Mode of action of theophylline on sodium efflux in barnacle muscle fibers

Summary The response of the Na efflux in unpoisoned barnacle fibers to 10mm theophylline is biphasic; i.e., inhibition is followed by stimulation. The stimulatory response is unaffected by ouabain. Fibers pretreated with ouabain show no transitory inhibition when 10mm theophylline is applied, but show prompt stimulation the magnitude of which is comparable to that observed with unpoisoned fibers. The same holds true for lower concentrations of theophylline. Prior injection of 500mm EGTA completely abolishes the biphasic action of 10mm theophylline. External application of 10mm theophylline following removal of external Ca²⁺ fails to bring about a biphasic effect. Ca²⁺ restoration, however, results in a moderate rise in the Na efflux. External application of 10mm theophylline stimulates the Na efflux into Ca²⁺-free artificial seawater (ASW) when the test fibers are pretreated with ouabain. Injection of the protein inhibitor of Walsh leads to reduced stimulation by 10mm theophylline of the Na efflux in unpoisoned fibers. Injection of the protein inhibitor of Corbin into unpoisoned fibers leads to reduced stimulation by 10mm theophylline. Injection of cAMP into ouabainpoisoned fibers, following internal application of Corbin's inhibitor and external application of 10mm theophylline, fails to cause a marked rise in the ouabain-insensitive Na efflux. Injection of Corbin's inhibitor into ouabain-poisoned fibers, following the onset of peak stimulation by 10mm theophylline, fails to reduce the Na efflux. Fibers injected with 1mm and 100mm EGTA and exposed to 10mm theophylline show a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP when the concentration of theophylline is 10mm. A poor response to injected cAMP is also seen in fibers bathed in Ca-free ASW containing 10mm theophylline. Theophylline (10mm) fails to cause an enhanced stimulation of the ouabain-insensitive Na efflux into Ca-free 3mm-HEPES ASW or 10mm-Ca²⁺-3mm-HEPES ASW following the addition of protons to the bathing medium. An enhanced response is similarly not observed with injected cAMP following the addition of theophylline to the bathing medium. Injection of 8-fluorotheophylline, 3-isobutyl-1-methylxanthine and doxantrazole leads to a marked reduction in the response of the ouabain-insensitive Na efflux to injected cAMP. Contraction always takes place upon injecting these substances. These results are in keeping with the theory that theophylline acts chiefly by reducing myoplasmic pCa (pCa-log₁₀[Ca²⁺]), and that a reduced pCa leads to stimulation of the ouabain-insensitive Na efflux as the result of activation of the cGMP-dependent protein kinase system by newly formed cGMP.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

ATP-dependent calcium transport in rat parotid basolateral membrane vesicles is modulated by membrane potential

Summary The ATP-dependent Ca²⁺ transport activity (T. Takuma, B.L. Kuyatt and B.J. Baum,Biochem. J. 227:239–245, 1985) exhibited by inverted basolateral membrane vesicles isolated from rat parotid gland was further characterized. The activity was dependent on Mg²⁺. Phosphate (5mm), but not oxalate (5mm), increased maximum Ca²⁺ accumulation by 50%. Half-maximal Ca²⁺ transport was achieved at 70nm Ca²⁺ in EGTA-buffered medium while maximal activity required >1 m Ca²⁺ (V _max=54 nmol/mg protein/min). Optimal rates of Ca²⁺ transport were obtained in the presence of KCl, while in a KCl-free medium (mannitol or sucrose) 40% of the total activity was achieved, which could not be stimulated by FCCP. The initial rate of Ca²⁺ transport could be significantly altered by preimposed membrane potentials generated by K⁺ gradients in the presence of valinomycin. Compared to the transport rate in the absence of membrane potential, a negative (interior) potential stimulated uptake by 30%, while a positive (interior) potential inhibited uptake. Initial rates of Ca²⁺ uptake could also be altered by imposing pH gradients, in the absence of KCl. When compared to the initial rate of Ca²⁺ transport in the absence of a pH gradient, pH _i =7.5/pH _o =7.5; the activity was 60% higher in the presence of an outwardly directed pH gradient, pH _i =7.5/pH _o =8.5; while it was 80% lower when an inwardly directed pH gradient was imposed, pH _i =7.5/pH _o =6.2. The data show that the ATP-dependent Ca²⁺ transport in BLMV can be modulated by the membrane potential, suggesting therefore that there is a transfer of charge into the vesicle during Ca²⁺ uptake, which could be compensated by other ion movements.     

