Measurement of Na-K pump current in acinar cells of rat lacrimal glands.

Isolated cells from rat lacrimal glands were voltage clamped using the tight-seal whole-cell recording technique. The intracellular solution contained ATP and an elevated Na concentration (70 mM). Removing external K ions elicited an inward current shift. Ouabain (0.5 mM) induced an inward current shift of identical amplitude, but with slower kinetics. In the presence of ouabain, removal of K ions did not alter the cell current. The potassium- and ouabain-sensitive current was outward between -120 and +20 mV, and its amplitude decreased below -60 mV. This current was highly sensitive to temperature, and was not affected by blockers of the K channels which are present in these cells. It was attributed to an inhibition of the Na-K pump. The Na-K pump current was estimated to be 15 pA for an average acinar cell at physiological temperature, with 70 mM internal Na ions and 20 mM external K ions. Implications of this value in terms of electrolyte secretion are discussed.     

Correlation of the effect of Ca+2 on Na+ and K+ permeability and membrane potential of Ehrlich ascites tumor cells

The effect of Ca+2 on the transport and intracellular distribution of Na+ and K+ in Ehrlich ascites tumor cells was investigated in an effort to establish the mechanism of Ca+2-induced hyperpolarization of the cell membrane. Inclusion of Ca+2 (2 mM) in the incubation medium leads to reduced cytoplasmic concentrations of Na+, K+ and Cl- in steady cells. In cells inhibited by ouabain, Ca+2 causes a 41% decrease in the rate of net K+ loss, but is without effect on the rate of net Na+ accumulation. Net K+ flux is reduced by 50%, while net Na+ flux is unchanged in the transport-inhibited cells. The membrane potential of cells in Ca+2-free medium (-13.9 +/- 0.8 mV) is unaffected by the addition of ouabain. However, the potential of cells in Ca+2-containing medium (-23.3 +/- 1.2 mV) declines in one hour after the addition of ouabain to values comparable to those of control cells (-15.2 +/- 0.7 mV). The results of these experiments are consistent with the postulation that Ca+2 exerts two effects on Na+ and K+ transport. First, Ca+2 reduces the membrane permeability to K+ by 25%. Second, Ca+2 alters the coupling of the Na/K active transport mechanism leading to an electrogenic hyperpolarization of the membrane.     

Direct measurement of the membrane potential of Ehrlich ascites tumor cells: lack of effect of valinomycin and ouabain.

The membrane potential of Ehrlich ascites tumor cells and the effects of valinomycin and ouabain upon it have been determined. The membrane potential in control cells was 12.0 mV, inside negative. Neither valinomycin nor ouabain alone affected this value. However, valinomycin and ouabain in combination resulted in a slight hyperpolarization of the membrane. Concomitant determinations of cellular Na+, K+ and Cl- showed that valinomycin induced net losses of K+ and Cl- and a net gain in Na+ when compared to ouabain-inhibited cells. K+ permeability was increased by approximately 30% in the presence of valinomycin. In addition, valinomycin caused a rapid depletion of cellular ATP. Inhibition of Na/K transport by ouabain was without sparing effect on the rate of ATP depletion. Possible mechanisms for the electroneutral increase in K+ permeability induced by valinomycin are discussed.     

Potassium, sodium, water, and Donnan potential changes in the myofibrils of intact muscle fibres after exposure to ouabain and K-free Ringer

Frog sartorius muscles were superfused for 40 min with solutions of K-free Ringer, normal Ringer containing ouabain, or K-free Ringer containing ouabain. Changes in myoplasmic K and Na were measured with ion-selective microelectrodes; changes in total fibre K and Na were measured by means of atomic absorption spectroscopy; and changes in total fibre water content were obtained from wet and dry weights. Application of a two-compartment model permitted one to calculate (i) the K, Na, and water changes in the myofibrils and in the surrounding myoplasm (extramyofibrillar space); (ii) the changes in the transmyofibrillar Donnan potential (ED); and (iii) the changes in the ratio of the apparent association constants (kNa/kK) of the myofilament charge sites to Na and K. In the resting fibres, the K, Na, and water content of the myofibrils were calculated to be 82, 87, and 80% of total fibre content, respectively; ED was calculated as -4.5 mV; kNa/kK was calculated as 1.4. After a 40-min ouabain treatment, 12 mmol (per kg fibre water) of intrafibre K exchanged with 7.5 mmol of extrafibre Na, 6.4 mmol of myofibrillar K exchanged with an equal amount of extramyofibrillar Na, ED increased to -8.3 mV, and kNa/kK remained relatively constant. After a 40-min K-free treatment, the fibres gained 5.5 mmol of Na without any change in fibre K or water, the myofibrils shifted 9.3% of their water into the extramyofibrillar space instead of exchanging K for Na, ED increased to -10.7 mV, and kNa/kK decreased to 0.47.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of potassium-free media and ouabain on epithelial cell composition in toad urinary bladder studied with X-ray microanalysis

Summary The technique of X-ray microanalysis was used to study the composition of toad urinary bladder epithelial cells incubated in Na Ringer's and K-free medium, with and without ouabain. Following incubation under short-circuit conditions, portions of tissue were coated with an external albumin standard and plunge-frozen. Cryosections were freeze-dried and analyzed. In Na Ringer's, granular and basal cells, and also the basal portion of the goblet cells, had similar water and ion compositions. In contrast, mitochondria-rich cells contained less Cl and Na. On average, the granular cells and a subpopulation of the basal cells lost K and gained Na after ouabain and in K-free medium alone. However, there was considerable variation from cell to cell in the responses, indicating differences between cells in the availabilities of ion pathways, either as a consequence of differences in the numbers of such pathways or in their control. In contrast, the compositions of both the low Cl, mitochondria-rich cells and a sub-population of the basal cells were little affected by the different incubation conditions. This is consistent with a comparatively low Na permeability of these cells. The results also indicate that (i) much, if not all, of the K in the dominant cell type, the granular cells, is potentially exchangeable with serosal medium Na, and (ii) Na is accumulated from the serosal medium under K-free conditions. They also provide information about the role of the (Na–K)-ATPase in the maintenance of cellular K in K-free medium, being consistent with other evidence that removal of serosal medium K inhibits transepithelial Na transport by decreasing Na entry to the cells from the mucosal medium, rather than solely by inhibiting the basolateral membrane (Na–K)-ATPase.     

Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism

Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.     

Electron microprobe analysis of frog skin epithelium: Evidence for a syncytial sodium transport compartment

Summary For elucidation of the functional organization of frog skin epithelium with regard to transepithelial Na transport, electrolyte concentrations in individual epithelial cells were determined by electron microprobe analysis. The measurements were performed on 1-m thick freeze-dried cryosections by an energy-dispersive X-ray detecting system. Quantification of the electrolyte concentrations was achieved by comparing the X-ray intensities obtained in the cells with those of an internal albumin standard.The granular, spiny, and germinal cells, which constitute the various layers of the epithelium, showed an identical behavior of their Na and K concentrations under all experimental conditions. In the control, both sides of the skin bathed in frog Ringer's solution, the mean cellular concentrations (in mmole/kg wet wt) were 9 for Na and 118 for K. Almost no change in the cellular Na occurred when the inside bathing solution was replaced by a Na-free isotonic Ringer's solution, whereas replacing the outside solution by distilled water resulted in a decrease of Na to almost zero in all layers. Inhibition of the transepithelial Na transport by ouabain (10^–4 m) produced an increase in Na to 109 and a decrease in K to 16. The effect of ouabain on the cellular Na and K concentrations was completely cancelled when the Na influx from the outside was prevented, either by removing Na or adding amiloride (10^–4 m). When, after the action of ouabain, Na was removed from the outside bathing solution, the Na and K concentration in all layers returned to control values. The latter effect could be abolished by amiloride.The other cell types of the epithelium showed under some experimental conditions a different behavior. In the cornified cells and the light cells, which occurred occasionally in the stratum granulosum, the electrolyte concentrations approximated those of the outer bathing meium under all experimental conditions. In the mitochondria-rich cells, the Na influx after ouabain could not be, prevented by adding amiloride. In the gland cells, only a small change in the Na and K concentrations could be detected after ouabain.The results of the present study are consistent with a two-barrier concept of transepithelial Na transport. The Na transport compartment comprises all living epithelial layers. Therefore, with the exception of some epithelial cell types, the frog skin epithelium can be regarded as a functional syncytium for Na.     

The influence of cellular amino acids and the Na+ : K+ pump on the membrane potential of the Ehrlich ascites tumor cell.

