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.     

Whole cell K and Cl currents in dissociated eccrine secretory coil cells during stimulation

Using the whole-cell voltage clamp (to determine the membrane current) and current clamp (to determine membrane potential) methods in conjunction with the nystatin-perforation technique, we studied the effect of methacholine (MCh) and other secretagogues on whole cell K and Cl currents in dissociated rhesus palm eccrine sweat clear cells. Application of MCh by local superfusion induced a net outward current (at a holding potential of ?60 mV and a clamp voltage of 0 mV), and a transient hyperpolarization by 5.6 mV, suggesting the stimulation of K currents. The net outward current gradually changed to the inward (presumably Cl) currents over the next 1 to 2 min of continuous MCh stimulation. During this time the membrane potential also changed from hyperpolarization to depolarization. The inward currents were increasingly more activated than outward (presumably K) currents during repeated MCh stimulations so that a net inward current (at ?60 mV) was observed after the fourth or fifth MCh stimulation. Ionomycin (10 μm) also activated both inward and outward current. The observed effect of MCh was abolished by reducing extracellular [Ca] to below 1 nm (Ca-free + 1 mm EGTA in the bath). MCh-activated outward currents were inhibited by 5 mm Ba and by 0.1 mm quinidine, although these agents also suppressed the inward currents. Bi-ionic potential measurements indicated that the contribution of Na to the membrane potential was negligible both before and after MCh or ISO (isoproterenol) stimulations and that the observed membrane current was carried mainly by K and Cl. MCh increased the bi-ionic potential by step changes in external K and Cl concentrations, further supporting that MCh-induced outward and inward currents represent K and Cl currents, respectively. Stimulation with ISO or FK (forskolin) resulted in a depolarization by about 55 mV and a net inward (most likely Cl) current independent of external Ca. CT-cAMP mimicked the effects of FK and ISO. The bi-ionic potential, produced by step changes in the external Cl concentration, increased during ISO stimulation, whereas that of K decreased. This indicates that the ISO-induced inward current is due to Cl current and that K currents were unchanged or slightly decreased during stimulation with ISO or 10 μm FK. Both myoepithelial and dark cells responded only to MCh (but not to FK) with a marked depolarization of the membrane potential due to activation of Cl, but not K, currents. We conclude that MCh stimulates Ca-dependent K and Cl currents, whereas ISO stimulates cAMP-dependent Cl currents in eccrine clear cells.     

An investigation into the effects of stimulators of fluid production on Locusta Malpighian tubule intracellular elemental composition

The intracellular elemental concentrations of Na, K, P, S, Cl and Mg in the type 1 cells of Malpighian tubules of Locusta migratoria L. have been measured using electron probe X-ray microanalysis. The effects of in vitro stimulation with 1 mM cAMP and corpora cardiaca extract (CC-extract) on the elemental concentrations have been quantified. The distribution of elements, particularly Na, K and Cl is not homogeneous in control cells, and concentration gradients exist within the cytoplasm. Dibutyryl-cAMP (DB-cAMP) caused a decrease in [K]_i without disrupting the gradient which increased from the basal to the apical surface, the apical [Na]_i was increased as was the [Cl]_i. In contrast, in vitro application of CC-extract did not cause changes to the intracellular elemental composition as compared with control cells These data are consistent with the interpretation that exogenous cAMP only partially activated the full stimulatory response of Malpighian tubule cells observed with CC-extract. The changes observed in the density and elemental composition of the `dark bodies' in response to DB-cAMP and CC-extract stimulation suggest that these structures have a role in the ionic economy of Malpighian tubule cells. Accepted: 6 April 1999     

The effects of salt depletion on blood and tissue ion concentrations in the freshwater mussel,Ligumia subrostrata (Say)

Summary The extracellular and intracellular fluid volumes of pondwater acclimatedLigumia subrostrata are equal (3.9 ml/g dry tissue). Total blood solute is 47 mOsm and is composed primarily of Na (19.1 mM), Cl (10.6 mM), HCO₃ (12.7 mM), Ca (4.3 mM), and K (0.5 mM). Major intracellular solutes are K (14.0 mM), Na (7.0 mM) and Cl (2.4 mM).L. subrostrata continuously exposed to deionized water at 20°C exhibit a maximum decrease of 23% in extracellular fluid total solute within 30 days. The maximum [Na] and [Cl] losses are 40% and 76% respectively, while [Ca] and [HCO₃] increase by 44% and 37% respectively. No apparent change in extracellular [K] occurs. Intracellular [Na] decreases 53% and [Cl] decreases 79%, but [K] declines only 15%. Intracellular fluid volume, extracellular fluid volume, and total body water decrease 17%, 31%, and 22% respectively. Inulin clearance is 0.41 ml/g dry tissue·h for pondwater acclimated mussels and declines to 0.24 ml/g dry tissue·h during salt depletion. When salt depleted mussels are returned to solutions containing Na or Cl, they experience a net uptake of salt. The accumulated ions are about equally distributed in the extra- and intracellular compartments.     

