Effects of potassium on the anion and cation contents of primary cultures of mouse astrocytes and neurons

Inastrocytes, as [K⁺]_o was increased from 1.2 to 10 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. As [K⁺]_o was increased from 10 to 60 mM, intracellular concentration of these three ions showed no significant change. When [K⁺]_o was increased from 60 to 122 mM, an increase in [K⁺]_i and [Cl^–]_i and a decrease in [Na⁺]_i were observed.Inneurons, as [K⁺]_o was increased from 1.2 to 2.8 mM, [Na⁺]_i and [Cl^–]_i were decreased, whereas [K⁺]_i was increased. As [K⁺]_o was increased from 2.8 to 30 mM, [K⁺]_i, [Na⁺]_i and [Cl^–]_i showed no significant change. When [K⁺]_o was increased from 30 to 122 mM, [K⁺]_i and [Cl^–]_i were increased, whereas [Na⁺]_i was decreased. Inastrocytes, pH_i increased when [K⁺]_o was increased. Inneurons, there was a biphasic change in pH_i. In lower [K⁺]_o (1.2–2.8 mM) pH_i decreased as [K⁺]_o increased, whereas in higher [K⁺]_o (2.8–122 mM) pH_i was directly related to [K⁺]_o. In bothastrocytes andneurons, changes in [K⁺]_o did not affect the extracellular water content, whereas the intracellular water content increased as the [K⁺]_o increased. Transmembrane potential (E_m) as measured with Tl-204 was inversely related to [K⁺]_o between 1.2 and 90 mM, a ten-fold increase in [K⁺]_o depolarized the astrocytes by about 56 mV and the neurons about 52 mV. The E_m values measured with Tl-204 were close to the potassium equilibrium potential (E_k) except those in neurons at lower [K⁺]_o. However, they were not equal to the chloride equilibrium potential (E_Cl) at [K⁺]_o lower than 30 mM in both astrocytes and neurons. Results of this study demonstrate that alteration of [K⁺]_o produced different changes in [K⁺]_i, [Na⁺]_i, [Cl^–]_i, and pH_i in astrocytes and neurons. The data show that astrocytes can adapt to alterations in [K⁺]_o, in such a way to maintain a more suitable environment for neurons.     

Role of external calcium in sodium and chloride transport in the gills of seawater-adaptedMugil capito

Summary When the mulletMugil capito is transferred to medium lacking Ca⁺⁺ (either Ca⁺⁺-free seawater or distilled water) the passive permeability of the gill to Na⁺ and Cl^– is increased and the activating effect of external K⁺ on the Na⁺ and Cl^– effluxes in hyposaline media is inhibited. The permeability of the gill increases progressively in proportion to the time of Ca⁺⁺ deprivation; it declines when Ca⁺⁺ is added again to the external medium. The active mechanisms for ion excretion are not reversible. At external Ca⁺⁺ concentrations from 0.1 to 10 mM the Na⁺ permeability is constant but the activation of Na⁺ efflux by K⁺ shows a maximum at a Ca⁺⁺ concentration of about 1 mM. For activation of Cl^– efflux external bicarbonate must be present, in addition to Ca⁺⁺, suggesting the existence of a Cl^–/HCO ₃ ^– exchange. The mechanism by which Ca⁺⁺ controls the passive branchial permeability is thus probably different from that involved in K⁺ activation of ion excretion. The Ca⁺⁺ effect on the K⁺ sensitive ionic excretory mechanisms seems to be related to intracellular Ca⁺⁺ movements. Thus, on the one hand, substances such as Ruthenium Red and La⁺⁺⁺ which both inhibit Ca⁺⁺ exchange, in media containing Ca⁺⁺ and HCO ₃ ^– also inhibit K⁺ activation of Na⁺ and Cl^– effluxes; on the other hand, the ionophore A 23187, a stimulator of Ca⁺⁺ exchange, when added to these media, activates the Na⁺ and Cl^– effluxes; its maximal effect on the Na⁺ flux occurs at 2 mM Ca⁺⁺.Abbreviations ASW-Ca artificial seawater minus calcium - DW deionised water - DWCa deionised water with 1 mM Ca⁺⁺ added - DWCaHCO ₃ DW with calcium plus bicarbonate - DWHCO ₃ DW with 1 mM sodium bicarbonate added - FW freshwater (tap water) - FWK freshwater with K⁺ added - P. D. potential difference - SW seawater The experiments reported in this paper were done with Jean Maetz who tragically died in August 1977. It is the last report about several years of friendly collaboration     