The membrane potential of the Ehrlich ascites tumor cell was shown to be influenced by its amino acid content and the activity of the Na+ :K+ pump. The membrane potential (monitored by the fluorescent dye, 3,3'-dipropylthiodicarbocyanine iodide) varied with the size of the endogenous amino acid pool and with the concentration of accumulated 2-aminoisobutyrate. When cellular amino acid content was high, the cells were hyperpolarized; as the pool declined in size, the cells were depolarized. The hyperpolarization seen with cellular amino acid required cellular Na+ but not cellular ATP. Na+ efflux was more rapid from cells containing 2-aminoisobutyrate than from cells low in internal amino acids. These observations indicate that the hyperpolarization recorded in cells with high cellular amino acid content resulted from the electrogenic co-efflux of Na+ and amino acids. Cellular ATP levels were found to decline rapidly in the presence of the dye and hence the influence of the pump was seen only if glucose was added to the cells. When the cells contained normal Na+ (approx. 30mM), the Na+ :K+ pump was shown to have little effect on the membrane potential (the addition of ouabain had little effect on the potential). When cellular Na+ was raised to 60mM, the activity of the pump changed the membrane potential from the range -25 to -30 mV to -44 to -63 mV. This hyperpolarization required external K+ and was inhibited by ouabain.     

Cellular permeation pathways in a leaky epithelium: the human amniochorion

The presence of cellular permeation pathways in human fetal membranes at term was evaluated. Electrical parameters (transepithelial potential [TEP], and conductance [Gt], and intracellular potentials [cell PD]), and water and urea diffusional coefficients (Pdw, Pdu), were determined in Ussing-like chambers. In amniochorion, the TEP was practically 0 (0.1 +/- 0.03 mV), and the Gt very high (144 +/- 14 mS/cm2). The Cell PD of amnion cells was -37 +/- 3 mV. Increasing the [K+] of the amniotic perfusate between 5.8 and 125.8 mM depolarized the cells with a slope of 23 mV. The deletion of Na+ hyperpolarized the cells, whereas amiloride and ouabain depolarized them. The Pdw and Pdu were determined in intact amnion and chorion and in their epithelial cell layers. The Pdw/Pdu ratio in amnion was 4.0, and 7.0 in its cell layer; the ratio in chorion was 2.5, and 3.3 in its cell layer. The amniochorion is a leaky structure, but its cellular layers possess definite transcellular permeation pathways. The ionic conductances in amnion cells are complex, with the Cell PD being determined by at least K+ and Na+ conductances, and ouabain- and amiloride-sensitive pathways. The amnion is a more effective diffusional barrier to water and urea than chorion is; its diffusional characteristics are comparable to those of nystatin-treated lecithin: cholesterol bilayers and the membranes of human erythrocytes.     

Ion transport by mitochondria-rich cells in toad skin

Summary The optical sectioning video imaging technique was used for measurements of the volume of mitochondria-rich (m.r.) cells of the isolated epithelium of toad skin. Under short-circuit conditions, cell volume decreased by about 14% in response to bilateral exposure to Cl-free (gluconate substitution) solutions, apical exposure to ouabain resulted in a large increase in volume, which could be prevented either by the simultaneous application of amiloride in the apical solution or by the exposure of the epithelium to bilateral Cl-free solutions. Unilateral exposure to a Cl-free solution did not prevent ouabain-induced cell swelling. It is concluded that m.r. cells have an amiloride-blockable Na conductance in the apical membrane, a ouabain-sensitive Na pump in the basolateral membrane, and a passive Cl permeability in both membranes. From the initial rate of ouabain-induced cell volume increase the active Na current carried by a single m.r. cell was estimated to be 9.9±1.3 pA. Voltage clamping of the preparation in the physiological range of potentials (0 to –100 mV, serosa grounded) resulted in a cell volume increase with a time course similar to that of the stimulation of the voltage-dependent activation were prevented by exposure of the tissue to a Cl-free apical solution. The steady-state volume of the m.r. cells increased with the clamping voltage, and at –100 mV the volume was about 1.15 times that under short-circuit conditions. The rate of volume increase during current passage was significantly decreased by lowering the serosal K concentration (K _i ) to 0.5mm, but was independent of whether K _i was 2.4, 5, or 10mm. This indicates that the K conductance of the serosal membrane becomes rate limiting for the uptake of KCl when K _i is significantly lower than its physiological value. It is concluded that the voltage-activated Cl currents flow through the m.r. cells and that swelling is caused by an uptake of Cl ions from the apical bath and K ions from the serosal bath. Bilateral exposure of the tissue to hypo- or hypertonic bathing solutions changed cell volume without detectable changes in the Cl conductance. The volume response to external osmotic perturbations followed that of an osmometer with an osmotically inactive volume of 21%. Using this value and the change in cell volume in response to bilateral Cl-free solutions, we calculated an intracellular steady-state Cl concentration of 19.8±1.7mm (n=6) of the short-circuited cell.     

