Modulation of caffeine contractures in mammalian skeletal muscles by variation of extracellular potassium

Caffeine contractures were induced after K⁺ -conditioning of skeletal muscles from pigs and mice. K⁺ -conditioning is defined as the partial depolarization caused by increasing external potassium (K) with [K⁺]×[Cl^?] constant. Conditioning depolarizations that rendered muscles refractory to brief electrical stimulation still enhanced the contracture tension elicited by subsequent direct caffeine stimulation of sarcoplasmic reticulum (SR) calcium release. The effects of K⁺ -conditioning on caffeine-induced contractures of intact cell bundles reached a maximum at 15–30 mM K and then progressively declined at higher [K⁺]₀. Conditioning with 30 mM K⁺ for 5 min, which inactivates excitation-contraction (EC) coupling in response to action potentials, both increased the magnitude of caffeine contractures 2–10-fold and shifted the contracture threshold toward lower caffeine concentrations. Enhanced sensitivity to caffeine was inhibited by dantrolene (20 μM) and its watersoluble analogue azumolene (150 μM). These drugs decreased caffeine-induced contractures following depolarization with 4–15 mM K⁺ to 25–50% of control tension. The inorganic anion perchlorate (CIO), which like caffeine potentiates twitches, increased caffeine-induced contractures ～? twofold after K⁺ -conditioning (>4 mM). The results suggest that CIO and dantrolene, in addition to caffeine, also influence SR calcium release either directly or by mechanism(s) subsequent to depolarization of the sarcolemma. Moreover, since CIO is known to shift the voltage-dependence of intramembrane charge movement, CIO may exert effects on the transverse-tubule voltage sensors as well as the SR. © 1995 Wiley-Liss, Inc.     

Regulation of changes in cytosolic Ca2+ and Na+ concentrations in rat submandibular gland acini exposed to carbachol and ATP

The relationship between cytosolic concentrations of Ca²⁺ (Ca) and Na⁺ (Na) were studied in preparations of rat submandibular and pancreatic acini loaded with the Ca²⁺-sensitive dye Fura-2 or the Na⁺-sensitive dye SBFI. Pancreatic acini showed no changes in Na during either transient or persistent changes in Ca. Increases in Ca produced by exposure of submandibular gland acini to carbachol, a muscarinic cholinergic agonist, were followed by an increase in Na after a delay of 5–10 s. When Ca²⁺ stores were mobilized without Ca²⁺ influx Na also increased, but in acini loaded with BAPTA, a nonfluorescent Ca²⁺ chelator, the transient increase in Ca²⁺ caused by mobilization of stored Ca²⁺ was virtually abolished, as was the increase in Na. In the presence of ionomycin, increases in Ca were followed by increases in Na. Ca²⁺-dependent increases in Na were abolished in Na⁺-free buffer and by the presence of furosemide, a blocker of Na⁺-K⁺-2Cl⁻ cotransport. In other studies, extracellular ATP (ATP_o) produced an increase in Ca and Na. The steady-state increase in Ca was reduced by increasing extracellular Na⁺ concentrations (Na) in dose-dependent fashion (IC₅₀ = 16.4 ± 4.7 mM Na⁺). Likewise, increasing Na reduced ATP_o-stimulated ⁴⁵Ca²⁺ uptake at steady state (IC₅₀ = 15.8 ± 9.2 mM Na⁺). Changing Na had no effect on carbachol-stimulated increases in Ca. We conclude that, in rat submandibular gland acini, ATP_o promotes an increase in Ca and Na via a common influx pathway and that, under physiologic conditions, Na⁺ significantly limits the ATP_o-stimulated increase in Ca. In the presence of carbachol, however, Na rises in Ca-dependent fashion in submandibular gland acini via stimulation of Na⁺-K⁺-2Cl⁻ cotransport. © 1996 Wiley-Liss, Inc.     

