Volume regulation by Amphiuma red blood cells. The membrane potential and its implications regarding the nature of the ion-flux pathways

After osmotic perturbation, the red blood cells of Amphiuma exhibited a volume-regulatory response that returned cell volume back to or toward control values. After osmotic swelling, cell-volume regulation (regulatory volume decrease; RVD) resulted from net cellular loss of K, Cl, and osmotically obliged H2O. In contrast, the volume-regulatory response to osmotic shrinkage (regulatory volume increase; RVI) was characterized by net cellular uptake of Na, Cl, and H2O. The net K and Na fluxes characteristic of RVD and RVI are increased by 1-2 orders of magnitude above those observed in studies of volume-static control cells. The cell membrane potential of volume-regulating and volume-static cells was measured by impalement with glass microelectrodes. The information gained from the electrical and ion-flux studies led to the conclusion that the ion fluxes responsible for cell-volume regulation proceed via electrically silent pathways. Furthermore, it was observed that Na fluxes during RVI were profoundly sensitive to medium [HCO3] and that during RVI the medium becomes more acid, whereas alkaline shifts in the suspension medium accompany RVD. The experimental observations are explained by a model featuring obligatorily coupled alkali metal-H and Cl-HCO3 exchangers. The anion- and cation-exchange pathways are separate and distinct yet functionally coupled via the net flux of H. As a result of the operation of such pathways, net alkali metal, Cl, and H2O fluxes proceed in the same direction, whereas H and HCO3 fluxes are cyclic. Data also are presented that suggest that the ion-flux pathways responsible for cell-volume regulation are not activated by changes in cell volume per se but by some event associated with osmotic perturbation, such as changes in intracellular pH.     

Activation of electroneutral K flux in Amphiuma red blood cells by N- ethylmaleimide. Distinction between K/H exchange and KCl cotransport

Exposure of Amphiuma red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) resulted in net K loss. In order to determine whether net K loss was conductive or was by electroneutral K/H exchange or KCl cotransport, studies were performed evaluating K flux in terms of the thermodynamic forces to which K flux by the above pathways should couple. The direction and magnitude of the NEM-induced net K flux did not correspond with the direction and magnitude of the forces relevant to K conductance or electroneutral KCl cotransport. Both the magnitude and direction of the NEM-activated K flux responded to the driving force for K/H exchange. We therefore conclude that NEM-induced K loss, like that by osmotically swollen Amphiuma red blood cells, is by an electroneutral K/H exchanger. In addition to the above studies, we evaluated the kinetic behavior of the volume- and NEM-induced K/H exchange flux pathways in media where Cl was replaced by SCN, NO3, para-aminohippurate (PAH), or gluconate. The anion replacement studies did not permit a distinction between K/H exchange and KCl cotransport, since, depending upon the anion used as a Cl replacement, partial inhibition or stimulation of volume-activated K/H exchange fluxes was observed. In contrast, all anions used were stimulatory to the NEM-induced K loss. Since, on the basis of force-flow analysis, both volume-and NEM-induced K loss are K/H exchange, it was necessary to reevaluate assumptions (i.e., anions serve as substrates and therefore probe the translocation step) associated with the use of anion replacement as a means of flux route identification. When viewed together with the force-flow studies, the Cl replacement studies suggest that anion effects upon K/H exchange are indirect. The different anions appear to alter mechanisms that couple NEM exposure and cell swelling to the activation of K/H exchange, as opposed to exerting direct effects upon K and H translocation.     

Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture

We studied the ionic mechanisms underlying the regulatory volume increase of rat hepatocytes in primary culture by use of confocal laser scanning microscopy, conventional and ion-sensitive microelectrodes, cable analysis, microfluorometry, and measurements of 86Rb+ uptake. Increasing osmolarity from 300 to 400 mosm/liter by addition of sucrose decreased cell volumes to 88.6% within 1 min; thereafter, cell volumes increased to 94.1% of control within 10 min, equivalent to a regulatory volume increase (RVI) by 44.5%. This RVI was paralleled by a decrease in cell input resistance and in specific cell membrane resistance to 88 and 60%, respectively. Ion substitution experiments (high K+, low Na+, low Cl-) revealed that these membrane effects are due to an increase in hepatocyte Na+ conductance. During RVI, ouabain-sensitive 86Rb+ uptake was augmented to 141% of control, and cell Na+ and cell K+ increased to 148 and 180%, respectively. The RVI, the increases in Na+ conductance and cell Na+, as well as the activation of Na+/K(+)-ATPase were completely blocked by 10(-5) mol/liter amiloride. At this concentration, amiloride had no effect on osmotically induced cell alkalinization via Na+/H+ exchange. When osmolarity was increased from 220 to 300 mosm/liter (by readdition of sucrose after a preperiod of 15 min in which the cells underwent a regulatory volume decrease, RVD) cell volumes initially decreased to 81.5%; thereafter cell volumes increased to 90.8% of control. This post-RVD-RVI of 55.0% is also mediated by an increase in Na+ conductance. We conclude that rat hepatocytes in confluent primary culture are capable of RVI as well as of post-RVD-RVI. In this system, hypertonic stress leads to a considerable increase in cell membrane Na+ conductance. In concert with conductive Na+ influx, cell K+ is then increased via activation of Na+/K(+)-ATPase. An additional role of Na+/H+ exchange in the volume regulation of rat hepatocytes remains to be defined.     

Volume regulation by flounder red blood cells in anisotonic media

The nucleated high K, low Na red blood cells of the winter flounder demonstrated a volume regulatory response subsequent to osmotic swelling or shrinkage. During volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation the net water flow was secondary to net inorganic cation flux. Volume regulation after osmotic swelling is referred to as regulatory volume decrease (RVD) and was characterized by net K and water loss. Since the electrochemical gradient for K is directed out of the cell there is no need to invoke active processes to explain RVD. When osmotically shrunken, the flounder erythrocyte demonstrated a regulatory volume increase (RVI) back toward control cell volume. The water movements characteristic of RVI were a consequence of net cellular NaCl and KCl uptake with Na accounting for 75 percent of the increase in intracellular cation content. Since the Na electrochemical gradient is directed into the cell, net Na uptake was the result of Na flux via dissipative pathways. The addition of 10(-4)M ouabain to suspensions of flounder erythrocytes was without effect upon net water movements during volume regulation. The presence of ouabain did however lead to a decreased ration of intracellular K:Na. Analysis of net Na and K fluxes in the presence and absence of ouabain led to the conclusion that Na and K fluxes via both conservative and dissipative pathways are increased in response to osmotic swelling or shrinkage. In addition, the Na and K flux rate through both pump and leak pathways decreased in a parallel fashion as cell volume was regulated. Taken as a whole, the Na and K movements through the flounder erythrocyte membrane demonstrated a functional dependence during volume regulation.     

Further studies of the volume-regulatory response of Amphiuma red cells in hypertonic media. Evidence for amiloride-sensitive Na/H exchange

When Amphiuma red cells are shrunken in hypertonic media, they return toward their original volume by gaining Na through an amiloride-sensitive pathway. As cells recover their volume during this volume-regulatory increase (VRI) response, acid is extruded into the medium. Medium acidification is correlated with cell Na uptake. Both medium acidification and cell Na uptake are blocked by 10(-3) M amiloride or by replacing medium Na with K or choline. Perturbations that increase cell Na uptake (such as increasing medium osmolality) also increase medium acidification. As the medium becomes more acidic, the cells become more alkaline. These changes in cell and medium pH are increased if pH equilibration across the cell membrane is prevented by inhibiting the anion exchanger with SITS (4-acetamido-4'-isothiocyano-2,2'-stilbene disulfonic acid). The quantity of acid extruded by SITS-treated cells is the same as the quantity of Na gained, which strongly suggests 1:1 exchange of Na for H. Cell enlargement in SITS-treated cells results from the exchange of osmotically active Na ions for H ions that are not osmotically active when combined with cellular buffers. Previous evidence indicates that the normal VRI response involves an increase in the cellular content of Cl as well as Na. We show that SITS completely blocks net Cl uptake, which suggests that Cl enters via the anion exchanger. SITS also slows Na entry, presumably as a result of the above-mentioned increase in cell pH caused by SITS. We suggest that the initial event in the VRI response is net Na uptake via a Na/H exchanger, and that net Cl uptake results from secondary Cl/HCO3 exchange via the anion exchanger.     