Regulation of maxi-K+ channels on pancreatic duct cells by cyclic AMP-dependent phosphorylation

Summary Using the patch-clamp technique we have identified a Ca²⁺-sensitive, voltage-dependent, maxi-K⁺ channel on the basolateral surface of rat pancreatic duct cells. The channel had a conductance of 200 pS in excised patches bathed in symmetrical 150mm K⁺, and was blocked by 1mm Ba²⁺. Channel openstate probability (P _o ) on unstimulated cells was very low, but was markedly increased by exposing the cells to secretin, dibutyryl cyclic AMP, forskolin or isobutylmethylxanthine. Stimulation also shifted theP _o /voltage relationship towards hyperpolarizing potentials, but channel conductance was unchanged. If patches were excised from stimulated cells into the inside-out configuration,P _o remained high, and was not markedly reduced by lowering bath (cytoplasmic) Ca²⁺ concentration from 2mm to 0.1 m. However, activated channels were still blocked by 1mm Ba²⁺. ChannelP _o was also increased by exposing the cytoplasmic face of excised patches to the purified catalytic subunit of cyclic AMP-dependent protein kinase., We conclude that cyclic AMP-dependent phosphorylation can activate maxi-K⁺ channels on pancreatic duct cells via a stable modification of the channel protein itself, or a closely associated regulatory subunit, and that phosphorylation alters the responsiveness of the channels to Ca²⁺. Physiologically, these K⁺ channels may contribute to the basolateral K⁺ conductance of the duct cell and, by providing a pathway for current flow across the basolateral membrane, play an important role in pancreatic bicarbonate secretion.     

The dual effect of rubidium ions on potassium efflux in depolarized frog skeletal muscle

Summary The effects of external Rb⁺ on the efflux of⁴²K⁺ from whole frog sartorius muscles loaded with 305mm K⁺ and 120mm Cl^– were studied. K⁺ efflux is activated by [Rb⁺] _o less than about 40mm according to a sigmoid relation similar to that for activation by [K⁺] _o . At [Rb⁺]_o greater than 40mm, K⁺ efflux declines, although at [Rb⁺] _o =300mm it is still greater than at [Rb⁺] _o =0mm. For low concentrations, the increment in K⁺ efflux over that in K⁺- and Rb⁺-free solution, k, is described by the relation k=a[X⁺] _o ⁿ , for both K⁺ and Rb⁺. The value ofa is larger for Rb⁺ than for K⁺, while the values ofn are similar; the activation produced by a given [Rb⁺] _o is larger than that by an equal [K⁺] _o for concentrations less than about 40mm. Adding a small amount of Rb⁺ to a K⁺-containing solution has effects on K⁺ efflux which depend on [K⁺] _o . At low [K⁺] _o , adding Rb⁺ increases K⁺ efflux, the effect being greatest near [K⁺] _o =30mm and declining at higher [K⁺] _o ; at [K⁺] _o above 40mm, addition of Rb⁺ decreases K⁺ efflux. At [K⁺] _o above 75mm, where K⁺ efflux is largely activated, Rb⁺ reduces K⁺ efflux by a factorb, described by the relationb=1/(1+c[Rb⁺] _o ). Activation is discussed in terms of binding to at least two sites in the membrane, and the reduction in K⁺ efflux by Rb⁺ at high [K⁺] _o in terms of association with an additional inhibitory site.     

