22Na+ Uptake and Catecholamine Secretion by Primary Cultures of Adrenal Medulla Cells

The uptake of 22Na+ and secretion of catecholamines by primary cultures of adrenal medulla cells under the influence of a variety of agonists and antagonists were determined. Veratridine, batrachotoxin, scorpion venom, and nicotine caused a parallel increase in 22Na+ uptake and Ca2+-dependent catecholamine secretion. Ba2+, depolarizing concentrations of K+, and the Ca2+ ionophore Ionomycin stimulated secretion of catecholamines but did not increase the uptake of 22Na+. Tetrodotoxin inhibited both 22Na+ uptake and catecholamine secretion evoked by veratridine, batrachotoxin, and scorpion venom, but had no effect on 22Na+ uptake and catecholamine secretion caused by nicotine. On the other hand, histrionicotoxin, which blocks the acetylcholine receptor-linked ion conductance channel, blocked nicotine-stimulated 22Na+ uptake and catecholamine secretion, but only partially inhibited veratridine-stimulated catecholamine secretion and had no effect on veratridine-stimulated 22Na+ uptake. The combination of veratridine plus tetrodotoxin, which has been shown to prevent nicotine-stimulated secretion of catecholamines by adrenal medulla cells, also prevented nicotine-stimulated 22Na+ uptake by the primary cultures. These studies demonstrate the presence of tetrodotoxin-sensitive Na+ channels in adrenal medulla cells which are functionally linked to Ca2+-dependent catecholamine secretion. However, These channels are not utilized for Na+ entry upon activation of nicotinic receptors; in this case Na+ entry occurs through the receptor-associated ion conductance channel.     

Interaction of steroidal alkaloid toxins with calcium channels in neuronal cell lines

Depolarization with 50 mM K+ increased 45Ca2+ uptake into neuronal clonal cell lines NG108-15, N1E-115 and NH15-CA2. In each cell line this depolarization-induced uptake was blocked by inorganic and organic blockers of voltage sensitive calcium channels. However, tetrodotoxin (10(-6) M) was ineffective. Moreover, in the presence of tetrodotoxin, neither batrachotoxin nor veratridine inhibited the depolarization-induced uptake. The novel dihydropyridine BAY K8644 enhanced depolarization-induced 45Ca2+ uptake into each cell line in a nitrendipine reversible fashion. In the presence of tetrodotoxin, the BAY K8644/50 mM K+ stimulated uptake could be partially inhibited by batrachotoxin (10(-6) M) and veratridine (5 X 10(-5) M). These effects were not altered by the presence of scorpion venom (1 microgram/ml). The results indicate that both batrachotoxin and veratridine can modulate the effects of dihydropyridines on the gating properties of voltage sensitive calcium channels.     

Alkaloid neurotoxins-dependent sodium transport in insect synaptic nerve-ending particles

1. Sodium uptake associated with the activation of voltage-sensitive sodium channels by alkaloid activators, batrachotoxin, veratridine, and aconitine in presynaptic nerve terminals isolated from the central nervous system of cockroach (Periplaneta americana) was investigated. 2. Batrachotoxin (K0.5, 0.2 microM) was full agonist as for most effective activator of Na+ uptake; veratridine (K0.5, 2.5 microM) and aconitine (K0.5, 7.6 microM) produced a maximal stimulation of 22Na+ uptake that were 71% and 43% respectively of that produced by batrachotoxin. 3. Veratridine-dependent 22Na+ uptake was completely inhibited by tetrodotoxin (I0.5, 11 nM), a specific inhibitor of the nerve membrane sodium channels. 4. The present study describes appropriate conditions for measuring neurotoxins--stimulated sodium transport in insect central nervous system synaptosomes. The data show that voltage-sensitive sodium channels as defined by specific activation by the alkaloid neurotoxins are qualitatively distinct in insect synaptosomes than those previously described for vertebrate brain synaptosomes, cultured neuronal cell, nerve membrane vesicles and neuroblastoma cells.     

Calcium-induced insulin release in monolayer culture of the endocrine pancreas. Studies with ionophore A23187.

