Alterations by saponins of passive Ca2+ permeability and Na+-Ca2+ exchange activity of canine cardiac sarcolemmal vesicles

Saponins can both permeabilize cell plasma membranes and cause positive inotropic effects in isolated cardiac muscles. Different saponins vary in their relative abilities to cause each effect suggesting that different mechanisms of action may be involved. To investigate this possibility, we have compared the effects of seven different saponins on the passive Ca2+ permeability and Na+-Ca2+ exchange activity of isolated canine cardiac sarcolemmal membranes. Saponins having hemolytic activity reversibly increased the passive efflux of Ca2+ from sarcolemmal vesicles preloaded with 45Ca2+ with the following order of potency: echinoside-A greater than echinoside-B greater than holothurin-A greater than holothurin-B greater than sakuraso-saponin. Ginsenoside-Rd and desacyl-jego-saponin, which lack hemolytic activity, had no significant effect on this variable. The saponins also stimulated Na+-Ca2+ exchange activity measured as Na+-dependent Ca2+ uptake by sarcolemmal vesicles. Ginsenoside-Rd and desacyl-jego-seponin, which did not affect passive Ca2+ permeability, stimulated the uptake, while in contrast, echinoside-A and -B only slightly increased or decreased this latter variable. Thus, the abilities of these compounds to enhance Na+-Ca2+ exchange activity seem to be inversely related to their abilities to increase the Ca2+ permeability. Effects by the echinosides on Na+-Ca2+ exchange may be masked by the loss of Ca2+ from the vesicles due to the increased permeability. These results suggest that the saponins interact with membrane constituent(s) that can influence the passive Ca2+ permeability and the Na+-Ca2+ exchange activity of cardiac sarcolemmal membranes.     

Effect of vanadate and of removal of extracellular Ca2+ and Na+ on tension development and 45Ca efflux in rat and frog myocardium

Vanadate in the range 0-5 mM has positive inotropic effects on myocardial strips of frog and to a lesser extent on those of rat. Inhibiting the sarcolemmal Na+, Ca2+ exchange by a solution free of Ca2+ and Na+ caused a drop in 45Ca efflux and a transient increase in resting tension. These effects were more expressed for the frog than for the rat myocardium, which suggests that the Na+ for Ca2+ exchange across the cell membrane is more important in the frog than in the rat myocardium. A subsequent addition of vanadate at 2 or 5 mM had no effect on 45Ca efflux, while it increased the resting tension. This increase was higher for the frog than for the rat myocardium. These results suggest that the inotropic effects of vanadate may be due to an effect on membrane-bound Ca2+-ATPase.     

Modulation of Na+-Ca2+ exchange and Ca2+ permeability in cardiac sarcolemmal vesicles by doxylstearic acids

We examine the effects of 5-, 12- and 16-doxylstearic acids on the Na+-Ca2+ exchange and passive Ca2+ permeability of cardiac sarcolemmal vesicles. Stearic acid is a weak stimulator of Na+-Ca2+ exchange. A doxyl moiety potentiates stimulation with the order of increasing potency being 5-, 12- and then 16-doxylstearic acid. Stearic acid has little effect on vesicle Ca2+ permeability but again the doxylstearates are more effective. The sequence of potency is reversed, however, from that for increasing Na+-Ca2+ exchange. 5-Doxylstearic acid most markedly exchanges passive Ca2+ flux followed by the 12-, and then 16-doxylstearic acids. Methyl esters of the doxylstearates have no effect on either Na+-Ca2+ exchange or Ca2+ permeability. We model the results as follows. For a fatty acid to stimulate Na+-Ca2+ exchange activity, an anionic charge is required to interact with the exchanger protein at the membrane surface. Stimulation is potentiated by a perturbation (such as provided by a doxyl group) within the lipid bilayer. The perturbation is most effective at a location towards the center of the bilayer. To increase passive Ca2+ permeability an anionic charge is again essential. Disorder within the bilayer is also important, but now the most important site is near the membrane surface. Results of experiments with linolenic and gamma-linolenic acid and previous studies with other fatty acids also support this model.     