Variable coupling of active NA+ + K+ transport in Ehrlich ascites tumor cells: Regulation by external NA+ and K+

The effects of altered external sodium and potassium concentrations on steady state, active Na⁺ + K⁺ transport in Ehrlich ascites tumor cells have been investigated. Membrane permeability to Na⁺ and K⁺, intracellular [Na⁺] and [K⁺], and membrane potential were measured. Active cation fluxes were calculated as equal and membrane potential were measured. Active cation fluxes were calculated as equal and opposite to the net, diffusional leak fluxes. Elevation of external K⁺ (6–60 Mm)by equivalent replacement of Na⁺ (154–91 mM) inhibits both active Na⁺ and K⁺ fluxes, but not proportionally. This results in a decrease of the coupling ratio (r_p = -J_k^p/J) as external K⁺ is increased. Elevation of external K⁺ (3–68 mM) at constant Na⁺ (92mM) inbibits J, but is without effect on J. The coupling ratio declines from 1.01 ± 0.14 to 0.07 ± 0.05, a 14-fold alteration. Reduction of external Na⁺ (154–25 mM) at constant K⁺ (6mM) depresses J, but is without effect on J. The coupling ratio increases from 0.63 ± 0.04 at 154 mM Na⁺ to 4.5 ± 2.04 at 25 mM Na⁺. The results of this investigation are consistent with the independent regulation of active cation fluxes by the transported species. Kinetic analysis of the data indicates that elevation of external sodium stimulates active sodium efflux by interacting at “modifier sites” at the outer cell surface. Similarly, external potassium inhibits active potassium influx by interaction at separate modifier sites.     

Regulation of the Na+-K+(NH)-2Cl− cotransporter of rat submandibular glands

A cellular suspension from rat submandibular glands was exposed to different concentrations of NH₄Cl, and the variations of the intracellular concentration of calcium ([Ca²⁺]_i) and the intracellular pH (pH_i) were measured using fura-2 and 2′,7′-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein. More than 5 mmol/l NH₄Cl significantly increased the [Ca²⁺]_i without affecting the response to 100 µmol/l carbachol. When exposed to 1 and 5 mmol/l NH₄Cl, the cells acidified immediately. At 30 mmol/l, NH₄Cl first alkalinized the cells and the pH_i subsequently dropped. This drop reflects the uptake of NH ions that dissociate to NH₃ and H⁺ in the cytosol. These protons are exchanged for extracellular sodium by the Na⁺/H⁺ exchanger because the presence of an inhibitor of the exchanger in the medium increased the acidification induced by 1 mmol/l NH₄Cl. Ouabain partly blocked the uptake of NH. In the combined presence of ouabain and bumetanide (an inhibitor of the Na⁺-K⁺-2Cl⁻ cotransporter), 1 mmol/l NH₄Cl alkalinized the cells. The contribution of the Na/K ATPase and the Na⁺-K⁺-2Cl⁻ cotransporter in the uptake of NH was independent of the presence of calcium in the medium. Isoproterenol increased the uptake of NH by the cotransporter. Conversely, 1 mmol/l extracellular ATP blocked the basal uptake of NH by the cotransporter. This inhibition was reversed by extracellular magnesium or Coomassie Blue. It was mimicked by benzoyl-ATP but not by CTP, GTP, UTP, ADP, or ADPβS. ATP only slightly inhibited the increase of cyclic AMP (−22%) by isoproterenol but fully blocked the stimulation of the cotransporter by the β-adrenergic agonist. ATP increased the release of ³H-arachidonic acid from prelabeled cells but SK&F 96365, an imidazole-based cytochrome P450 inhibitor, did not affect the inhibition by ATP. It is concluded that the activation of a purinoceptor inhibits the basal and the cyclic AMP-stimulated activity of the Na⁺-K⁺-2Cl⁻ cotransporter. J. Cell. Physiol. 180:422–430, 1999. © 1999 Wiley-Liss, Inc.     