Cat Heart Muscle In Vitro : IV. Inhibition of transport in quiescent muscles

Cellular concentrations, [K]_i, [Na]_i, and [Cl]_i, and cell water contents were measured in vitro at 27°C in cat papillary muscles. Measurements were made with and without ouabain at varying concentrations of K and ouabain, at pH 5.2 and 9.0, in absence of O₂, and in NaCl-free solution. Large losses of cell K and increases of cell Na occurred in presence of ouabain, at 2–3°C, and in K-free medium. The dependence of inhibition of cation transport by ouabain on external K concentration, studied at constant initial [K]_i, was consistent with a competition between K and ouabain localized to the external face of the membrane. In NaCl-free sucrose solution [K]_i remained at its physiological value and was not affected by exposure to ouabain or low temperature, except when Ca was also omitted. Ouabain inhibition persisted at pH 9.0 and in Ca-poor media. Cells swelled and lost K at pH 5.2, and residual ouabain effect was small. At pH 9.0, or in absence of O₂, or in Ca-poor solutions cells became permeable to mannitol. The ion movements observed after inhibition of active transport are compatible either with a passive K distribution and a primary inhibition of Na extrusion or with inhibition of a coupled active transport of both K and Na.     

The Role of Calcium in the Regulation of the Steady-State Levels of Sodium and Potassium in the HeLa Cell

Studies on HeLa cells in spinner culture at pH 7.0 and 37° have shown that [Na]_i decreased and [K]_i increased with increasing [Ca]_o. In Na-free (choline) medium [K]_i remained high whether or not Ca was present in the medium. [Na]_i and [K]_i approached a new steady state within 1 min after transfer to Ca-free medium and returned to the initial values within 15 min upon readdition of Ca. 40% of the cell Ca exchanged within 1 min followed by a slow exchange of the remaining Ca over several hours. [Ca]_i increased with decreasing [Na]_o but was independent of [K]_o. Equimolar Mg did not substitute for Ca in maintaining low [Na]_i and high [K]_i. Under steady-state conditions about 50% of the cell Na exchanged in accordance with a single rate constant. The initial Na influx was 270, 100, and 2.5 µM/liter of cell water/sec for 0, 0.10, and 1.0 mM [Ca]_o, respectively. When Na transport was inhibited with strophanthidin and [Na]_i and [K]_i allowed to reach a steady state, Na influx was more rapid for cells incubated in Ca-free medium than for cells incubated in medium containing 1.0 mM Ca. These results suggest that Ca competes with Na at the cell membrane and thus controls the passive diffusion of Na into the cell.     

Changes in intracellular electrolyte concentrations during apoptosis induced by UV irradiation of human myeloblastic cells

Decreases in the intracellular concentrations of both K⁺ and Cl^– have been implicated in playing a major role in the progression of apoptosis, but little is known about the temporal relationship between decreases in electrolyte concentration and the key events in apoptosis, and there is no information about how such decreases affect different intracellular compartments. Electron probe X-ray microanalysis was used to determine changes in element concentrations (Na, P, Cl, and K) in nucleus, cytoplasm, and mitochondria in U937 cells undergoing UV-induced apoptosis. In all compartments, the initial stages of apoptosis were characterized by decreases in [K] and [Cl]. The largest decreases in these elements were in the mitochondria and occurred before the release of cytochrome c. Initial decreases in [K] and [Cl] also preceded apoptotic changes in the nucleus. In the later stages of apoptosis, the [K] continued to decrease, whereas that of Cl began to increase toward control levels and was accompanied by an increase in [Na]. In the nucleus, these increases coincided with poly(ADP-ribose) polymerase cleavage, chromatin condensation, and DNA laddering. The cytoplasm was the compartment least affected and the pattern of change of Cl was similar to those in other compartments, but the decrease in [K] was not significant until after active caspase-3 was detected. Our results support the concept that normotonic cell shrinkage occurs early in apoptosis, and demonstrate that changes in the intracellular concentrations of K and Cl precede apoptotic changes in the cell compartments studied. sodium; potassium; chloride; cell shrinkage     

