Inhibition by divalent cations and sulphydryl reagents of the passive Ca2+ transport in human red blood cells observed in the presence of vanadate

The uptake of 45Ca2+ by human red blood cells induced by vanadate was found to be inhibited by a number of divalent cations. The following order of potencies was determined (in parentheses, IC50 in mmol/l): Cu2+ (0.006), Zn2+ (0.014), Cd2+ (0.030), Co2+ (0.20), Ni2+ (0.25), Mn2+ (8.0), Ba2+ (9.0), Sr2+ (14.0). The effects of Cu2+, Zn2+ and Cd2+ were biphasic--over a critical concentration their inhibitory potencies decreased, and finally, were lost. Besides Ca2+, Sr2+, Ba2+ and Mn2+ were also taken up, but only Ca2+ and Sr2+ were capable of eliciting the Gárdos effect. Ni2+ was not taken up. Several HS reagents also inhibited 45Ca2+ uptake. The following order of potencies was determined (in parentheses, IC50 in mmol/l): mersalyl (0.0025), 5,5'-dithiobis(2,2'-dinitrobenzoic acid) (0.011), p-chloromercuric acid (0.042), N-ethylmaleimide (2.0). The effects of all HS reagents except N-ethylmaleimide were biphasic. The biphasicity of the actions of the indicated agents was caused by the opening of a new pathway for 45Ca2+ entry which is different from that observed in the presence of vanadate alone, and is inhibited by low concentrations of these agents. The modified form of the anion channel seems to be identical with the former pathway. The last one is mediated by a transport protein which has an ionic specificity similar to Ca2+ channels in excitable tissues, and contains an HS group which is essential for the transport function.     

Inhibition of protein kinase C phosphorylation by mono- and divalent cations

The effect of a matrix of concentrations of Ca2+ (0.01, 0.1, 0.5, 5 mM), Mg2+ (0.2, 0.5, 1, 2, 5, 10 mM), and Na+ (50, 100, 150 mM) on the phosphorylation of histone H-1 by protein kinase C was measured in the presence of 5 mol % diacylglycerol and Mg-ATP in both phosphatidylserine micelles and liposomes formed from a 1:4 mixture of phosphatidylserine and phosphatidylcholine. Monovalent cations (150 mM) reduced activity by 60 and 84% in the micelle and liposome assay systems, respectively. Inhibition was also observed with 5 mM Ca2+ and 10 mM Mg2+. The phosphorylating activity was compared with computer calculations of the negative electrostatic potentials (psi o) of the phospholipid membranes in the presence of the cations.     

Simple shifts in the voltage dependence of sodium channel gating caused by divalent cations

The effect of elevated divalent cation concentration on the kinetics of sodium ionic and gating currents was studied in voltage-clamped frog skeletal muscle fibers. Raising the Ca concentration from 2 to 40 mM resulted in nearly identical 30-mV shifts in the time courses of activation, inactivation, tail current decay, and ON and OFF gating currents, and in the steady state levels of inactivation, charge immobilization, and charge vs. voltage. Adding 38 mM Mg to the 2 mM Ca bathing a fiber produced a smaller shift of approximately 20 mV in gating current kinetics and the charge vs. voltage relationship. The results with both Ca and Mg are consistent with the hypothesis that elevated concentrations of these alkali earth cations alter Na channel gating by changing the membrane surface potential. The different shifts produced by Ca and Mg are consistent with the hypothesis that the two ions bind to fixed membrane surface charges with different affinities, in addition to possible screening.     

Inhibition of intestinal copper absorption by divalent cations and low-molecular-weight ligands in the rat

l-histidine (His) has been shown to enhance the inhibitory effect of zinc on intestinal copper absorption. This study was aimed at examining whether this effect of His was also extended to the interactions of other divalent cations: ferrous iron, tin, and cobalt, using an in vivo perfusion system in rats. Copper absorption and intestinal content of this element significantly decreased in the presence of 2 mM His and ferrous iron. Iron accumulation was greater when His was present than when omitted. A fivefold excess of tin inhibited copper absorption only when His was present. Citrate, at the same concentration as His, had no effect on copper absorption, but hepatic copper levels were increased, as compared to the absence of either His or citrate. Addition of 0.5 or 1.0 mM cobaltous salt plus His resulted in a sharp decrease in copper intestinal absorption, with an increase in intestinal tissue retention. These results confirm earlier findings with zinc and His, and suggest that a general phenomenon, either accelerating the removal of copper from the intestinal lumen or increasing, the retention of this element by the intestinal tissue, is a common feature of the interaction between cations of similar electronic configuration to copper and a high-affinity ligand, such as His.     