The role of Ca2+ on insulin release has been studied by the use of ionophore A23187. The ionophore complexes divalent cations and permits Ca2+ entry into cells by acting as a carrier in the plasma membranes. Cultured cells obtained by enzymatic digestion of pancreases from newborn rats were studied on the 3rd day of culture. With Ca2+ in the incubation medium the ionophore induced sustained insulin release even in the absence of glucose. Optimal effects of the ionophore were observed at 3 and 10 mug per ml in the presence of 0.3 to 1.0 mM Ca-2+. Under these conditions the insulin release was greater than that caused by 16.7 mM glucose. A graded response was observed to changes in Ca-2+ concentration from 0.1 to 1.0 mM Ca-2+. Higher Ca-2+ concentrations caused a large amount of insulin to be released promptly, but the release was not sustained. Mg-2+ and Sr-2+ were not found to substitute for Ca-2+. Ba-2+ at 0.3 mM stimulated insulin release even in the absence of ionophore. Cyclic adenosine 3':5'-monophosphate was able to increase ionophore-induced insulin release. The alpha-adrenergic effect of epinephrine to inhibit insulin release was not observed in the presence of Ca-2+ and the ionophore, and a stimulatory effect of epinephrine was seen. This unusual stimulatory effect of epinephrine was blocked by propranolol indicating a beta-adrenergic mechanism for epinephrine. It is concluded that Ca-2+, which plays an essential role in the stimulus-secretion coupling, can alone initiate and cause sustained insulin release.     

Sodium channels in presynaptic nerve terminals. Regulation by neurotoxins

Regulation of Na+ channels by neurotoxins has been studied in pinched- off nerve endings (synaptosomes) from rat brain. Activation of Na+ channels by the steroid batrachotoxin and by the alkaloid veratridine resulted in an increase in the rate of influx of 22Na into the synaptosomes. In the presence of 145 mM Na+, these agents also depolarized the synaptosomes, as indicated by increased fluorescence in the presence of a voltage-sensitive oxacarbocyanine dye [diO-C5(3)]. Polypeptide neurotoxins from the scorpion Leiurus quinquestriatus and from the sea anemone Anthopleura xanthogrammica potentiated the stimulatory effects of batrachotoxin and veratridine on the influx of 22Na into synaptosomes. Saxitoxin and tetrodotoxin blocked the stimulatory effects of batrachotoxin and veratridine, both in the presence and absence of the polypeptide toxins, but did not affect control 22Na influx or resting membrane potential. A three-state model for Na+ channel operation can account for the effects of these neurotoxins on Na+ channels as determined both by Na+ flux measurements in vitro and by electrophysiological experiments in intact nerve and muscle.     

Sodium transport by the acetylcholine receptor of cultured muscle cells.

Activation of the acetylcholine receptors of cultured muscle cells by carbamylcholine increases the rate of passive 22-Na+ uptake into the muscle cells up to 20-fold. The Na+ transport activity of the receptor desensitizes during exposure to carbamylcholine. The rate and extent of desensitization is reduced by lowering the assay temperature from 36 degrees to 2 degrees, allowing accurate measurements of initial rates of Na+ transport by the receptor. Activation of the receptor by carbamylcholine and acetylcholine is significantly cooperative (Hill coefficients of 1.4 to 2.0). Inhibition by D-tubocurarine is not cooperative. The carbamylcholine-induced Na+ transport activity of the receptor is inhibited 50% by 4 muM D-tubocurarine, 100 muM atropine, or 1.6 nM diiodo-alpha-bungarotoxin but is not affected by tetrodotoxin. The initial rate of Na+ transport by the receptor is temperature-independent between 2 degrees and 36 degrees. Receptor Na+ transport is saturable by Na+ at 2 degrees with an apparent Km of 150 plus and minus 20 mM. Saturation by Na+ not observed at 36 degrees at the concentrations tested. Saturation by Na+ is observed at 2 degrees both under conditions of net Na+ influx and under conditions of isotopic exchange at equilibrium. The receptor does not catalyze obligatory exchange diffusion at a detectable rate. Comparison of binding of [125-I]diiodo-alpha-bungarotoxin with rates of Na+ transport indicates a turnover number of 2 times 10-7 ions per min per receptor. These results are discussed in terms of the mechanism of Na+ transport by the receptor.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ

A fluorescence assay for measuring Na channel activation in liposomes containing voltage-sensitive Na channels isolated from Electrophorus electricus is described. The assay is based on transport of a heavy-metal cation, T1+, through the activated channel to quench fluorescence of an internalized, water-soluble chromophore. The channel is "locked" in a chronically opened configuration with alkaloid neurotoxins such as veratridine or batrachotoxin. Diffusion potentials are used to amplify the signal, and enlarged liposomes (greater than 8000 A) result in time courses extended to the range of seconds. Analysis of the kinetics of quenching yields parameters that behave as linear functions of channel activation and reflect vesicle size and channel abundance. The k1/2's for activation by veratridine and batrachotoxin were 5 microM and 169 nM, respectively, and that for tetrodotoxin blockade was 4 nM. Externally applied QX-222 and tetrodotoxin each acted to partially block the stimulated signal, as expected for compounds that act on oppositely oriented channels in the membrane. Single-channel conductances estimated with either veratridine or batrachotoxin ranged between 0.6 and 40.7 pS, corresponding to transport numbers of (1.2 X 10(5)) to (8.1 X 10(6)) ions s-1 channel-1 under the conditions of assay. The assay is approximately 100-fold more sensitive than radiotracer influx assays, requiring 1 fmol of protein per time course.     

Effects of monovalent and divalent cations and of guanine nucleotides on binding of vasopressin to the rat mesenteric vasculature

The rat mesenteric vasculature contains high affinity binding sites specific for [3H]Arg8-vasopressin which mediate its vasoconstrictor action. We have investigated the in vitro effect of monovalent and divalent cations and guanine nucleotides on the interactions between [3H]Arg8-vasopressin and its receptor in this preparation. Binding was increased by divalent cations from fourfold in the presence of Mg2+ at 5 mM to ninefold in the presence of Mn2+ at 5 mM. The potency order of divalent cations to increase binding was Mn2+ greater than Co2+ greater than Ni2+ greater than Mg2+ greater than Ca2+ approximately equal to control without cations. Addition of Na2+ or other monovalent cations (K+, Li+, and NH4+) in the presence or absence of divalent cations reduced binding significantly. Analysis of saturation binding curves showed a single high affinity site. In the presence of 5 mM Mn2+, binding capacity (Bmax) increased to 139 +/- 23 fmol/mg protein. Receptor affinity was enhanced (KD decreased to 0.33 +/- 0.07 nM). In presence of 5 mM Mg2+ or 150 mM Na+, Bmax and affinity were reduced. The addition of 100 microM GTP or its nonhydrolyzable analogue, Gpp(NH)p, reduced receptor affinity in the presence of Mn2+ + Na+, Mg2+, and Mg2+ + Na+, but not in the presence of Mn2+ alone. Computer modeling of competition binding curves demonstrated that in contrast with saturation studies, the data were best explained by a two-site model with high affinity, low capacity sites and low affinity, high capacity sites. Mn2+ or Mn2+ + Na+ with or without guanine nucleotides resulted in a predominance of high affinity sites. GTP or Gpp(NH)p in the presence of Mg2+ or Mg2+ + Na+ induced a reduction of affinity of the high affinity binding sites and the number of these sites. In the presence of Mg2+ + Na+ and guanine nucleotides, high affinity sites were maximally decreased. An association kinetic study indicated that the association rate constant (K+1) was increased by divalent cations and reduced by guanine nucleotides, without change in the dissociation rate constant (K-1). The equilibrium dissociation constant (KD) calculated with these rate constants (K-1/K+1) was similar to that obtained in saturation experiments at steady state. Dissociation kinetics were biphasic, indicating the presence of two receptor states, one of high and one of low affinity, associated with a slow and a rapid dissociation rate. Cations and guanine nucleotides interact with one or more sites closely associated with vasopressin receptors, including possibly with a GTP-sensitive regulatory protein, to modulate receptor affinity for vasopressin.     