High capacity Na+/H+ exchange activity in mineralizing osteoblasts

Osteoblasts synthesize bone in polarized groups of cells sealed by tight junctions. Large amounts of acid are produced as bone mineral is precipitated. We addressed the mechanism by which cells manage this acid load by measuring intracellular pH (pHi) in non‐transformed osteoblasts in response to weak acid or bicarbonate loading. Basal pHi in mineralizing osteoblasts was ～7.3 and decreased by ～1.4 units upon replacing extracellular Na⁺ with N‐methyl‐D ‐glucamine. Loading with 40 mM acetic or propionic acids, in normal extracellular Na⁺, caused only mild cytosolic acidification. In contrast, in Na⁺‐free solutions, weak acids reduced pHi dramatically. After Na⁺ reintroduction, pHi recovered rapidly, in keeping with Na⁺/H⁺ exchanger (NHE) activity. Sodium‐dependent pHi recovery from weak acid loading was inhibited by amiloride with the Ki consistent with NHEs. NHE1 and NHE6 were expressed strongly, and expression was upregulated highly, by mineralization, in human osteoblasts. Antibody labeling of mouse bone showed NHE1 on basolateral surfaces of all osteoblasts. NHE6 occurred on basolateral surfaces of osteoblasts mainly in areas of mineralization. Conversely, elevated HCO alkalinized osteoblasts, and pH recovered in medium containing Cl^?, with or without Na⁺, in keeping with Na⁺‐independent Cl^?/HCO exchange. The exchanger AE2 also occurred on the basolateral surface of osteoblasts, consistent with Cl^?/HCO exchange for elimination of metabolic carbonate. Overexpression of NHE6 or knockdown of NHE1 in MG63 human osteosarcoma cells confirmed roles of NHE1 and NHE6 in maintaining pHi. We conclude that in mineralizing osteoblasts, slightly basic basal pHi is maintained, and external acid load is dissipated, by high‐capacity Na⁺/H⁺ exchange via NHE1 and NHE6. J. Cell. Physiol. 226: 1702–1712, 2011. © 2010 Wiley‐Liss, Inc.     

Multiple Ca2+ signaling pathways regulate intracellular Ca2+ activity in human cardiac fibroblasts

Ca²⁺ signaling pathways are well studied in cardiac myocytes, but not in cardiac fibroblasts. The aim of the present study is to characterize Ca²⁺ signaling pathways in cultured human cardiac fibroblasts using confocal scanning microscope and RT‐PCR techniques. It was found that spontaneous intracellular Ca²⁺ (Ca) oscillations were present in about 29% of human cardiac fibroblasts, and the number of cells with Ca oscillations was increased to 57.3% by application of 3% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry. Ca oscillations were abolished by the store‐operated Ca²⁺ (SOC) entry channel blocker La³⁺, the phospholipase C inhibitor U‐73122, and the inositol trisphosphate receptors (IP3Rs) inhibitor 2‐aminoethoxydiphenyl borate, but not by ryanodine. The IP3R agonist thimerosal enhanced Ca oscillations. Inhibition of plasma membrane Ca²⁺ pump (PMCA) and Na⁺–Ca²⁺ exchanger (NCX) also suppressed Ca oscillations. In addition, the frequency of Ca oscillations was reduced by nifedipine, and increased by Bay K8644 in cells with spontaneous Ca²⁺ oscillations. RT‐PCR revealed that mRNAs for IP3R1‐3, SERCA1‐3, Ca_V1.2, NCX3, PMCA1,3,4, TRPC1,3,4,6, STIM1, and Orai1‐3, were readily detectable, but not RyRs. Our results demonstrate for the first time that spontaneous Ca oscillations are present in cultured human cardiac fibroblasts and are regulated by multiple Ca²⁺ pathways, which are not identical to those of the well‐studied contractile cardiomyocytes. This study provides a base for future investigations into how Ca²⁺ signals regulate biological activity in human cardiac fibroblasts and cardiac remodeling under pathological conditions. J. Cell. Physiol. 223: 68–75, 2010. © 2009 Wiley‐Liss, Inc.     