Direct inhibitory action of EGTA-Ca complex on reverse-mode Na/Ca exchange inMyxicola giant axons

Summary Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing²²Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca²⁺] _o , was measured using dialysis to maintain [Na⁺] _i at 100mm, which enhances the [Ca²⁺] _o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca²⁺] _i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca²⁺] _i to activate Na/Ca exchange. The addition of EGTA inhibited Ca _o -dependent Na efflux even when a large enough excess of [Ca²⁺] _i was present to saturate the EGTA and still produce elevated values of [Ca²⁺] _i . Control experiments showed that these high values of [Ca²⁺] _i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.     

Modification of Ca2+-activated K+ channels in cultured medullary thick ascending limb cells by N-bromoacetamide

Summary Ca²⁺-activated K⁺ channels were studied in cultured medullary thick ascending limb (MTAL) cells using the patch-clamp technique in the inside-out configuration. The Ca²⁺ activation site was modified using N-bromoacetamide (NBA). 1mm NBA in the bath solution, at 2.5 m Ca²⁺ reduces the open probability,P _o , of the channel to <0.01, without an effect on single-channel conductance. NBA-modified channels are still Ca²⁺-sensitive, requiring 25mm Ca²⁺ to raiseP _o to 0.2. Both before and after NBA modification channel openings display at least two distributions, indicative of more than one open state. High Ca²⁺ (1mm) protects the channels from modification. Also presented is a second class of Ca²⁺-activated K⁺ channels which are normally present in MTAL cells which open infrequently at 10 m Ca²⁺ (P _o =0.01) but have aP _o of 0.08 at 1mm Ca²⁺. We can conclude (i) that NBA modifies the channel by shifting Ca²⁺-sensitivity to very high Ca²⁺, (ii) that NBA acts on a site involved in Ca²⁺ gating, and (iii) that a low affinity channel is present in the apical cell membrane with characteristics similar to those of normal channels modified with NBA.     

Influence of external barium and potassium on potassium efflux in depolarized frog sartorius muscles

Summary Efflux of⁴²K⁺ was measured in frog sartorius muscles equilibrated in depolarizing solutions with external K⁺ concentrations ([K⁺] _o ) between 75 and 300mm and NaCl concentrations of 60, 120, or 240mm. For several combinations of KCl and NaCl, steady-state internal potentials (V _i) were the same for different [K⁺] _o . For the range ofV _i examined, K⁺ efflux occurs principally through the K⁺ inward rectifier channels. When external K⁺ is removedV _i remains constant for 2 to 3 hr because of the high membrane conductance to Cl^–, but K⁺ efflux drops by about one order of magnitude.External Ba²⁺ in the presence or absence of external K⁺ produces an inhibition of K⁺ efflux described by a relation of the formu=(u₁/(1+C)[Ba²⁺] _o ))+u ₂, whereu is the uninhibited fraction of K⁺ efflux;u ₁, u₂ andC are constants; andu ₁+u₂=1.C depends both on [K⁺] _o andV _i. When [K⁺] _o 75mm, increasing [K⁺] _o at constantV _i reduces Ba²⁺ sensitivity. For constantV _i–30 mV, Ba²⁺ sensitivity is less when [K⁺] _o =0 than when [K⁺] _o 75mm. When [K⁺] _o =0, Ba²⁺ sensitivity decreases asV _i is made more positive. The dependence of the Ba²⁺ sensitivity onV _i at constant [K⁺] _o is greater when [K⁺] _o =0 than when [K⁺] _o 75mm.Both the activation of K⁺ efflux by external K⁺ and the Ba²⁺ inhibition of K⁺ efflux can be explained on the basis of two membrane control sites associated with each channel. When both sites are occupied by K⁺, the channels are in a high flux state. When one or both sites are empty, the channels are in a low, nonzero flux state. When Ba²⁺ occupies either site, K⁺ efflux is further reduced. The reduction of Ba²⁺-sensitivity by increasing [K⁺] _o at high [K⁺] _o is attributable to the displacement of Ba²⁺ from the control sites by K⁺. The increased Ba²⁺ sensitivity produced by going from [K⁺] _o =0 to [K⁺] _o >-75mm whenV _i–30 mV is attributable to states in which Ba²⁺ occupies one site and K⁺ the other when [K⁺] _o 0. The smallerV _i dependence of the Ba²⁺ sensitivity when [K⁺] _o 75mm compared to [K⁺] _o =0 is attributable to the necessity that Ba²⁺ displace K⁺ at the control sites when [K⁺] _o is high but not when [K⁺] _o =0.     