Mouse brain synaptosomal sodium channels: activation by aconitine, batrachotoxin, and veratridine, and inhibition by tetrodotoxin

Batrachotoxin, veratridine and aconitine, activators of the voltage-dependent sodium channel in excitable cell membranes, increase the rate of 22Na+ uptake by mouse brain synaptosomes. Batrachotoxin was both the most potent (K0.5, 0.49 microM) and most effective activator of specific 22Na+ uptake. Veratridine (K0.5, 34.5 microM) and aconitine (K0.5, 19.6 microM) produced maximal stimulations of 22Na+ uptake that were 73% and 46%, respectively, of that produced by batrachotoxin. Activation of 22Na+ uptake by veratridine was completely inhibited by tetrodotoxin (I50, 6 nM ), a specific blocker of nerve membrane sodium channels. These results identify appropriate conditions for measuring sodium channel-dependent 22Na+ flux in mouse brain synaptosomes. The pharmacological properties of mouse brain synaptosomal sodium channels described here are distinct from those previously described for sodium channels in rat brain synaptosomes and mouse neuroblastoma cells.     

The role of Ca-2+ and cyclic adenosine 3':5'-monophosphate in insulin release induced in vitro by the divalent cation ionophore A23187.

Insulin release from isolated perifused pancreatic islets was stimulated by the divalent ionophore A23187 in the absence of exogenous glucose. In addition, A23187 produced a 2-fold elevation of cyclic adenosine 3':5'-monophosphate (cAMP) levels in isolated perifused islets. The elevation of cAMP levels coincided with peak insulin release. Ionophore-induced insulin release was unaffected by pretreatment of the islets with theophylline (5 mM). Stimulation of insulin release produced by the ionophore occurred either in the presence or absence of extracellular Ca-2+; however, cAMP accumulation required the presence of extracellular Ca-2+. The ionophore (10 muM) had no effect on adenylate cyclase activity of homogenates of isolated islets. The results of this study are interpreted as indicating that intracellular Ca-2+ has an essential role in the insulin releasing mechanism, whereas the cAMP system has a modulatory effect on this process.     

Negative surface charge near sodium channels of nerve: divalent ions, monovalent ions, and pH.

Evidence is given for a high density of negative surface charge near the sodium channel of myelinated nerve fibres. The voltage dependence of peak sodium permeability is measured in a voltage clamp. The object is to measure voltage shifts in sodium activation as the following external variables are varied: divalent cation concentration and type, monovalent concentration, and pH. With equimolar substitution of divalent ions the order of effectiveness for giving a positive shift is: Ba equals Sr less than Mg less than Ca less than Co approximately equal to Mn less than Ni less than Zn. A tenfold increase of concentration of any of these ions gives a shift of +20 to +25 mV. At low pH, the shift with a tenfold increase in Ca-2+ is much less than at normal pH, and conversely for high pH. Soulutions with no added divalent ions give a shift of minus 18 mV relative to 2 mM Ca-2+. Removal of 7/8 of the cations from the calcium-free solution gives a further shift of minue 35 mV. All shifts are explained quantitatively by assuming that changes in an external surface potential set up by fixed charges near the sodium channel produce the shifts. The model involves a diffuse double layer of counterions at the nerve surface and some binding of H+ions and divalent ions to the fixed charges. Three types of surface groups are postulated: (1) an acid pKa equals 2.88 charge density minus 0.9 nm- minus 2; (i) an acid pKa equals 4.58, charge density minus 0.58 nm- minus 2; (3) a base pKa equals 6.28, charge density +0.33 nm- minus 2. The two acid groups also bind Ca-2+ ions with a dissociation constant K equals 28 M. Reasonable agreement can also be obtained with a lower net surface charge density and stronger binding of divalent ions and H+ ions.     