Relations between intracellular ion activities and extracellular osmolarity inNecturus gallbladder epithelium

Summary The interactions between ion and water fluxes have an important bearing on osmoregulation and transepithelial water transport in epithelial cells. Some of these interactions were investigated using ion-selective microelectrodes in theNecturus gallbladder. The intracellular activities of K⁺ and Cl^– in epithelial cells change when the epithelium is adapted to transport in solutions of a low osmolarity. In order to achieve new steady states at low osmolarities, cells lost K⁺, Cl^– and some unidentified anions. Surprisingly, the apparent K⁺ concentration remained high: at an external osmolartity of 64 mOsm the intracellular K⁺ concentration averaged 95mm. This imbalance was sensitive to anoxia and ouabain. The effects of abrupt changes in the external osmolarities on the intracellular activities of Na⁺, K⁺ and Cl^– were also investigated. The gradients were effectuated by mannitol. The initial relative rates of change of the intracellular activities of Na⁺ and Cl^– were equal. The data were consistent with Na⁺ and Cl^– ions initially remaining inside the cell and a cell membraneL _p of 10^–3 cm sec^–1 osm^–1, which is close to the values determine by Spring and co-workers (K.R. Spring, A. Hope & B.-E. Persson, 1981.In: Water Transport Across Epithelia. Alfred Benzon Symposium 15. pp. 190–200. Munskgaard, Copenhagen). The initial rate of change of the intracellular activity of K⁺ was only 0.1–0.2 times the change observed in Na⁺ and Cl^– activities, and suggests that K⁺ ions leave the cell during the osmotically induced H₂O efflux and enter with an induced H₂O influx. The coupling is between 98 and 102 mmoles liter^–1. Various explanations for the anomalous behavior of intracellular K⁺ ions are considered. A discussion of the apparent coupling between K⁺ and H₂O, observed in nonsteady states, and its effects on the distribution of K⁺ and H₂O across the cell membrane in the steady states, is presented.     

Na+, K+, Cl− cotransport and its regulation in Ehrlich ascites Tumor cells. Ca2+/Calmodulin and protein kinase C dependent pathways

Summary Net Cl^– uptake as well as unidirectional³⁶Cl influx during regulatory volume increase (RVI) require external K⁺. Half-maximal rate of bumetanide-sensitive³⁶Cl uptake is attained at about 3.3mm external K⁺. The bumetanide-sensitive K⁺ influx found during RVI is strongly dependent on both Na⁺ and Cl^–. The bumetanide-sensitive unidirectional Na⁺ influx during RVI is dependent on K⁺ as well as on Cl^–. The cotransporter activated during RVI in Ehrlich cells, therefore, seems to transport Na⁺, K⁺ and Cl^–. In the presence of ouabain and Ba⁺ the stoichiometry of the bumetanide-sensitive net fluxes can be measured at 1.0 Na⁺, 0.8 K⁺, 2.0 Cl^– or approximately 1 : Na, 1 : K, 2 : Cl. Under these circumstances the K⁺ and Cl^– flux ratios (influx/efflux) for the bumetanide-sensitive component were estimated at 1.34 ±0.08 and 1.82 ± 0.15 which should be compared to the gradient for the Na⁺, K⁺, 2Cl^– cotransport system at 1.75 ± 0.24.Addition of sucrose to hypertonicity causes the Ehrlich cells to shrink with no signs of RVI, whereas shrinkage with hypertonic standard medium (all extracellular ion concentrations increased) results in a RVI response towards the original cell volume. Under both conditions a bumetanide-sensitive unidirectional K⁺ influx is activated. During hypotonic conditions a small bumetanide-sensitive K⁺ influx is observed, indicating that the cotransport system is already activated.The cotransport is activated 10–15 fold by bradykinin, an agonist which stimulates phospholipase C resulting in release of internal Ca²⁺ and activation of protein kinase C.The anti-calmodulin drug pimozide inhibits most of the bumetanide-sensitive K⁺ influx during RVI. The cotransporter can be activated by the phorbol ester TPA. These results indicate that the stimulation of the Na⁺, K⁺, Cl^– cotransport involves both Ca²⁺/calmodulin and protein kinase C.     