Intracellular Cl regulates Na-K-Cl cotransport activity in human trabecular meshwork cells

The trabecular meshwork (TM) of the eye plays a central role inmodulating intraocular pressure by regulating aqueous humor outflow,although the mechanisms are largely unknown. We and others have shownpreviously that aqueous humor outflow facility is modulated byconditions that alter TM cell volume. We have also shown that theNa-K-Cl cotransport system is a primary regulator of TM cell volume andthat its activity appears to be coordinated with net efflux pathways tomaintain steady-state volume. However, the cellular mechanisms thatregulate cotransport activity and cell volume in TM cells have yet tobe elucidated. The present study was conducted to investigate thehypothesis that intracellular Cl concentration([Cl]_i) acts toregulate TM cell Na-K-Cl cotransport activity, as has been shownpreviously for some other cell types. We demonstrate here that thehuman TM cell Na-K-Cl cotransporter is highly sensitive to changes in[Cl]_i. Our findingsreveal a marked stimulation of Na-K-Cl cotransport activity, assessedas ouabain-insensitive, bumetanide-sensitive K influx, in TM cells following preincubation of cells with Cl-free medium as a means ofreducing [Cl]_i. Incontrast, preincubation of cells with media containing elevated Kconcentrations as a means of increasing [Cl]_i results ininhibition of Na-K-Cl cotransport activity. The effects of reducing[Cl]_i, as well aselevating [Cl]_i, onNa-K-Cl cotransport activity are concentration dependent. Furthermore, the stimulatory effect of reduced[Cl]_i is additive withcell-shrinkage-induced stimulation of the cotransporter. Our studiesalso show that TM cell Na-K-Cl cotransport activity is altered by avariety of Cl channel modulators, presumably through changes in[Cl]_i. These findingssupport the hypothesis that regulation of Na-K-Cl cotransport activity,and thus cell volume, by[Cl]_i may participatein modulating outflow facility across the TM.

     

Influence of the explantation milieu on intranuclear (Na), (K) and (Mg) of Chironomus thummi salivary gland cells

Intranuclear Na, K and Mg concentrations were determined in cells of salivary glands incubated for 1^h in selected NaCl/KCl/MgCl₂ media. By variation of the external milieu beyond “physiological” limits the intranuclear electrolytes can be shifted between ca 100 and 280 mM [K]ⁱ, between ca 8 and 100 mM [Na]ⁱ and between ca 5 and 75 mM [Mg]ⁱ. No significant competition or interactions of the 3 ionic species are apparent. The relationships [K]^e : [K]ⁱ and [Na]^e : [Na]ⁱ can best be described by a positive and linear, that between [Mg]^e : [Mg]ⁱ by a negative and exponential function. Regression parameters are given which permit a computation of intranuclear [Na], [K] and [Mg] as induced by NaCl/KCl/MgCl₂ in any binary or triple combination that is tolerated by the explanted gland without visible damage.     

Na+/H+ exchange is increased in sickle cell anemia and young normal red cells

Summary Red cell volume regulation is important in sickle cell anemia because the rate and extent of HbS polymerization are strongly dependent on initial hemoglobin concentration. We have demonstrated that volume-sensitive K:Cl cotransport is highly active in SS whole blood and is capable of increasing MCHC. We now report that Na⁺/H⁺ exchange (Na/H EXC), which is capable of decreasing the MCHC of erythrocytes with pH_i<7.2, is also very active in the blood of patients homozygous for HbS. The activity of Na/H EXC (maximum rate) was determined by measuring net Na⁺ influx (mmol/liter cell·hr=FU) driven by an outward H⁺ gradient in oxygenated, acidloaded (pH_i 6.0), DIDS-treated SS cells. The Na/H EXC activity was 33±3 FU (mean±se) (n=19) in AA whites, 37±8 FU (n=8) in AA blacks, and 85±15 FU (n=14) in SS patients (P<0.005). Separation of SS cells into four density-defined fractions by density gradient revealed mean values of Na/H EXC four to five times higher in reticulocytes (SS1), discocytes (SS2) and dense discocytes (SS3), than in the fraction containing irreversibly sickled cells and dense discocytes (SS4). In contrast to K:Cl cotransport, which dramatically decreases after reticulocyte maturation, Na/H EXC persists well after reticulocyte maturation. In density-defined, normal AA red cells, Na/H EXC decreased monotonically as cell density increased. In SS and AA red cells, the magnitude of stimulation of Na/H EXC by cell shrinkage varied from individual to individual. We conclude that Na/H EXC is highly expressed in SS and AA young red cells and decays slowly after reticulocyte maturation.     

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.     

Functional interaction of the K-Cl cotransporter (KCC1) with the Na-K-Cl cotransporter in HEK-293 cells

We have studiedthe regulation of the K-Cl cotransporter KCC1 and its functionalinteraction with the Na-K-Cl cotransporter. K-Cl cotransporter activitywas substantially activated in HEK-293 cells overexpressing KCC1(KCC1-HEK) by hypotonic cell swelling, 50 mM external K, andpretreatment with N-ethylmaleimide(NEM). Bumetanide inhibited ⁸⁶Rbefflux in KCC1-HEK cells after cell swelling [inhibition constant (K_i) ~190µM] and pretreatment with NEM(K_i ~60 µM).Thus regulation of KCC1 is consistent with properties of the red cellK-Cl cotransporter. To investigate functional interactions between K-Cland Na-K-Cl cotransporters, we studied the relationship between Na-K-Clcotransporter activation and intracellular Cl concentration([Cl]_i). Without stimulation, KCC1-HEK cells had greater Na-K-Cl cotransporter activitythan controls. Endogenous Na-K-Cl cotransporter of KCC1-HEK cells wasactivated <2-fold by low-Cl hypotonic prestimulation, compared with10-fold activation in HEK-293 cells and >20-fold activation in cellsoverexpressing the Na-K-Cl cotransporter (NKCC1-HEK). KCC1-HEK cellshad lower resting[Cl]_i than HEK-293cells; cell volume was not different among cell lines. We found a steeprelationship between[Cl]_i and Na-K-Clcotransport activity within the physiological range, supporting aprimary role for [Cl]_iin activation of Na-K-Cl cotransport and in apical-basolateral crosstalk in ion-transporting epithelia.     

Effects of external sodium and cell membrane potential on intracellular chloride activity in gallbladder epithelium

Summary Conventional and Cl-selective liquid ion-exchanger intracellular microelectrodes were employed to study the effects of extracellular ionic substitutions on intracellular Cl activity (aCl _i ) inNecturus gallbladder epithelium. As shown previously (Reuss, L., Weinman, S.A., 1979;J. Membrane Biol. 49:345), when the tissue was exposed to NaCl-Ringer on both sidesaCl _i was about 30mm, i.e., much higher than the activity predicted from equilibrium distribution (aCl_eq) across either membrane (5–9mm). Removal of Cl from the apical side caused a reversible decrease ofaCl _i towards the equilibrium value across the basolateral membrane. A new steady-stateaCl _i was reached in about 10 min. Removal of Na from the mucosal medium or from both media also caused reversible decreases ofaCl _i when Li, choline, tetramethylammonium or N-methyl-d-glucamine (NMDG) were employed to replace Na. During bilateral Na substitutions with choline the cells depolarized significantly. However, no change of cell potential was observed when NMDG was employed as Na substitute. Na replacements with choline or NMDG on the serosal side only did not changeaCl _i . When K substituted for mucosal Na, the cells depolarized andaCl _i rose significantly. Combinations of K for Na and Cl for SO₄ substitutions showed that net Cl entry during cell depolarization can take place across either membrane. The increase ofaCl _i in depolarized cells exposed to K₂SO₄-Ringer on the mucosal side indicates that the basolateral membrane Cl permeability, (P _Cl) increased. These results support the hypothesis that NaCl entry at the apical membrane occurs by an electroneutral mechanism, driven by the Na electrochemical gradient. In addition, we suggest that Cl entry during cell depolarization is downhill and involves an increase of basolateral membraneP _Cl.     

Interaction of (Na + K + 2 Cl) cotransport and the Na/K pump in cultured chick cardiac myocytes

We have recently reported the presence of an electroneutral (Na + K + 2 Cl) cotransport mechanism that is bumetanide-sensitive and maintains Cl_i above its electrochemical equilibrium in cultured chick heart cells. In steady state, (Na + K + 2 Cl) cotransport is inwardly directed and so contributes to the Na influx that must be counterbalanced by the activity of the Na/K pump to maintain Na_i homeostasis. We now show that manipulating (Na + K + 2 Cl) cotransport by restoring Cl_o to a Cl-free solution indirectly influences Na/K pump activity because the bumetanide-sensitive recovery of a _infNa ^supi to its control level and the accompanying hyperpolarization could be blocked by 10^–4M ouabain. In another protocol, when the Na/K pump was reactivated by restoring K_o (from 0.5 mM to 5.4 mM) and removing ouabain, the recovery of a_Na was attenuated by 10^–4M bumetanide. The relatively slow rate of ouabain dissociation coupled with the activation of Na influx by (Na + K + 2 Cl) cotransport clearly establishes the interaction of these transport mechanisms in regulating Na_i. Although (Na + K + 2 Cl) cotransport is electroneutral, secondary consequences of its activity can indirectly affect the electrophysiological properties of cardiac cells.     

Anomalous influence of reduced internal ATP levels on sodium efflux inMyxicola giant axons

Summary Giant axons from the marine annelid,Myxicola infundibulum, were internally dialyzed with ATP-free media and with media with lower than normal ATP levels in an attempt to determine quantitatively the ATP requirement of the Na pump in these cells. This was accomplished by using²²Na ions to measure Na efflux. When [ATP] _i in dialysis fluid fell to values within the range of 20–40 m, a marked stimulation of Na efflux was observed even though an essentially normal ouabain sensitivity of Na efflux persisted; when axons were dialyzed with ATP-free solutions with ouabain present in the external medium throughout the dialysis period, the stimulation of Na efflux still occurred. The stimulation of Na efflux produced by low [ATP] _i levels could be reversed by reintroducing normal ATP levels into the dialysis medium. Reversibility was complete provided axons were not depleted of ATP for periods longer than about 1 hr. Longer periods of ATP depletion led to larger and ultimately irreversible increases in Na efflux. The increases in Na efflux occasioned by ATP depletion either prevented or obscured the decrease in Na efflux expected to occur from unfueling the Na pump. Since [ATP] _i levels required to significantly unfuel the Na pump lie below the levels at which the Na efflux stimulation occurred, it is problematic to quantitatively assess the influence of [ATP] _i levels on Na pump rate by measurements of Na efflux in this preparation. Substitutes for ATP failed to prevent increases in Na efflux. The large increases in Na efflux observed at low [ATP] _i occurred with no important changes in the resting membrane potential, and also occurred in Na-free and Ca-free external media. At least part of the increased Na efflux under these conditions may be due to a Na/Na exchange component, as a significant dependence of Na efflux on [Na] _o appropriate for this kind of exchange was observed in the ATP-depleted axons. Whether the highly reproducible anomalous effect on Na efflux inMyxicola axons has some fundamental significance in its own right is a matter for future investigation. A few possible explanations of the anomalous effect of reduced ATP levels are discussed.     

Role of Apical H-K Exchange and Basolateral K Channel in the Regulation of Intracellular pH in Rat Distal Colon Crypt Cells

An apical membrane ouabain-sensitive H-K exchange and a barium-sensitive basolateral membrane potassium channel are present in colonic crypt cells and may play a role in both K absorption and intracellular pH (pH_i) regulation. To examine the possible interrelationship between apical membrane H-K exchange and basolateral membrane K movement in rat distal colon in the regulation of pH_i, experiments were designed to assess whether changes in extracellular potassium can alter pH_i. pH_i in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF-AM (2′,7′-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester). After loading with the dye, crypts were superfused with a Na-free solution which resulted in a rapid and reversible fall in pH_i (7.36 ± 0.02 to 6.98 ± 0.03). Following an increase in extracellular [K] to 20 mm, in the continued absence of Na, there was a further decrease in pH_i (0.20 ± 0.02, P < 0.01). K-induced acidification was blocked both by 2 mm bath barium, a K channel blocker, and by 0.5 mm lumen ouabain. K-induced acidification was also observed when intracellular acidification was induced by a NH₄Cl prepulse. These observations suggest that increased basolateral K movement increases intracellular [K] resulting in a decrease in pH_i that is mediated by a ouabain-sensitive apical membrane H,K-ATPase. Our results demonstrate an interrelationship between basolateral K movement and apical H-K exchange in the regulation of pH_i and apical K entry in rat distal colon. Received: 31 March 1998/Revised: 8 September 1998     