Caclium uptake and associated adenosine triphosphatase activity in fragmented sarcoplasmic reticulum. Requirement for potassium ions.

The effects of monovalent cations on calcium uptake by fragmented sarcoplasmic reticulum have been clarified. Homogenization of muscle tissue in salt-containing solutions leads to contamination of this subcellular fraction with actomyosin and mitochondrial membranes. When, in addition, inorganic cations are contributed by the microsomal suspension and in association with nucleotide triphosphate substrates there is an apparent inhibition of the calcium transport system by potassium and other cations. However, when purified preparations were obtained after homogenization in sucrose medium followed by centrifugation on a sucrose density gradient in a zonal rotor, calcium uptake and the associated adenosine triphosphatase activity were considerably activated by potassium and other univalent cations. When plotted against the log of the free calcium concentration there was only a slight increase in calcium uptake and ATPase activity in the absence of potassium ions but sigmoid-shaped curves were obtained in 100 mM K+ with half-maximal stimulation occurring at 2 muM Ca2+ for both calcium uptake and ATPase activity. The augmentation in calcium uptake was not due to an ionic strength effect as Tris cation at pH 6.6 was shown to be inactive in this respect. Other monovalent cations were effective in the order K+ greater than Na+ greater than NH4+=Rb+=Cs+ greater than Li+ with half-maximal stimulation in 11 mM K+, 16 mM Na+, 25 mM NH4+, Rb+, and Cs+ and in 50 mM Li+. There was nos synergistic action between K+ AND Na+ ions and both calcium uptak and associated ATPase were insensitive to ouabain. Thallous ions stimulate many K+-requiring enzymes and at one-tenth the concentration were nearly as effective as K+ ions in promoting calcium uptake. The ratio of Ca2+ ions transported to P1 released remained unchanged at 2 after addition of K+ ions indicating an effect on the rate of calcium uptake rather than an increased efficiency of uptake. In support of this it was found that during the stimulation of calcium uptake by Na+ ions there was a reduction in the steady state concentration of phosphorylated intermediate formed from [gamma-32P]ATP. It is considered that there is a physiological requirement for potassium ions in the relaxation process.     

Regulation of the (Na+ + K+)-ATPase in cultured chick skeletal muscle. Modulation of expression by the demand for ion transport

The levels of (Na+ + K+)-ATPase expression during muscle development and in response to modulation of demand for ion transport were studied in chick skeletal muscle cells in culture. The number of (Na+ + K+)-ATPase molecules on the myogenic cell surface, quantified with 125I-labeled monoclonal antibodies, increased 20-fold during muscle differentiation, with a substantial increase in (Na+ + K+)-ATPase molecules/unit area of membrane. The demand for sodium ion transport by the (Na+ + K+)-ATPase was modulated by activating voltage-sensitive sodium channels with veratridine or exposing cultures to low [K+]o (0.5 mM). Exposure to veratridine (10 microM) resulted in a 60-100% increase in cell surface and a smaller increase in intracellular (Na+ + K+)-ATPase over a 24-36-h period. Neither high [K+]o (50 mM) nor Ca2+ ionophore A23187 (1 microM) produced any such change, suggesting that neither membrane depolarization nor elevated cytosolic calcium was mediating the effect of veratridine. Veratridine stimulated up-regulation was specific for the (Na+ + K+)-ATPase, blocked by tetrodotoxin, and completely reversible. The kinetics of the reversal (down-regulation) process were much faster (t1/2 = 3 h) than those of up-regulation (t1/2 = 18 h). Up-regulation of the (Na+ + K+)-ATPase by veratridine occurred by a combination of two mechanisms: the first an early phase involving a stimulated biosynthesis of the (Na+ + K+)-ATPase and a later phase in which the biosynthetic rate returned to approximately control levels while the degradation rate slowed (t1/2 control = 31 h, t1/2 veratridine = 64 h).     