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.     

Metabolic control of the K+ channel of human red cells

Summary The effects of cAMP, ATP and GTP on the Ca²⁺-dependent K⁺ channel of fresh (1–2 days) or cold-stored (28–36 days) human red cells were studied using atomic absorption flame photometry of Ca²⁺-EGTA loaded ghosts which had been resealed to monovalent cations in dextran solutions. When high-K⁺ ghosts were incubated in an isotonic Na⁺ medium, the rate constant of Ca²⁺-dependent K⁺ efflux was reduced by a half on increasing the theophylline concentration to 40mm. This effect was observed in ghosts from both fresh and stored cells, but only if they were previously loaded with ATP. The inhibition was more marked when Mg²⁺ was added together with ATP, and it was abolished by raising free Ca²⁺ to the micromolar level. Like theophylline, isobutyl methylxanthine (10mm) also affected K⁺ efflux. cAMP (0.2–0.5mm), added both internally and externally (as free salt, dibutyryl or bromide derivatives), had no significant effect on K⁺ loss when the ghost free-Ca²⁺ level was below 1 m, but it was slightly inhibitory at higher concentrations. The combined presence of cAMP (0.2mm) plus either theophylline (10mm), or isobutyl methylxanthine (0.5mm), was more effective than cAMP alone. This inhibition showed a strict requirement for ATP plus Mg²⁺ and it, was not overcome by raising internal Ca²⁺. Ghosts from stored cells seemed more sensitive than those from fresh cells, to the combined action of cAMP and methylxanthines. Loading ATP into ghosts from fresh or stored cells markedly decreased K⁺ loss. Although this effect was observed in the absence of added Mg²⁺ (0.5mm EDTA present), it was potentiated upon adding 2mm Mg²⁺. The K⁺ efflux from ATP-loaded ghosts was not altered by dithio-bis-nitrobenzoic acid (10mm) or acridine orange (100 m), while it was increased two-to fourfold by incubating with MgF₂ (10mm), or MgF₂ (10mm)+theophylline (40mm), respectively. By contrast, a marked efflux reduction was obtained by incorporating 0.5mm GTP into ATP-containing ghosts. The degree of phosphorylation obtained by incubating membranes with (-³²P)ATP under various conditions affecting K⁺ channel activity, was in direct correspondence to their effect on K⁺ efflux. The results suggest that the K⁺ channel of red cells is under complex metabolic control, via cAMP-mediated and nonmediated mechanisms, some which require ATP and presumably, involve phosphorylation of the channel proteins.     

Ca2+-activated K+ conductance of the human red cell membrane: Voltage-dependent Na+ block of outward-going currents

Summary Human red cells were prepared with various cellular Na⁺ and K⁺ concentrations at a constant sum of 156mm. At maximal activation of the K⁺ conductance,g _K(Ca), the net efflux of K⁺ was determined as a function of the cellular Na⁺ and K⁺ concentrations and the membrane potential,V _m , at a fixed [K⁺]_ex of 3.5mm.V _m was only varied from (V _m –E _K)25 mV and upwards, that is, outside the range of potentials with a steep inward rectifying voltage dependence (Stampe & Vestergaard-Bogind, 1988).g _K(Ca) as a function of cellular Na⁺ and K⁺ concentrations atV _m =–40, 0 and 40 mV indicated a competitive, voltage-dependent block of the outward current conductance by cellular Na⁺. Since the present Ca²⁺-activated K⁺ channels have been shown to be of the multi-ion type, the experimental data from each set of Na⁺ and K⁺ concentrations were fitted separately to a Boltzmann-type equation, assuming that the outward current conductance in the absence of cellular Na⁺ is independent of voltage. The equivalent valence determined in this way was a function of the cellular Na⁺ concentration increasing from 0.5 to 1.5 as this concentration increased from 11 to 101mm. Data from a previous study of voltage dependence as a function of the degree of Ca²⁺ activation of the channel could be accounted for in this way as well. It is therefore suggested that the voltage dependence ofg _K(Ca) for outward currents at (V _m –E _K)>25 25 mV reflects a voltage-dependent Na⁺ block of the Ca²⁺-activated K⁺ channels.     

Asymmetric effects of divalent cations and protons on active Ca2+ efflux and Ca2+-ATPase in intact red blood cells

Summary The influence of the asymmetric addition of various divalent cations and protons on the properties of active Ca²⁺ transport have been examined in intact human red blood cells. Active Ca²⁺ efflux was determined from the initial rate of⁴⁵Ca²⁺ loss after CoCl₂ was added to block Ca²⁺ loading via the ionophore A23187. Ca²⁺-ATPase activity was measured as phosphate production over 5 min in cells equilibrated with EGTA-buffered free Ca²⁺ in the presence of A23187. The apparent Ca affinity of active Ca²⁺ efflux (K _0.5=30–40 mol/liter cells) was significantly lower than that measured by the Ca²⁺-ATPase assay (K _0.5=0.4 m). Possible reasons for this apparent difference are considered. Both active Ca²⁺ efflux and Ca²⁺-ATPase activity were reduced to less than 5% of maximal levels (20 mmol/liter cells · hr) in Mg²⁺-depleted cells, and completely restored by reintroduction of intracellular Mg²⁺. Active Ca²⁺ efflux was inhibited almost completely by raising external CaCl₂ (but not MgCl₂) to 20mm, probably by interaction of Ca²⁺ at the externally oriented E₂P conformation of the pump. Cd²⁺ was more potent than Ca²⁺ in this inhibition, while Mn²⁺ was less potent and 10mm Ba²⁺ was without effect. A Ca²⁺: proton exchange mechanism for active Ca²⁺ efflux was supported by the results, as external protons (pH 6–6.5) stimulated active Ca²⁺ efflux at least twofold above the efflux rate at pH 7.8 Ca²⁺ transport was not affected by decreasing the membrane potential across the red cell.     

Calcium efflux from human erythrocyte ghosts

Summary The passive Ca efflux from human red cell ghosts was studied in media of differing ion compositions and compared to the ATP-dependent Ca efflux. Cells were loaded with⁴⁵Ca during reversible hemolysis, and the loss of radioactivity into the non-radioactive incubation medium was measured, usually for 3 hr at 37°C. Analysis of the efflux curves revealed that⁴⁵Ca efflux followed the kinetics of a simple two-compartment system. In the concentration range between 0 and 1mm Ca in the external solution ([Ca⁺⁺] _o ), the rate constant of passive Ca efflux (k min^–1, fraction of⁴⁵Ca lost per minute into the medium) increased from 0.00732 to 0.0150 min^–1. There was no further increase at higher [Ca⁺⁺] _o . The relation between the rate constant of Ca efflux and [Ca⁺⁺] _o is thus characterized by saturation kinetics. The passive transfer system for Ca could also be activated by Sr. The alkali metal ions Na, K and Li did not seem to have any significant influence on passive Ca transfer. The passive Ca efflux was slightly inhibited by Mg and strongly inhibited by Pb. Under most experimental conditions, a fraction of 15 to 50% of the intracellular Ca seemed to be inexchangeable. The inexchangeable fraction decreased with increasing [Ca⁺⁺] _o and increased with increasing [Ca⁺⁺] _i . It was not influenced by alkali metal ions, CN or Pb, but it could be completely removed from the cells by the addition of 0.1mm Mersalyl to the incubation medium or by hemolysis with addition of a detergent. The active ATP-dependent Ca transport differed characteristically from passive transfer; the rate constant decreased with increasing [Ca⁺⁺] _o , and the inexchangeable Ca fraction increased with increasing [Ca⁺⁺] _o . The experimental results suggest that there exists a carrier-mediated Ca–Ca exchange diffusion in the erythrocyte membrane and that only a fraction of the ghost cell population participates in the Ca exchange diffusion.     

