Ouabain-insensitive salt and water movements in duck red cells. II. The role of chloride in the volume response

This paper describes the effect of external chloride on the typical swelling response induced in duck red cells by hypertonicity or norepinephrine. Lowering chloride inhibits swelling and produces concomitant changes in net movements of sodium and potassium in ouabain-treated cells, which resemble the effect of lowering external sodium or potassium. Inhibition is the same whether chloride is replaced with gluconate or with an osmotic equivalent of sucrose. Since changes in external chloride also cause predictable changes in cell chloride, pH, and water, these variables were systematically investigated by varying external pH along with chloride. Lowering pH to 6.60 does not abolish the response if external chloride levels are normal, although the cells are initially swollen due to the increased acidity. Cells deliberately preswollen in hypotonic solutions with appropriate ionic composition can also respond to norepinephrine by further swelling. These results rule out initial values of cell water, chloride, and pH as significant variables affecting the response. Initial values of the chloride equilibrium potential do have marked effect on the direction and rate of net water movement. If chloride is lowered by replacement with the permeant anion, acetate, E(Cl) is unchanged and a normal response to norepinephrine, which is inhibited by furosemide, is observed. Increasing internal sodium by the nystatin technique also inhibits the response. A theory is developed which depicts that the cotransport carrier proposed in the previous paper (W.F. Schmidt and T.J. McManus. 1977b. J. Gen. Physiol. 70:81-97) moves in response to the net electrochemical potential difference driving sodium and potassium across the membrane. Predictions of this theory fit the data for both cations and anions.     

Ouabain-insensitive salt and water movements in duck red cells. I. Kinetics of cation transport under hypertonic conditions

Duck red cells in hypertonic media experience rapid osmotic shrinkage followed by gradual reswelling back toward their original volume. This uptake of salt and water is self limiting and demands a specific ionic composition of the external solution. Although ouabain (10(-4)M) alters the pattern of cation accumulation from predominantly potassium to sodium, it does not affect the rate of the reaction, or the total amount of salt or water taken up. To study the response without the complications of active Na-K transport, ouabain was added to most incubations. All water accumulated by the cells can be accounted for by net salt uptake. Specific external cation requirements for reswelling include: sufficient sodium (more than 23 mM), and elevated potassium (more than 7 mM). In the absence of external potassium cells lose potassium without gaining sodium and continue to shrink instead of reswelling. Adding rubidium to the potassium- free solution promotes an even greater loss of cell potassium, yet causes swelling due to a net uptake of sodium and rubidium followed by chloride. The diuretic furosemide (10(-3)M) inhibits net sodium uptake which depends on potassium (or rubidium), as well as inhibits net sodium uptake which depends on sodium. As a result, cell volume is stabilized in the presence of this drug by inhibition of shrinkage, at low, and of swelling at high external potassium. The response has a high apparent energy of activation (15-20 kcal/mol). We propose that net salt and water movements in hypertonic solutions containing ouabain are mediated by direct coupling or cis-interaction, between sodium and potassium so that the uphill movement of one is driven by the downhill movement of the other in the same direction.     

Outward sodium and potassium cotransport in human red cells

Summary This paper reports some kinetic properties of Na–K cotransport in human red cells. All fluxes were measured in the presence of 10^–4 M ouabain. We measured Na and K efflux from cells loaded by the PCMBS method to contain different concentrations of these ions into a medium that contained neither Na nor K (MgCl₂-sucrose substitution) in the absence and presence of furosemide. Furosemide inhibited 30–60% of the total efflux depending on the internal ion concentration and the individual subject. We took the furosemide-sensitive fluxes to be a measure of Na–K cotransport. The ratio of Na to K cotransport was 1 over the entire range of internal Na and K concentrations studied. When Na was substituted for K as the only internal cation, cotransport was maximally activated when the Na and K concentrations were between 20 and 90 mmol/liter cells. The concentration of internal Na required to produce half-maximal cotransport was about 13±4 mmol/liter cells (n=4), while the comparable concentration of K was somewhat lower. The activation curve was definitely sigmoid in character, suggesting that at least two Na ions are involved in the transport process. The maximum of Na–K cotransport was about 0.5±0.15 mmol/liter cells × hr (n=5); it had a flat maximum in the medium at about pH 7.0, decreasing in both the acid and alkaline sides. furosemide-resistant effluxes were found to be linear functions of internal Na and K concentrations and to yield rate coefficients of 0.019±0.002 hr^–1 and 0.014±0.002 hr^–1 (n=7), respectively. These values are of the same order of magnitude expected of ions moving across phospholipid bilayers.Charge de Recherches CNRS.     

