KCl Loss Research Articles

Coordinate modulation of Na-K-2Cl cotransport and K-Cl cotransport by cell volume and chloride.

Na-K-2Cl cotransporter (NKCC) and K-Cl cotransporter (KCC) play key roles in cell volume regulation and epithelial Cl(-) transport. Reductions in either cell volume or cytosolic Cl(-) concentration ([Cl(-)](i)) stimulate a corrective uptake of KCl and water via NKCC, whereas cell swelling triggers KCl loss via KCC. The dependence of these transporters on volume and [Cl(-)](i) was evaluated in model duck red blood cells. Replacement of [Cl(-)](i) with methanesulfonate elevated the volume set point at which NKCC activates and KCC inactivates. The set point was insensitive to cytosolic ionic strength. Reducing [Cl(-)](i) at a constant driving force for inward NKCC and outward KCC caused the cells to adopt the new set point volume. Phosphopeptide maps of NKCC indicated that activation by cell shrinkage or low [Cl(-)](i) is associated with phosphorylation of a similar constellation of Ser/Thr sites. Like shrinkage, reduction of [Cl(-)](i) accelerated NKCC phosphorylation after abrupt inhibition of the deactivating phosphatase with calyculin A in vivo, whereas [Cl(-)] had no specific effect on dephosphorylation in vitro. Our results indicate that NKCC and KCC are reciprocally regulated by a negative feedback system dually modulated by cell volume and [Cl(-)]. The major effect of Cl(-) on NKCC is exerted through the volume-sensitive kinase that phosphorylates the transport protein.

Relationship between intracellular pH and chloride in Xenopus oocytes expressing the chloride channel ClC-0.

During maturation of oocytes, Cl(-) conductance (G(Cl)) oscillates and intracellular pH (pH(i)) increases. Elevating pH(i) permits the protein synthesis essential to maturation. To examine whether changes in G(Cl) and pH(i) are coupled, the Cl(-) channel ClC-0 was heterologously expressed. Overexpressing ClC-0 elevates pH(i), decreases intracellular Cl(-) concentration ([Cl(-)](i)), and reduces volume. Acute acidification with butyrate does not activate acid extrusion in ClC-0-expressing or control oocytes. The ClC-0-induced pH(i) change increases after overnight incubation at extracellular pH 8.5 but is unaltered after incubation at extracellular pH 6.5. Membrane depolarization did not change pH(i). In contrast, hyperpolarization elevates pH(i). Thus neither membrane depolarization nor acute activation of acid extrusion accounts for the ClC-0-dependent alkalinization. Overnight incubation in low extracellular Cl(-) concentration increases pH(i) and decreases [Cl(-)](i) in control and ClC-0 expressing oocytes, with the effect greater in the latter. Incubation in hypotonic, low extracellular Cl(-) solutions prevented pH(i) elevation, although the decrease in [Cl(-)](i) persisted. Taken together, our observations suggest that KCl loss leads to oocyte shrinkage, which transiently activates acid extrusion. In conclusion, expressing ClC-0 in oocytes increases pH(i) and decreases [Cl(-)](i). These parameters are coupled via shrinkage activation of proton extrusion. Normal, cyclical changes of oocyte G(Cl) may exert an effect on pH(i) via shrinkage, thus inducing meiotic maturation.

Understanding salivary fluid and protein secretion.

Understanding salivary fluid and protein secretion.

Purinergic activation of BK channels in clonal kidney cells (Vero cells).