Characterization of calcium signaling pathways in human preadipocytes

Intracellular free Ca²⁺ (Ca) is an important regulator of many cellular activities; however, Ca²⁺ signaling is not well studied in human preadipocytes. The purpose of the present study was to characterize Ca²⁺ signal pathways using a confocal scanning technique and RT‐PCR. It was found that spontaneous Ca oscillations were observed in 12.1% preadipocytes, and number of cells with Ca²⁺ oscillations was increased to 47.9% by 1% fetal bovine serum. Ca oscillations were dependent on Ca²⁺ entry mainly via stored‐operated Ca²⁺ (SOC) entry. They were suppressed by the SOC entry channel blocker La³⁺, the phospholipase C (PLC) inhibitor U73122, the inositol trisphosphate receptor (IP3R) blocker 2‐amino‐ethoxydiphenyl borate, or the sarcoplasmic/endoplasmic reticulum Ca²⁺ pump (SERCA) inhibitors thapsigargin and cyclopiazonic acid, but not by ryanodine. The IP3R activator thimerosal increased Ca oscillations. In addition, the plasma membrane Ca²⁺ pump (PMCA) inhibitor carboxyeosin and Na⁺–Ca²⁺ exchanger (NCX) inhibitor Ni²⁺ both suppressed Ca²⁺ oscillations. RT‐PCR revealed that the mRNAs for IP3R1‐3, SERCA1,2, NCX3 and PMCA1,3,4, Ca_V1.2, and TRPC1,4,6, STIM1 and Orai1 (for SOC entry channels) were significant in human preadipocytes. The present study demonstrates that multiple Ca²⁺ signal pathways are present in human preadipocytes, and provides a basis for investigating how Ca²⁺ signals regulate biological and physiological activities of human preadipocytes. J. Cell. Physiol. 220: 765–770, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of H+ on the kinetics of Na+-dependent amino acid transport in ehrlich ascites tumor cells: Evidence for H+ as an alternative substrate

The effects of H⁺ on the kinetics of α-aminoisobutyric acid (AIB) influx in Ehrlich ascites tumor cells have been investigated at different external Na⁺ concentrations. Elevation of [H⁺] in the presence of both high (154 mEq/l) and low (10 mEq/l) external Na⁺ leads to decreases in the maximum influx (J) and increases in the apparent Michaleis-Menten constant (K) for influx of AIB. In the virtual absence of external Na⁺ (0.96 ± 0.04 mEq/l), alterations in [H⁺] are without measurable effect on AIB flux. Furthermore, addition of AIB (10 mM) to cell suspensions (pH 5.90) stimulates H⁺ uptake by the cells in either the presence or absence of Na⁺. The data are consistent with two kinetic models for Na⁺-dependent amino acid transport: an order bireactant (Na⁺-binding necessary before AIB binding) system or a random bireactant system. Both models require that H⁺ serve as an alternative substrate for Na⁺. The consistency of the models was tested by fit to data from the present study (not used to evaluate the kinetic parameters) and by prediction of the pH dependence of Na⁺-dependent amino acid transport compared to earlier studies.     

Comparative biochemical and cytochemical studies on superoxide and peroxide in mouse macrophages

Maximal rates of superoxide (O) release, and the cytochemical locales of peroxide staining in resident, elicited, and activated macrophages have been determined. Macrophages elicited into the peritoneum with either casein (1.2% w/v) or proteose-peptone (10.0% w/v) release about twice as much O as macrophages activated by infection of the animals with either Listeria monocytogenes, or Bacille Calmette-Guerin (BCG) followed by immune boosting with Purified Protein Derivative (PPD) (i.e., about 35 vs. 14–18 nmol O/min/10⁷ cells). Macrophages elicited with thioglycollate (3.0% w/v) and resident macrophages produce negligible amounts of O upon stimulation with PMA. These data are compared with those reported by other investigators who used different procedures. A cytochemical procedure for localizing peroxide has been modified for use with murine macrophages. No production of H₂O₂ by macrophages is detected cytochemically in the absence of stimulation. Upon exposure to PMA, resident macrophages are still largely unresponsive. Approximately 20% of the casein elicited macrophages and BCG-PPD activated macrophages exhibit H₂O₂ staining, which is largely restricted to the cytoplasmic vesicles and channels induced by PMA in these cells. The only exception to this staining pattern is a small population (about 2%) of activated macrophages which exhibits H₂O₂ staining in the cytoplasmic vesicles and channels and on the plasmalemma as well.     

Rab11b and its effector Rip11 regulate the acidosis‐induced traffic of V‐ATPase in salivary ducts