Effect of membrane potential on furosemide-inhibitable sodium influxes in human red blood cells

Summary Furosemide-inhibitable Na influx (a measure of Na/K/Cl cotransport) was determined as a function of membrane potential in human red blood cells. The membrane potential was varied from –42 to +118 mV using valinomycin and gradients of K. The furosemide-inhibitable, unidirectional Na influx was independent of membrane potential over the entire range of potentials. The change in flux per mV, 0.443 mol/(liter cells·hr· mV), was not significantly different from zero. The mean flux was 153±16mol/(liter cells·hr) (±sem,n=71). The ouabain and furosemide-resistant influexes of Na and K were also measured as functions of membrane potential using either valinomycin and K or a chloride-free, tartrate flux medium to vary membrane potential. The unidirectional Na influx decreased slightly as the membrane potential was increased from negative potentials to about +10 mV. At higher membrane potentials Na influx rose dramatically with potential. This increase was not reversible and was also observed with K influx.     

Ionic basis of volume regulation in mammalian cells following osmotic shock

Previous studies with mammalian cultured cells have shown that volume regulation in hypotonic medium requires active Na transport. In the present study, determinations of intracellular Na and K content were made in cultured mouse lymphoblasts during the process of swelling and subsequent shrinking (volume regulation) in hypotonic medium. Na and K content were measured in cells in which the shrinking phase was inhibited by the cardiac glycoside, ouabain. In osmotically-shocked cells, an initial permeability increase to K, and not Na, was observed, which allowed K to diffuse out rapidly, down its gradient. Na, meanwhile, rapidly flowed inward with water entry during the swelling process, and was later lost with the same kinetics as the cell shrinkage. This loss of Na was prevented in the presence of ouabain. The results imply that volume regulation is achieved by pumping Na gained during swelling out of the cells, while any K taken up by the pump is rapidly lost through a more permeable membrane. The loss of osmotically active Na, presumably with accompanying anions, allows water to passively diffuse down its osmotic gradient, reducing cell volume subsequent to the initial passive swelling, during which K was rapidly lost.     

Intracellular gradients of ion activities in the epithelial cells of theNecturus gallbladder recorded with ion-selective microelectrodes

In Necturus gallbladder epithelial cells the intracellular electrical potential, as recorded with microelectrodes, varied from -28 mV in the mucosal end to about -50 mV in the serosal end of the transporting cell. The Na+ activity varied concurrently from about 39 mM to between 8 and 19 mM. Thus, within the cell both the recorded electrical and chemical gradients caused Na+ to move towards the serosal end. Serosal addition of ouabain (5 X 10(-4) M) caused the intracellular Na+ activity to attain electrochemical equilibrium within 30 min. However, the intracellular electrical potential gradient was only slowly affected. In cells from animals stored at 5 degrees C, the Cl- activity varied from about 55 mM in the mucosal end to 28 mM in the serosal end, and the K+ activity from 50 mM to between 95 and 131 mM. Both ions were close to electrochemical equilibrium within the cytoplasm but were too concentrated to be in equilibrium with the mucosal solution. Bubbling CO2 through the mucosal solution caused the intracellular gradients to vanish. When Na+ in the bathing solutions was exchanged for K+, the intracellular electrical potential became roughly constant at about -5 mV. The Cl- activity became constant in 65 mM, and the K+ activity became constant at 109 mM, both close to equilibrium with the mucosal solution. The Na+ activity was reduced to about 1 mM. The ratio of cytoplasmic resistivities between cells bathed in K+-rich saline to cells bathed in Na+-rich saline was measured by means of triple-barreled electrodes and compared to the same ratio as assessed from the activity measurements. The two values were equal only if one assumes the mobility of Na+ inside the cell to be less than 1/10 of the mobility of K+ or Cl-. The same conclusion was reached by comparing the intracellular Na+ flux calculated from the gradient of electrochemical potential to that flux assess from the net solute absorption. Animals kept at 15 degrees C had lower intracellular Na+ activities, higher Cl- and K+ activities, and higher rates of absorption than animals stored at 5 degrees C. Finally, the degree to which the intracellularly recorded electrical and chemical potentials could reflect an electrode artefact is discussed.     