p-Chloromercuribenzenesulfonic acid stimulation of chloride-dependent sodium and potassium transport in human red blood cells

The organic mercurial p-chloromercuribenzensulfonic acid (PCMBS) reversibly increases fluxes of sodium and potassium across the human red blood cell membrane. We examined the effect of different monovalent anions on cation fluxes stimulated by PCMBS. A substantial portion of the fluxes of both cations was found to have a specific anion requirement for chloride or bromide, and was not observed when chloride was replaced by nitrate, acetate or methylsulfate. The chloride-dependent component of the cation fluxes was only observed when the cells were exposed to PCMBS concentrations of 0.5 mM or greater. Furosemide (1 mM) did not inhibit the PCMBS-stimulated cation fluxes. The observed anion specificity is directly associated with the transport process rather than PCMBS binding to the membrane. A portion of the potassium transport stimulated by PCMBS appears to involve K+-K+ exchange; however, Na+ + K+ cotransport is not stimulated by this sulfhydryl reagent.     

The sodium and potassium activated ATPase. II. Comparative study of intestinal epithelium and red cells

The Na-K ATPase found in sedimentable fractions of intestinal epithelium of rats hydrolyzed cytidine triphosphate nearly as well as ATP (25% to 50%); was active only in presence of divalent cations, with specificity for Mg (100%), Mn (50%) and Ca (10%); showed a plateau of activation when Mg concentrations were in excess of substrate; and was inhibited by a second divalent cation (Zn > Mn > Ca), and by 3 × 10^?4 M ouabain (50%). Parallel assays of rat red cell ghosts showed differences in substrate specificity (CTP was not utilized), in activation kinetics (activation peak with Mg) and in greater specificity to Mg (Mn was a weaker activator and Zn was a weaker inhibitor). Stabilities also differed in the two preparations: Na? K ATPase of intestinal epithelium was activated by sucrose extraction and denatured during cytolysis at room temperature, while that of red cell fragments was denatured during sucrose extraction and preserved by hemolysis at room temperature. Other properties of Na? K ATPase studied in the two tissues included activation by monovalent cations (optimum at 160 mM Na, 15 mM K), specificity to monovalent cations, and sensitivity to lipid solvents and to some drugs. The data were discussed in terms of comparative properties of Na? K ATPases of various cells. Residual ATPase activities of intestinal epithelium and red cell ghosts were shown to differ in substrate specificity, inhibition and activation. “Residual ATPase” from intestinal epithelium was a zinc-activated nucleoside polyphosphate phosphohydrolase, while ghosts contained Mg? ATPase. Only the latter enzyme was specific to ATP and Mg, activated by Ca in presence of Mg, and sensitive to inhibition by PCMB and Zn.     

Active sodium and potassium transport in high potassium and low potassium sheep red cells

The kinetic characteristics of the ouabain-sensitive (Na + K) transport system (pump) of high potassium (HK) and low potassium (LK) sheep red cells have been investigated. In sodium medium, the curve relating pump rate to external K is sigmoid with half maximal stimulation (K_1/2) occurring at 3 mM for both cell types, the maximum pump rate in HK cells being about four times that in LK cells. In sodium-free media, both HK and LK pumps are adequately described by the Michaelis-Menten equation, but the K_1/2 for HK cells is 0.6 ± 0.1 mM K, while that for LK is 0.2 ± 0.05 mM K. When the internal Na and K content of the cells was varied by the PCMBS method, it was found that the pump rate of HK cells showed a gradual increase from zero at very low internal Na to a maximum when internal K was reduced to nearly zero (100% Na). In LK cells, on the other hand, no pump activity was detected if Na constituted less than 70% of the total (Na + K) in the cell. Increasing Na from 70 to nearly 100% of the internal cation composition, however, resulted in an exponential increase in pump rate in these cells to about ⅙ the maximum rate observed in HK cells. While changes in internal composition altered the pump rate at saturating concentrations of external K, it had no effect on the apparent affinity of the pumps for external K. These results lead us to conclude that the individual pump sites in the HK and LK sheep red cell membranes must be different. Moreover, we believe that these data contribute significantly to defining the types of mechanism which can account for the kinetic characteristics of (Na + K) transport in sheep red cells and perhaps in other systems.     