Importance of extracellular divalent cations to polarisation of polymorphonuclear leukocytes induced by plasma

The roles of extracellular calcium and magnesium ions in the polarisation of human peripheral blood polymorphonuclear leukocytes (PMN) induced by autologous fresh heparinised plasma were investigated by studying the effects of 5 mM chelators of divalent cations [ethylenediamine tetra-acetic acid (EDTA), ethylenebis-(oxyethylene-nitrilo)-tetra-acetic acid (EGTA) or disodium hydrogen citrate]. In addition, the effects of a blocker of membrane calcium channels (verapamil) were studied. Polarisation of PMN suspended in plasma (84.1 +/- 11.9%) was reduced by each chelating agent over 30 min (to 20.0 +/- 15.6% by EDTA, to 42.5 +/- 19.3% by EGTA and to 29.4 +/- 22.9% by citrate). Polarisation of PMN suspended in plasma treated with EDTA or EGTA was restored by inclusion of equimolar additional Ca2+ ions, and in plasma treated with EDTA, EGTA or citrate, by equimolar additional Mg2+ ions. Additional Mg2+ had no effect on the spherical shape of PMN in Hanks' solution and additional cations had no effects on the polarisation of PMN induced by fMLP. Cells rendered spherical by each chelating agent in plasma for 30 min retained their ability to polarise on addition of fMLP to the plasma-chelator medium. Verapamil (10(-4) M) markedly reduced polarisation in plasma (to 52 +/- 11.3%) but the same drug (10(-5) M) had no such effect. In contrast to the polarisation of cells in plasma, the polarisation response of PMN to N-formyl-methionyl-leucyl-phenylalanine (fMLP, 10(-8) M) in buffered Hanks' solution was not affected by any of the chelating agents or by verapamil, even in high concentration. These results indicate that extracellular divalent cations are necessary for the polarisation of PMN suspended in autologous plasma and that the mechanism of polarisation of PMN in plasma may be different to that of polarisation induced by fMLP.     

Block by extracellular divalent cations of Drosophila big brain channels expressed in Xenopus oocytes

Drosophila Big Brain (BIB) is a transmembrane protein encoded by the neurogenic gene big brain (bib), which is important for early development of the fly nervous system. BIB expressed in Xenopus oocytes is a monovalent cation channel modulated by tyrosine kinase signaling. Results here demonstrate that the BIB conductance shows voltage- and dose-dependent block by extracellular divalent cations Ca(2+) and Ba(2+) but not by Mg(2+) in wild-type channels. Site-directed mutagenesis of negatively charged glutamate (Glu(274)) and aspartate (Asp(253)) residues had no effect on divalent cation block. However, mutation of a conserved glutamate at position 71 (Glu(71)) in the first transmembrane domain (M1) altered channel properties. Mutation of Glu(71) to Asp introduced a new sensitivity to block by extracellular Mg(2+); substitutions with asparagine or glutamine decreased whole-cell conductance; and substitution with lysine compromised plasma membrane expression. Block by divalent cations is important in other ion channels for voltage-dependent function, enhanced signal resolution, and feedback regulation. Our data show that the wild-type BIB conductance is attenuated by external Ca(2+), suggesting that endogenous divalent cation block might be relevant for enhancing signal resolution or voltage dependence for the native signaling process in neuronal cell fate determination.     

Sodium transport through the amiloride-sensitive Na-Mg pathway of hamster red cells

Previous work showed that in hamster red cells the amiloride-sensitive (AS) Na⁺ influx of 0.8 mmol/liter cells/hr is not mediated by Na-H exchange as in other red cells, but depends upon intracellular Mg²⁺ and can be increased by 40-fold by loading cells with Mg²⁺ to 10 mm. The purpose of this study was to verify the connection of AS Na⁺ influx with Na-dependent, amiloride-sensitive Mg²⁺ efflux and to utilize AS Na⁺ influx to explore that pathway.Determination of unidirectional influx of Na⁺ and net loss of Mg²⁺ in parallel sets of cells showed that activation by extracellular [Na⁺] follows a simple Michaelis-Menten relationship for both processes with a K _m of 105–107 mm and that activation of both processes is sigmoidally dependent upon cytoplasmic [Mg²⁺] with a [Mg²⁺]_0.5 of 2.1–2.3 mm and a Hill coefficient of 1.8. Comparison of V_max for both sets of experiments indicated a stoichiometry of 2 Na: l Mg. Amiloride inhibits Na⁺ influx and Mg²⁺ extrusion in parallel (K _i = 0.3 mm). Like Mg²⁺ extrusion, amiloride-sensitive Na⁺ influx shows an absolute requirement for cytoplasmic ATP and is increased by cell swelling. Hence, amiloride-sensitive Na⁺ influx in hamster red cells appears to be through the Na-Mg exchange pathway.There was no amiloride-sensitive Na⁺ efflux in hamster red cells loaded with Na⁺ and incubated with high [Mg²⁺] in the medium with or without external Na⁺, nor with ATP depletion. Hence, this is not a simple Na-Mg exchange carrier.     

Sodium fluxes in human fibroblasts: kinetics of serum-dependent and serum-independent pathways

Sodium influx in serum-deprived human fibroblasts is by way of a pathway which shows saturation kinetics. A plot of initial Na influx versus [Na]0 ([Na]i approximately equal to 10 mM) gives a simple Michaelis-Menten type of curve with a K1/2 = 70.0 +/- 8.1 mM and a Vmax = 14.5 +/- 1.9 mumol/g prot/min. A similar plot of initial Na influx versus [Na]0 in the presence of 10% fetal bovine serum (FBS) gives a nonsaturating curvilinear response which appears to be biphasic. A plot of the serum-dependent Na influx versus [Na]0 (obtained by subtracting the curve in the absence of FBS from the curve in the presence of 10% FBS) shows that there is a linear relationship between serum-induced Na influx and external [Na]. At physiological Na concentrations, in the presence of FBS, the serum-induced Na influx is equal to the amiloride-sensitive Na flux, whereas in the absence of serum amiloride inhibits less than 10% of the Na influx. The effect of intracellular Na on Na flux was tested by preloading cells with Na in a digitoxin-containing medium prior to measurement of Na flux. A plot of steady-state Na exchange flux versus [Na]0 ([Na]i approximately equal to [Na]0) in the absence of serum gives a curve that appears to saturate at approximately 100 mM Na (flux = 100 mumol/g prot/min) and then declines with increasing [Na] (flux = 40 mumol/g prot/min at 150 mM). In contrast to Na influx in control serum-deprived cells, Na flux in Na-loaded cells in dramatically inhibited by the presence of amiloride. Since the peak Na exchange flux of 100 mumol/g prot/min is greatly in excess of the Vmax for Na influx in control serum-deprived cells and the enhanced Na flux is amiloride-sensitive, elevating intracellular Na must somehow activate the amiloride-sensitive Na transport system, which is normally only minimally active in the absence of serum.     

Inhibition of trichocyst exocytosis and calcium influx in Paramecium by amiloride and divalent cations

Summary— Regulated exocytosis of defensive secretory organelles, the trichocysts, as well as a transient Ca²⁺-influx can be induced in Paramecium by aminoethyldextran (Kerb?uf and Cohen, J Cell Biol (1990) 111, 2527). Knoll et al (Febs Lett (1992) 304, 265) reported that veratridine was also a secretagogue for Paramecium. Here we show that, like aminoethyldextran, veratridine induces a transient Ca²⁺-influx. Both aminoethyldextran-and veratridine-induced exocytosis and associated Ca²⁺-influx were: i) blocked in the nd12 thermosensitive mutant at the non-permissive temperature; and ii) inhibited by amiloride and four divalent cations, Ba²⁺, Mg²⁺, Sr²⁺ and Co²⁺. This suggests that, although of different chemical nature, aminoethyldextran and veratridine act through the same physiological pathway. In addition, the inhibitory doses are comparable to the ones found to inhibit a hyperpolarization-sensitive Ca²⁺-current described in Paramecium (Preston et al (1992) J Gen Physiol 100, 233). The possibility that the activation of this Ca²⁺-current by the secretagogue represents an early step in the regulation of trichocyst exocytosis is discussed.     

Effect of anions on amiloride-sensitive,active sodium transport across rabbit colon,in Vitro

Summary replacement of Cl in the solutions bathing partial mucosal strips of rabbit descending colon with sulfate, isethionate, hydroxypropane-sulfonate and, to a lesser degree, ethanesulfonate stimulates active Na absorption (J _net ^Na ) when the baso-lateral pump mechanism is not saturated. These effects are rapid in onset and are readily reversible. Our findings indicate that these stimulatory anions decrease the resistance of the amiloridesensitive Na entry step at the mucosal membrane (R _Na ^m ). However, when the active Na pump mechanism at the baso-lateral membrane is saturated these stimulatory anions do not decrease the resistance of the Na entry process. These findings suggest the presence of a negative feedback between the activity of the pump mechanism and the resistance of the Na entry step which may be mediated by the size of the intracellular Na transport pool. In other words, it seems that when the baso-lateral pump is operating at its maximal rate the resistance to Na entry across the mucosal membrane through the amiloride-sensitive pathway is at a minimum and cannot be further decreased.     