Effects of an hypo-osmotic shock on Na+, K+ and Cl? levels in isolated axons ofCarcinus maenas

Summary Application of an hypo-osmotic shock to isolated axons ofCarcinus maenas induces a decrease in the intracellular content of K⁺, Na⁺ and Cl^–. The changes in Na⁺ and Cl^– levels are only transitory while the K⁺ level reaches new steady-state value much lower than the control. The modification of K⁺ concentration cannot be ascribed only to a simple dilution process and it is proposed that the regulation of intracellular K⁺ plays an important role in limiting the swelling which occurs in this tissue.Hypo-osmotic conditions also induce an increase in Na⁺ permeability.The results are discussed in relation to changes in ion transport and interactions with intracellular organic compounds that could arise during the process of volume regulation.     

Intracellular compartmentation of Na+, K+ and Cl− in the Ehrlich ascites tumor cell: Correlation with the membrane potential

Summary The intracellular distribution of Na⁺, K⁺, Cl^– and water has been studied in the Ehrlich ascites tumor cell. Comparison of the ion and water contents of whole cells with those of cells exposed to La³⁺ and mechanical stress indicated that La³⁺ treatment results in selective damage to the cell membrane and permits evaluation of cytoplasmic and nuclear ion concentrations. The results show that Na⁺ is sequestered within the nucleus, while K⁺ and Cl^– are more highly concentrated in the cell cytoplasm. Reduction of the [Na⁺] of the incubation medium by replacement with K⁺ results in reduced cytoplasmic [Na⁺], increased [Cl^–] and no change in [K⁺]. Nuclear concentrations of these ions are virtually insensitive to the cation composition of the medium. Concomitant measurements of the membrane potential were made. The potential in control cells was –13.7 mV. Reduction of [Na⁺] in the medium caused significant depolarization. The measured potential is describable by the Cl^– equilibrium potential and can be accounted for in terms of cation distributions and permeabilities. The energetic implications of the intracellular compartmentation of ions are discussed.     

Salt tolerance inNitellopsis obtusa

Summary The mechanism of salt tolerance was studied using isolated internodal cells of the charophyteNitellopsis obtusa grown in fresh water. When 100 mM NaCl was added to artificial pond water (0.1 mM each of NaCl, KC1, CaCl₂), no cell survived for more than one day. Within the first 30 minutes, membrane potential (E_m) depolarized and membrane resistance (R_m) decreased markedly. Simultaneously, cytoplasmic Na⁺ increased and K⁺ decreased greatly. At steady state the increase in Na⁺ content was roughly equal to the decrease in K⁺ content. The Cl^– content of the cytoplasm did not change. These results suggest that Na⁺ enters the cytoplasm by exchange with cytoplasmic K⁺. Both the entry of Na⁺ and the exit of K⁺ are assumed to be passive and the latter being caused by membrane depolarization. Vacuolar K⁺, Na⁺, and Cl^– remained virtually constant, suggesting that rapid influx of Na⁺ from the cytoplasm did not occur.In 100 mM NaCl containing 10 mM CaCl₂, membrane depolarization, membrane resistance decrease and changes in cytoplasmic [Na⁺] and [K⁺] did not occur, and cells survived for many days. When cells treated with 100 mM NaCl were transferred within 1 hour to 100 mM NaCl containing 10 mM CaCl₂, Em decreased, Rm increased, cytoplasmic Na⁺ and K⁺ returned to their initial levels, and cells survived. Two possible mechanisms for the role of Ca²⁺ in salt tolerance inNitellopsis are discussed; one a reduction in plasmalemma permeability to Na⁺ and the other a stimulation of active Na⁺-extrusion.     