Activation of Na+/H+ exchange in lymphocytes by osmotically induced volume changes and by cytoplasmic acidification

After swelling in hypotonic solutions, peripheral blood mononuclear cells (PBM) shrink toward their original volumes. Upon restoration of isotonicity, the cells initially shrink but then regain near-normal size again. This regulatory volume increase (RVI) is abolished by removal of Na+o or Cl-o or by addition of amiloride. RVI is unaffected by removal of K+o or by ouabain and is only partially inhibited by 1 mM furosemide. As a result of increased influx, the cells gain both Na+ and K+ during reswelling. In contrast, only Na+ content increases in the presence of ouabain. Amiloride largely eliminates the changes in the content of both cations. Using diS-C3-(5), no significant membrane potential changes were detected during RVI, which suggests that the fluxes are electroneutral. The cytoplasmic pH of volume-static cells was measured with 5,6-dicarboxyfluorescein. After acid loading, the addition of extracellular Na+ induced an amiloride-inhibitable alkalinization, which is consistent with Na+/H+ exchange. Cytoplasmic pH was not affected by cell shrinkage itself, but an internal alkalinization, which was also amiloride sensitive and Na+ dependent, developed during reswelling. In isotonic lightly buffered solutions without HCO-3, an amiloride-sensitive acidification of the medium was measurable when Na+ was added to shrunken PBM. K+ was unable to mimic this effect. The observations are compatible with the model proposed by Cala (J. Gen. Physiol. 1980. 76:683-708), whereby an electroneutral Na+o/H+i exchange is activated by osmotic shrinking. Cellular volume gain occurs as Cl-o simultaneously exchanges for either HCO-3i or OH-i. Na+i is secondarily replaced by K+ through the pump, but this step is not essential for RVI.     

Volume-regulating behavior of human platelets

Human platelets exposed to hypotonic media undergo an initial swelling followed by shrinking (regulatory volume decrease [RVD]). If the RVD is blocked, the degree of swelling is in accord with osmotic behavior. The cells could swell at least threefold without significant lysis. Two methods were used to follow the volume changes, electronic sizing and turbidimetry. Changes in shape produced only limited contribution to the measurements. The RVD was very rapid, essentially complete in 2 to 8 minutes, with a rate proportional to the degree of initial cell swelling. RVD involved a loss of KCl via volume-activated conductive permeability pathways for K+ and anions, presumably Cl-. In media containing greater than 50 mM KCl, the shrinking was inhibited and with higher concentrations was reversed (secondary swelling), suggesting that it is driven by the net gradient of K+ plus Cl-. The K+ pathway was specific for Rb+ and K+ compared to Li+ and Na+. The Cl- pathway accepted NO-3 and SCN- but not citrate or SO4(2-). In isotonic medium, the permeability of platelets to Cl- appeared to be low compared to that of K+. After hypotonic swelling both permeabilities were increased, but the Cl- permeability exceeded that of K+. The Cl- conductive pathway remained open as long as the cells were swollen. RVD was incomplete unless amiloride, an inhibitor of Na+/H+ exchange, was present or unless Na+ was replaced by an impermeant cation. In addition, acidification of the cytoplasm occurred upon cell swelling. This reduction in pHi appeared to activate Na+/H+ exchange, with a resultant uptake of Na+ and reduction in the rate and amount of shrinking. Like other cells, platelets responded to hypertonic shrinking with activation of Na+/H+ exchange, but regulatory volume increase was not detectable.     

Role of separate K+ and Cl- channels and of Na+/Cl- cotransport in volume regulation in Ehrlich cells

Cells resuspended in hypotonic medium initially swell as nearly perfect osmometers, but later recover their volume with an associated KCl loss. This regulatory volume decrease (RVD) is unaffected when nitrate is substituted for Cl- or if bumetanide or 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS) is added. It is inhibited by quinine, Ba2+, low pH, anticalmodulin drugs, and depletion of intracellular Ca2+. It is accelerated by the Ca2+ ionophore A23187, or by a sudden increase in external Ca2+ and at high pH. A net KCl loss is also seen after addition of ionophore A23187 in isotonic medium. Similarities are demonstrated between the KCl loss seen after addition of A23187 and the KCl loss seen during RVD. It is proposed that separate conductive K+ and Cl- channels are activated during RVD by release of Ca2+ from internal stores, and that the effect is mediated by calmodulin. After restoration of tonicity the cells shrink initially, but recover their volume with an associated KCl uptake. This regulatory volume increase (RVI) is inhibited when NO3- is substituted for Cl-, and is also inhibited by furosemide or bumetanide, but it is unaffected by DIDS. The unidirectional Cl-flux ratio is compatible with either a coupled uptake of Na+ and Cl-, or an uptake via a K+/Na+/2Cl- cotransport system. No K+ uptake was found, however, in ouabain-poisoned cells where a bumetanide-sensitive uptake of Na+ and Cl- in nearly equimolar amounts was demonstrated. Therefore, it is proposed that the primary process during RVI is an activation of an otherwise quiescent Na+/Cl- cotransport system with subsequent replacement of Na+ by K+ via the Na+/K+ pump. There is a marked increase in the rate of pump activity in the absence of a detectable increase in intracellular Na+ concentration.     

Activation of Na+/H+ and K+/H+ exchange by calyculin A in Amphiuma tridactylum red blood cells: implications for the control of volume-induced ion flux activity

Alteration in cell volume of vertebrates results in activation of volume-sensitive ion flux pathways. Fine control of the activity of these pathways enables cells to regulate volume following osmotic perturbation. Protein phosphorylation and dephosphorylation have been reported to play a crucial role in the control of volume-sensitive ion flux pathways. Exposing Amphiuma tridactylu red blood cells (RBCs) to phorbol esters in isotonic medium results in a simultaneous, dose-dependent activation of both Na(+)/H(+) and K(+)/H(+) exchangers. We tested the hypothesis that in Amphiuma RBCs, both shrinkage-induced Na(+)/H(+) exchange and swelling-induced K(+)/H(+) exchange are activated by phosphorylation-dependent reactions. To this end, we assessed the effect of calyculin A, a phosphatase inhibitor, on the activity of the aforementioned exchangers. We found that exposure of Amphiuma RBCs to calyculin-A in isotonic media results in simultaneous, 1-2 orders of magnitude increase in the activity of both K(+)/H(+) and Na(+)/H(+) exchangers. We also demonstrate that, in isotonic media, calyculin A-dependent increases in net Na(+) uptake and K(+) loss are a direct result of phosphatase inhibition and are not dependent on changes in cell volume. Whereas calyculin A exposure in the absence of volume changes results in stimulation of both the Na(+)/H(+) and K(+)/H(+) exchangers, superimposing cell swelling or shrinkage and calyculin A treatment results in selective activation of K(+)/H(+) or Na(+)/H(+) exchange, respectively. We conclude that kinase-dependent reactions are responsible for Na(+)/H(+) and K(+)/H(+) exchange activity, whereas undefined volume-dependent reactions confer specificity and coordinated control.     

Volume-regulatory responses of Amphiuma red cells in anisotonic media. The effect of amiloride

Amphiuma red cells were incubated for several hours in hypotonic or hypertonic media. They regulate their volume in both media by using ouabain-insensitive salt transport mechanisms. After initially enlarging osmotically, cells in hypotonic media return toward their original size by losing K, Cl, and H2O. During this volume-regulatory decrease (VRD) response, K loss results from a greater than 10-fold increase in K efflux. Cells in hypertonic media initially shrink osmotically, but then return toward their original volume by gaining Na, Cl, and H2O. The volume-regulatory increase (VRI) response involves a large (greater than 100-fold) increase in Na uptake that is entirely blocked by the diuretic amiloride (10(-3) M). Na transport in the VRI response shares many of the characteristics of amiloride-sensitive transport in epithelia: (a) amiloride inhibition is reversible; (b) removal of amiloride from cells pretreated with amiloride enhances Na uptake relative to untreated controls; (c) amiloride appears to act as a competitive inhibitor (Ki = 1-3 microM) of Na uptake; (d) Na uptake is a saturable function of external Na (Km approximately 29 mM); (e) Li can substitute for Na but K cannot. Anomalous Na/K pump behavior is observed in both the VRD and the VRI responses. In the VRD response, pump activity increases 3-fold despite a decrease in intracellular Na concentration, while in the VRI response, a 10-fold increase in pump activity is observed when only a doubling is predicted from increases in intracellular Na.     

pH regulatory Na/H exchange by Amphiuma red blood cells

In Amphiuma red blood cells, the Na/H exchanger has been shown to play a central role in the regulation of cell volume following cell shrinkage (Cala, P. M. 1980. Journal of General Physiology. 76:683- 708.) The present study was designed to evaluate the existence of pH regulatory Na/H exchange in the Amphiuma red blood cell. The data illustrate that when the intracellular pHi was decreased below the normal value of 7.00, Na/H exchange was activated in proportion to the degree of acidification. Once activated, net Na/H exchange flux persisted until normal intracellular pH (6.9-7.0) was restored, with a half time of approximately 5 min. These observations established a pHi set point of 7.00 for the pH-activated Na/H exchange of Amphiuma red blood cell. This is in contrast to the behavior of osmotically shrunken Amphiuma red blood cells in which no pHi set point could be demonstrated. That is, when activated by cell shrinkage the Na/H exchange mediated net Na flux persisted until normal volume was restored regardless of pHi. In contrast, when activated by cell acidification, the Na/H exchanger functioned until pHi was restored to normal and cell volume appeared to have no effect on pH-activated Na/H exchange. Studies evaluating the kinetic and inferentially, the molecular equivalence of the volume and pHi-induced Amphiuma erythrocyte Na/H exchanger(s), indicated that the apparent Na affinity of the pH activated cells is four times greater than that of shrunken cells. The apparent Vmax is also higher (two times) in the pH activated cells, suggesting the involvement of two distinct populations of the transporter in pH and volume regulation. However, when analyzed in terms of a bisubstrate model, the same data are consistent with the conclusion that both pH and volume regulatory functions are mediated by the same transport protein. Taken together, these data support the conclusion that volume and pH are regulated by the same effector (Na/H exchanger) under the control of as yet unidentified, distinct and cross inhibitory volume and pH sensing mechanisms.     