Induction of 86Rb fluxes by Ca2+ and volume changes in thymocytes and their isolated membranes

Cell swelling and elevated intracellular Ca2+ increase K+ permeability in lymphocytes. Experiments were performed to test whether these effects can also be elicited in isolated plasma membrane vesicles. Rabbit thymocytes, used as a source of membrane vesicles, were found to regain their volume after swelling in hypotonic, low-K+ media. This regulatory volume decrease (RVD) was inhibited by quinine and trifluoperazine, but not affected by ouabain. Both efflux and uptake of K+ (86Rb) were stimulated by hypotonicity. Addition of A23187 plus Ca2+ also increased 86Rb fluxes. Ca2+- and volume-induced 86Rb fluxes were also studied in isolated membranes. A plasma membrane-rich vesicle fraction, enriched over 11-fold in 5'-nucleotidase, was isolated from thymocytes. The vesicles were about 35% inside-out and trapped 86Rb in an osmotically active compartment of approximately 1.3 microliter/mg protein. Equilibrium exchange fluxes of 86Rb in the vesicles were unaffected by Ca2+ with or without A23187. Calmodulin had no effect on 86Rb permeability but stimulated ATP-dependent Ca2+ accumulation. Hypotonic swelling increased both uptake and efflux of 86Rb from vesicles. However, this increase was not blocked by either quinine or trifluoperazine, was not specific for K+ (86Rb), and is probably unrelated to RVD. It is concluded that components essential for the volume- and Ca2+-induced changes in K+ permeability are lost or inactivated during membrane isolation. An intact cytoarchitecture may be required for RVD.     

Stimulation of sodium-calcium exchange by cholesterol incorporation into isolated cardiac sarcolemmal vesicles

Modification of the cholesterol content of highly purified cardiac sarcolemma from dog ventricles was accomplished by incubation with phosphatidylcholine liposomes containing various amounts of cholesterol. The degree of cholesterol enrichment could be varied by changing the liposomal cholesterol/phospholipid ratio or varying the liposome-membrane incubation time. Na+-Ca2+ exchange measured in cholesterol-enriched sarcolemmal vesicles was increased up to 48% over control values. The stimulation of Na+-Ca2+ exchange was associated with an increased affinity of the exchanger for Ca2+ (Km = 17 microM compared with Km = 22 microM for control preparations). Na+-Ca2+ exchange measured in cholesterol-depleted membrane preparations was decreased by 15%. This depressed activity was associated with a decreased affinity of the exchanger for Ca2+ (Km = 27 microM). These changes were not due to either a change in membrane permeability to Ca2+ or an increase in the amount of Ca2+ bound to sarcolemmal vesicles. The stimulating effect of cholesterol enrichment was specific to the Na+-Ca2+ exchange process since sarcolemmal Ca2+-Mg2+ ATPase activity was depressed 40% by cholesterol enrichment. Further, K+-p-nitrophenylphosphatase and Na+-K+ ATPase activities were depressed in both cholesterol-depleted and cholesterol-enriched sarcolemmal vesicles. In situ oxidation of membrane cholesterol completely eliminated Na+-Ca2+ exchange. These results suggest that cholesterol is intimately associated with Na+-Ca2+ exchange and may interact with the exchange protein and modulate its activity.     

Effects of fatty acids on Na+-Ca2+ exchange and Ca2+ permeability of cardiac sarcolemmal vesicles

We have previously reported that anionic phospholipids (Philipson, K.D., and Nishimoto, A.Y. (1984) J. Biol. Chem. 259, 16-19) and other anionic amphiphiles (Philipson, K.D. (1984) J. Biol. Chem. 259, 13999-14002) stimulate Na+-Ca2+ exchange in cardiac sarcolemmal vesicles. To further these studies, we have now investigated the effects of a variety of fatty acids on both Na+-Ca2+ exchange and passive Ca2+ permeability. Na+-Ca2+ exchange was stimulated by fatty acids by up to 150%. Unsaturated fatty acids were more potent than saturated fatty acids, and the stimulation was primarily due to a decrease in the apparent KM (Ca2+). There was a positive correlation between the ability of a fatty acid to stimulate Na+-Ca2+ exchange and to increase passive Ca2+ permeability. The methyl esters of fatty acids had no effects on either exchange or permeability indicating the importance of anionic charge. We conclude that the combination of local lipid disorder and anionic charge regulate Na+-Ca2+ exchange. Perturbations of the bilayer hydrophobic region and increased negative surface charge are both required for fatty acids to increase passive Ca2+ flux. Na+-Ca2+ exchange is stimulated when the ratio of membrane free fatty acid to phospholipid is about 5%. This level of fatty acid is achieved during 1 h of myocardial ischemia (Chien, K. R., Han, A., Sen, A., Buja, L. M., and Willerson, J. T. (1984) Circ. Res. 54, 313-322), indicating that ischemia could induce altered sarcolemmal Ca2+ transport due to fatty acid accumulation.     