Stimulation by HCO 3 − of Na+ transport in rabbit gallbladder

Summary Bicarbonate presence in the bathing media doubles Na⁺ and fluid transepithelial transport and in parallel significantly increases Na⁺ and Cl^– intracellular concentrations and contents, decreases K⁺ cell concentration without changing its amount, and causes a large cell swelling. Na⁺ and Cl^– lumen-to-cell influxes are significantly enhanced, Na⁺ more so than Cl^–. The stimulation does not raise any immediate change in luminal membrane potential and cannot be due to a HCO ₃ ^– -ATPase in the brush border. The stimulation goes together with a large increase in a Na⁺-dependent H⁺ secretion into the lumen. All of these data suggests that HCO ₃ ^– both activates Na⁺–Cl^– cotransport and H⁺–Na⁺ countertransport at the luminal barrier.Thiocyanate inhibits Na⁺ and fluid transepithelial transport without affecting H⁺ secretion and HCO ₃ ^– -dependent Na⁺ influx. It reduces Na⁺ and Cl^– concentrations and contents, increases the same parameters for K⁺, causes a cell shrinking, and abolishes the lumen-to-cell Cl^– influx. It enters the cell and is accumulated in the cytoplasm with a process which is Na⁺-dependent and HCO ₃ ^– -activated. Thus, SCN^– is likely to compete for the Cl^– site on the cotransport carrier and to be slowly transferred by the cotransport system itself.     

Intracellular chloride and potassium ions in relation to excitability ofChara membrane

Summary Internodal cells ofChara australis were made tonoplast-free by replacing the cell sap with EGTA-containing media; then the involvement of internal Cl^– and K⁺ in the excitation of the plasmalemma was studied.[Cl^–] _i was drastically decreased by perfusing the cell interior twice with a medium lacking Cl^–. The lowered [Cl^–] _i was about 0.01mm. Cells with this low [Cl^–] _i generated action potential and showed anN-shapedV–I curve under voltage clamped depolarization like Cl^–-rich cells containing 13 or 29mm Cl^–.E _m at the peak of the action potential was constant at [Cl^–] _i between 0.01 and 29mm. The possibility that the plasmalemma becomes as permeable to other anions as to Cl^– during excitation is discussed.At [Cl^–] _i higher than 48mm, cells were inexcitable. When anions were added to the perfusion medium to bring the K⁺ concentration to 100mm, NO ₃ ^– , F^–, SO ₄ ^2– , acetate, and propionate inhibited the generation of action potentials like Cl^–, while methane sulfonate, PIPES, and phosphate did not inhibit excitability.The duration of the action potential depended strongly on the intracellular K⁺ concentration. It decreased as [K⁺] _i (K-methane sulfonate) increased. Increase in [Na⁺] _i (Na-methane sulfonate) also caused its decrease, although this effect was weaker than that of K⁺. The action of these monovalent cations on the duration of the action potential is the opposite of their action on the membrane from the outside (cf. Shimmen, Kikuyama & Tazawa, 1976,J. Membrane Biol. 30:249).     

Relationship between coupled Na+−K+−Cl− transport and water absorption across the seawater eel intestine