We have studied the activation of a high-conductance channel in clonal kidney cells from African green monkey (Vero cells) using patch-clamp recordings and microfluorometric (fura-2) measurements of cytosolic Ca2+. The single-channel conductance in excised patches is 170 pS in symmetrical 140 mM KCl. The channel is highly selective for K+ and activated by membrane depolarization and application of Ca2+ to the cytoplasmatic side of the patch. The channel is, thus, a large-conductance Ca2+-activated K+ channel (BK channel). Cell-attached recordings revealed that the channel is inactive in unstimulated cells. Extracellular application of less than 0.1 microM ATP transiently increased the cytosolic Ca2+ concentration ([Ca2+]i) to about 550 nM, and induced membrane hyperpolarization caused by Ca2+-activated K+ currents. ATP stimulation also activated BK channels in cell-attached patches at both the normal-resting potential and during membrane hyperpolarization. The increase in [Ca2+]i was owing to Ca2+ release from internal stores, suggesting that Vero cells express G-protein-coupled purinergic receptors (P2Y) mediating IP3-induced release of Ca2+. The P2Y receptors were sensitive to both uracil triphosphate (UTP) and adenosine diphosphate (ADP), and the rank of agonist potency was ATP >> UTP >/= ADP. This result indicates the presence of both P2Y1 and P2Y2 receptors or a receptor subtype with untypical agonist sensitivity. It has previously been shown that hypotonic challenge activates BK channels in both normal and clonal kidney cells. The subsequent loss of KCl may be an important factor in cellular volume regulation. Our results support the idea of an autocrine role of ATP in this process. A minute release of ATP induced by hypotonically evoked membrane stretch may activate the P2Y receptors, subsequently increasing [Ca2+]i and thus causing K+ efflux through BK channels.

Volume changes in cardiac ventricles from Aplysia brasiliana upon exposure to hyposmotic shock

We investigated the possible role of ion channels and transporters in cell volume control using Aplysia brasiliana ventricular tissues exposed to a 26% hyposmotic shock, by assessing changes in wet weight, intracellular water and ionic contents. Thirty minutes after the shock, the wet weight of isolated ventricles increase about 20% above control levels and then attain near original weight within 60 min after the shock. At the time when the wet weight returned to control values, intracellular water and KCl contents are decreased by 22 and 20%, respectively. The K + channel blockers, 4-AP and TEA, but not the cotransport blockers, hydrochlorothiazide and furosemide, greatly affect the magnitude of wet weight gain and the time course of weight recovery, indicating that KCl loss occur through conductive pathways. Intracellular recordings performed on ventricular myocytes during exposure to the osmotic shock showed an immediate membrane hyperpolarization and blockade of spontaneous electrical activity; diastolic membrane potential recover over time and spontaneous action potentials are completely restored 60 min after the hyposmotic shock. Because significant weight loss is observed during the exposure of ventricular tissues to 26% hypo-ionic, but isosmotic saline, it is suggested that ventricular volume restoration is accomplished by two distinct but simultaneously occurring processes: a volume-dependent and a volume-independent mechanism. Because wet weight restoration is completely prevented by exposing ventricular tissue to a Ca 2+-free hyposmotic solution, we postulate that both processes involved in A. brasiliana ventricular weight restoration are Ca 2+-dependent mechanisms.

Volume control in sickle cells is facilitated by the novel anion conductance inhibitor NS1652

A low cation conductance and a high anion conductance are characteristic of normal erythrocytes. In sickle cell anemia, the polymerization of hemoglobin S (HbS) under conditions of low oxygen tension is preceded by an increase in cation conductance. This increase in conductance is mediated in part through Ca(++)-activated K(+) channels. A net efflux of potassium chloride (KCl) leads to a decrease in intracellular volume, which in turn increases the rate of HbS polymerization. Treatments minimizing the passive transport of ions and solvent to prevent such volume depletion might include inhibitors targeting either the Ca(++)-activated K(+) channel or the anion conductance. NS1652 is an anion conductance inhibitor that has recently been developed. In vitro application of this compound lowers the net KCl loss from deoxygenated sickle cells from about 12 mmol/L cells/h to about 4 mmol/L cells/h, a value similar to that observed in oxygenated cells. Experiments performed in mice demonstrate that NS1652 is well tolerated and decreases red cell anion conductance in vivo. (Blood. 2000;95:1842-1848)

Expansion of the cellular content of ribonucleoside triphosphates induces cell shrinkage and KCl loss in Ehrlich ascites tumor cells and in Chinese hamster ovary cells

The conversion to corresponding triphosphate derivatives of various ribonucleosides has been studied in Ehrlich ascites tumor cells and in Chinese hamster ovary cells under conditions that are optimal for cellular uptake of orthophosphate. The initial cellular uptake of orthophosphate is followed by a cellular loss of Cl − which might be consistent with a H 2PO 4 −/Cl − exchange mechanism. Subsequent addition of ribonucleosides to the medium leads to cellular accumulation of the corresponding triphosphate and to a concomitant loss of KCl and to sustained cell volume reduction. The latter two events are quite unspecific with regard to the nucleobase moiety of the ribonucleoside triphosphate accumulated (adenine, guanine and purine being almost equally effective) and they depend in a rather simple way on the increase of the cellular content of these compounds. The KCl loss seems to depend on opening of the separate K + and Cl − channels. The pharmacological profile of the putative ion channels could not be identified in spite of experiments with conventional blockers. In the case of purine riboside the accumulation of the corresponding triphosphate and concomitant loss of KCl and cell water may be followed by a regain of cell volume due to a continued purine riboside triphosphate accumulation, which apparently depends on the uptake of orthophosphate by cotransport with Na + and which for osmotic reasons is accompanied by the uptake of water and hence volume increase. The possibility that the nucleoside triphosphate induced opening of a putative Cl − channel may be due to a direct effect of triphosphate on a channel protein is discussed.

Upregulation of Na(+)-K(+)-2Cl- cotransporter activity in rat parotid acinar cells by muscarinic stimulation.

1. The effects of fluid secretory stimuli on the Na(+)-K(+)-2Cl- cotransporter in rat parotid acini were investigated. Cotransporter activity was measured using NH4+ as a K+ surrogate and following cotransporter-mediated NH4+ fluxes by monitoring intracellular pH. 2. A dramatic upregulation (15- to 20-fold) of acinar Na(+)-K(+)-2Cl- cotransporter activity was induced by muscarinic, alpha 1-adrenergic and peptidergic stimuli. A half-maximal effect of the muscarinic agonist carbachol was observed at approximately 0.5 microM. 3. Our results indicate that the rise in intracellular calcium concentration ([Ca2+]i) which accompanies these stimuli is both a necessary and a sufficient condition for this effect; but it is not a consequence of the KCl loss and concomitant isotonic shrinkage caused by increased [Ca2+]i as it persists when these effects are prevented. 4. The effect of muscarinic stimulation on the cotransporter can, however, be blocked by inhibitors of phospholipase A2 (4-bromophenacylbromide and manoalide), by a general inhibitor of arachidonic acid metabolism (5,8,11,14-eicosatetraynoic acid) and by specific inhibitors of the cytochrome P450 pathway (methoxsalen and ketoconazole). 5. These latter results argue strongly for the involvement of a product of the cytochrome P450 pathway of arachidonic acid metabolism in upregulation of the salivary Na(+)-K(+)-2Cl- cotransporter. 6. Owing to the complexity of the arachidonic acid cascade a wide variety of agents could potentially interfere with this upregulation of the cotransporter, and thereby result in decreased salivary fluid production. We suggest that such an effect could underlie the dry mouth (xerostomia) that occurs as an unexplained side-effect of many commonly prescribed medications.

Role of LTD4 in the regulatory volume decrease response in Ehrlich ascites tumor cells.

Stimulation with leukotriene D4 (LTD4) (3-100 nM) induces a transient increase in the free intracellular Ca2+ concentration ([Ca2+]i) in Ehrlich ascites tumor cells. The LTD4-induced increase in [Ca2+]i is, however, significantly reduced in Ca2+-free medium (2 mM EGTA), and under these conditions stimulation with a low LTD4 concentration (3 nM) does not result in any detectable increase in [Ca2+]i. Addition of LTD4 (3-100 nM) moreover accelerates the KCl loss seen during Regulatory Volume Decrease (RVD) in cells suspended in a hypotonic medium. The LTD4-induced (100 nM) acceleration of the RVD response is also seen in Ca2+-free medium and also at 3 nM LTD4, indicating that LTD4 can open K+- and Cl--channels without any detectable increase in [Ca2+]i. Buffering cellular Ca2+ with BAPTA almost completely blocks the LTD4-induced (100 nM) acceleration of the RVD response. Thus, the reduced [Ca2+]i level after BAPTA-loading or buffering of [Ca2+]i seems to inhibit the LTD4-induced stimulation of the RVD response even though the LTD4-induced cell shrinkage is not necessarily preceded by any detectable increase in [Ca2+]i. The LTD4 receptor antagonist L649, 923 (1 microM) completely blocks the LTD4-induced increase in [Ca2+]i and inhibits the RVD response as well as the LTD4-induced acceleration of the RVD response. When the LTD4 receptor is desensitized by preincubation with 100 nM LTD4, a subsequent RVD response is strongly inhibited. In conclusion, the present study supports the notion that LTD4 plays a role in the activation of the RVD response. LTD4 seems to activate K+ and Cl- channels via stimulation of a LTD4 receptor with no need for a detectable increase in [Ca2+]i.