Calcium dependence of the thrombin-induced increase in endothelial albumin permeability

We examined whether the increase in endothelial albumin permeability induced by alpha-thrombin is dependent on extracellular Ca2+ influx. Permeability of 125I-albumin across confluent monolayers of cultured bovine pulmonary artery endothelial cells was measured before and after the addition of 0.1 microM alpha-thrombin. In the presence of normal extracellular Ca2+ concentration ([Ca2+]o, 1000 microM), alpha-thrombin produced a 175 +/- 10% increase in 125I-albumin permeability. At lower [Ca2+]o (100, 10, 1, or less than 1 microM), alpha-thrombin caused a 140% increase in permeability (P less than 0.005). LaCl3 (1 mM), which competes for Ca2+ entry, blunted 38% of the increase in permeability. Preloading endothelial monolayers with quin2 to buffer cytosolic Ca2+ (Cai2+) produced a dose-dependent inhibition of the increase in 125I-albumin permeability. Preincubation with nifedipine or verapamil was ineffective in reducing the thrombin-induced permeability increase. A 60 mM K+ isosmotic solution did not alter base-line endothelial permeability. alpha-Thrombin increased [Ca2+]i in a dose-dependent manner and the 45Ca2+ influx rate. Extracellular medium containing 60 mM K+ did not increase 45Ca2+ influx, and nifedipine did not block the rise in 45Ca2+ influx caused by alpha-thrombin. Ca2+ flux into endothelial cells induced by alpha-thrombin does not occur through voltage-sensitive channels but may involve receptor-operated channels. In conclusion, the increase in endothelial albumin permeability caused by alpha-thrombin is dependent on Ca2+ influx and intracellular Ca2+ mobilization.     

Investigation of the relationship between cell-surface calcium-ion gating and phosphatidylinositol turnover by comparison of the effects of elevated extracellular potassium ion concentration on ileium smooth muscle and pancreas.

Incubation of fragments of guinea-pig ileum smooth muscle in the presence of an elevated extracellular K+ concentration, which causes an increase in cell-surface Ca2+ permeability and thus leads to contraction, caused a marked increase in phosphatidylinositol turnover, as assessed by incorporation of 32Pi. This response was not diminished by atropine or propylbenzilycholine mustard, two muscarinic cholinergic antagonists, and was therefore not caused by the release of endogenous acetylcholine within the tissue. In contrast, exposure of guinea-pig pancreas fragments to high extracellular [K+], which does not increase cell-surface Ca2+ permeability or evoke secretion, did not cause an increase in phosphatidylinositol turnover, even though such an increase was triggered by carbamoylcholine, which is a secretagogue. These observations are consistent with a suggested function for phosphatidylinositol breakdown in the mechanisms of cell-surface Ca2+ gates.     

Effects of vanadate, menadione and menadione analogs on the Ca2+-activated K+ channels in human red cells. Possible relations to membrane-bound oxidoreductase activity

The modulation of the Ca2+- (or Pb2+-)activated K+ permeability in human erythrocytes by vanadate, menadione and chloro-substituted menadione analogs was investigated by measurements of K+ fluxes and single-channel currents. Vanadate and menadione stimulate the K+ permeability by increasing the probability of channel openings; the menadione analogs, on the other hand, inhibit the K+ permeability by increasing the probability of channel closings. The compounds used in these experiments also interact with oxidoreductases; it is demonstrated that menadione analogs in contrast to menadione strongly inhibit the membrane-bound dehydrogenase in the erythrocytes. Concentrations of Pb2+ above 10 mumol/l, but not of Ca2+, inhibit the enzyme activity as well as the K+ permeability. The parallel effects on dehydrogenase activity and the K+ channels suggest a direct relationship between these two systems in the membrane of erythrocytes.     