Redistribution of acid‐base transporters is a crucial regulatory mechanism for many types of cells to cope with extracellular pH changes. In epithelial cells, however, translocation of acid‐base transporters ultimately leads to changes in vectorial transport of H⁺ and HCO. We have previously shown that the bicarbonate‐secreting epithelium of salivary ducts responds to changes of systemic acid‐base balance by adaptive redistribution of H⁺ and HCO transporters, thereby influencing the ionic composition and buffering capacity of saliva. However, the specific proteins involved in regulated vesicular traffic of acid‐base transporters are largely unknown. In the present study we have investigated the impact of Rab11 family members on the acidosis‐induced trafficking of the vacuolar‐type H⁺‐ATPase (V‐ATPase) in salivary duct cells in vitro using the human submandibular cell line of ductal origin HSG as an experimental model. The results show that Rab11b is expressed in salivary ducts and exhibits a significantly higher co‐localization with V‐ATPase than Rab11a and Rab25. We also show that Rab11 but not Rab25 interacts with the ε subunit of V‐ATPase. Extracellular acidosis up‐regulates Rab11b expression and protein abundance in HSG cells and causes translocation of the V‐ATPase from intracellular pools toward the plasma membrane. Loss‐of‐function experiments using specific siRNA either against Rab11b or against its effector Rip11 prevent acidosis‐induced V‐ATPase translocation. These data introduce Rab11b as a crucial regulator and Rip11 as mediator of acidosis‐induced V‐ATPase traffic in duct cells of submandibular gland. J. Cell. Physiol. 226: 638–651, 2011. © 2010 Wiley‐Liss, Inc.     

Kinetic mechanism of Na+, K+, Cl−-cotransport as studied by Rb+ influx into HeLa cells: Effects of extracellular monovalent ions

Summary Ouabain-insensitive, furosemide-sensitive Rb⁺ influx (J _Rb) into HeLa cells was examined as functions of the extracellular Rb⁺, Na⁺ and Cl^– concentrations. Rate equations and kinetic parameters, including the apparent maximumJ _Rb, the apparent values ofK _m for the three ions and the apparentK _i for K⁺, were derived. Results suggested that one unit molecule of this transport system has one Na⁺, one K⁺ and two Cl^– sites with different affinities, one of the Cl^– sites related with binding of Na⁺, and the other with binding of K⁺(Rb⁺). A 11 stoichiometry was demonstrated between ouabain-insensitive, furosemidesensitive influxes of²²Na⁺ and Rb⁺, and a 12 stoichiometry between those of Rb⁺ and³⁶Cl^–. The influx of either one of these ions was inhibited in the absence of any one of the other two ions. Monovalent anions such as nitrate, acetate, thiocyanate and lactate as substitutes for Cl^– inhibited ouabain-insensitive Rb⁺ influx, whereas sulfamate and probably also gluconate did not inhibitJ _Rb. From the present results, a general model and a specialized cotransport model were proposed: 1) In HeLa cells, one Na⁺ and one Cl^– bind concurrently to their sites and then one K⁺ (Rb⁺) and another Cl^– bind concurrently. 2) After completion of ion bindings Na⁺, K⁺(Rb^–) and Cl^– in a ratio of 112 show synchronous transmembrane movements.     

Lymphocyte CFTR promotes epithelial bicarbonate secretion for bacterial killing

The expression of cystic fibrosis transmembrane conductance regulator (CFTR) in lymphocytes has been reported for nearly two decades; however, its physiological role remains elusive. Here, we report that co‐culture of lymphocytes with lung epithelial cell line, Calu‐3, promotes epithelial HCO production/secretion with up‐regulated expression of carbonic anhydrase 2 and 4 (CA‐2, CA‐4) and enhanced bacterial killing capability. The lymphocyte‐enhanced epithelial HCO secretion and bacterial killing activity was abolished when Calu3 cells were co‐cultured with lymphocytes from CFTR knockout mice, or significantly reduced by interfering with E‐cadherin, a putative binding partner of CFTR. Bacterial lipopolysaccharide (LPS)‐induced E‐cadherin and CA‐4 expression in the challenged lung was also found to be impaired in CFTR knockout mice compared to that of the wild‐type. These results suggest that the interaction between lymphocytes and epithelial cells may induce a previously unsuspected innate host defense mechanism against bacterial infection by stimulating epithelial HCO production/secretion, which requires CFTR expression in lymphocytes. J. Cell. Physiol. 227: 3887–3894, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.     

Uridine transport in Novikoff rat hepatoma cells and other cell lines and its relationship to uridine phosphorylation and phosphorolysis.