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.     

Evidence for two modes of Ca2+ entry following muscarinic stimulation of a human salivary epithelial cell line

Summary We have investigated muscarinic receptor-operated Ca²⁺ mobilization in a salivary epithelial cell line, HSG-PA, using an experimental approach which allows independent evaluation of intracellular Ca²⁺ release and extracellular Ca²⁺ entry. The carbachol (Cch) dose response of intracellular Ca²⁺ release indicates the involvement of a single, relatively low-affinity, muscarinic receptor site (K _0.510 or 30 m, depending on the method for [Ca²⁺] _i determination). However, similar data for Ca²⁺ entry indicate the involvement of two Cch sites, one consistent with that associated with Ca²⁺ release and a second higher affinity site withK _0.52.5 m. In addition, the Ca²⁺ entry response observed at lower concentrations of Cch (2.5 m) was completely inhibited by membrane depolarization induced with high K⁺ (>55mm) or gramicidin D (1 m), while membrane depolarization had little or no effect on Ca²⁺ entry induced by 100 m Cch. Another muscarinic agonist, oxotremorine-M (100 m; Oxo-M), like Cch, also induced an increase in the [Ca²⁺] _i of HSG-PA cells (from 72±2 to 104±5nm). This response was profoundly blocked (75%) by the inorganic Ca²⁺ channel blocker La³⁺ (25–50 m) suggesting that Oxo-M primarily mobilizes Ca²⁺ in these cells by increasing Ca²⁺ entry. Organic Ca²⁺ channel blockers (verapamil or diltiazem at 10 m, nifedipine at 1 m), had no effect on this response. The Oxo-M induced Ca²⁺ mobilization response, like that observed at lower doses of Cch, was markedly inhibited (70–90%) by membrane depolarization (high K⁺ or gramicidin D). At 100 m Cch the formation of inositol trisphosphate (IP₃) was increased 55% above basal levels. A low concentration of carbachol (1 m) elicited a smaller change in IP₃ formation (25%), similar to that seen with 100 m Oxo-M (20%). Taken together, these results suggest that there are two modes of muscarinic receptor-induced Ca²⁺ entry in HSG-PA cells. One is associated with IP₃ formation and intracellular Ca²⁺ release and is independent of membrane potential; the other is less dependent on IP₃ formation and intracellular Ca²⁺ release and is modulated by membrane potential. This latter pathway may exhibit voltage-dependent gating.     

Effect of ionophore A23187 upon membrane function and ion movement in human and toad erythrocytes

Summary Addition of 0.1–0.3 m A23187, a divalent cation ionophore, to human erythrocytes suspended in a 1.0mm ⁴⁵Ca²⁺-containing buffer results in a small ( two fold) increase in [Ca²⁺] _i , a significant decrease in osmotic fragility, and a decrease in intracellular K⁺ (100 mmoles/liter of cells to 70 mmoles/liter cells) without significant alteration of intracellular [Na⁺]. This decrease in [K⁺] _i is associated with a significant decrease in packed cell volume and correlates directly with the observed alteration is osmotic fragility. Increasing extracellular K⁺ to 125mm prevents the A23187-induced changes in osmotic fragility, K⁺ content and cell volume, but does not prevent the ionophore-induced uptake of⁴⁵Ca²⁺. Addition of 0.1–0.3 m A23187 to toad erythrocytes leads to an increase in⁴⁵Ca²⁺ uptake comparable to that observed in human erythrocytes, but does not alter osmotic fragility, cell volume or K⁺ content. Higher concentrations of ionophore (3.0–10.0 m) cause a 30- to 50-fold increase in⁴⁵Ca²⁺ uptake and concomitant change in K⁺ content, cell volume and osmotic fragility. These changes in cell properties can be prevented by increasing extracellular [K⁺] to 90mm. The difference in sensitivity of the two cell types to A23187 is attributed to the presence of additional intracellular calcium pools within toad erythrocytes that prevent an increase in cytoplasmic Ca²⁺ until Ca²⁺ uptake is increased substantially at the higher concentrations of A23187.     