Interaction of calcium with bovine plasma protein C

The binding of 45Ca2+ to bovine plasma protein C (PC) and to activated bovine plasma protein C (APC) has been examined by equilibrium ultrafiltration at pH 7.4 and 25 degrees C. Under these conditions, PC possesses 16.0 plus or minus 2.0 equivalent Ca2+ binding sites, of average KD (8.7 plus or minus 1.5) x 10(-4) M, and APC contains 9.0 plus or minus 1.0 equivalent Ca2+ binding sites, with an average KD of (4.3 plus or minus 1.1) x 10(-4) M. Both Mn2+ and Sr2+ were capable of ready displacement of Ca2+ from a Ca2+-PC complex, while Mg2+ was less effective in this regard. The alpha-thrombin-catalyzed activation of PC was inhibited by the presence of Ca2+. A kinetic analysis of this effect demonstrated that it was, in large part, due to an increase in the Km of the reaction. Addition of other divalent cations, e.g. Mn2+, Sr2+, and Mg2+, in place of Ca2+ also resulted in inhibition of the alpha-thrombin-catalyzed activation of PC in a manner which paralleled their ability to displace Ca2+ from a Ca2+-PC complex. On the other hand, the activation of PC by the coagulant protein from Russell's Viper venom was augmented by the presence of Ca2+. Other divalent metal ions, such as Sr2+ and Mn2+, in the absence of Ca2+, also weakly stimulated this reaction. Mg2+ was without notable effect.     

Interaction of valinomycin and monovalent cations with the (Ca2+,Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum

The interactions of monovalent cations and of the K+-specific ionophore, valinomycin, with the Ca2+-ATPase of skeletal muscle of sarcoplasmic reticulum have been studied in the absence of cation gradients by their effects on enzyme turnover and on the ATP plus Ca2+-dependent enhanced fluorescence of the ATP analogue, 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)-adenosine 5'-triphosphate (TNP-ATP) (Watanabe, T., and Inesi, G. (1982) J. Biol. Chem. 257, 11510-11516). Monovalent cations decreased turnover-dependent TNP-ATP fluorescence in the series K+ greater than Rb+ approximately equal to Cs+ greater than Na+ greater than Li+ (K0.5 = 49, 73, 75, 94, and 246 mM, respectively), consistent with the known specificity of the monovalent cation binding site that stimulates turnover and E-P hydrolysis. Valinomycin (200 nmol/mg), in the absence of monovalent cations, decreased ATPase activity by 30% and abolished the stimulatory effects of 150 mM KCl or NaCl on turnover. The ionophore alone enhanced TNP-ATP fluorescence by 20% and altered the specificity and affinity of the site that inhibited TNP-ATP fluorescence to Cs+ greater than Rb+ greater than K+ approximately equal to Na+ greater than Li+ (K0.5 = 79, 111, 134, 136, and 270 mM, respectively), which follows the Hofmeister series for effectiveness of monovalent lyotropic cations. TNP-ATP binding was not affected by either monovalent cations or valinomycin. Inhibition of turnover-dependent TNP-ATP fluorescence appears to be a useful parameter for monitoring monovalent cation binding to the Ca2+-ATPase. It is concluded that the ionophore interacts directly with the Ca2+-ATPase, independent of its K+ conductance effects on the lipid bilayer, and modifies the affinity and specificity of the monovalent cation site, either by direct interaction or by the formation of a valinomycin-monovalent cation-enzyme complex.     

Divalent metal is required for both phosphate transport and phosphate binding to phosphorin, a proteolipid isolated from brush-border membrane vesicles

The Na+-dependent phosphate transport system in the brush border of rabbit kidney exhibits a positive requirement for a divalent metal ion. Treatment of the brush-border membrane vesicles (BBMV) with a divalent metal chelator in combination with the divalent metal ionophore A23187 dramatically and selectively decreased the Na+-dependent uptake of phosphate; Na+-independent uptake of phosphate was not affected. The combination of chelator plus A23187 also inhibited uptake of phosphate in the presence of Na+ but in the absence of a gradient for sodium across the BBMV. This indicates that the inhibitor is not a result of an alteration in the Na+ gradient by chelator plus ionophore. The inhibited Na+ gradient-dependent transport of phosphate was restored by removing the chelator and adding Mn2+ to the BBMV. The phosphate-binding proteolipid (phosphorin) isolated from rabbit kidney BBMV binds inorganic phosphate with high affinity and specificity. Binding of phosphate to phosphorin is also inhibited by divalent metal chelators and can be restored by addition of a divalent metal. We conclude that a divalent metal ion is required both for the Na+-dependent phosphate transport in BBMV and for the binding of phosphate to the proteolipid phosphorin. These findings are consistent with our suggestion that phosphorin is a component of the Na+-dependent phosphate transport system in renal brush-border membranes.     