Effect of inhibition of Na+/K(+)-ATPase on the vasopressin-induced increase in intracellular Na+ concentration in vascular smooth muscle cells.

The effect of inhibition of Na+/K(+)-ATPase by ouabain on the arginine vasopressin (AVP)-induced increase in intracellular Na+ concentration [( Na+]i) was examined in cultured rat vascular smooth muscle cells (VSMC) by the direct measurement of [Na+]i using a fluorescent indicator dye. AVP at a concentration of 1 x 10(-9) M or higher increased [Na+]i in a dose-dependent manner in cultured rat VSMC. The preincubation of cells with 1 x 10(-4) M ouabain for 1 hr at 37 degrees C did not affect the basal [Na+]i but enhanced the 1 x 10(-6) M AVP-induced increase in [Na+]i. The preincubation was not necessary because similar results were obtained after the simultaneous administration of AVP and ouabain. The treatment with ouabain did not affect the intracellular pH changes induced by AVP. These results therefore indicate that the inhibition of Na+/K(+)-ATPase enhances the AVP-induced increase in [Na+]i by decreasing cellular Na+ efflux in cultured rat VSMC.     

Properties of sodium pumps in internally perfused barnacle muscle fibers

To study the properties of the Na extrusion mechanism, giant muscle fibers from barnacle (Balanus nubilus) were internally perfused with solutions containing tracer 22Na. In fibers perfused with solutions containing adenosine 5'-triphosphate (ATP) and 30 mM Na, the Na efflux into 10 mM K seawater was approximately 25-30 pmol/cm2.s; 70% of this efflux was blocked by 50-100 microM ouabain, and approximately 30% was blocked by removal of external K. The ouabain-sensitive and K-dependent Na effluxes were abolished by depletion of internal ATP and were sigmoid-shaped functions of the internal Na concentration ([Na]i), with half-maxima at [Na]i approximately or equal to 20 mM. These sigmoid functions fit the Hill equation with Hill coefficients of approximately 3.5. Ouabain depolarized ATP-fueled fibers by 1.5-2 mV ([Na]i greater than or equal to 30 mM) but had very little effect on the membrane potential of ATP-depleted fibers; ATP depletion itself caused a 2-2.5- mV depolarization. When fueled fibers were treated with 3,4- diaminopyridine or Ba2+ (to reduce the K conductance and increase membrane resistance), application of ouabain produced a 4-5 mV depolarization. These results indicate that an electrogenic, ATP- dependent Na-K exchange pump is functional in internally perfused fibers; the internal perfusion technique provides a convenient method for performing transport studies that require good intracellular solute control.     

Characterization of ouabain-resistant mutants of a canine kidney cell line, MDCK

Madin-Darby canine kidney (MDCK) cells were mutagenized and variants resistant to 10, 160, and 2000 times the ouabain lethal dose for wild type cells selected. The phenotypes were stable in the absence of selection. The frequencies with which variants were recovered were consistent with genetic alterations being responsible for drug resistance. It was shown that 50% of the (Na+, K+)-ATPase activity present in mutant cells had a higher Kd for ouabain than normal while 50% remained wild type for ouabain binding. Wild type MDCK cells were measured to have 2 X 10(6) ouabain binding sites per cell with a Kd for the drug of 0.6-1.0 X 10(-7) M. The novel (Na+, K+)-ATPase activities in the mutants demonstrated Kd values for ouabain of 10(-5) M, 3 X 10(-4) M, or 3 X 10(-3) M for the different mutant classes tested. The rate of synthesis of the (Na+, K+)-ATPase as well as the total amount of enzyme per unit of cell protein was unaltered in the mutants. Comparison of the alpha subunit of the enzyme, known to contain the ouabain-binding site, by sodium dodecyl sulfate-gel electrophoresis did not reveal any difference in the size of this subunit in mutant versus wild type cells.     