Actin-based cytoskeleton regulates a chloride channel and cell volume in a renal cortical collecting duct cell line.

The regulatory volume decrease (RVD) of a renal cortical collecting duct cell line (RCCT-28A) exposed to a hypotonic solution was studied using electronic cell sizing to measure cell volume and the patch clamp technique to measure Cl- channel activity. Results demonstrate that RVD was mediated in part by KCl loss through separate K+ and Cl- channels. The Cl- channel had a conductance of 305 pS and was activated by cell swelling, membrane stretch, and disruption of F-actin by dihydrocytochalasins. In contrast, stabilizing F-actin with phalloidin prevented swelling and stretch activation of the Cl- channel and inhibited the RVD. Thus, the state of actin polymerization regulates the probability of the 305 pS Cl- channel being open. Short actin filaments activate whereas long actin filaments inactivate the channel. Taken together, our studies suggest that RVD in this renal collecting duct cell line cell is mediated in part by a 305 pS Cl- channel, which is activated, during cell swelling, by a signaling pathway that includes disruption of F-actin.

Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca(2+)-mediated secretagogue-induced volume decrease in small-intestinal crypts.

The cellular volume of crypts isolated from 2- to 3-week-old mouse small intestine has been measured to assess the capacity of the epithelial cells to respond to secretagogues. Vasoactive intestinal polypeptide (VIP) or carbachol, respectively cAMP- and calcium-mediated secretagogues, produced a reduction crypt volume attributed to KCl loss through channels activated by the agonists. Consistent with the participation of separate chloride channels, 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) blocked the carbachol- but not the VIP-induced volume decrease, whilst glibenclamide abolished the VIP effect without affecting the carbachol-induced volume decrease. Animals homozygous for a disrupted cftr gene, introduced by gene targeting, were also used as the source for crypt isolation. In these CFTR (-/-) crypts. VIP failed to elicit any reduction in cellular volume, while the response to carbachol was indistinguishable from that seen in crypts from age-matched control animals. These results are consistent with murine CFTR being a cAMP-activated chloride channel inhibited by glibenclamide and resistant to DIDS. A separate chloride conductance activated by calcium mobilization in small-intestinal crypts appears to be independent of CFTR.

The role of calcium in cell shrinkage and intracellular alkalinization by bradykinin in Ha-ras oncogene expressing cells

In ras oncogene expressing cells, bradykinin leads to intracellular alkalinization by activation of the Na +/H + exchanger. This effect is paralleled by oscillatory increase of intracellular calcium activity and cell shrinkage. Staurosporine (1 μmol/l) is not sufficient to prevent bradykinin induced intracellular alkalinization, thus pointing to a protein kinase C independent pathway for the activation of Na +/H + exchange. The present study has been performed to elucidate, whether the increase of intracellular calcium contributes to cell shrinkage and activation of the Na +/H + exchanger. To this end, the effects of the calcium ionophore ionomycin have been tested. Ionomycin leads to a dose dependent increase of intracellular calcium activity. At 100 nmol/l ionomycin intracellular calcium is increased from 114 ± 17 nmol/l to 342 ± 24 nmol/l ( n = 9), a value within the range of intracellular calcium concentrations following application of bradykinin. The calcium increase is paralleled by a decrease of cell volume by 12 ± 2% ( n = 5) and an increase of intracellular pH from 6.78 ± 0.02 to 6.90 ± 0.03 ( n = 11), values similar to those following application of bradykinin. The alkalinizing effect of ionomycin is completely abolished in the presence of the novel Na +/H + exchange inhibitor HOE 694 (10 μmol/l), but is not inhibited by 1 μmol/l staurosporine. Inhibition of K + and Cl − channels by barium (5 mmol/l) and ochratoxin-A (5 μmol/l) prevents both ionomycin induced cell shrinkage and protein kinase C independent intracellular alkalinization. It is concluded that bradykinin leads to intracellular alkalinization mainly by increasing intracellular calcium concentration. Calcium triggers calcium sensitive K + channels, and presumably Cl − channels, the subsequent loss of cellular KCl leads to cell shrinkage which, in turn, activates Na +/H + exchange.

Regulatory volume increase after hypertonicity- or vasoactive-intestinal-peptide-induced cell-volume decrease in small-intestinal crypts is dependent on Na(+)-K(+)-2Cl- cotransport.

The volume of intact crypts isolated from guinea-pig small intestine has been measured to assess the capacity of the cells to regulate their volume after hypertonic shock or vasoactive-intestinal-peptide (VIP)-induced shrinkage. Crypts exposed to anisotonic medium initially behave as perfect osmometers. Continued exposure to a hypertonic (400 mosmol/l) medium was followed by regulatory volume increase (RVI), which led to complete volume recovery in about 20 min. VIP produced a volume reduction, attributed to KCl loss through channels activated by the secretagogue, without any recovery during exposure to the polypeptide. Removal of VIP led to an increase of cellular volume towards control levels. This volume recovery after secretagogue-induced shrinkage is termed SVI. Both RVI and SVI were abolished by removal of Na+ or Cl- from the bathing solution, by addition of the loop diuretic bumetanide (1 microM), but not by addition of ethylisopropylamiloride (10 microM) or amiloride (1 mM). Cell shrinkage was also observed when tonicity was increased by addition of 100 mM NaCl or 200 mM D-mannitol, but RVI was seen only when NaCl was the added osmolyte. The ion dependence, pharmacological sensitivity and thermodynamic considerations of these effects are consistent with the operation of a Na(+)-K(+)-2Cl- cotransport mechanism activated by cell shrinkage and the secretagogue action of VIP.

Regulatory volume decrease in Ehrlich ascites tumor cells is not mediated by a rise in intracellular calcium

Ehrlich ascites tumor cells suspended in hyposmotic solution initially swell and then shrink back towards normal volume, a process known as regulatory volume decrease (RVD), RVD is characterized by a specific loss of KCl, although the mechanism for this is currently unknown. The hypothesis that a rise in intracellular calcium ([Ca 2+] i) activates calcium-sensitive ion conductances to initiate RVD was investigated. The results indicate that in the Ehrlich cell no rise in [Ca 2+] i occurs when the extracellular osmolality is reduced from 300 mosM to 180 mosM. These findings were substantiated by the lack of sensitivity of RVD to the Ca 2+-sensitive K + channel blockers charybdotoxin (CTX) and nifedipine. In contrast, the ionophore ionomycin induced a cell shrinkage that was sensitive to CTX and nifedipine indicating that a rise in [Ca 2+] i could play a role in cell volume reduction but that this occurred by a mechanism different from that observed in RVD. The conclusion from these experiments is that Ca 2+ does not act as a second messenger for RVD in the Ehrlich cell.

Oxygenation-activated K fluxes in trout red blood cells.

The effect of oxygenation on the dissipative fluxes of K in trout red blood cells has been determined. Unidirectional influx under low oxygen tension (PO2 = 1 kPa) was 0.56 +/- 0.07 mmol.l-1 packed cells.h-1. Within a few minutes of equilibration with high oxygen tension (PO2 = 120 kPa), influx was increased 14-fold, and this was associated with a progressive loss of KCl and a cell shrinkage. K influx progressively declined over the following 3 h to levels close to those characteristic of cells at low oxygen tension. Replacement of medium Cl by NO3- or methane sulfonate inhibited the stimulation due to high oxygen as did furosemide and low extracellular pH. The oxygenation-stimulated influx was highly volume sensitive, being increased by up to 100% by osmotic swelling and decreased by osmotic shrinkage. By contrast, the small influx under low oxygen tension was unaffected by either Cl replacement or by shrinkage and increased only with extreme swelling. Thus high oxygen tension activated a Cl-dependent and furosemide-sensitive K flux. Once activated, the mechanism was rapidly deactivated on transfer back to low oxygen tension but slowly deactivated when maintained at high PO2. The oxygenation-stimulated flux mechanism promotes a rapid and more complete volume regulatory decrease than in cells at low oxygen tension.

Cl- fluxes related to fluid secretion by the rat parotid: involvement of Cl(-)-HCO3- exchange.

Muscarinic-induced 36Cl- and 86Rb+ (K+ substitute) fluxes were studied in rat parotid acini. Stimulation resulted in a rapid [half time (t1/2) less than 30 s] decrease in both Cl- and Rb+ content (approximately 50 and 30%, respectively) followed by a slower partial recovery (t1/2 approximately 3-4 min) to approximately 80% of resting levels for both ions. Cl- loss was inhibited by the venom of Leiurus quinquestriatus, which contains the maxi-K+ channel blocker charybdotoxin. Cl- recovery was blunted in the presence of bumetanide, an inhibitor of Na(+)-K(+)-Cl- cotransport, or on HCO3- removal and was completely blocked in the presence of bumetanide and 4,4' diisothiocyanostilbene-2,2' disulfonic acid (DIDS), an inhibitor of Cl(-)-HCO3- exchange. In HCO3(-)-containing medium a rapid (t1/2 less than 1 min), DIDS-inhibitable cytoplasmic alkalinization (approximately 0.4 pH unit) was observed in acini switched to a Cl(-)-free solution. This alkalinization was not seen in HCO3(-)-free medium but persisted in the absence of Na+, consistent with the presence of a potent Na(+)-independent Cl(-)-HCO3- exchanger. Kinetic studies indicated that the half-maximal effect of this exchanger for extracellular Cl- was approximately 18 mM. These results are consistent with the hypothesis that secretagogue-induced KCl loss by salivary acinar cells occurs via electrically coupled K+ and Cl- channels. In addition, they provide strong evidence that Cl- entry into, and thus fluid secretion by, these cells is mediated by both Cl(-)-HCO3- exchange and Na(+)-K(+)-Cl- cotransport.

Regulatory volume decrease by cultured non-pigmented ciliary epithelial cells

Cells (ODM C1-2/SV40) derived from human non-pigmented ciliary epithelial cells were studied by electronic cell sizing. The time course of the cell volume (vc) was monitored after suspending cells in paired experimental and control, isosmotic and hyposmotic solutions of identical ionic composition. Following anisosmotic cell swelling, the cells displayed the regulatory volume decrease (RVD) previously described. The RVD primarily reflects loss of cell KCl since: (1) the K(+)-channel blockers quinidine and Ba2+ both inhibit the RVD; and (2) replacement of external Cl- with gluconate or addition of the Cl- channel blocker NPPB also inhibits the RVD. Bicarbonate has previously been reported to speed the RVD. This action likely reflects pH dependence of the channels since: (1) increasing the external pH speeds the RVD, whether or not HCO3- is present; and (2) DIDS (a blocker of Cl- channels and of Cl-/HCO3- exchange) is an effective inhibitor of the RVD, even after blocking Cl-/HCO3- exchange by removing external HCO3-. The RVD could also be inhibited by reducing the availability of Ca2+, either by omitting Ca2+ from the external medium or by blocking mobilization of intracellular Ca2+ with TMB-8. Furthermore, the RVD was slowed and incomplete in the presence of the calcium/calmodulin blocker trifluoperazine. We conclude that anisosmotic swelling triggers a series of events, mediated at least in part by calcium/calmodulin, leading to the extrusion of KCl through parallel K+ and Cl- channels.

Movement of cellular ions during stimulation with isoproterenol in simian eccrine clear cells

The ionic mechanism of beta-adrenergic sweating is unknown. In isolated rhesus eccrine secretory coils, K efflux was determined by an extracellular K electrode and cellular monovalent ions by X-ray microanalysis. Isoproterenol (Iso) induced a small (dose-dependent propranolol-inhibitable) K efflux followed by net K reuptake. Similar K response was seen with forskolin, theophylline, or isobutyl-methylxanthine (IBMX). The net K uptake often exceeded the net K efflux, causing a small net accumulation of K. Bumetanide (BT) and ouabain not only abolished the Iso (with or without IBMX) -induced net K uptake but increased the Iso-induced initial K efflux about threefold. BT and ouabain drastically decreased K and Cl concentrations in the clear cell (by X-ray microanalysis) only in the presence of Iso plus IBMX, suggesting that adenosine 3',5'-cyclic monophosphate (cAMP) may simultaneously stimulate both KCl efflux (by unknown mechanisms) and K reuptake (presumably by BT-sensitive cotransporters and ouabain-sensitive Na pumps). Thus the cAMP-mediated ion movement is different from the cholinergic mechanism that is characterized by the net KCl loss, cell shrinkage (Saga et al., J. Membr. Biol. 107: 13-24, 1989; Takemura et al., J. Membr. Biol. In press), and no augmentation of methacholine-induced K efflux by BT or ouabain.

Activation by N-Ethylmaleimide of a Cl− -Dependent K+ Flux in Isolated Trout Hepatocytes

Isolated trout hepatocytes when swollen in hypotonic medium undergo a regulatory volume decrease (RVD), which occurs via KCl loss. The system shows characteristics similar to those of the transporter described in red cells. This led us to investigate, in trout hepatocytes, the effect of another signal known to activate this flux in red cells, i.e. treatment with the sulphhydryl-group reagent N-ethylmaleimide (NEM). NEM treatment resulted in a striking increase in ouabain-resistant K+ uptake measured by an isotope pulse uptake technique. The time course of the response to NEM was similar to that obtained with a hypotonic shock, indicating that the effect of NEM was immediate and transient. The NEM-stimulated K+ influx demonstrated the same anion sensitivity as the volume-induced K+ influx, i.e. a specific requirement for Br- or Cl-. Efflux experiments showed that NEM treatment produced a stimulation of both K+ and Cl- effluxes leading to a substantial net loss (10%) of cellular KCl, as confirmed by analysis of ionic contents. This KCl loss is consistent with the rapid cell shrinkage observed after addition of NEM. The Cl--dependent K+ influx was found to be independent of external Na+; in addition, NEM had no effect on Na+ content, indicating that Na+ is not implicated in this process. The effect of loop diuretics was tested on the NEM-stimulated K+ influx. As observed for the volume-induced K+ flux, a high concentration of furosemide (10(-3) mol l-1) is required for full inhibition of this flux; no effect was obtained with bumetanide (10(-4) mol l-1). Consequently, NEM appears to activate a KCl cotransport similar to the one induced in hypotonically swollen cells. Finally, the combination of the two treatments, NEM and hypotonic shock, was found to increase the K+ fluxes even further, suggesting additivity of the two stimuli by mutual positive interaction.

Actions of mercurials on cell volume regulation of dissociated MDCK cells

The mercurial, p-chloromercuribenzoylsulfonate (PCMBS), blocks volume recovery of dissociated, osmotically swollen, Madin-Darby canine kidney cells (MDCK) and, at higher concentrations, induces substantial swelling. In the absence of Na+ the rate of volume recovery is, in contrast, substantially increased. PCMBS does not inhibit the "normal" volume-regulating pathways, K+ and Cl- conductances. Rather, its blocking action is due to substantial activation of Na+ and K+ permeabilities, especially the former. Consequently, the normal reshrinking mechanism, loss of KCl, is counterbalanced by PCMBS-induced gains of NaCl. In isotonic cells, PCMBS, at higher concentrations, induces cell swelling, indicating that Cl- permeability is also increased, a conclusion confirmed by direct measurement of 36Cl- fluxes. HgCl2 produces similar effects except that it is more potent and more rapid in its action. Activation of conductive ion permeabilities to Na+, K+, and Cl- are associated with appropriate changes in membrane potential. A small bumetanide-sensitive swelling component (Na(+)-Cl- cotransport) is activated by HgCl2 but not by PCMBS. Another effect is elevation of cytoplasmic Ca2+, apparently by mobilization from internal stores. Some of the functional sites (Na+ and K+) appear to be located externally, rapidly accessible to both HgCl2 and PCMBS, whereas others (Cl- and Ca2+) appear to be internal, rapidly accessible to the permeant HgCl2 but slowly to relatively impermeant PCMBS. In conclusion, the disturbances of volume regulation are largely due to the increases in conductive ion fluxes.