等渗NaCL和KCL胁迫对高梁幼苗生长和气体交换的影响

本文比较研究了等渗NaCl和KCl胁迫下,高粱幼苗生长及叶片离子含量、质膜相对透性和有关气体交换参数的变化。结果表明,在低浓度NaCl和KCl胁迫7天时,高粱生长、含水量和质膜相对透性与对照相比没有明显变化,而净光合速率、蒸腾速率和气孔导度已明显下降,叶肉细胞间隙CO2浓度明显增加。NaCl胁迫下叶片Na+含量成倍增加,而K+和Ca2+含量无明显变化。KCl胁迫时叶片K+含量明显增加,Ca2+含量明显下降,而Na+含量没有明显变化。随着NaCl或KCl浓度的增加,幼苗生长和叶片含水量明显下降,质膜透性和细胞间隙CO2浓度明显增加,净光合速率、蒸腾速率和气孔导度进一步下降。 NaCl胁迫下叶片Na+含量进一步增加,K+和Ca2+进一步下降,而KCl胁迫下叶片K+含量进一步 增加,Na+和Ca2+含量进一步下降。KCl对高粱生长抑制、质膜透性、Ca2+含量下降及光合气体交换参数的影响均明显大于等渗的NaCl。     

3':5'-AMP-dependent phosphorylation of muscular membrane proteins and calcium transport

The data are presented concerning the role of cAMP-dependent phosphorylation of sarcoplasmic reticulum and sarcolemma proteins in the transport of Ca2+ through the cardiac skeletal and smooth muscle membranes. Phosphorylation of membrane proteins by soluble and membrane-bound cAMP-dependent protein kinases is shown to have a regulating effect on Mn2+-, Ca2+-ATPase activity and to change membrane permeability for Ca2+. The molecular mechanisms and the character of the interaction between the transport systems and the phosphorylating protein substrates have not been yet established.     

Arachidonic acid inhibits thyrotropin-releasing hormone-induced elevation of cytoplasmic free calcium in GH3 pituitary cells

We have shown that arachidonic acid stimulates 45Ca2+ efflux from prelabeled rat pituitary mammotropic (GH3) cells resuspended in "Ca2+-free" medium (Kolesnick, R. N., Mussachio, I., Thaw, C., and Gershengorn, M. C. (1984) Am. J. Physiol. 246, E458-E462). In this study, we further characterize the effects of arachidonic acid on Ca2+ homeostasis in GH3 cells and demonstrate its antagonism of changes induced by thyrotropin-releasing hormone (TRH). At below 5 microM, arachidonic acid stimulated intracellular for extracellular Ca2+ exchange without affecting cell Ca2+ content. Above 5 microM, arachidonic acid decreased membrane-bound Ca2+, as monitored by chlortetracycline, and decreased total cell 45Ca2+ content by depleting nonmitochondrial and mitochondrial pools. However, arachidonic acid did not elevate cytoplasmic free Ca2+ concentration ([Ca2+]i). Arachidonic acid inhibited TRH-induced 45Ca2+ efflux, loss of membrane-bound Ca2+, mobilization of nonmitochondrial Ca2+, and elevation of [Ca2+]i. Arachidonic acid also lowered elevated [Ca2+]i caused by release of mitochondrial Ca2+ with an uncoupler or by influx of extracellular Ca2+ stimulated with K+ depolarization. Hence, arachidonic acid stimulates Ca2+ extrusion from and depletes Ca2+ stores within GH3 cells. We suggest that arachidonic acid may be an important regulator of cellular Ca2+ homeostasis which may inhibit TRH-induced elevation of [Ca2+]i.     

Tentoxin-induced energy-independent adenine nucleotide exchange and ATPase activity with chloroplast coupling factor 1.