Time courses of [³H]uridine uptake as a function of uridine concentration were determined at 25° in untreated and ATP-depleted wild-type and uridine kinase-deficient Novikoff cells and in mouse L and P388 cells, Chinese hamster ovary cells and human HeLa cells. Short term uptake was measured by a rapid sampling technique which allows sampling of cell suspensions in intervals as short as one and one-half seconds. The initial segments of the time courses were the same in untreated, wild-type cells in which uridine is rapidly phosphorylated and in cells in which uridine phosphorylation was prevented due to lack of ATP or uridine kinase. The initial rates of uptake, therefore, reflected the rate of uridine transport. Uridine uptake, however, was approximately linear for only five to ten seconds at uridine concentrations from 20–160 μM and somewhat longer at higher concentrations. In phosphorylating cells the rate of uridine uptake (at 80 μM) then decreased to about 20–30% of the initial rate and this rate was largely determined by the rate of phosphorylation rather than transport. At uridine concentrations below 1 μM, however, the rate of intracellular phosphorylation in Novikoff cells approached the transport rate. The apparent substrate saturation of phosphorylation suggests the presence of a low K_m uridine phosphorylation system in these cells. The “zero-trans” (zt) K_m for the facilitated transport of uridine as estimated from initial uptake rates fell between 50 and 240 μM for all cell lines examined. The zero-trans V_max values were also similar for all the lines (4–15 pmoles/μ1 cell H₂O.sec). The time courses of uridine uptake by CHO cells and the kinetic constants for transport were about the same whether the cells were propagated (and analyzed for uridine uptake) in suspension or monolayer culture. When Novikoff cells were preloaded with 10 μM uridine the apparent K_m and V_max values (infinite-trans) were two to three times higher than the corresponding zero-trans values. Uridine transport was inhibited in a simple competitive manner by several other ribo- and deoxyribonucleosides. All nucleosides seem to be transported by the same system, but with different efficiencies. Uridine transport was also inhibited by hypoxanthine, adenine, thymine, Persantin, papaverin, and o-nitrobenzylthioinosine, and by pretreatment of the cells with p-chloromercuri-benzoate, but not by high concentrations of cytosine, D-ribose or acronycin. The inhibition of uridine transport by Persantin involved changes in both V and K. Because of the rapidity of transport, some loss of intracellular uridine occurred when cells were rinsed in buffer solution to remove extracellular substrate, even at 0°. This loss was prevented by the presence of a transport inhibitor, Persantin, in the rinse fluid or by separating suspended cells from the medium by centrifugation through oil. Metabolic conversion of intracellular uridine were also found to continue during the rinse period. The extent of artifacts due to efflux and metabolism during rinsing increased with duration of the rinse.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.     

Furosemide-sensitive K+ channel in glioma cells but not neuroblastoma cells in culture

A furosemide-sensitive, ouabain-insensitive [⁸⁶Rb⁺] uptake is described in glioma cells in culture which is dependent upon external Na⁺, K⁺, and Cl^? concentrations. This transport activity was also inhibited by bumetanide at 100-fold lower concentrations than furosemide. Furosemide-sensitive swelling of glioma cells is demonstrated and this activity is dependent upon external Na⁺ and K⁺ in a manner similar to [⁸⁶Rb⁺] uptake. This transport activity was not detected in neuroblastoma cells and the possible relevance of these findings to extracellular K⁺ buffering by glia is discussed.     

Thiol-Dependent passive K/Cl transport in sheep red cells: IV. Furosemide inhibition as a function of external Rb+, Na+, and Cl−

Summary The effect of the loop diuretic furosemide (4-chloro-N-furfuryl-5-sulfamoyl-anthranilic acid) on the thiol-dependent, ouabain-insensitive K(Rb)/Cl transport in low K⁺ sheep red cells was studied at various concentrations of extracellular Rb⁺, Na⁺ and Cl^–. In Rb⁺-free NaCl media, 2×10^–3 m furosemide inhibited only one-half of thiol-dependent K⁺ efflux. In the presence of 23mm RbCl, however, the concentration of furosemide to produce 50% K⁺ efflux inhibition (IC₅₀) was 5×10^–5 m. In Rb⁺ containing NaCl media, the inhibitory effect of 10^–3 m furosemide was equal to that caused by NO ₃ ^– replacement of Cl^– in the medium. The apparent synergistic action of furosemide and external Rb⁺ on K⁺ efflux was also seen in the ouabain-insensitive Rb⁺ influx. A preliminary kinetic analysis suggests that furosemide binding alters both maximal K⁺(Rb⁺) transport and apparent external Rb⁺ affinity. In the presence of external Rb⁺, Na⁺ (as compared to choline) exerted a small but significant augmentation of the furosemide inhibition of K⁺(Rb⁺) fluxes. There was no effect of Cl^– on the IC₅₀ value of furosemide. As there is no evidence for coupled Na⁺K⁺ cotransport in low K⁺ sheep red cells, furosemide may modify thiol-dependent K⁺(Rb⁺/Cl flux or Rb⁺ (and to a slight degree Na⁺) modulate the effect of furosemide.     

Insulin action on cardiac glucose transport Studies on the role of the Na+/K+ pump

Isolated muscle cells from adult rat heart have been used to study the relationship between myocardial glucose transport and the activity of the Na⁺/K⁺ pump. ⁸⁶Rb⁺-uptake by cardiac cells was found to be linear up to 2 min with a steady-state reached by 40–60 min, and was used to monitor the activity of the Na⁺/K⁺ pump. Ouabain (10^?3 mol/I) inhibited the steady-state uptake of ⁸⁶Rb⁺ by more than 90%. Both, the ouabain-sensitive and ouabain-insensitive ⁸⁶Rb⁺-uptake by cardiac cells were found to be unaffected by insulin treatment under conditions where a significant stimulation of 3-O-methylglucose transport occurred. ⁸⁶Rb⁺-uptake was markedly reduced by the presence of calcium and/or magnesium, but remained unresponsive towards insulin treatment. Inhibition of the Na⁺/K⁺ pump activity by ouabain and a concomitant shift in the intracellular Na⁺:K⁺ ratio did not affect basal or insulin stimulated rates of 3-O-methylglucose transport in cardiac myocytes. The data argue against a functional relationship between the myocardial Na⁺/K⁺ pump and the glucose transport system.     

Na+/K+/Cl− cotransport in cultured vascular smooth muscle cells: Stimulation by angiotensin II and calcium ionophores,inhibition by cyclic AMP and calmodulin antagonists

Summary The specific activity of the Na⁺/K⁺/Cl^– cotransporter was assayed by measuring the initial rates of furosemide-inhibitable⁸⁶Rb⁺ influx and efflux. The presence of all three ions in the external medium was essential for cotransport activity. In cultured smooth muscle cells furosemide and bumetanide inhibited influx by 50% at 5 and 0.2 m, respectively. The dependence of furosemide-inhibitable⁸⁶Rb⁺ influx on external Na⁺ and K⁺ was hyperbolic with apparentK _m values of 46 and 4mm, respectively. The dependence on Cl^– was sigmoidal. Assuming a stoichiometry of 112 for Na⁺/K⁺/Cl^–, aK _m of 78mm was obtained for Cl^–. In quiescent smooth muscle cells cotransport activity was approximately equal to Na⁺ pump activity with each pathway accounting for 30% of total⁸⁶Rb⁺ influx. Growing muscle cells had approximately 3 times higher cotransport activity than quiescent ones. Na⁺ pump activity was not significantly different in the gorwing and quiescent cultures. Angiotensin II (ANG) stimulated cotransport activity as did two calcium-transporting ionophores, A23187 and ionomycin. The removal of external Ca²⁺ prevented A23187, but not ANG, from stimulating the cotransporter. Calmodulin antagonists selectively inhibited⁸⁶Rb⁺ influx via the cotransporter. Beta-adrenoreceptor stimulation with isoproterenol, like other treatments which increase cAMP, inhibited cotransport activity. Cultured porcine endothelial cells had 3 times higher cotransport activity than growing muscle cells. Calmodulin antagonists inhibited cotransport activity, but agents which increase cAMP or calcium had no effect on cotransport activity in the endothelial cells.     

Einfluß extracellulärer Kaliuminoenkonzentrationen auf die celluläre Natriumionenkonzentration von Lodderomyces elongisporus D

It was found that the cellular Na⁺-concentration (C) of Lodderomyces elongisporus D is depended on the extracellular K⁺-concentration (C). The relationship can be described by an equation in the form The function of the natrium ion seem to be to support the utilisation rate of potassium ion at lower extracellular K⁺-concentration.