Furosemide-sensitive K+ (Rb+) transport in human erythrocytes: Modes of operation,dependence on extracellular and intracellular Na+, kinetics,pH dependency and the effect of cell volume and N-ethylmaleimide

Summary The effect of extracellular and intracellular Na⁺ (Na _o ⁺ , Na _i ⁺ ) on ouabain-resistant, furosemide-sensitive (FS) Rb⁺ transport was studied in human erythrocytes under varying experimental conditions. The results obtained are consistent with the view that a (1 Na⁺+1 K⁺+2 Cl^–) cotransport system operates in two different modes: modei) promoting bidirectional 11 (Na⁺–K⁺) cotransport, and modeii) a Na _o ⁺ -independent 11 K _o ⁺ /K _i ⁺ exchange requiring Na _i ⁺ which, however, is not extruded. The activities of the two modes of operation vary strictly in parallel to each other among erythrocytes of different donors and in cell fractions of individual donors separated according to density. Rb⁺ uptake through Rb _o ⁺ /K _i ⁺ exchange contributes about 25% to total Rb⁺ uptake in 145mm NaCl media containing 5mm RbCl at normal Na _i ⁺ (pH 7.4). Na⁺–K⁺ cotransport into the cells occurs largely additive to K⁺/K⁺ exchange. Inward Na⁺–Rb⁺ cotransport exhibits a substrate inhibition at high Rb _o ⁺ . With increasing pH, the maximum rate of cotransport is accelerated at the expense of K⁺/K⁺ exchange (apparent pK close to pH 7.4). The apparentK _m ^Rb _o ⁺ of Na⁺–K⁺ cotransport is low (2mm) and almost independent of pH, and high for K⁺/K⁺ exchange (10 to 15mm), the affinity increasing with pH. The two modes are discussed in terms of a partial reaction scheme of (1 Na⁺+1 K⁺+2 Cl^–) cotransport with ordered binding and debinding, exhibiting a glide symmetry (first on outside = first off inside) as proposed by McManus for duck erythrocytes (McManus, T.J., 1987,Fed. Proc., in press). N-ethylmaleimide (NEM) chemically induces a Cl^–-dependent K⁺ transport pathway that is independent of both Na _o ⁺ and Na _i ⁺ . This pathway differs in many properties from the basal, Na _o ⁺ -independent K⁺/K⁺ exchange active in untreated human erythrocytes at normal cell volume. Cell swelling accelerates a Na _o ⁺ -independent FS K⁺ transport pathway which most probably is not identical to basal K⁺/K⁺ exchange. K _o ⁺ o ⁺

o ⁺ o ²⁺ reduce furosemide-resistant Rb⁺ inward leakage relative to choline _o ⁺ .     

Charybdotoxin blocks with high affinity the Ca-activated K+ channel of Hb A and Hb S red cells: Individual differences in the number of channels