Cell volume regulation in nonrenal epithelia

Cell volume regulation occurs in both tight, Na+-transporting epithelia (e.g., frog skin) and in leaky. NaCl-transporting epithelia (e.g. amphibian gallbladder). In tight epithelia volume regulation occurs only in response to cell swelling, i.e. only regulatory volume decrease (RVD) is observed, whereas in leaky epithelia cell volume regulation has been observed in response to osmotic challenges that either swell or shrink the cells. In other words, both RVD and regulatory volume increase (RVI) are present. Both volume regulatory responses involve stimulation of ion transport in a polarized fashion: in RVD the response is basolateral KCl efflux, whereas in RVI it is apical membrane NaCl uptake. The loss of KCl during RVD appears to result in most instances from increases in basolateral electrodiffusive K+ and Cl-permeabilities. In gallbladder, concomitant activation of coupled KCl efflux may also occur. The RVI response includes activation of apical membrane cation (Na+/H+) and anion (Cl-/HCO-3) exchangers. It is presently unclear whether the net ion fluxes resulting from activation of these transporters, during either RVD or RVI, account for the measured rates of restoration of cell volume. In gallbladder epithelium, RVD is inhibited by agents which disrupt microfilaments or interfere with the Ca2+-calmodulin system. These pharmacologic effects are absent in RVI. Some steps in the chain of events resulting in either RVI or RVD have been established, but the signals involved remain largely unknown. There is reason to suspect a role of intracellular pH in the case of RVI and of membrane insertion of transporters in the case of RVD, possibly with causal roles of both intracellular Ca2+ and the cytoskeleton in the latter.     

Regulatory volume decrease in the presence of HCO3- by single osteosarcoma cells UMR-106-01.

The technique for the simultaneous recording of cell volume changes and pHi in single cells was used to study the role of HCO3- in regulatory volume decrease (RVD) by the osteosarcoma cells UMR-106-01. In the presence of HCO3-, steady state pHi is regulated by Na+/H+ exchange, Na+ (HCO3-)3 cotransport and Na(+)-independent Cl-/HCO3- exchange. Following swelling in hypotonic medium, pHi was reduced from 7.16 +/- 0.02 to 6.48 +/- 0.02 within 3.4 +/- 0.28 min. During this period of time, the cells performed RVD until cell volume was decreased by 31 +/- 5% beyond that of control cells (RVD overshoot). Subsequently, while the cells were still in hypotonic medium, pHi slowly increased from 6.48 +/- 0.02 to 6.75 +/- 0.02. This increase in pHi coincided with an increase in cell volume back to normal (recovery from RVD overshoot or hypotonic regulatory volume increase (RVI)). The same profound changes in cell volume and pHi after cell swelling were observed in the complete absence of Cl- or Na+, providing HCO3- was present. On the other hand, depolarizing the cells by increasing external K+ or by inhibition of K+ channels with quinidine, Ba2+ or tetraethylammonium prevented the changes in pHi and RVD. These findings suggest that in the presence of HCO3-, RVD in UMR-106-01 cells is largely mediated by the conductive efflux of K+ and HCO3-. Removal of external Na+ but not Cl- prevented the hypotonic RVI that occurred after the overshoot in RVD. Amiloride had no effect, whereas pretreatment with 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) strongly inhibited hypotonic RVI. Thus, hypotonic RVI is mediated by a Na+(out)-dependent, Cl(-)-independent and DIDS-inhibitable mechanism, which is indicative of a Na+(HCO3-)3 cotransporter. This is the first evidence for the involvement of this transporter in cell volume regulation. The present results also stress the power of the new technique used in delineating complicated cell volume regulatory mechanisms in attached single cells.     

Volume regulation of Chinese hamster ovary cells in anisoosmotic media

Chinese hamster ovary (CHO) cells when suspended in anisoosmotic media regulate their volumes by the activation of specific ion transport pathways. In hypoosmotic media the cells first swell and then return to their isoosmotic volumes by the loss of cellular KCl and osmotically obliged water. This regulatory volume decrease (RVD) is insensitive to ouabain or bumetanide but is blocked by quinine, cetiedil and oligomycin C. Based on cell volume and membrane potential measurements under various experimental conditions, we conclude that hypoosmotic shock activates independent, conductive transport pathways for K+ and for Cl-, respectively. The anion pathway can also transport NO3- and SCN- but not gluconate- anions. Osmotic shrinkage of CHO cells does not produce a regulatory volume increase (RVI) unless the cells have previously undergone a cycle of RVD. RVI is a Na+-dependent, amiloride-sensitive, but ouabain- and oligomycin-insensitive process, probably involving a Na+-H+ exchange system. Internal acidification of isoosmotic cells by addition of a permeable weak acid also activates an amiloride-sensitive Na+-H+ exchange, producing a volume increase. Both RVD and RVI in CHO cells seem to involve molecular mechanisms similar to those described for the volume regulation of lymphocytes, indicating the prevalence of these phenomena in nucleated mammalian cells. Cultured CHO cell lines may provide a basis for a genetic characterization of the volume-regulatory transport pathways.     

Different patterns of cell volume regulation in hyposmotic media between attached and suspended HeLa cells.

Both attached and suspended HeLa cells swelled in a medium of a hypotonic osmolality of 235 mosmol/kg H2O. When the osmolality was further decreased to 166 mosmol/kg H2O, attached cells instantly swelled and then rapidly lost water and K+, followed by slow gains of them. Suspended cells instantly swelled and then K+ loss and regulatory volume decrease (RVD) occurred. Neither 0.1 mM ouabain nor 10 mM TEA changed the water loss of attached cells, whereas ouabain inhibited RVD of suspended cells. Quinine (1 mM) inhibited water losses from both cells and comparison of the losses implies stronger activation of K+ channel in attached cells than in suspended cells. Omission of medium Ca2+ or addition of 10 mM BaCl2 inhibited RVD in part. These results suggest that hyposmotic stress induces net water loss from attached cells, associated with K+ release through the Ca(2+)-dependent K+ channel. Suspended cells osmotically swell, followed by RVD with K+ and Na+ releases through the K+ channel and Na(+)-pump, respectively. The different patterns of volume changes may relate to the difference of activity or time of activation of the K+ channel between both cells.     

22Na+ fluxes in thymic lymphocytes. I. Na+/Na+ and Na+/H+ exchange through an amiloride-insensitive pathway

The Na+ transport pathways of normal rat thymocytes were investigated. Na+ conductance was found to be lower than K+ conductance, which is consistent with reported values of membrane potential. In contrast, the isotopically measured Na+ permeability was greater than 10-fold higher than that of K+, which indicates that most of the flux is electroneutral. Cotransport with Cl- (or K+ and Cl-) and countertransport with Ca2+ were ruled out by ion substitution experiments and use of inhibitors. Countertransport for Na+ or H+ through the amiloride-sensitive antiport accounts for only 15-20% of the resting influx. In the presence of amiloride, 22Na+ uptake was increased in Na+-loaded cells, which suggests the existence of Na+/Na+ countertransport. Cytoplasmic pH determinations using fluorescent probes indicated that under certain conditions this amiloride-resistant system will also exchange Na+ for H+, as evidenced by an internal Na+- dependent acidification is proportional to internal [Na+] but inversely related to extracellular [Na+]. Moreover, 22Na+ uptake is inhibited by increasing external [H+]. The results support the existence of a substantial amiloride-insensitive, electroneutral cation exchange system capable of transporting Na+ and H+.     

Theoretical effects of transmembrane electroneutral exchange on membrane potential

Transmembrane electroneutral transport mechanisms [e.g., Na/H exchange, Cl/HCO3 exchange, (K + Cl) cotransport] have recently been identified in a wide variety of cell types. If these exchanges sum to give a net electroneutral Na/K exchange, they may hyperpolarize the membrane potential beyond the value calculated from the Mullins-Noda equation, provided the cell maintains steady state intracellular ionic concentrations. In extreme circumstances, the membrane potential could hyperpolarize beyond the potassium reversal potential. This effect is mediated by the electrogenic Na/K pump. If either Na or K exchanges electroneutrally against a third ion (e.g., Na/Ca exchange), then the exchange may depolarize the membrane potential.     

Volume regulation by human lymphocytes. Role of calcium

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++- containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).     

Simultaneous recording of cell volume changes and intracellular pH or Ca2+ concentration in single osteosarcoma cells UMR-106-01.

