Sodium channels contribute to action potential generation in canine and human pancreatic islet B cells

Summary Pancreatic islet B cells depolarize and display trains of action potentials in response to stimulatory concentrations of glucose. Based on data from rodent islets these action potentials are considered to be predominantly Ca²⁺ dependent. Here we describe Na⁺-dependent action potentials and Na⁺ currents recorded from canine and human pancreatic islet B cells. Current-clamp recording using the nystatin perforated-patch technique demonstrates that B cells from both species display tetrodotoxin-sensitive Na⁺ action potentials in response to modest glucose-induced depolarization. In companion whole-cell voltage-clamp experiments on canine B cells, the underlying Na⁺ current displays steep voltage-dependent activation and inactivation over the range of –50 to –40 mV. The Na⁺ current is sensitive to tetrodotoxin block with aK ₁=3.2nm and has a reversal potential which changes with [Na⁺] _o as predicted by the Nernst equation. These results suggest that a voltage-dependent Na⁺ current may contribute significantly to action potential generation in some species outside the rodent family.     

Effect of Extracellular pH on Presteady-State and Steady-State Current Mediated bythe Na<Superscript>+</Superscript>/K<Superscript>+</Superscript> Pump

A ouabain sensitive inward current occurs in Xenopus oocytes in Na⁺ and K⁺ -free solutions. Several laboratories have investigated the properties of this current and suggested that acidic extracellular pH (pH_o) produces a conducting pathway through the Na⁺/K⁺ pump that is permeable to H⁺ and blocked by [Na⁺]_o. An alternative suggestion is that the current is mediated by an electrogenic H⁺-ATPase. Here we investigate the effect of pH_o and [Na⁺]_o on both transient and steady-state ouabain-sensitive current. At alkaline or neutral pH_o the relaxation rate of pre-steady-state current is an exponential function of voltage. Its U-shaped voltage dependence becomes apparent at acidic pH_o, as predicted by a model in which protonation of the Na⁺/K⁺ pump reduces the energy barrier between the internal solution and the Na⁺ occluded state. The model also predicts that acidic pH_o increases steady-state current leak through the pump. The apparent pK of the titratable group(s) is 6, suggesting that histidine is involved in induction of the conductance pathway. ²²Na efflux experiments in squid giant axon and current measurements in oocytes at acidic pH_o suggest that both Na⁺ and H⁺ are permeant. The acid-induced inward current is reduced by high [Na⁺]_o, consistent with block by Na⁺. A least squares analysis predicts that H⁺ is four orders of magnitude more permeant than Na⁺, and that block occurs when 3 Na⁺ ions occupy a low affinity binding site (K _0.5=130±30 mM) with a dielectric coefficient of 0.23±0.03. These data support the conclusion that the ouabain-sensitive conducting pathway is a result of passive leak of both Na⁺ and H⁺ through the Na⁺/K⁺ pump.     

Chemically modified cardiac Na+ channels and their sensitivity to antiarrhythmics: Is there a hidden drug receptor?

Elementary Na⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na⁺ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na⁺ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na⁺ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O₁–O₂ reaction (_open(1) 1.4±0.3 msec; _open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (_{open 3.0±0.5} msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP _o significantly can interact with both types of noninactivating Na⁺ channels to reduce the dwell time in the conducting configuration. lodate-modified Na⁺ channels became drug sensitive during the O₂ state. At –40 mV, for example, lidocaine reduced _open(2) to 62±5% of the control without detectable changes in _open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na⁺ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce _open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, a_lidocaine was 0.64×10⁶ mol^–1 sec^–1 and a_diprafenone 13.6×10⁶ mol^–1 sec^–1. Trypsin-modified Na⁺ channels also appear capable of discriminating among these antiarrhythmics, the ratio a_diprafenone/a_lidocaine even exceeded the value in iodate-modified Na⁺ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na⁺ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na⁺ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn.     

Solubilization and reconstitution of a vanadate-sensitive H+-ATPase from the plasma membrane ofBeta vulgaris

Summary A vanadate-sensitive H⁺-translocating ATPase isolated from red beet plasma membrane has been solubilized in active form and successfully reconstituted into artificial proteoliposomes. The H⁺-ATPase was solubilized in active form with deoxycholate, CHAPSO or octylglucoside in the presence of glycerol. Following detergent removal by gel filtration and reconstitution into proteoliposomes, ATP:Mg-dependent H⁺ transport could be measured as ionophore-reversible quenching of acridine orange fluorescence. Solubilization resulted in a three-to fourfold purification of the plasma membrane ATPase, with some additional enrichment of specific activity following reconstitution. H⁺ transport activity was inhibited half-maximally between 1 and 5 M vanadate (Na₃VO₄) and nearly abolished by 100 M vanadate. ATPase activity of native plasma membrane showed aK _i for vanadate inhibition of 9.5 M, and was inhibited up to 80% by 15 to 20 M vanadate (Na₃VO₄). ATPase activity of the reconstituted vesicles showed aK _i of 2.6 M for vanadate inhibition. The strong inhibition by low concentrations of vanadate indicates a plasma membrane rather than a mitochondrial or tonoplast origin for the reconstituted enzyme.     

Cloning,sequencing and expression of the nhaA and nhaR genes from Salmonella entiritidis

Na⁺/H⁺ antiporter activity is wide-spread and plays essential physiological roles. We found that several Enterobacteriaceae share conserved sequences with nhaA, the gene coding for an E. coli antiporter. A nhaA strain which is sensitive to Na⁺ and Li⁺, was used to clone by complementation a DNA fragment from Salmonella enteritidis which confers resistance to the ions. The cloned fragment increased Na⁺/H⁺ antiport activity in membranes isolated from strains carrying the respective hybrid plasmid. DNA sequence analysis of the insert revealed two open reading frames. Both encode putative polypeptides which are closely homologous to the nhaA and nhaR gene products from Escherichia coli. The antiporter activity displays properties very similar to that of the E. coli NhaA, namely, it is activiated by alkaline pH and recognizes Li⁺ with high affinity.Abbreviations _H ⁺ Proton electrochemical potential - pH transmembrane pH gradient - _Na ⁺ Sodium electrochemical potential - SDS Sodium dodecyl sulfate - CIP Calf intestine alkaline phosphates - ORF open reading frame     

Characterization of inside-out oriented H+-ATPases in cholate-pretreated renal brush-border membrane vesicles

Summary Exposure of porcine renal brush-border membrane vesicles to 1.2% cholate and subsequent detergent removal by dialysis reorients almost all N-ethylmaleimide (NEM)-sensitive ATPases from the vesicle inside to the outside. ATP addition to cholate-pretreated, but not to intact, vesicles causes H⁺ uptake as visualized by the pH indicator, acridine organge. The reoriented H⁺-pump is electrogenic because permeant extravesicular anions or intravesicular K⁺ plus valinomycin enhance H⁺ transport. ATP stimulates H⁺ uptake with an apparentK _m of 93 m. Support of H⁺ uptake andP _i liberation by ATP>GTPITP> UTP indicates a preference for ATP and utilization of other nucleotides at lower efficiency. ADP is a potent, competitive inhibitor of ATP-driven H⁺ uptake,(K _i , 24 m). Mg²⁺ and Mn^2– support ATP-driven H⁺ uptake, but Ca²⁺, Ba²⁺ and Zn²⁺ do not. Imm Zn²⁺ inhibits MgATP-driven H⁺ transport completely. NEM-sensitiveP _i liberation is stimulated by Mg²⁺ and Mg^2– and, unlike H⁺ uptake, also by Ca²⁺ suggesting Ca²⁺-dependent ATP hydrolysis unrelated to H⁺ transport. The inside-out oriented H⁺-pump is relatively insensitive toward oligomycin, azide, N,N-dicyclohexylcarbodiimide (DCCD) and vanadate, but efficiently inhibited by NEM (apparentK _i , 0.77 m), and 4-chloro-7-nitro-benzoxa-1,3-diazole (NBD-Cl; apparentK _i , 0.39 m). Taken together, the H⁺-ATPase of proximal tubular brush-border membranes exhibits characteristics very similar to those of vacuolar type (V-type) H⁺-ATPases. Hence,V-type H⁺-ATPases occur not only in intracellular organelles but also in specialized plasma membrane areas.     