Summary Simultaneous measurements of net ion and water fluxes were made in the stripped intestine of the seawater eel, and the relationship between Na⁺, K⁺, Cl^– and water transport were examined in the presence of mucosal KCl and serosal NaCl Ringer (standard condition). When Cl^– was removed from both sides of the intestine, net K⁺ flux from mucosa to serosa was reduced, accompanied by complete blockage of water absorption. Since it has been shown that net Cl^– and water fluxes depend on K⁺ transport under the standard condition (Ando 1983), the interdependence of K⁺ and Cl^– transport suggests the existence of a coupled KCl transport system, while the parallelism between the net Cl^– and water fluxes suggests that water absorption is linked to the coupled KCl transport. The coupled KCl and water transport were inhibited by treatment with ouabain or with Na⁺-free Ringer solutions, suggesting the existence of a Na⁺-dependent KCl transport system and linkage of water absorption to the coupled Na⁺–K⁺–Cl^– transport. Since ouabain blocked the active Na⁺–K⁺–Cl^– transport almost completely, the permeability coefficients for K⁺ and Na⁺ through the paracellular shunt pathway were estimated as P_K=0.076 and P_Na=0.058 cm/h, and P_Cl was calculated as 0.005 cm/h. Although Na⁺-independent K⁺ and Cl^tt- fluxes were observed again in the present study, these fluxes were not inhibited by CN^–, ouabain or diuretics, and evoked even after blocking the Na⁺–K⁺–Cl^– transport completely with ouabain. These results indicate that the Na⁺-independent K⁺ and Cl^– fluxes are distinct from the active Na⁺–K⁺–Cl^– transport and are not themselves active.     

Inability of Ehrlich ascites tumor cells to volume regulate following a hyperosmotic challenge

Summary Ehrlich cells shrink when the osmolality of the suspending medium is increased and behave, at least initially, as osmometers. Subsequent behavior depends on the nature of the hyperosmotic solute but in no case did the cells exhibit regulatory volume increase. With hyperosmotic NaCl an osmometric response was found and the resultant volume maintained relatively constant. Continuous shrinkage was observed, however, with sucrose-induced hyperosmolality. In both cases increasing osmolality from 300 to 500 mOsm initiated significant changes in cellular electrolyte content, as well as intracellular pH. This was brought about by activation of the Na⁺/H⁺ exchanger, the Na/K pump, the Na⁺+K⁺+2Cl^– cotransporter and by loss of K⁺ via a Ba-sensitive pathway. The cotransporter in response to elevated [Cl^–] _i (100mm) and/or the increase in the outwardly directed gradient of chemical potential for Na⁺, K⁺ and Cl^–, mediated net loss of ions which accounted for cell shrinkage in the sucrose-containing medium. In hyperosmotic NaCl, however, the net Cl^– flux was almost zero suggesting minimal net cotransport activity.We conclude that volume stability following cell shrinkage depends on the transmembrane gradient of chemical potential for [Na⁺+K⁺+Cl^–], as well as the ratio of intra- to extracellular [Cl^–]. Both factors appear to influence the activity of the cotransport pathway.     

On the interrelations of the Na+-and Cl?-influxes in the pulmonate freshwater snailLymnaea stagnalis

Summary In the freshwater snailLymnaea stagnalis the influxes of Na⁺ and Cl^– were studied at different external concentrations of these ions. The characteristies of the Na⁺- and Cl^–-influxes are similar with respect to saturation kinetics,K _m (0.1 mM) and activation by low-salt adaptation. In short-term experiments the Na⁺- and Cl^–-influxes are independent. Because of the counter-ions (H⁺ and HCO ₃ ^– ) involved, this indicates a potential acid-base regulatory capacity. Low-salt adaptation, due to either Na⁺-or Cl^–-depletion, activates both the Na⁺- and the Cl^–-influx. It is suggested that under both conditions the number of active integumental pumps, involved in Na⁺- as well as in Cl^–-uptake, is increased.     

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more¹²⁵I^– than³⁶Cl^– from the medium. The average cell/medium ratio of¹²⁵I^– of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of³⁶Cl^– of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both¹²⁵I^– and³⁶Cl^– calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting¹²⁵I^– uptake whereas furosemide is more effective in inhibiting³⁶Cl^– uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na⁺]_o but was not significantly affected by changes of [Cl^–]_o or [HCO ₃ ^– ]_o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl^–]_o and [HCO ₃ ^– ]_o and was not affected [Na⁺]_o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na⁺]_o between 60 and 140 mM and inversely related to [HCO ₃ ^– ]_o between 10 and 40 mM, but it was not affected by [Cl^–]_o. Radiochloride uptake by neurons was directly related to [Cl^–]_o and to [Na⁺]_o between 60 and 140 mM and was not affected by [HCO ₃ ^– ]_o. However, in sodium-free medium both¹²⁵I^– and³⁶Cl^– uptakes into neurons were higher than those in [Na⁺]_o between 5 and 60 mM. These results indicate that uptake of¹²⁵I^– and³⁶Cl^– into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.Special issue dedicated to Dr. Paola S. Timiras     