1. The effect of energy transfer inhibitors on energy-dependent exchange of tightly bound adenine nucleotides with washed, broken spinach thylakoids has been studied. Energy transfer inhibitors that inhibit the ATPase activity of soluble chloroplast coupling factor 1 (CF1) (e.g. phloridzin and tentoxin) do not inhibit energy-dependent adenine nucleotide exchange. Energy transfer inhibitors that block proton flux through the hydrophobic protein proton channel (CF0) (e.g. dicyclohexylcarbodiimide and triphenyltin chloride) also block light-dependent adenine nucleotide exchange. 2. Tentoxin, at relatively high concentrations, stimulates an energy-independent exchange of adenosine diphosphate. 3. High concentrations of tentoxin elicit a Ca2+-dependent ATPase activity with soluble CF1, but has no effect on the Ca2+-dependent ATPase activity of membrane-bound CF1. 4. The trypsin-activated, Ca2+-dependent, membrane-bound ATPase is not affected by high concentrations of tentoxin, whereas the dithiothreitol-activated, Mg2+-dependent ATPase is markedly inhibited. 5. The reconstitution of chloroplasts, partially depleted in CF1, with soluble CF1 is correlated with the loss of tentoxin-induced, Ca2+-dependent ATPase activity associated with soluble CF1.     

A Na(+)-dependent Ca2+ exchanger generates the sustained increase in intracellular Ca2+ required for T cell activation.

Movement of extracellular Ca2+ is required for the sustained increase in [Ca2+]i necessary for T cell activation. However, the mechanisms mediating mitogen-stimulated Ca2+ movement into T cells have not been completely delineated. To explore the possibility that a Na(+)-dependent Ca2+ (Na+/Ca2+) exchanger might play a role in the mitogen-induced increases in [Ca2+]i required for T cell activation, the effects of inhibitors of this exchanger were examined. Inhibitors of Na+/Ca2+ exchange suppressed the sustained increase in [Ca2+]i stimulated by ligation of the CD3-TCR complex, but did not affect mobilization of intracellular Ca2+ stores. Consistent with the importance of this prolonged increase in [Ca2+]i in T cell activation, Na+/Ca2+ exchange inhibitors, but not inhibitors of the Na+/H+ antiporter, inhibited DNA synthesis stimulated by immobilized anti-CD3 mAb. Inhibition only occurred when the agents were present during the first hours after stimulation. These agents also inhibited IL-2 production, but not expression of the IL-2R or of an early activation Ag, 4F2. Inhibition of IL-2 production did not account for the inhibition of T cell proliferation as addition of exogenous IL-2 or phorbol ester (PDB) did not overcome the inhibition. In contrast, activation pathways that are not thought to require an increase in [Ca2+]i such as IL-1 + PDB or engagement of CD28 in the presence of PDB were less sensitive to the suppressive effects of inhibitors of Na+/Ca2+ exchange. Thus, proliferation induced by these stimuli was not suppressed by low concentrations of these inhibitors and IL-2 production induced by mAb to CD28 + PDB was not inhibited by any concentration of inhibitors of Na+/Ca2+ exchange. These results suggest that stimulation of a Ca2+ transporter with the same spectrum of inhibition as the Na+/Ca2+ exchanger in other tissues mediates the sustained increase in [Ca2+]i required for T cell activation after CD3 ligation.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

Phorbol ester-induced changes in cytoplasmic Ca2+ in human neutrophils. Involvement of a pertussis toxin-sensitive G protein