We present a new technique for the simultaneous measurement of cell volume changes and intracellular ionic activities in single cells. The technique uses measurement of changes in the concentration of intracellularly trapped fluorescent dyes to report relative cell volume. By using pH- or Ca(2+)-sensitive dyes and recording at the ion-sensitive and -insensitive (isosbestic) wavelengths, the method can measure both cell volume changes and intracellular ionic activities. The technique was used to study the mechanisms of regulatory volume decrease (RVD) in the osteosarcoma cell line UMR-106-01 grown on cover slips. Swelling cells in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-buffered hypotonic medium was followed by stable cytosolic acidification and a decrease in cell volume back toward normal. The recovery of cell volume could be blocked by depolarization, treatment with ouabain, or depletion of cell Cl-. These suggest the conductive efflux of K+ and Cl- during RVD. The cytosolic acidification that accompanied cell swelling was not blocked by amiloride, bafilomycin A, or removal of Cl- and could not be reproduced by depletion of cellular ATP. These findings exclude Na+/H+ and Cl-/HCO-3 exchange, intracellularly generated acid, or increased metabolism, respectively, as the cause of the acidification. The cell swelling-induced acidification was inhibited by depolarization, suggesting the involvement of an electrogenic pathway. The acidification, as well as RVD, was inhibited by short incubation with deoxyglucose, and these effects could not be reversed by valinomycin. Thus, the anionic pathway(s) participating in RVD and the acidification are sensitive to the cellular level of ATP. Together, these studies indicate that RVD in UMR-106-01 cells in HEPES-buffered medium is mediated by the conductive efflux of K+, Cl-, and OH-.     

Effect of arachidonic acid,fatty acids,prostaglandins, and leukotrienes on volume regulation in Ehrlich ascites tumor cells

Summary Arachidonic acid inhibits the cell shrinkage observed in Ehrlich ascites tumor cells during regulatory volume decrease (RVD) or after addition of the Ca ionophore A23187 plus Ca. In Na-containing media, arachidonic acid increases cellular Na uptake under isotonic as well as under hypotonic conditions. Arachidonic acid also inhibits KCl and water loss following swelling in Na-free, hypotonic media even when a high K conductance has been ensured by addition of gramicidin. In isotonic, Na-free medium arachidonic acid inhibits A23187 + Ca-induced cell shrinkage in the absence but not in the presence of gramicidin. It is proposed that inhibition of RVD in hypotonic media by arachidonic acid is caused by reduction in the volume-induced Cl and K permeabilities as well as by an increase in Na permeability and that reduction in A23187 + Ca-induced cell shrinkage is due to a reduction in K permeability and an increase in Na permeability. The A23187 + Ca-activated Cl permeability in unaffected by arachidonic acid. PGE₂ inhibits RVD in Na-containing, hypotonic media but not in Na-free, hypotonic media, indicating a PGE₂-induced Na uptake. PGE₂ has no effect on the volume-activated K and Cl permeabilities. LTB₄, LTC₄ and LTE₄ inhibit RVD insignificantly in hypotonically swollen cells. LTD₄, more-over, induces cell shrinkage in steady-state cells and accelerates the RVD following hypotonic exposure. The effect of LTD₄ even reflects a stimulating effect on K and Cl transport pathways. Thus none of the leukotrienes show the inhibitory effect found for arachidonic acid on the K and Cl permeabilities. The RVD response in hypotonic, Na-free media is, on the other hand, also inhibited by addition of the unsaturated oleic, linoleic, linolenic and palmitoleic acid, even in the presence of the cationophor gramicidin. The saturated arachidic and stearic acid had no effect on RVD. It is, therefore, suggested that a minor part of the inhibitory effect of arachidonic acid on RVD in Na-containing media is via an increased synthesis of prostaglandins and that the major part of the arachidonic acid effect on RVD in Na-free media, and most probably also in Na-containing media, is due to the inhibition of the volume-induced K and Cl transport pathways, caused by a nonspecific detergent effect of an unsaturated fatty acid.