Molecular species of membrane phospholipids containing arachidonic acid and linoleic acid contribute to the interindividual variability of red blood cell Na+-Li+ countertransport: In vivo and in vitro evidence

Summary Previous studies indicate a particular sensitivity of red blood cell Na⁺-Li⁺ countertransport activity to small variations in the fatty acid composition of membrane phospholipids. To assess whether the interindividual variability of Na⁺-Li⁺ countertransport is related to differences in the species pattern of erythrocyte phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vivo, the molecular species composition of PC and PE as well as the kinetics of Na⁺-Li⁺ countertransport were analyzed in parallel in normo- and hyperlipidemic donors. Both in diacyl PC and in diacyl-PE the species 160/204 and 160/182 were, respectively, positively and negatively related to the apparent maximal velocity of Na⁺-Li⁺ countertransport. The sum of all species with 204 at sn₂ of diacyl-PE exhibited a strong positive (r = 0.82, 2p < 0.001), and those containing 182 a negative correlation (r = –0.63, 2p < 0.01) to the transport activity. Essentially similar connections were observed between these species and the apparent affinity of the transport system for intracellular Na⁺. To evaluate whether the associations between molecular species of membrane phospholipids and Na⁺-Li⁺ countertransport activity were indicative of a causal relationship, the species 160/204-PC and 160/182-PC were selectively introduced into the erythrocyte membrane by means of the PC-specific transfer protein. Replacement of 11% of native PC by 160/182-PC inhibited the transport rate by about 25%. Exchange of 6 and 9% of PC with 160/204-PC, in contrast, accelerated the transport rate by 30 and 60%, respectively. The accordance between the in vivo relations and the results of the in vitro modification strongly suggests that elevations and reductions in the arachidonic acid and linoleic acid content of membrane PC and PE contribute to the interindividual variability of red blood cell Na⁺-Li⁺ counter-transport activity and its acceleration in hyperlipidemias.The authors wish to thank Dr. W.O. Richter (II. Medizinische Klinik, Klinikum Großhadern, Universität München) for selection of the patients and Dr. T. Brosche (Universität ErlangenNürnberg) for gaschromatographic analyses. This study was supported in part by a grant of the Wilhelm-Sander-Stiftung to B.E.     

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.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Uptake of lysine and prolinevia separate α-neutral amino acid transport pathways inMytilus gill brush border membranes

Summary Brush border membrane vesicles (BBMV) were prepared from the gills of the marine mussel,Mytilus edulis. These membranes contained two distinct pathways for cotransport of Na⁺ and -neutral amino acids. The major pathway in mussel gill BBMV was the alanine-lysine (AK) pathway, which had a high affinity for alanine and for the cationic amino acid, lysine. The AK pathway was inhibited by nonpolar -neutral amino acids and cationic amino acids, but was not affected by -neutral amino acids or imino acids. The kinetics of lysine transport were consistent with a single saturable process, with aJ _max of 550 pmol/mg-min and aK _t of 5 m. The AK pathway did not have a strict requirement for Na⁺, and concentrative transport of lysine was seen in the presence of inwardly directed gradients of Li⁺ and K⁺, as well as Na⁺. Harmaline inhibited the transport of lysine in solutions containing either Na⁺ or K⁺. The alanine-proline (AP) pathway transported both alanine and proline in mussel gill BBMV. The AP pathway was strongly inhibited by nonpolar -neutral amino acids, proline, and -(methylamino)isobutyric acid (Me-AIB). The kinetics of proline transport were described by a single saturable process, with aJ _max of 180 pmol/mg-min andK _t of 4 m. In contrast to the AK pathway, the AP pathway appeared to have a strict requirement for Na⁺. Na⁺-activation experiments with lysine and proline revealed sigmoid kinetics, indicating that multiple Na⁺ ions are involved in the transport of these substrates. The transport of both lysine and proline was affected by membrane potential in a manner consistent with electrogenic transport.     

Characterization of a Na+/glucose cotransporter cloned from rabbit small intestine

Summary The Na⁺/glucose cotransporter from rabbit intestinal brush border membranes has been cloned, sequenced, and expressed inXenopus oocytes. Injection of cloned RNA into oocytes increased Na⁺/sugar cotransport by three orders of magnitude. In this study, we have compared and contrasted the transport properties of this cloned protein expressed inXenopus oocytes with the native transporter present in rabbit intestinal brush borders. Initial rates of¹⁴C--methyl-d-glucopyranoside uptake into brush border membrane vesicles andXenopus oocytes were measured as a function of the external sodium, sugar, and phlorizin concentrations. Sugar uptake into oocytes and brush borders was Na⁺-dependent (Hill coefficient 1.5 and 1.7), phlorizin inhibitable (K _i 6 and 9 m), and saturable (-methyl-d-glucopyranosideK _m 110 and 570 m). The sugar specificity was examined by competition experiments, and in both cases the selectivity wasd-glucose>-methyl-d-glucopyranoside>d-galactose>3-O-methyl-d-glucoside. In view of the close similarity between the properties of the cloned protein expressed in oocytes and the native brush border transporter, we conclude that we have cloned the classical Na⁺/glucose cotransporter.     

Apical sodium entry in split frog skin: Current-voltage relationship

Summary Apical Na⁺ entry into frog skin epithelium is widely presumed to be electrodiffusive in nature, as for other tight epithelia. However, in contrast to rabbit descending colon andNecturus urinary bladder, the constant field equation has been reported to fit the apical sodium current (N _Na)-membrane potential (^mc) relationship over only a narrow range of apical membrane potentials or to be inapplicable altogether. We have re-examined this issue by impaling split frog skins across the basolateral membrane and examining the current-voltage relationships at extremely early endpoints in time after initiating pulses of constant transepithelial voltage. In this study, the rapid transient responses in ^mc were completed within 0.5 to 3.5 msec. Using endpoints to 1 to 25 msec, the Goldman equation provided excellent fits of the data over large ranges in apical potential of 300 to 420 mV, from approximately –200 to about +145 mV (cell relative to mucosa). Split skins were also studied when superfused with high serosal K⁺ in order to determine whether theI _Na-^mc relationship could be generated purely by transepithelial measurements. Under these conditions, the basolateral membrane potential was found to be –10±3 mV (cell relative to serosa, mean±se), the basolateral fractional resistance was greater than zero, and the transepithelial current was markedly and reversibly reduced. For these reasons, use of high serosal K⁺ is considered inadvisable for determining theI _Na-^mc relationship, at least in those tissues (such as frog skin) where more direct measurements are technically feasible. Analysis of theI _Na-^mc relationships under baseline conditions provided estimates of intracellular Na⁺ concentration and of apical Na⁺ permeability of 9 to 14mm and of 3 × 10^–7 cm · sec^–1, respectively, in reasonable agreement with estimates obtained by different techniques.     

Interaction among anion,cation and glucose transport proteins in the human red cell

Summary The time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4-dibenzamido-2,2-stilbene disulfonate), to band 3 can be measured by the stopped-flow method. We have previously used the reaction time constant, _DBDS, to obtain the kinetic constants for binding and, thus, to report on the conformational state of the band 3 binding site. To validate the method, we have now shown that the ID₅₀ (0.3±0.1 m) for H₂-DIDS (4,4-diisothiocyano-2,2-dihydrostilbene disulfonate) inhibition of _DBDS is virtually the same as the ID₅₀ (0.47±0.04 m) for H₂-DIDS inhibition of red cell Cl^– flux, thus relating _DBDS directly to band 3 anion exchange. The specific glucose transport inhibitor, cytochalasin B, causes significant changes in _DBDS, which can be reversed with intracellular, but not extracellular,d-glucose. ID₅₀ for cytochalasin B modulation of _DBDS is 0.1±0.2 m in good agreement withK _D =0.06±0.005 m for cytochalasin B binding to the glucose transport protein. These experiments suggest that the glucose transport protein is either adjacent to band 3, or linked to it through a mechanism, which can transmit conformational information. Ouabain (0.1 m), the specific inhibitor of red cell Na⁺,K⁺-ATPase, increases red cell Cl^– exchange flux in red cells by a factor of about two. This interaction indicates that the Na⁺,K⁺-ATPase, like the glucose transport protein, is either in contact with, or closely linked to, band 3. These results would be consistent with a transport proteincomplex, centered on band 3, and responsible for the entire transport process, not only the provision of metabolic energy, but also the actual carriage of the cations and anions themselves.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

K+ transport in ‘Tight’ epithelial monolayers of MDCK cells

Summary Bidirectional transepithelial K⁺ flux measurements across high-resistance epithelial monolayers of MDCK cells grown upon millipore filters show no significant net K⁺ flux.Measurements of influx and efflux across the basal-lateral and apical cell membranes demonstrate that the apical membranes are effectively impermeable to K⁺.K⁺ influx across the basal-lateral cell membranes consists of an ouabain-sensitive component, an ouabain-insensitive component, an ouabain-insensitive but furosemide-sensitive component, and an ouabain-and furosemide-insensitive component.The action of furosemide upon K⁺ influx is independent of (Na⁺–K⁺)-pump inhibition. The furosemide-sensitive component is markedly dependent upon the medium K⁺, Na⁺ and Cl^– content. Acetate and nitrate are ineffective substitutes for Cl^–, whereas Br^– is partially effective. Partial Cl^– replacement by NO₃ gives a roughly linear increase in the furosemide-sensitive component. Na⁺ replacement by choline abolishes the furosemide-sensitive component, whereas Li⁺ is a partially effective replacement. Partial Na⁺ replacement with choline gives an apparent affinity of 7mm Na, whereas variation of the external K⁺ content gives an affinity of the furosemide-sensitive component of 1.0mm.Furosemide inhibition is of high affinity (K _1/2=3 m). Piretanide, ethacrynic acid, and phloretin inhibit the same component of passive K⁺ influx as furosemide; amiloride, 4,-aminopyridine, and 2,4,6-triaminopyrimidine partially so. SITS was ineffective.Externally applied furosemide and Cl^– replacement by NO ₃ ^– inhibit K⁺ efflux across the basal-lateral membranes indicating that the furosemide-sensitive component consists primarily of KK exchange.     

Role of the furosemide-sensitive Na+/K+ transport system in determining the steady-state Na+ and K+ content and volume of human erythrocytesin vitro andin vivo

Summary To study the physiological role of the bidirectionally operating, furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes, the effect of furosemide on red cell cation and hemoglobin content was determined in cells incubated for 24 hr with ouabain in 145mm NaCl media containing 0 to 10mm K⁺ or Rb⁺. In pure Na⁺ media, furosemide accelerated cell Na⁺ gain and retarded cellular K⁺ loss. External K⁺ (5mm) had an effect similar to furosemide and markedly reduced the action of the drug on cellular cation content. External Rb⁺ accelerated the Na⁺ gain like K⁺, but did not affect the K⁺ retention induced by furosemide. The data are interpreted to indicate that the furosemide-sensitive Na⁺/K⁺ transport system of human erythrocytes mediates an equimolar extrusion of Na⁺ and K⁺ in Na⁺ media (Na⁺/K⁺ cotransport), a 1:1 K⁺/K⁺ (K⁺/Rb⁺) and Na⁺/Na⁺ exchange progressively appearing upon increasing external K⁺ (Rb⁺) concentrations to 5mm. The effect of furosemide (or external K⁺/Rb⁺) on cation contents was associated with a prevention of the cell shrinkage seen in pure Na⁺ media, or with a cell swelling, indicating that the furosemide-sensitive Na⁺/K⁺ transport system is involved in the control of cell volume of human erythrocytes. The action of furosemide on cellular volume and cation content tended to disappear at 5mm external K⁺ or Rb⁺. Thein vivo red cell K⁺ content was negatively correlated to the rate of furosemide-sensitive K⁺ (Rb⁺) uptake, and a positive correlation was seen between mean cellular hemoglobin content and furosemide-sensitive transport activity. The transport system possibly functions as a K⁺ and waterextruding mechanism under physiological conditiosin vivo. The red cell Na⁺ content showed no correlation to the activity of the furosemide-sensitive transport system.     

Agonist-induced activation of Na+/H+ exchange in rat parotid acinar cells

Summary The present studies were designed to test our previous suggestion that Na⁺/H⁺ exchange was activated by muscarinic stimulation of rat parotid acinar cells. Consistent with this hypothesis, we demonstrate here that intact rat parotid acini stimulated with the muscarinic agonist carbachol in HCO ₃ ^– -free medium show an enhanced recovery from an acute acid load as compared to similarly challenged untreated preparations. Amiloride-sensitive²²Na uptake, due to Na⁺/H⁺ exchange, was also studied in plasma membrane vesicles prepared from rat parotid acini pretreated with carbachol. This uptake was stimulated twofold relative to that observed in vesicles from control (untreated) acini. This stimulation was time dependent, requiring 15 min of acinar incubation with carbachol to reach completion, and ws blocked by the presence of the muscarinic antagonist atropine (2×10^–5 m) in the pretreatment medium. The effect of carbachol was dose dependent withK _0.53×10^–6 m. Stimulation of the exchanger was also seen in vesicles prepared from acini pretreated with the -adrenergic agonist epinephrine, but not with the -adrenergic agonist isoproterenol, or with substance P. Kinetic analysis indicated that the stimulation induced by carbachol was due to an alkaline shift in the pH responsiveness of the exchanger in addition to an increasedapparent transport capacity. Taken together with previous results from this and other laboratories, these results strongly suggest that the Na⁺/H⁺ exchanger and its regulation are intimately involved in the fluidsecretory response of the rat parotid.     

Voltage dependence of the Ca2+-activated K+ conductance of human red cell membranes is strongly dependent on the extracellular K+ concentration

Summary The conductance of the Ca²⁺-activated K⁺ channel (g _K(Ca)) of the human red cell membrane was studied as a function of membrane potential (V _m ) and extracellular K⁺ concentration ([K⁺]_ex). ATP-depleted cells, with fixed values of cellular K⁺ (145mm) and pH (7.1), and preloaded with 27 m ionized Ca were transferred, with open K⁺ channels, to buffer-free salt solutions with given K⁺ concentrations. Outward-current conductances were calculated from initial net effluxes of K⁺, correspondingV _m , monitored by CCCP-mediated electrochemical equilibration of protons between a buffer-free extracellular and the heavily buffered cellular phases, and Nernst equilibrium potentials of K ions (E _K) determined at the peak of hyperpolarization. Zero-current conductances were calculated from unidirectional effluxes of⁴²K at (V _m –E _K)0, using a single-file flux ratio exponent of 2.7. Within a [K⁺]_ex range of 5.5 to 60mm and at (V _m –E _K) 20 mV a basic conductance, which was independent of [K⁺]_ex, was found. It had a small voltage dependence, varying linearly from 45 to 70 S/cm² between 0 and –100 mV. As (V _m –E _K) decreased from 20 towards zero mVg _K(Ca) increased hyperbolically from the basic value towards a zero-current value of 165 S/cm². The zero-current conductance was not significantly dependent on [K⁺]_ex (30 to 156mm) corresponding toV _m (–50 mV to 0). A further increase ing _K(Ca) symmetrically aroundE _K is suggested as (V _m –E _K) becomes positive. Increasing the extracellular K⁺ concentration from zero and up to 3mm resulted in an increase ing _K(Ca) from 50 to 70 S/cm². Since the driving force (V _m –E _K) was larger than 20 mV within this range of [K⁺]_ex this was probably a specific K⁺ activation ofg _K(Ca). In conclusion: The Ca²⁺-activated K⁺ channel of the human red cell membrane is an inward rectifier showing the characteristic voltage dependence of this type of channel.     

An Amiloride-sensitive Na+ conductance in the basolateral membrane of toad urinary bladder

Summary Exposing the apical membrane of toad urinary bladder to the ionophore nystatin lowers its resistance to less than 100 cm². The basolateral membrane can then be studied by means of transepithelial measurements. If the mucosal solution contains more than 5mm Na⁺, and serosal Na⁺ is substituted by K⁺, Cs⁺, or N-methyl-d-glucamine, the basolateral membrane expresses what appears to be a large Na⁺ conductance, passing strong currents out of the cell. This pathway is insensitive to ouabain or vanadate and does not require serosal or mucosal Ca²⁺. In Cl-free SO ₄ ^2– Ringer's solution it is the major conductive pathway in the basolateral membrane even though the serosal side has 60mm K⁺. This pathway can be blocked by serosal amiloride (K _i=13.1 m) or serosal Na⁺ ions (K _i 10 to 20mm). It also conducts Li⁺ and shows a voltage-dependent relaxation with characteristic rates of 10 to 20 rad sec^–1 at 0 mV.     

Rabit distal colon epithelium: II. Characterization of (Na+, K+, Cl−)-cotransport and [3H]-bumetanide binding

Summary Loop diuretic-sensitive (Na⁺,K⁺,Cl^–)-cotransport activity was found to be present in basolateral membrane vesicles of surface and crypt cells of rabbit distal colon epithelium. The presence of grandients of all three ions was essential for optimal transport activity (Na⁺,K⁺) gradien-driven³⁶Cl fluxes weree half-maximally inhibited by 0.14 m bumetanide and 44 m furosimide. While⁸⁶Rb uptake rates showed hyperbolic dependencies on Na⁺ and K⁺ concentrations with Hill coefficients of 0.8 and 0.9, respectively, uptakes were sigmoidally related to the Cl^– concentration, Hill coefficient 1.8, indicating a 1 Na⁺: 1 K⁺:2 Cl^– stoichiometry of ion transport.The interaction of putative (Na⁺, K⁺, Cl^–)-cotransport proteins with loop diuretics was studied from equilibrium-binding experiments using [³H]-bumetanide. The requirement for the simulataneous presence of Na⁺,K⁺, and Cl^–, saturability, reversibility, and specificity for diuretics suggest specific binding to the (Na⁺, K⁺, Cl^–)-cotransporter. [³H]-bumetanide recognizes a minimum of two classes of diuretic receptors sites. high-affinity (K _D1=0.13 m;B _max1 =6.4 pmol/mg of protein) and low-affinity (K _D2=34 m;B _max2=153 pmol/mg of protein) sites. The specific binding to the high-affinity receptor was found to be linearly competitive with Cl^– (K ₁=60mm), whereas low-affinity sites seem to be unaffected by Cl^–. We have shown that only high-affinity [³H]-bumetanide binding correlates with transport inhibition raising questions on the physiological significance of diuretic receptor site heterogeneity observed in rabbit distal colon epithelium.