Growth characteristic and ion contents of rice callus under the influence of NaCl and hydroxyproline

Calli of salt tolerant (Bhoora rata) and salt susceptible (GR₁₁) rice varieties were cultured on Linsmaeir and Skoog’s medium containing LD₅₀ concentration of NaCl (200 mM) and hydroxyproline (10 mM). Growth rate of callus and Na⁺, K⁺, Cl⁻, Mg⁺², and Ca⁺² contents of the cultured rice tissues were determined at the end of 0, 2, 4 and 6 weeks of incubation. Hydroxyproline resistant calli of both rice varieties when cultured on Linsmaeir and Skoog’s medium containing both NaCl and hydroxyproline showed increased dry weight and enhanced intracellular levels of K⁺, Mg⁺² and Ca⁺². The accumulation of Na⁺ and Cl⁻ ions was less in the hydroxyproline resistant calli.     

Response of sugarcane to different types of salt stress

Summary Due to climatic conditions and prevailing water regime the yield and sucrose recovery in sugarcane are high in South Western India. However, excessive irrigation, poor drainage and luxuriant use of fertilizers have resulted in conversion of large fertile areas into saline lands. The salinity is due to the excess of Na⁺, Ca⁺⁺, Mg⁺⁺, SO₄ ^– and Cl^– ions. Individual salts of NaCl, Na₂SO₄, MgCl₂ and MgSO₄ were employed in culture experiments to study salt stress effect on sugarcane variety Co 740. It was observed that sulphate salinity was more toxic to sugarcane than the chloride one. Sulphate salts caused more inhibition of growth, chlorophyll synthesis, PEPCase activity, decreased the uptake of K⁺ and Ca⁺⁺ ions but stimulated nitrate reductase. The stress did not result in proline accumulation in the sugarcane cultivar Co 740. The degree of toxicity of different ions in decreasing order in sugarcane cultivar Co 740 is SO₄ ^–>Na⁺>Cl^–>Mg⁺⁺.     

Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): Effects of K+, Na+ and Mg2+

Summary Patch-clamp techniques were used to study the permeability to ions of an ATP-sensitive channel in membranes from the pancreatic B-cell line (RINm5F). With patches in the outside-out configuration, theI-V curves for different Na⁺–K⁺ mixtures in the bath and 140 mM K⁺ in the pipette were almost linear, and crossed the zero-current axis at voltages that indicated a variable permeability ratio. When K⁺ was added symmetrically, the plot of the conductancevs. K⁺ activity exhibited saturation, with aG _max of about 160 pS and a half-maximal activity of 216 mM. TheI-V behavior for different K⁺–Na⁺ mixtures in the bath could be accurately described with a model based on Eyring theory, assuming two sites and one-ion occupancy. For K⁺, the dissociation constants (K_K) of the two sites were 290 and 850 mM, the lower value pertaining to the site close to the intracellular medium. In experiments with inside-out patches, both Na⁺ and Mg²⁺, when present in the bath, induced a voltagedependent block of the outward current. Fitting the data with the model suggested that for these ions only one of the two sites binds significantly, the corresponding dissociation constants being (mM): 46 for Na⁺ and 34 for Mg²⁺. Blocking by Na⁺ and Mg²⁺ may account for the low outward current seen in intact cells. This hypothesis is consistent with the observation that such current is further reduced by addition of 2,4-DNP, since metabolism inhibitors are expected to lower the ATP level, thereby liberating Mg²⁺ from the Mg²⁺-ATP complex, as well as inducing accumulation of Na⁺ by decreasing the rate of the Na⁺–K⁺ pump.     