Potassium fluxes in dialyzed squid axons

Measurements have been made of K influx in squid giant axons under internal solute control by dialysis. With [ATP]_i = 1 µM, [Na]_i = 0, K influx was 6 ± 0.6 pmole/cm² sec; an increase to [ATP]_i = 4 mM gave an influx of 8 ± 0.5 pmole/cm² sec, while [ATP]_i 4, [Na]_i 80 gave a K influx of 19 ± 0.7 pmole/cm² sec (all measurements at ∼16°C). Strophanthidin (10 µM) in seawater quantitatively abolished the ATP-dependent increase in K influx. The concentration dependence of ATP-dependent K influx on [ATP]_i, [Na]_i, and [K]_o was measured; an [ATP]_i of 30 µM gave a K influx about half that at physiological concentrations (2–3 mM). About 7 mM [Na]_i yielded half the K influx found at 80 mM [Na]_i. The ATP-dependent K influx responded linearly to [K]_o from 1–20 mM and was independent of whether Na, Li, or choline was the principal cation of seawater. Substances tested as possible energy sources for the K pump were acetyl phosphate, phosphoarginine, PEP, and d-ATP. None was effective except d-ATP and this substance gave 70% of the maximal flux only when phosphoarginine or PEP was also present.     

Na/K Pump and Intracellular Monovalent Cations: Novel Mechanism of Excitation–Transcription Coupling Involved in Inhibition of Apoptosis

The Na/K pump plays a key role in the regulation of the intracellular concentrations of monovalent cations and related cell function leading to electrogenesis and excitation–contraction coupling. We focus this review on the analysis of recent data showing that (i) inhibition of the Na/K pump triggers a signaling cascade independently of modulation of the intracellular [Na⁺]_i/[K⁺]_i ratio; (ii) elevation of [Na⁺]_i under sustained inhibition of the Na/K pump leads to expression of a set of genes by [Ca²⁺]_i-dependent and independent pathways; (iii) [Na⁺]_i-sensitive genes are involved in the inhibition of programmed cell death (apoptosis) in vascular smooth muscle cells.     

Electrolyte Metabolism in HeLa Cells

Methods have been developed to study cellular Na, K, and Cl concentrations in HeLa cells. Cell [Na] and [K] are functions of the age of the culture. As the culture grows [K], expressed in mmols/liter cell H₂O, rises from an initial value of 121 to a peak of 206 at about 4 days, and thereafter falls until it has almost returned to the initial value by the 9th day. [Na] falls as [K] rises, but there is no fixed relationship between the cellular concentrations of the two cations. There is, however, a correlation between generation time and cellular [K]. Measurements of net K uptake and net Na extrusion were carried out during 1 hour incubation at 37°C of low K cells. Both net K uptake and net Na extrusion took place against chemical concentration gradients, so that at least one transport system must be active; if the Cl distribution is passive both net K uptake and net Na extrusion are active. Studies with inhibitors of respiration and glycolysis lead to the conclusion that respiration is not required for these net transports, which appear to derive their energy from glycolytic sources.     

Intracellular activities during volume regulation byNecturus gallbladder

Summary Necturus gallbladder epithelial cells regulate their volume after a change in solution osmolality. We determined the intracellular activities of Na, K and Cl when the mucosal bathing solution osmolality was increased 18% by the addition of mannitol. The gallbladder was mounted in a rapid flow chamber and punctured simultaneously with two single-barrelled microelectrodes. One electrode sensed membrane potential and the other was sensitive to the activity of Na, K or Cl. Cell volume measurements, made in previous studies utilizing quantitative light microscopy, indicated that hypertonicity of the mucosal bath first caused a cell shrinkage of 15% followed by volume readjustment. Some loss of Na, K and Cl was observed during shrinkage; subsequently during volume regulation, the intracellular quantities of all three ions increased. The loss of Na during the initial cell shrinkage could be blocked by ouabain and was therefore due to increased transport. K and Cl losses were probably related to the increase in their concentrations during shrinkage. The gain of Na, K and Cl during volume regulation was similar in magnitude to the loss of these solutes during cell shrinkage. The increase of Na, K and Cl during volume regulation accounted for about 60% of the increase of cell solutes during this period indicating that other solutes also contributed to the volume regulation response.