Induction of amino acid transport activity in chick embryo fibroblasts by replacement of extracellular sodium chloride with disaccharide

The activity of amino acid transport System A in avian fibroblasts was increased following incubation of the cells in a medium in which most of the NaCl normally present had been isoosmotically replaced by sucrose. This increase was detectable after 2 h of incubation, reached a maximum at about 4 h, and remained constant thereafter. Transfer of treated cells back to a normal medium resulted in decay of the induced transport activity, with a half-life of less than 2 h. Kinetic analysis revealed that the increase in transport activity arose from an increase in Vmax, with little change in Km. This induction of System A activity did not occur if an inhibitor of either RNA or protein synthesis was present in the modified medium. The use of various different solutes as replacements for NaCl in the incubation medium showed that, although each replacement caused a decrease in both cellular Na+ content and protein synthesis, only disaccharides produced the increase in amino acid transport activity. In addition, estimates of cell volume indicated that, even under iso-osmotic conditions, incubation in the sucrose-containing medium caused initial cell shrinkage, followed by swelling. It is concluded that this induction of System A activity is associated with a volume regulatory process and that this process probably accounts for the parallel responses previously observed when cells were incubated in hyperosmolar media. Induction of amino acid transport activity by this process is distinct from adaptive regulation, caused by amino acid starvation; but the two processes are not strictly additive, and so appear to converge at some step.     

Mechanisms of TNF-alpha stimulation of amiloride-sensitive sodium transport across alveolar epithelium

Because tumor necrosis factor (TNF)-alpha can upregulate alveolar fluid clearance (AFC) in pneumonia or septic peritonitis, the mechanisms responsible for the TNF-alpha-mediated increase in epithelial fluid transport were studied. In rats, 5 microg of TNF-alpha in the alveolar instillate increased AFC by 67%. This increase was inhibited by amiloride but not by propranolol. We also tested a triple-mutant TNF-alpha that is deficient in the lectinlike tip portion of the molecule responsible for its membrane conductance effect; the mutant also has decreased binding affinity to both TNF-alpha receptors. The triple-mutant TNF-alpha did not increase AFC. Perfusion of human A549 cells, patched in the whole cell mode, with TNF-alpha (120 ng/ml) resulted in a sustained increase in Na(+) currents from 82 +/- 9 to 549 +/- 146 pA (P < 0.005; n = 6). The TNF-alpha-elicited Na(+) current was inhibited by amiloride, and there was no change when A549 cells were perfused with the triple-mutant TNF-alpha or after preincubation with blocking antibodies to the two TNF-alpha receptors before perfusion with TNF-alpha. In conclusion, although TNF- alpha can initiate acute inflammation and edema formation in the lung, TNF-alpha can also increase AFC by an amiloride-sensitive, cAMP-independent mechanism that enhances the resolution of alveolar edema in pathological conditions by either binding to its receptors or activating Na(+) channels by means of its lectinlike domain.     

Rectification of cystic fibrosis transmembrane conductance regulator chloride channel mediated by extracellular divalent cations.

We report here distinct rectification of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel reconstituted in lipid bilayer membranes. Under the symmetrical ionic condition of 200 mM KCl (with 1 mM MgCl2 in cis intracellular and 0 MgCl2 in trans extracellular solutions, pH in both solutions buffered at 7.4 with 10 mM HEPES), the inward currents (intracellular-->extracellular chloride movement) through a single CFTR channel were approximately 20% larger than the outward currents. This inward rectification of the CFTR channel was mediated by extracellular divalent cations, as the linear current-voltage relationship of the channel could be restored through the addition of millimolar concentrations of MgCl2 or CaCl2 to the trans solution. The dose responses for [Mg]zero and [Ca]zero had half-dissociation constants of 152 +/- 72 microM and 172 +/- 40 microM, respectively. Changing the pH buffer from HEPES to N-tris-(hydroxymethyl)methyl-2-aminoethanesulfonic acid did not alter rectification of the CFTR channel. The nonlinear conductance property of the CFTR channel seemed to be due to negative surface charges on the CFTR protein, because in pure neutral phospholipid bilayers, clear rectification of the channel was also observed when the extracellular solution did not contain divalent cations. The CFTR protein contains clusters of negatively charged amino acids on several extracellular loops joining the transmembrane segments, which could constitute the putative binding sites for Ca and Mg.