Sidium-dependent ion cotransport in steady-state Ehrlich ascites tumor cells

Summary The Ehrlich tumor cell possesses and anion-cation cotransport system which operates as a bidirectional exchanger during the physiological steady state. This cotransport system, like that associated with the volume regulatory mechanism (i.e. coupled net uptake of Cl^–+Na⁺ and/or K⁺) is Cl^–-selective and furosemide-sensitive, suggesting the same mechanism operating in two different modes. Since Na⁺ has an important function in the volume regulatory response, its role in steady-state cotransport was investigated. In the absence of Na⁺, ouabain-insensitive K⁺ and DIDS-insensitive Cl^– transport (KCl cotransport) are low and equivalent to that found in 150mm Na⁺ medium containing furosemide. Increasing the [Na⁺] results in parallel increases in K⁺ and Cl^– transport. The maximum rate of each (18 to 20 meq/(kg dry wt)·min) is reached at about 20mm Na⁺ and is maintained up to 55mm. Thus, over the range 1 to 55mm Na⁺ the stoichiometry of KCl cotransport is 11. In contrast to K⁺ and Cl^–, furosemide-sensitive Na⁺ transport is undetectable until the [Na⁺] exceeds 50mm. From 50 to 150mm Na⁺, it progressively rises to 7 meq/(kg dry wt)·min, while K⁺ and Cl^– transport decrease to 9 and 16 meq/(kg dry wt)·min, respectively. Thus, at 150mm Na⁺ the stoichiometric relationship between Cl^–, Na⁺ and K⁺ is 211. These results are consistent with the proposal that the Cl^–-dependent cation cotransport system when operating during the steady state mediates the exchange of KCl for KCl or NaCl for NaCl; the relative proportion of each determined by the extracellular [Na⁺].     

Effect of anisotonic media on volume,ion and amino-acid content and membrane potential of kidney cells (MDCK) in culture

Summary Effects of anisotonic media on a monolayer of confluent kidney cells in culture (MDCK) were studied by measuring: cell thickness and cross-section changes, ion and amino-acid content and membrane potential. The volume was also determined with cells in suspension. When cells in a monolayer were incubated in hypotonic media, the lateral and the apical membranes were rapidly stretched. Afterwards the lateral membranes returned to their initial state while the apical membranes remained stretched. This partial regulatory volume decrease (RVD) was verified with cells in suspension. RVD was accompanied by a loss of K⁺, Cl^– and amino acids, but there was no loss of inorganic phosphate. Also a transient hyperpolarization of the membrane potential was observed, suggesting an increase of the K⁺ conductance during RVD. Upon restoring the isotonic medium, a regulatory volume increase (RVI) was observed accompanied by a rapid Na⁺ and Cl^– increase and followed by a slow recovery of the initial K⁺ and Na⁺ content while amino acids remained at their reduced content. A transient depolarization of the membrane potential was measured during this RVI, suggesting that Na⁺ and Cl^– conductance could have increased. In hypertonic media, only a small and slow RVI was observed accompanied by an increase in K⁺ and Cl^– content but without any change of membrane potential. Quinine partly inhibited RVD in hypotonic media with cells in a monolayer while inhibiting RVD completely with cells in suspension. Incubation during four hours in a Ca²⁺ free medium had no effect on RVD. Furosemide and amiloride had no effect on RVD and RVI. Volume regulation, RVD or RVI, was not affected by replacing Cl^– by nitrate. When cells in a monolayer were incubated in a hypotonic K₂SO₄ medium, no RVD was observed. From these results, it seems that MDCK cells in a confluent monolayer regulate their volume by activating specific ion and amino-acid transport pathways. Selective K⁺ and Na⁺ conductances are activated during RVD and RVI, while the activated anion conductance has a low selectivity. The controlling mechanism might not be the free intracellular Ca²⁺ concentration.     