Inhibition of Na+,K+-ATPase by ouabain triggers epithelial cell death independently of inversion of the [Na+]i/[K+]i ratio

Treatment with ouabain led to massive death of principal cells from collecting ducts (C7-MDCK), indicated by cell swelling, loss of mitochondrial function, an irregular pattern of DNA degradation, and insensitivity to pan-caspase inhibitor. Equimolar substitution of extracellular Na(+) by K(+) or choline(+) sharply attenuated the effect of ouabain on intracellular Na(+) and K(+) content but did not protect the cells from death in the presence of ouabain. In contrast to ouabain, inhibition of the Na(+)/K(+) pump in K(+)-free medium increased Na(+)(i) content but did not affect cell survival. In control and K(+)-free medium, ouabain triggered half-maximal cell death at concentrations of approximately 0.5 and 0.05 microM, respectively, which was consistent with elevation of Na(+)/K(+) pump sensitivity to ouabain in K(+)-depleted medium. Our results show for the first time that the death of ouabain-treated renal epithelial cells is independent of the inhibition of Na(+)/K(+) pump-mediated ion fluxes and the [Na(+)](i)]/[K(+)](i) ratio.     

Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system.

Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.     

Electron probe X-ray microanalysis of cellular ions in the eccrine secretory coil cells during methacholine stimulation

Summary Intracellular concentrations of Na, K, Cl ([Na], [K] and [Cl], respectively) and other elements were determined in isolated monkey eccrine sweat secretory coil cells using quantitative electron probe X-ray microanalysis of freeze dried cryosections. The validity of the methodology was partially supported by qualitative agreement of the X-ray microanalysis data with those obtained by micro-titration with a helium glow spectrophotometer. [Na], [K] and [Cl] of the cytoplasm were the same as those in the nucleus in both clear and dark cells. [Na], [K], and [Cl] of the clear cells were also the same as those of the dark cells at rest and after stimulation with methacholine (MCh), suggesting that these two cell types behave like a functional syncytium. MCh stimulation induced a pharmacologically specific, dose-dependent decrease in [K] and [Cl] (as much as 65%), and a 3.7-fold increase in [Na]. In myoepithelial cells, a similar change in [Na] and [K] was noted after MCh stimulation although the decrease in [Cl] was only 20%. The MCh-induced change in [Na], [K] and [Cl] was almost completely inhibited by removal of Ca²⁺ from the medium. 10^–4 m bumetanide inhibited the MCh-induced increase in [Na], reduced the decrease in [K] by about 50%, but slightly augmented the MCh-induced decrease in [Cl]. 10^–4 m ouabain increased [Na] and decreased [K] as did MCh; however, unlike MCh, ouabain increased [Cl] by 56% after 30 min of incubation. Thus the data may be best interpreted to indicate that Ca-dependent K efflux and (perhaps also Ca-dependent) Cl efflux are the predominat initial ionic movement in muscarinic cholinergic stimulation of the eccrine sweat secretory coils and that the ouabain-sensitive Na pump plays an important role in maintenance of intracellular ions and sweat secretion.     

Na+, K+ and Cl- transport in isolated small intestinal cells from guinea pig. Evidences for the existence of a second Na+ pump

Isolated small intestinal epithelial cells, after incubation at 4 degrees C for 30 min, reach ion concentrations (36 mM K+, 113 mM Na+ and 110 mM Cl-) very similar to those of the incubation medium. Upon rewarming to 37 degrees C, cells are able to extrude Na+, Cl- and water and to gain K+. Na+ extrusion is performed by two active mechanisms. The first mechanism, transporting Na+ by exchanging it for K+, is inhibited by ouabain and is insensitive to ethacrynic acid. It is the classical Na+ pump. The second mechanism transports Na+ with Cl- and water, is insensitive to ouabain but is inhibited by ethacrynic acid. Both mechanisms are inhibited by dinitrophenol and anoxia. The second Na+ extruding mechanism could be the Na+/K+/2Cl- cotransport system. However, this possibility can be ruled out because the force driving cotransport would work inwards, and because Na+ extrusion with water loss continues after substitution of Cl- by NO3-. We propose that enterocytes have a second Na+ pump, similar to that proposed in proximal tubular cells.