Volume-responsive sodium and proton movements in dog red blood cells

Shrinkage of dog red blood cells (RBC) activates a Na transport pathway that is Cl dependent, amiloride sensitive, and capable of conducting Na- proton counterflow. It is possible to establish transmembrane gradients for either Na or protons and to demonstrate that each cation species can drive reciprocal movements of the other. The nature of the coupling between Na and proton movements was investigated using the fluorescent probe diS-C3(5) and also by an indirect method in which K movements through valinomycin channels were used to draw inferences about the membrane potential. No evidence was found to suggest that the Na-proton pathway activated by shrinkage of dog RBC is a conductive one. By exclusion, it is presumed that the coupling between the counterflow of Na and protons is electroneutral. The volume-activated Na-proton fluxes in dog RBC have certain properties that distinguish them from similar transport pathways in other cell types.     

Ouabain-insensitive sodium/lithium exchange and the effect of anti-L in low potassium sheep erythrocytes

The activity of the ouabain-insensitive Na⁺/Na⁺ exchange system was assessed by measurements of Li⁺ net-uptake in LK and HK sheep erythrocytes in the absence and presence of the L-antibody and various inhibitors. N-ethylmaleimide, p-chloromercuribenzoic sulfonate and phloretin inhibited the exchange by about 50%. Anti-L, while stimulating the K⁺ pump flux in LK cells, did not alter Na⁺/Li⁺ countertransport. The activity of the exchange system with fully saturated internal and external loading sites was estimated to be identical in LK and HK sheep red cells. Hence the Na⁺/Na⁺ exchange system seems to be molecularly unrelated to the ouabain-sensitive Na⁺K⁺ pump in these cells and not under genetic control of the HK/LK and M/L genes.     

Heterogeneity in dog red blood cells: sodium and potassium transport

After incubation in isotonic KCl, dog red blood cells can be separated by centrifugation into subgroups which assume different cell volumes and possess different transport characteristics. Those red cells which swell in isotonic KCl exhibit a higher permeability to K and possess a greater volume dependence for transport of K than those red cells which shrink. A high Na permeability characterizes cells which shrink in isotonic KCl and these cells exhibit a larger volume-dependent Na flux than those red cells which swell. These two subgroups of red cells do not seem to represent two cell populations of different age. The results indicate that the population of normal cells is evidently heterogeneous in that the volume-dependent changes in Na and K permeability are distributed between differnt cell types rather than representing a single cell type which reciprocally changes its selectivity to Na and K.     

Norepinephrine stimulation of sodium transport in Necturus urinary bladder

Norepinephrine alters the transepithelial electrical properties of an open-circuited urinary bladder from the mud puppy, Necturus maculosus. When 10(-5) M norepinephrine is superfused over the serosa of the epithelium, the transepithelial voltage (Vt) and short-circuit current (Isc) increase as the resistance (Rt) decreases. The norepinephrine-mediated changes are reversed by the addition of amiloride (5.10(-5) M) to the mucosal Ringer's solution. The serosal adrenoceptors mediating the Na+ transport are more sensitive to norepinephrine (EC50 = 1.2.10(-6) M) than to epinephrine or isoproterenol. Since the Isc is blocked selectively by the antagonist, phenoxybenzamine, stimulation of active transepithelial Na(+)-flux by catecholamines is mediated by an alpha-adrenoceptor. The apical cell membrane voltage (Va) and fractional resistance (fRa) were recorded using conventional KCl-filled microelectrodes. Untreated tissues have Va close to 0 mV while the basolateral membrane voltage (Vb) is between -85 and -95 mV. About 90% of Rt is apical cell membrane resistance (fRa). When amiloride inhibits sodium transport, Va becomes negative, Vb hyperpolarizes slightly and fRa increases to 97%. On the other hand, if the bladders are treated with norepinephrine, fRa decreases to 79% as Va becomes positive and Vb depolarizes. When Rt changes, the resistance of the paracellular pathway (Rp) is unaltered. Changes in the electrical properties of the tissue appear to be mediated primarily by alterations in Ra. Since the Necturus bladder does not respond to antidiuretic hormone, this study implies that biogenic amines regulate Na+ transport in the epithelium.