Effects of antidiuretic hormone on cellular conductive pathways in mouse medullary thick ascending limbs of Henle: I. ADH increases transcellular conductance pathways

Summary This paper reports experiments designed to assess the relations between net salt absorption and transcellular routes for ion conductance in single mouse medullary thick ascending limbs of Henle microperfusedin vitro. The experimental data indicate that ADH significantly increased the transepithelial electrical conductance, and that this conductance increase could be rationalized in terms of transcellular conductance changes. A minimal estimate (G _c ^min ) of the transcellular conductance, estimated from Ba⁺⁺ blockade of apical membrane K⁺ channels, indicated thatG _c ^min was approximately 30–40% of the measured transepithelial conductance. In apical membranes, K⁺ was the major conductive species; and ADH increased the magnitude of a Ba⁺⁺-sensitive K⁺ conductance under conditions where net Cl^– absorption was nearly abolished. In basolateral membranes, ADH increased the magnitude of a Cl^– conductance; this ADH-dependent increase in basal Cl^– conductance depended on a simultaneous hormone-dependent increase in the rate of net Cl^– absorption. Cl^– removal from luminal solutions had no detectable effect onG _e , and net Cl^– absorption was reduced at luminal K⁺ concentrations less than 5mm; thus apical Cl^– entry may have been a Na⁺,K⁺,2Cl^– cotransport process having a negligible conductance. The net rate of K⁺ secretion was approximately 10% of the net rate of Cl^– absorption, while the chemical rate of net Cl^– absorption was virtually equal to the equivalent short-circuit current. Thus net Cl^– absorption was rheogenic; and approximately half of net Na⁺ absorption could be rationalized in terms of dissipative flux through the paracellular pathway. These findings, coupled with the observation that K⁺ was the principal conductive species in apical plasma membranes, support the view that the majority of K⁺ efflux from cell to lumen through the Ba⁺⁺-sensitive apical K⁺ conductance pathway was recycled into cells by Na⁺,K⁺,2Cl^– cotransport.     

Effects of anions on cellular volume and transepithelial Na+ transport across toad urinary bladder

Summary The effects of complete substitution of gluconate for mucosal and/or serosal medium Cl^– on transepithelial Na⁺ transport have been studied using toad urinary bladder. With mucosal gluconate, transepithelial potential difference (V _T) decreased rapidly, transepithelial resistance (R _T) increased, and calculated short-circuit current (I _sc) decreased. CalculatedE _Na was unaffected, indicating that the inhibition of Na⁺ transport was a consequence of a decreased apical membrane Na⁺ conductance. This conclusion was supported by the finding that a higher amiloride concentration was required to inhibit the residual transport. With serosal gluconateV _T decreased,R _T increased andI _sc fell to a new steady-state value following an initial and variable transient increase in transport. Epithelial cells were shrunken markedly as judged histologically. CalculatedE _Na fell substantially (from 130 to 68 mV on average). Ba²⁺ (3mm) reduced calculatedE _Na in Cl^– Ringer's but not in gluconate Ringer's. With replacement of serosal Cl^– by acetate, transepithelial transport was stimulated, the decrease in cellular volume was prevented andE _Na did not fall. Replacement of serosal isosmotic Cl^– medium by a hypo-osmotic gluconate medium (one-half normal) also prevented cell shrinkage and did not result in inhibition of Na⁺ transport. Thus the inhibition of Na⁺ transport can be correlated with changes in cell volume rather than with the change in Cl^– per se. Nystatin virtually abolished the resistance of the apical plasma membrane as judged by measurement of tissue capacitance. With K⁺ gluconate mucosa, Na⁺ gluconate serosa, calculated basolateral membrane resistance was much greater, estimated basolateral emf was much lower, and the Na⁺/K⁺ basolateral permeability ratio was much higher than with acetate media. It is concluded the decrease in cellular volume associated with substitution of serosal gluconate for Cl^– results in a loss of highly specific Ba²⁺-sensitive K⁺ conductance channels from the basolateral plasma membrane. It is possible that the number of Na⁺ pump sites in this membrane is also decreased.