumes, alkaline phosphatase, aminopeptidase M and Na+-H+ exchange were not affected by phenylglyoxal treatment. Inhibition of phosphate uptake was studied in more detail and showed that the chemical modification introduced by phenylglyoxal inhibited the overshoot of phosphate uptake caused by the Na+ gradient, and decreased the apparent maximal velocity of the phosphate-transport system in its interaction with Na+. Phosphate uptake measured in the absence of Na+ was not affected by phenylglyoxal. Shunting of the transmembrane electrical potential with K+ and valinomycin had no effect on phenylglyoxal inhibition, proving that the alteration of transmembrane electrical potential could not be responsible for this effect. Phenylglyoxal had no ionophoric effect on the Na+ gradients studied (1-100 mM). Na+ efflux was also unaffected by phenylglyoxal treatment. Na+, harmaline and amiloride were ineffective in protecting against phenylglyoxal inhibition, suggesting that the site modified was not an Na+-binding site. These results indicate the involvement of highly reactive arginine residues in phosphate, glucose and alanine uptake.     

Effect of potassium depletion of Hep 2 cells on intracellular pH and on chloride uptake by anion antiport

The effect of K+ depletion of Hep 2 cells on ion fluxes, internal pH, cell volume, and membrane potential was studied. The cells were depleted of K+ by incubation in K+-free buffer with or without a preceding exposure to hypotonic medium. Efflux of K+ in cells not exposed to hypotonic medium was inhibited by furosemide or by incubation in Na+-free medium, indicating that in this case at least part of the K+ efflux occurs by Na+/K+/Cl- cotransport. After exposure to hypotonic medium, K+ efflux was not inhibited by furosemide, whereas it was partly inhibited by 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS). Exposure to hypotonic medium induced acidification of the cytosol, apparently because of efflux of protons from intracellular acidic vesicles. When isotonicity was restored, a rebound alkalinization of the cytosol was induced, because of activation of the Na+/H+ antiporter. While hypotonic shock and a subsequent incubation in K+-free buffer rapidly depolarized the cells, depolarization occurred much more slowly when the K+ depletion was carried out by incubation in K+-free buffer alone. The cell volume was reduced in both cases. K+ depletion by either method strongly reduced the ability of the cells to accumulate 36Cl- by anion antiport, and K+-depleted cells were unable to increase the rate of 36Cl- uptake in response to alkalinization of the cytosol.     

Calcium regulates cAMP-induced potassium ion efflux in Dictyostelium discoideum

The chemoattractant cAMP elicits a transient efflux of K+ in cell suspensions of Dictyostelium discoideum. This cellular response displayed half-maximal activity at about 1 microM cAMP and saturated at 100 microM cAMP, cAMP-stimulated K+ efflux, measured with a K+-sensitive electrode, depended on the extracellular free Ca2+ concentration ([Ca2+]0) and was maximal in the presence of EGTA. Usually more than 90% of the K+ release could be inhibited by the addition of Ca2+. Half-maximal reduction occurred at about 2 microM [Ca2+]0. Inhibition was also observed in the presence of caffeine or A23187, drugs known to elevate the intracellular free Ca2+ concentration ([Ca2+]i). Under conditions where [Ca2+]0 was maintained at a low level, half-maximal inhibition was 1 mM for caffeine and 3 microM for A23187. These results indicate that Cai2+ is involved in the regulation of K+ efflux. Simultaneous measurements of Ca2+ uptake and K+ efflux induced by cAMP as well as free running oscillations of both ions revealed that initiation and termination of Ca2+ uptake slightly preceded those of K+ efflux.     

Mechanisms of taurocholate transport in canalicular and basolateral rat liver plasma membrane vesicles. Evidence for an electrogenic canalicular organic anion carrier

The driving forces for taurocholate transport were determined in highly purified canalicular (cLPM) and basolateral rat liver plasma membrane (LPM) vesicles. Alanine transport was also examined for comparison. Inwardly directed Na+ but not K+ gradients transiently stimulated [3H]taurocholate (1 microM) and [3H]alanine (0.2 mM) uptake into basolateral LPM 3-4- fold above their respective equilibrium values (overshoots). Na+ also stimulated [3H]taurocholate countertransport and tracer exchange in basolateral LPM whereas valinomycin-induced inside negative K+ diffusion potentials stimulated alanine uptake but had no effect on taurocholate uptake. In contrast, in the "right-side out" oriented cLPM vesicles, [3H]taurocholate countertransport and tracer exchange were not dependent on Na+. Efflux of [3H]taurocholate from cLPM was also independent of Na+ and could be trans-stimulated by extra-vesicular taurocholate. Furthermore, an inside negative valinomycin-mediated K+ diffusion potential inhibited taurocholate uptake into and stimulated taurocholate efflux from the cLPM vesicles. These studies provide direct evidence for a "carrier mediated" and potential-sensitive conductive pathway for the canalicular excretion of taurocholate. In addition, they confirm the presence of a possibly electroneutral Na+-taurocholate cotransport system in basolateral membranes of the hepatocyte.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased flow rate and papaverine increase K+ exchange in perfused rat hind-limb skeletal muscle.

This study tested the hypothesis that increases in perfusate flow rate result in increased rates of unidirectional and net K+ transport in rat hind-limb skeletal muscle at rest. Ten neurally and vascularly isolated hind limbs, with arterial and venous catheters placed proximal to the popliteal region, were perfused for 10-min periods at flow rates (presented in a random order) of 0.27, 0.42, 0.63, 0.84, or 1.05 mL x min(-1) x g(-1). Potassium extraction and unidirectional K+ influx were determined using 42K, and arterial perfusion pressure was measured continuously. Increases in flow rate resulted in decreases in K+ extraction and increases in unidirectional K+ influx, unidirectional K+ efflux, and net K+ efflux. The increases in K+ flux were associated with increases in oxygen uptake, glucose uptake, and lactate release. In separate experiments (n = 5), the vasodilator papaverine (10(-4) M) did not further vasodilate the vasculature of resting hind limbs, suggesting that the hind limbs in this preparation were fully vasodilated. Papaverine, at constant flow, resulted in a nearly 1.5-fold increase in K+ extraction, a doubling of unidirectional K+ influx, and increases in unidirectional K+ efflux and net K+ efflux. It is concluded that physiological increases in flow rate result in increases in K+ transport in isolated, perfused rat hind-limb skeletal muscle. Furthermore, papaverine appeared to induce an increase in skeletal muscle membrane permeability to K+.     

Assay of muscarinic acetylcholine receptor function in cultured cardiac cells by stimulation of 86Rb+ efflux

An assay for the increase in potassium permeability mediated by muscarinic acetylcholine receptors (mAChR) in cultured cardiac cells is described, using the K+ ion substitute 86Rb+ as the tracer ion. Cardiac cells accumulate 86Rb+ from the extracellular medium in a Na+/K+ ATPase-dependent manner. Subsequent efflux of 86Rb+ in the absence and presence of muscarinic agonists follows kinetics similar to those previously reported for 42K+. The mAChR agonist carbamylcholine (carbachol) stimulated 86Rb+ efflux with an EC50 of 50 nM. The half-time for efflux is reduced by greater than 40% at maximally effective concentrations of agonist. Stimulation of 86Rb+ efflux by carbachol is blocked by the mAChR antagonist atropine with an IC50 of 15 nM. The stimulation of 86Rb+ efflux by carbachol is not affected by the presence of the Na+/K+ ATPase inhibitor ouabain. This assay provides a method for quantitating the mAChR-mediated increase in K+ permeability in cardiac cells without the use of 42K+.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

Cell volume regulation in the perfused liver of a freshwater air-breathing catfishClarias batrachus under aniso-osmotic conditions: Roles of inorganic ions and taurine

The roles of various inorganic ions and taurine, an organic osmolyte, in cell volume regulation were investigated in the perfused liver of a freshwater air-breathing catfishClarias batrachus under aniso-osmotic conditions. There was a transient increase and decrease of liver cell volume following hypotonic (-80 mOsmol/l) and hypertonic (+80 mOsmol/l) exposures, respectively, which gradually decreased/increased near to the control level due to release/ uptake of water within a period of 25–30 min. Liver volume decrease was accompanied by enhanced efflux of K⁺ (9.45 ± 0.54 μmol/g liver) due to activation of Ba²⁺- and quinidine-sensitive K⁺ channel, and to a lesser extent due to enhanced efflux of Cl^- (4.35 ± 0.25 μmol/g liver) and Na⁺ (3.68 ± 0.37 μmol/g liver). Conversely, upon hypertonic exposure, there was amiloride- and ouabain-sensitive uptake of K⁺(9.78 ± 0.65 μmol/g liver), and also Cl^- (3.72 ± 0.25 μmol/g liver). The alkalization/acidification of the liver effluents under hypo-/hypertonicity was mainly due to movement of various ions during volume regulatory processes. Taurine, an important organic osmolyte, appears also to play a very important role in hepatocyte cell volume regulation in the walking catfish as evidenced by the fact that hypo- and hyper-osmolarity caused transient efflux (5.68 ± 0.38 μmol/g liver) and uptake (6.38 ± 0.45 μmol/g liver) of taurine, respectively. The taurine efflux was sensitive to 4,4′-di-isothiocyanatostilbene-2,2′-disulphonic acid (DIDS, an anion channel blocker), but the uptake was insensitive to DIDS, thus indicating that the release and uptake of taurine during volume regulatory processes are unidirectional. Although the liver of walking catfish possesses the RVD and RVI mechanisms, it is to be noted that liver cells remain partly swollen and shrunken during anisotonic exposures, thereby possibly causing various volume-sensitive metabolic changes in the liver as reported earlier.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

Formyl peptide stimulation of superoxide anion release from lung macrophages: sodium and potassium involvement

We examined the role of the monovalent cations Na+ and K+ in the events encompassing the release of O-2 by alveolar macrophages after stimulation with formyl methionyl phenylalanine (FMP). This was accomplished by determining the effect of changing the extracellular [Na+] and/or [K+] on FMP-stimulated O-2 production; and measuring 22Na+, 42K+ and 86Rb+ influx and efflux and intracellular [K+] for control and FMP-stimulated alveolar macrophages. Stimulated O-2 production was relatively insensitive to changes in extracellular K+ or Na+ concentrations until the [Na+] was decreased below 35 mM. At 4 mM [Na+], the rate of O-2 production remained at 75% of the maximal rate observed at physiological concentrations of [Na+]. Both influx and efflux of 22Na+ were stimulated above control rates by FMP. The increased rates of fluxes lasted for a few minutes suggesting a transient increase in membrane permeability to Na+. Ouabain partially inhibited 22Na+ efflux but had no effect on O-2 release. The influx of 86Rb+ and 42K+ was not altered by the addition of FMP but was virtually abolished in the presence of 10 microM ouabain or 1 mM quinine. In the presence of extracellular calcium, FMP-stimulated a prolonged (greater than 20 minutes) increase in 86Rb+ or 42K+ efflux which was inhibitable by 1 mM quinine. In the absence of extracellular calcium, FMP stimulation of K+ efflux was greatly diminished and was not affected by quinine, although quinine still inhibited O-2 production under these conditions. It was also observed that there was a loss of intracellular K+ when cells were stimulated by FMP in the presence of Ca+2, but not in the absence of Ca+2. Taken together, these results suggest a minimal direct role, if any, for K+ in the events that lead to FMP-stimulated O-2 release by alveolar macrophages.     

Taurine and cell volume maintenance in the shark rectal gland: cellular fluxes and kinetics

Tissue slices of shark rectal gland are studied to examine the kinetics of the cellular fluxes of taurine, a major intracellular osmolyte in this organ. Maintenance of high steady-state cell taurine (50 mM) is achieved by a ouabain-sensitive active Na+-dependent uptake process and a relatively slow efflux. Uptake kinetics are described by two saturable taurine transport components (high-affinity, Km 60 microM; and low-affinity, Km 9 mM). [14C]Taurine uptake is enhanced by external Cl-, inhibited by beta-alanine and unaffected by inhibitors of the Na+/K+/2Cl- co-transport system. Two cellular efflux components of taurine are documented. Incubation of slices in p-chloromercuribenzene sulfonate (1 mM) reduces taurine uptake, increases efflux of taurine and induces cell swelling. Studies of efflux in isotonic media with various cation and anion substitutions demonstrate that high-K+ markedly enhances taurine efflux irrespective of cell volume changes (i.e. membrane stretching is not involved). Moreover, iso-osmotic cell swelling induced in media containing propionate is not associated with enhanced efflux of taurine from the cells. It is suggested that external K+ exerts a specific effect on the cytoplasmic membrane to increase its permeability to taurine.