Cation selectivity characteristics of the reconstituted voltage-dependent sodium channel purified from rat skeletal muscle sarcolemma

In this report, the alkali metal cation selectivity of the purified, voltage-dependent sodium channel from rat skeletal muscle is described. Isolated sodium channel protein (980-2840 pmol of saxitoxin binding/mg of protein) was reconstituted into egg phosphatidylcholine vesicles, and channels were subsequently activated by either batrachotoxin (5 X 10(-6) M) or veratridine (5 X 10(-4) M). Activation of the reconstituted sodium channel by batrachotoxin permitted rapid specific influx of cations into channel-containing vesicles. Quenched flow kinetic techniques were adapted to allow resolution of the kinetics of cation movement. Uptake rates for 42K+, 86Rb+, and 137Cs+ were measured directly and half-times for equilibration at 18 degrees C were determined to be 350 ms, 2.5 s, and 10 s, respectively, in this vesicle population. 22Na+ equilibration occurred within the mimimum quenching time of the apparatus (90 ms) but an upper limit of 50 ms at 18 degrees C could be assigned to its half-time. Based on this upper estimate for Na+, cation selectivity ratios of the batrachotoxin-activated channel were Na+ (1):K+ (0.14):Rb+ (0.02):Cs+ (0.005). Toxin-stimulated influx could be blocked by saxitoxin with a Ki of approximately 5 X 10(-9) M at 18 degrees C. Rates of cation movement through veratridine-activated channels were much slower, with half-times of 1.0, 1.2, 2.0, and 2.6 min at 36 degrees C for Na+, K+, Rb+, and Cs+, respectively. The temperature dependences of batrachotoxin and veratridine-stimulated cation uptake were markedly different. The activation energies for 86Rb+ and 137Cs+ movement into batrachotoxin-activated vesicles were 7.6 and 6.1 kcal/mol, respectively, while comparable measurements for these two cations in veratridine-activated vesicles yielded activation energies of 31 kcal/mol. Measurements of cation exchange with batrachotoxin-activated channels may reflect characteristics of an open sodium channel while the process of channel opening itself may be rate-limiting when veratridine is used for activation.     

Role of voltage-dependent Na+ channels for rhythmic Ca2+ signalling in glucose-stimulated mouse pancreatic beta-cells.

The putative role of voltage-dependent Na+ channels for glucose induction of rhythmic Ca2+ signalling was studied in mouse pancreatic beta-cells with the use of the Ca2+ indicator fura-2. A rise in glucose from 3 to 11 mM resulted in slow oscillations of the cytoplasmic Ca2+ concentration ([Ca2+]i). These oscillations, as well as superimposed transients seen during forskolin-induced elevation of cAMP, remained unaffected in the presence of the Na+ channel blocker tetrodotoxin. During exposure to 1-10 microM veratridine, which facilitates the opening of voltage-dependent Na+ channels, the slow oscillations were replaced by repetitive and pronounced [Ca2+]i transients arising from the basal level. The effects of veratridine were reversed by tetrodotoxin. The veratridine-induced [Ca2+]i transients were critically dependent on the influx of Ca2+ and persisted after thapsigargin inhibition of the endoplasmic reticulum Ca2+-ATPase. Both tolbutamide and ketoisocaproate mimicked the action of glucose in promoting [Ca2+]i transients in the presence of veratridine. It is suggested that activation of voltage-dependent Na+ channels is a useful approach for amplifying Ca2+ signals for insulin release.     

Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy