Sucrose uptake by pinocytosis in Amoeba proteus and the influence of external calcium

The relationship between Ca++ and pinocytosis was investigated in Amoeba proteus. Pinocytosis was induced with 0.01% alcian blue, a large molecular weight dye which binds irreversibly to the cell surface. The time-course and intensity of pinocytosis was monitored by following the uptake of [3H]SUCROSE. When the cells are exposed to 0.01% alcian blue, there is an immediate uptake of sucrose. The cells take up integral of 10% of their initial volume during the time-course of pinocytosis. The duration of pinocytosis in the amoeba is integral of 50 min, with maximum sucrose uptake occurring 15 min after the induction of pinocytosis. The pinocytotic uptake of sucrose is reversibly blocked at 3 degrees C and a decrease in pH increases the uptake of sucrose by pinocytosis. The process of pinocytosis is also dependent upon the concentration of the inducer in the external medium. The association between Ca++ and pinocytosis in A. proteus was investigated initially by determining the effect of the external Ca++ concentration on sucrose uptake induced by alcian blue. In Ca++-free medium, no sucrose uptake is observed in the presence of 0.01% alcian blue. As the Ca++ concentration is increased, up to a maximum of 0.1 mM, pinocytotic sucrose uptake is also increased. Increases in the external Ca++ concentration above 0.1 mM brings about a decrease in sucrose uptake. Further investigations into the association between Ca++ and pinocytosis demonstrated that the inducer of pinocytosis displaces surface calcium in the amoeba. It is suggested that Ca++ is involved in two separate stages in the process of pinocytosis; an initial displacement of surface calcium by the inducer which may increase the permeability of the membrane to solutes and a subsequent Ca++ influx bringing about localized increases in cytoplasmic Ca++ ion activity.     

Depolarizing response of rat parathyroid cells to divalent cations

Membrane potentials were recorded from rat parathyroid glands continuously perfused in vitro. At 1.5 mM external Ca++, the resting potential averages -73 +/- 5 mV (mean +/- SD, n = 66). On exposure to 2.5 mM Ca++, the cells depolarize reversibly to a potential of -34 +/- 8 mV (mean +/- SD). Depolarization to this value is complete in approximately 2-4 min, and repolarization on return to 1.5 mM Ca++ takes about the same time. The depolarizing action of high Ca++ is mimicked by all divalent cations tested, with the following order of effectiveness: Ca++ greater than Sr++ greater than Mg++ greater than Ba++ for alkali-earth metals, and Ca++ greater than Cd++ greater than Mn++ greater than Co++ greater than Zn++ for transition metals. Input resistance in 1.5 mM Ca++ was 24.35 +/- 14 M omega (mean +/- SD) and increased by an average factor of 2.43 +/- 0.8 after switching to 2.5 mM Ca++. The low value of input resistance suggests that cells are coupled by low-resistance junctions. The resting potential in low Ca++ is quite insensitive to removal of external Na+ or Cl-, but very sensitive to changes in external K+. Cells depolarize by 61 mV for a 10- fold increase in external K+. In high Ca++, membrane potential is less sensitive to an increase in external K+ and is unchanged by increasing K+ from 5 to 25 mM. Depolarization evoked by high Ca++ may be slowed, but is unchanged in amplitude by removal of external Na+ or Cl-. Organic (D600) and inorganic (Co++, Cd++, and Mn++) blockers of the Ca++ channels do not interfere with the electrical response to Ca++ changes. Our results show remarkable parallels to previous observations on the control of parathormone (PTH) release by Ca++. They suggest an association between membrane voltage and secretion that is very unusual: parathyroid cells secrete when fully polarized, and secrete less when depolarized. The extraordinary sensitivity of parathyroid cells to divalent cations leads us to hypothesize the existence in their membranes of a divalent cation receptor that controls membrane permeability (possibly to K+) and PTH secretion.     

Effects of Na readmission on cellular45Ca fluxes in Na-Depleted guinea pig taenia coli

Summary The removal of Na from the medium causes a cellular Ca uptake in the smooth muscle of the guinea pig taenia coli which is rapidly reversed if medium Na is readmitted. This net extrusion was characterized in tissues which were first Na-depleted in a zero-Na (sucrose) solution. Li was able to substitute for Na in mediating this effect. K was also able to mimic Na in this respect if the depolarization-mediated Ca influx caused by the isotonic K solution was blocked with 10^–5 m D-600. The net Ca extrusion upon Na readmission was due to a small decrease in Ca influx, as well as a marked increase in the transmembrane Ca efflux rate, as revealed by⁴⁵Ca washout experiments. The increased⁴⁵Ca efflux upon Na readmission could be mimicked by Li, K, choline and tris. We conclude that the Na/Ca-exchange hypothesis is insufficient to explain these data, in that both Ca extrusion and⁴⁵Ca efflux can be stimulated in the absence of a Na gradient, or in the absence of any monovalent cationic gradient. These observations are discussed in terms of a possible intracellular competition of Ca and monovalent cations for anionic binding sites, as well as with regard to a possible direct stimulation of a plasmalemmal CaATPase by monovalent cations.     

Interactions of sodium transport, cell volume, and calcium in frog urinary bladder

The volume of individual cells in intact frog urinary bladders was determined by quantitative microscopy and changes in volume were used to monitor the movement of solute across the basolateral membrane. When exposed to a serosal hyposmotic solution, the cells swell as expected for an osmometer, but then regulate their volume back to near control in a process that involves the loss of KCl. We show here that volume regulation is abolished by Ba++, which suggests that KCl movements are mediated by conductive channels for both ions. Volume regulation is also inhibited by removing Ca++ from the serosal perfusate, which suggests that the channels are activated by this cation. Previously, amiloride was observed to inhibit volume regulation: in this study, amiloride-inhibited, hyposmotically swollen cells lost volume when the Ca++ ionophore A23187 was added to Ca++-replete media. We attempted to effect volume changes under isosmotic conditions by suddenly inhibiting Na+ entry across the apical membrane with amiloride, or Na+ exit across the basolateral membrane with ouabain. Neither of these Na+ transport inhibitors produced the expected results. Amiloride, instead of causing a decrease in cell volume, had no effect, and ouabain, instead of causing cell swelling, caused cell shrinkage. However, increasing cell Ca++ with A23187, in both the absence and presence of amiloride, caused cells to lose volume, and Ca++-free Ringer's solution (serosal perfusate only) caused ouabain-blocked cells to swell. Finally, again under isosmotic conditions, removal of Na+ from the serosal perfusate caused a loss of volume from cells exposed to amiloride. These results strongly suggest that intracellular Ca++ mediates cell volume regulation by exerting a negative control on apical membrane Na+ permeability and a positive control on basolateral membrane K+ permeability. They also are compatible with the existence of a basolateral Na+/Ca++ exchanger.     

Volume regulation by human lymphocytes. Role of calcium

Human peripheral blood lymphocytes regulate their volumes in hypotonic solutions. In hypotonic media in which Na+ is the predominant cation, an initial swelling phase is followed by a regulatory volume decrease (RVD) associated with a net loss of cellular K+. In media in which K+ is the predominant cation, the rapid initial swelling is followed by a slower second swelling phase. 86Rb+ fluxes increased during RVD and returned to normal when the original volume was approximately regained. Effects similar to those induced by hypotonic stress could also be produced by raising the intracellular Ca++ level. In isotonic, Ca++- containing media cells were found to shrink upon addition of the Ca++ ionophore A23187 in K+-free media, but to swell in K+-rich media. Exposure to Ca++ plus A23187 also increased 86Rb+ fluxes. Quinine (75 microM), an inhibitor of the Ca++-activated K+ pathway in other systems blocked RVD, the associated K+ loss, and the increase in 86Rb+ efflux. Quinine also inhibited the volume changes and the increased 86Rb fluxes induced by Ca++ plus ionophore. The calmodulin inhibitors trifluoperazine, pimozide and chlorpromazine blocked RVD as well as Ca++ plus A23187-induced volume changes. Trifluoperazine also prevented the increase in 86Rb+ fluxes and K+ loss induced by hypotonicity. Chlorpromazine sulfoxide, a relatively ineffective calmodulin antagonist, was considerably less potent as an inhibitor of RVD than chlorpromazine. It is suggested than an elevation in cytoplasmic [Ca++], triggered by cell swelling, increases the plasma membrane permeability to K+, the ensuing increased efflux of K+, associated anions, and osmotically obliged water, leading to cell shrinking (RVD).     

Effect of acetylcholine on postjunctional membrane permeability in eel electroplaque

Acetylcholine (ACh) was applied iontophoretically to the innervated face of isolated eel electroplaques while the membrane potential was being recorded intracellularly. At the resting potential (about -85 mV) application of the drug produced depolarizations (ACh potentials) of 20 mV or more which became smaller when the membrane was depolarized and reversed in polarity at about zero membrane potential. The reversal potential shifted in the negative direction when external Na+ was partially replaced by glucosamine. Increasing external K+ caused a shift of reversal potential in the positive direction. It was concluded that ACh increased the permeability of the postjunctional membrane to both ions. Replacement of Cl- by propionate had no effect on the reversal potential. In Na+-free solution containing glucosamine the reversal potential was positive to the resting potential, suggesting that ACh increased the permeability to glucosamine. Addition of Ca++ resulted in a still more positive reversal potential, indicating an increased permeability to Ca++ as well. Analysis of the results indicated that the increases in permeability of the postjunctional membrane to K+, Na+, Ca++, and glucosamine were in the ratios of approximately 1.0:0.9:0.7:0.2, respectively. With these permeability ratios, all of the observed shifts in reversal potential with changes in external ionic composition were predicted accurately by the constant field equation.     

Mechanism of depolarization of rat cortical synaptosomes at submicromolar external Ca2+ activity. The use of Ca2+ buffers to control the synaptosomal membrane potential.

Rat cortical synaptosomes responded to a reduction of external Ca2+ from pCa 3.5 to pCa 4.8 in the absence of MgCl2 with a slight decrease of internal K+ and an increase of Na+. The effects were prevented by tetrodotoxin or millimolar concentrations of MgCl2. Further lowering of external pCa to 7.7 with N-hydroxyethylethylenediaminetriacetate evoked a rapid fall of internal K+, which was specifically blocked by Ruthenium Red; tetrodotoxin and nifedipine were ineffective. A linear relationship was established between K+ and methyltriphenylphosphonium cation distribution ratios by varying external pCa between 4.8 and 7.7, indicating that K+ efflux resulted from a depolarization of the plasma membrane. An increase of Na+ permeability was suggested by the synaptosomes' gain of Na+ and the disappearance of the depolarization in an Na+-free sucrose medium. According to the constant field equation, the permeability ratio PNa/PK increased from 0.029 at pCa4.8 to 0.090 at pCa 7.7 with plasma membrane potentials of -74mV and -47mV, respectively. Since the plasma membrane responded to variation of external Ca2+ activities in the micromolar range with a graded and sustained depolarization, the use of Ca2+ buffers to control membrane potentials is suggested.     

The influence of external cations and membrane potential on Ca- activated Na efflux in Myxicola giant axons

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca- activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao- activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.     

Active calcium responses recorded optically from nerve terminals of the frog neurohypophysis

Voltage-sensitive dyes were used to record by optical means membrane potential changes from nerve terminals in the isolated frog neurohypophysis. Following the block of voltage-sensitive Na+ channels by tetrodotoxin (TTX) and K+ channels by tetraethylammonium (TEA), direct electric field stimulation of the nerve terminals still evoked large active responses. These responses were reversibly blocked by the addition of 0.5 mM CdCl2. At both normal and low [Na+]o, the regenerative response appeared to increase with increasing [Ca++]o (0.1-10 mM). There was a marked decrease in the size of the response, as well as in its rate of rise, at low [Ca++]o (0.2 mM) when [Na+]o was reduced from 120 to 8 mM (replaced by sucrose), but little if any effect of this reduction of [Na+]o at normal [Ca++]o. In normal [Ca++]o, these local responses most probably arise from an inward Ca++ current associated with hormone release from these nerve terminals. At low [Ca++]o, Na+ appears to contribute to the TTX-insensitive inward current.     

Thyroid hormone-induced changes in different ion-specific adenosine triphosphatase activities in liver of Singi fish Heteropneustes fossilis (Bloch)

A single injection of different doses of T3 (0.5, 5, 20, and 50 micrograms/g) to Singi fish caused an increase in Na+K+-ATPase activity in crude liver homogenate in a dose-dependent non-linear fashion on the 3rd d. Ca++- and Mg++-ATPase activity increased only with 20 and 50 micrograms/g of T3. Lowering the dose of T3 to 0.1 microgram and 0.25 microgram/g in a single injection had not effect on these enzyme activities. TETRAC (1, 2, and 4 micrograms/g) and TRIAC (2 and 4 micrograms/g) in a single injection enhanced the activities of Na+K+-ATPase, but Ca++- and Mg++-ATPase activities remained unchanged on the 3rd d. Immersion of Singi fish in thiourea-containing medium (1 mg/ml) for 30 d caused reduction in Na+K+-ATPase activity, but Ca++- and Mg++-ATPase activity remained unaltered. The reduced level of Na+K+-ATPase activity in the thiourea-treated hypothyroid fish was recovered and even brought above the control level by a single injection of T3 at the dose of 0.5 microgram/g. Differential sensitivity of various ion-specific ATPases to T3 in liver of Singi fish is thus documented.     

Ionic selectivity, saturation, and block in a K+-selective channel from sarcoplasmic reticulum

The open-channel conductance properties of a voltage-gated channel from sarcoplasmic reticulum were studied in planar phospholipid membranes. The channel is ideally selective for K+ over Cl- and for K+ over Ca++. In symmetrical 1 M solutions, the single-channel conductance (in pmho) falls in the order: K+ (214) > NH4+ (157) > Rb+ (125) > Na+ (72) > La+ (8.1) > Cs+ (< 3). In neutral bilayers, the channel conductance saturates with ion activity according to a rectangular hyperbolic relation, with half-saturation activities of 54 mM for K+ and 34 mM for Na+. Under symmetrical salt conditions, the K+:Na+ channel conductance ratio increases with salt activity, but the permeability ratio, measured by single-channel bi-ionic potentials, is constant between 20 mM and 2.5 M salt; the permeability ratio is equal to the conductance ratio in the limit of low-salt concentration. The channel conductance varies < 5% in the voltage range -100 to +70 mV. The maximum conductance varies K+ and Na+ is only weakly temperature dependent (delta H++ = 4.6 and 5.3 kcal/mol, respectively), but that of Li+ varies strongly with temperature (delta H++ = 13 kcal/mol). The channel's K+ conductance is blocked asymmetrically by Cs+, and this block is competitive with K+. The results are consistent with an Eyring-type barriers as it permeates the channel. The data conform to Lüger's (1973. Biochem. Biophys. Acta. 311:423-441) predictions for a "pure" single-ion channel.     

Effect of ionophore A23187 on the response to ADH in the ventral skin of Rana esculenta

The addition of the Ca++ ionophore A23187 (10 microM) to the inside solution of the frog skin induced a transient increase in the active Na+ transport in frog skin (Rana esculenta) which decayed to the control values 60 minutes after the addition. At the same time the skin resistance failed significantly; antidiuretic hormone addition resulted in no-more increase of the Na+ active transport; the skin resistance remained unchanged. To further investigate the role of intracellular calcium on the skin transepithelial permeability, the effect of A23187 ionophore on thiourea permeability has been tested. Increase in intracellular Ca++ concentration brought about by calcium ionophores have been shown to modify both basal and ADH-stimulated thiourea transport.     

Increased Ca++ or mg++ concentration reduces relative tight-junction permeability to Na+ in the cortical thick ascending limb of Henle's loop of rabbit kidney

We have tested whether increased Ca++ and Mg++ concentrations have an effect on transepithelial voltage (PDte) and transepithelial resistance (Rte) in isolated perfused cortical thick ascending limbs (cTAL) of rabbit kidney. The divalent cations added at 2.5, 5.0 and 10.0 mmol.l-1 to the lumen or peritubular bath perfusate led to a concentration-dependent increase in Rte. The maximal response in Rte was observed between 5 and 10 mmol.l-1. No significant change in active transepithelial potential difference (PDte) was observed. The increase in Rte still occurred when the transcellular current was reduced by Ba++ (3 mmol.l-1) added to the lumen perfusate. This suggests that the increase in Rte caused by Ca++ and Mg++ is due to a modification of the paracellular shunt pathway. In the absence of active transport, i.e. when furosemide (5.10(-5) mol.l-1) was added to the lumen perfusate. Ca++ and Mg++ reduced the transepithelial diffusion potential generated by a NaCl gradient established across the epithelium, and thus produced a reduction of the relative permeability for Na+ over Cl- (PNa+/PCl-) of the paracellular shunt pathway. This indicates that divalent cations increase Rte by reducing the sodium permeability of the tight junctions. The observed Ca++ and Mg++ induced reduction of the sodium permeability of the paracellular pathway corresponds to a decrease in net Na+ reabsorption by 5-10%. Since it has been demonstrated that peptide hormones such as parathyrin (PTH) modulate divalent cation and NaCl reabsorptions, in a second series of experiments we tested the effects of PTH (2-20 USP.l-1) and dbcAMP (10(-3) mol.l-1) on PDte and Rte of isolated perfused cTAL segments of rabbit nephron. Neither Rte nor PDte were affected by PTH or dbcAMP.     

Na+/K+/Cl- cotransport is stimulated by a Ca(++)-calmodulin-mediated pathway in BALB/c 3T3 fibroblasts.

In the present study, we investigated the role of intracellular Ca++ in the stimulation of the Na+/K+/Cl- cotransport in synchronized BALB/c 3T3 cells. The Na+/K+/Cl- cotransport was stimulated by the growth factors EGF, TGF-alpha, IGF-1, and IGF-2, which do not activate protein kinase C, but do induce a transient increase in free cytoplasmic Ca++. In addition, direct activation of protein kinase C by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) did not affect the Na+/K+/Cl- cotransport activity of quiescent cells. The Na+/K+/Cl- cotransport was also stimulated by the above mitogens in cells pretreated with the phorbol ester TPA. This treatment led to a progressive decline in the activity of cellular protein kinase C. This result implies that cells deficient in protein kinase C may still support stimulation of the Na+/K+/Cl- cotransport. Taken as a whole, these findings suggest that the Na+/K+/Cl- cotransport is stimulated predominantly by a protein kinase C-independent mechanism in BALB/c 3T3 fibroblasts. Both the intracellular Ca++ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) and two potent calmodulin antagonists, trifluoperazine (TFP) and chloropromazine (CP), blocked serum- and mitogen-stimulated Na+/K+/Cl- cotransport. These results suggest that the Na+/K+/Cl- cotransport is stimulated by an increase of intracellular Ca++ and subsequently by a Ca(++)-calmodulin-mediated pathway in the synchronized BALB/c 3T3 fibroblasts.     

The permeability of endplate channels to monovalent and divalent metal cations

The relative permeability of endplate channels to monovalent and divalent metal ions was determined from reversal potentials. Thallium is the most permeant ion with a permeability ratio relative to Na+ of 2.5. The selectivity among alkali metals is weak with a sequence, Cs+ greater than Rb+ greater than K+ greater than Na+ greater than Li+, and permeability ratios of 1.4, 1.3, 1.1, 1.0, and 0.9. The selectivity among divalent ions is also weak, with a sequence for alkaline earths of Mg++ greater than Ca++ greater than Ba++ greater than Sr++. The transition metal ions Mn++, Co++, Ni++, Zn++, and Cd++ are also permeant. Permeability ratios for divalent ions decreased as the concentration of divalent ion was increased in a manner consistent with the negative surface potential theory of Lewis (1979 J. Physiol. (Lond.). 286: 417--445). With 20 mM XCl2 and 85.5 mM glucosamine.HCl in the external solution, the apparent permeability ratios for the alkaline earth cations (X++) are in the range 0.18--0.25. Alkali metal ions see the endplate channel as a water-filled, neutral pore without high-field-strength sites inside. Their permeability sequence is the same as their aqueous mobility sequence. Divalent ions, however, have a permeability sequence almost opposite from their mobility sequence and must experience some interaction with groups in the channel. In addition, the concentrations of monovalent and divalent ions are increased near the channel mouth by a weak negative surface potential.     