Summary We have investigated the effect of a purified preparation of Charybdotoxin (CTX) on the Ca-activated K⁺ (Ca–K) channel of human red cells (RBC). Cytosolic Ca²⁺ was increased either by ATP depletion or by the Ca ionophore A23187 and incubation in Na⁺ media containing CaCl₂. The Ca–K efflux activated by metabolic depletion was partially (77%) inhibited from 15.8±2.4 mmol/liter cell · hr, to 3.7±1.0 mmol/liter cell · hr by 6nm CTX (n=3). The kinetic of Ca–K efflux was studied by increasing cell ionized Ca²⁺ using A23187 (60 mol/liter cell), and buffering with EGTA or citrate; initial rates of net K⁺ efflux (90 mmol/liter cell K⁺) into Na⁺ medium containing glucose, ouabain, bumetanide at pH 7.4 were measured. Ca–K efflux increased in a sigmoidal fashion (n of Hill 1.8) when Ca²⁺ was raised, with aK _m of 0.37 m and saturating between 2 and 10 m Ca²⁺. Ca–K efflux was partially blocked (71±7.8%, mean ±sd,n=17) by CTX with high affinity (IC₅₀0.8nm), a finding suggesting that is a high affinity ligand of Ca–K channels. CTX also blocked 72% of the Ca-activated K⁺ efflux into 75mm K⁺ medium, which counteracted membrane hyperpolarization, cell acidification and cell shrinkage produced by opening of the K⁺ channel in Na⁺ media. CTX did not block Valinomycin-activated K⁺ efflux into Na⁺ or K⁺ medium and therefore it does not inhibit K⁺ movement coupled to anion conductive permeability.TheV _max, but not theK _m–Ca of Ca–K efflux showed large individual differences varying between 4.8 and 15.8 mmol/liter cell · min (FU). In red cells with Hb A,V _max was 9.36±3.0 FU (mean ±sd,n=17). TheV _max of the CTX-sensitive, Ca–K efflux was 6.27±2.5 FU (range 3.4 to 16.4 FU) in Hb A red cells and it was not significantly different in Hb S (6.75±3.2 FU,n=8). Since there is larger fraction of reticulocytes in Hb S red cells, this finding indicates that cell age might not be an important determinant of theV _max of Ca–K⁺ efflux.Estimation of the number of CTX-sensitive Ca-activated K⁺ channels per cell indicate that there are 1 to 3 channels/per cell either in Hb A or Hb S red cells. The CTX-insensitive K⁺ efflux (2.7±0.9 FU) may reflect the activity of a different channel, nonspecific changes in permeability or coupling to an anion conductive pathway.     

Ca2+-activated K+ channels in the apical membrane ofNecturus choroid plexus

Summary The properties of Ca²⁺-activated K⁺ channels in the apical membrane of theNecturus choroid plexus were studied using single-channel recording techniques in the cell-attached and excised-patch configurations. Channels with large unitary conductances clustered around 150 and 220 pS were most commonly observed. These channels exhibited a high selectivity for K⁺ over Na⁺ and K⁺ over Cs⁺. They were blocked by high cytoplasmic Na⁺ concentrations (110mm). Channel activity increased with depolarizing membrane potentials, and with increasing cytoplasmic Ca²⁺ concentrations. Increasing Ca²⁺ from 5 to 500nm, increased open probability by an order of magnitude, without changing single-channel conductance. Open probability increased up to 10-fold with a 20-mV depolarization when Ca²⁺ was 500nm. Lowering intracellular pH one unit, decreased open probability by more than two orders of magnitude, but pH did not affect single-channel conductance. Cytoplasmic Ba²⁺ reduced both channel-open probability and conductance. The sites for the action of Ba²⁺ are located at a distance more than halfway through the applied electric field from the inside of the membrane. Values of 0.013 and 117mm were calculated as the apparent Ba²⁺ dissociation constants (K _d (0 mV) for the effects on probability and conductance, respectively. TEA⁺ (tetraethylammonium) reduced single-channel current. Applied to the cytoplasmic side, it acted on a site 20% of the distance through the membrane, with aK _d (0 mV)=5.6mm. A second site, with a higher affinity,K _d (0 mV)=0.23mm, may account for the near total block of chanel conductance by 2mm TEA⁺ applied to the outside of the membrane. It is concluded that the channels inNecturus choroid plexus exhibit many of the properties of maxi Ca²⁺-activated K⁺ channels found in other tissues.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.