Potassium and anion transport and activity of the Na+-pump in the erythrocyte membrane: 3 different mechanisms of regulation by intracellular calcium

A rise in intracellular Ca2+(Ca2+in) concentration from 1 to 100 microM is accompanied by a 100-fold increase of erythrocyte membrane permeability for k+ (opening of k+-channels) as well as by membrane hyperpolarization. Both effects are partly inhibited by trifluoroperazine and completely by calmidozolium (R24571). The Ca2+-dependencies of erythrocyte permeability for K+ and of Ca2+ binding to calmodulin are in good correlation. Within the same range of Ca2+in concentrations, i.e. 1-100 microM the activity of Na+-pump decreases by 90% despite the presence of trifluoroperazine and R24571. The permeability of erythrocytes for o-phosphate anions diminishes 15-fold after addition of the anionic exchanger SITS inhibitor. The SITS-inhibited component decreases 9-10 times with a rise in Ca2+in from 10 and 100 microM. In the presence of trifluoroperazine and R24571 the sensitivity of the anionic exchanger towards Ca2+ shows a 2-3 increase. The increase in Ca2+in up to 100 microM is concomitant with the activation of 32Pi incorporation into band 4.1 protein. The effect of Ca2+in on the phosphorylation of this protein is inhibited by calmodulin inhibitors. Addition of protein kinase C activator (4 beta-phorbol-12 beta-myristate-13-acetate) also leads to the increased incorporation of 32P into band 4.1 protein, whereas protein kinase A activator (dibutyryl-cAMP) causes 32P incorporation into bands 4.1 and 5 proteins. No effect of protein kinase activators on the activity of Na+-pump as well as on the permeability of erythrocyte membranes for K+ and anions was revealed. The data obtained point to the differences in the mechanisms of Ca2+in involvement in the regulation of the above ion transport systems. Presumably, none of the mechanisms is coupled with modification of the level of cytoskeleton protein phosphorylation. The effect of Ca2+ is mediated by the Ca2+ interaction with calmodulin only in the case of K+-channels.     

Serosal Na/Ca exchange and H+ and Na+ transport by the turtle and toad bladders

Summary A Na/Ca exchange system has been described in the plasma membrane of several tissues and seems to regulate the concentration of calcium in cytosol. Replacement of extracellular Na by sucrose increases calcium uptake into and decreases calcium efflux from the cell, leading to an increase in cytosolic calcium. The effect of an increase in cytosolic calcium mediated by the Na/Ca exchange system on H⁺ and Na transport in the turtle and toad bladder was investigated by replacing serosal Na isosmotically by sucrose or choline. Replacement of serosal by sucrose was associated with a significant inhibition of H⁺ secretion or Na transport which was reversible by addition of NaCl. Replacement of mucosal Na by sucrose failed to alter H⁺ secretion. Removal of serosal Na was associated with a significant increase in⁴⁵Ca uptake which could be blocked by pretreatment with lanthanum chloride. Pretreatment with lanthanum chloride blunted the inhibitory effect of replacement of serosal Na by sucrose on H⁺ and Na transport, thus suggesting that the increase in calcium uptake and the inhibition of transport are causally related. Under anaerobic conditions the rate of H⁺ or Na transport are linked to the rate of lactate production. The inhibition of Na or H⁺ transport by removal of serosal Na was accompanied by a proportional decrease in lactate production, thus suggesting that an increase in cytosolic calcium does not inhibit transport by uncoupling glycolysis from transport. Replacement of serosal Na by sucrose did not alter the force of the H⁺ or Na pump but led to an increase in resistance of the active pathway of H⁺ and Na transport. The inhibition of Na transport by replacement of serosal Na with sucrose could be reversed by addition of amphotericin B, an agent which increases luminal permeability to Na, thus suggesting that decreased Na entry across the apical membrane is the mechanism responsible for the inhibition of Na transport. The results of the present studies strongly suggest that an increase in cytosolic calcium through the serosal Na/Ca exchange system inhibits H⁺ and Na transport in the turtle and toad bladder probably by increasing the resistance of the luminal membrane.     

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.     

Binding of 3H-naloxone in the mouse brain: effect of ions and tolerance development

The binding of ³H-naloxone (spec. act. 5.2 Ci/mmol) in a crude mitochondrial fraction of the whole mouse brain was examined. Binding was reversed by the narcotic agonists levorphanol, morphine and 1-methadone but not by dextrorphan. Levorphanol sensitive (specific) ³H-naloxone binding was blocked by Na+, Li+, Ca++, Mg++ and Mn++ but not by K+. When the crude mitochondrial fraction was separated on a discontinuous sucrose gradient, the highest activity of specific binding was found in the nerve ending particle fraction. Animals made physically dependent by 3 day morphine pellet implantation did not show an increased binding affinity for ³H-nalovxone. The implantation of a 10 mg naloxone pellet increased the apparent total number of binding sites on the second and third day of implantation.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.