Activation of neutrophils by most soluble stimuli is associated with a marked increase in intracellular free Ca2+ ([Ca2+]i). However, under physiological conditions (Na+-rich media), the potent activator 12-O-tetradecanoylphorbol-13-acetate (TPA) causes no change or a decrease in [Ca2+]i. We report here that the [Ca2+]i response to phorbol esters varies depending on the ionic composition of the medium. A marked increase in [Ca2+]i was detected in Na+-free solutions. Maximal effects were observed when N-methyl-D-glucammonium+ or choline+ were substituted for Na+, whereas an intermediate response was recorded in K+ medium. The increase in [Ca2+]i was substantially (approximately 65%) inhibited by removal of external Ca2+. A [Ca2+]i increase was also elicited by other beta-phorbol diesters and by diacylglycerol, but not by unesterified phorbol or by alpha-phorbol diesters, indicating involvement of protein kinase C. The increase in [Ca2+]i observed in Na+-free media is not due to inhibition of Na+/Ca2+ exchange, since no change in [Ca2+]i in response to TPA was observed in: 1) cells suspended in Li+, which is not countertransported for Ca2+; 2) cells preloaded with Na+ to eliminate the driving force for Na+/Ca2+ exchange; and 3) cells treated with 3',4'-dichlorobenzamyl, an inhibitor of Na+/Ca2+ exchange. Similarly, the [Ca2+]i increase in Na+-free media is not linked to the absence of Na+/H+ exchange and the associated cytoplasmic acidification since: 1) it was not observed in Na+ media in the presence of inhibitors of the Na+/H+ antiport and 2) it was not mimicked by inducing acidification with nigericin. Pretreatment with pertussis toxin largely inhibited the phorbol ester-induced change in [Ca2+]i, while activation of protein kinase C under these conditions was unaffected. It is concluded that in the absence of extracellular Na+ (or Li+), activation of protein kinase C leads to a net Ca2+ influx into the cytoplasm through a process mediated by a GTP-binding or G protein. Opening of a Na+-sensitive Ca2+ channel could partially explain these observations. Alternatively, the nature of the monovalent cation could conceivably affect the conformation of a G protein or of an associated receptor, inducing the appearance of a site susceptible to an activating phosphorylation by protein kinase C.     

Independent elevation of cytosolic [Ca2+] and pH of mammalian sperm by voltage-dependent and pH-sensitive mechanisms

Previous work (Babcock, D. F., Rufo, G. A., and Lardy, H.A. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 1327-1331) established that increased cytosolic pH (pHi) promotes metabolic and swimming activity of bull sperm and that intracellular alkalinization results from elevated extracellular K+, presumably as a consequence of membrane depolarization. The present studies show that a persistent but reversible increase in [Ca2+]i accompanies the increase in pHi that similarly results from treatment of ram sperm with elevated [K+] in alkaline media. Because comparable increases in pHi occur in the presence or absence of external Ca2+ and because [Ca2+]i is unaltered by imposed changes in pHi alone, [Ca2+]i and pHi apparently are neither directly linked by transmembrane Ca2+/H+ exchange nor indirectly linked through Na+/H+ and Na+/Ca2+ exchange under these conditions. Instead, inhibition of K+-induced increases in [Ca2+]i (but not of increases in pHi) by prenylamine, diltiazem, nifedipine, or verapamil (C1/2 = 6, 20, 30, and 60 microM, respectively) indicates that voltage-dependent Ca2+ channels, distinct from previously described voltage-dependent effectors of pHi, operate in mammalian sperm to control [Ca2+]i. Treatment with Cs+ plus valinomycin (as an alternative method of membrane depolarization) increases pHi much more effectively than it increases [Ca2+]i, and thus also partially supports this contention. In contrast to an apparent insensitivity to pHi, K+-dependent increases in [Ca2+]i are promoted reversibly by elevation of pHo, probably reflecting local surface charge effects on channel activity (as suggested by patch-clamp studies in other systems). A selective increase in membrane permeability to Ca2+ that is induced by 12 mM NaF under nondepolarizing conditions is not a consequence of cellular aggregation, but is attenuated by the chelator deferoxamine, suggesting that GTP-binding protein additionally may couple sperm Ca2+ channels to surface receptors and promote channel opening during sperm capacitation, presumably in response to agonists produced within the mammalian female reproductive tract.     

Effect of cytochalasins on cytosolic-free calcium concentration and phosphoinositide metabolism in leukocytes

Cytochalasins are routinely used to stimulate a variety of functions in eukaryotic cells even though their precise mode of action remains to be elucidated. In the present work we used the fluorescent Ca2+ indicator quin2 to study the effect of various cytochalasins, cytochalasins A, B, C, D, E (CA, CB, CC, CD, CE) and dihydrocytochalasin B (dhCB) on the intracellular Ca2+ concentration ([Ca2+]i) in various types of leukocytes, viz, neutrophils and lymphocytes. In human neutrophils, cytochalasins increase [Ca2+]i mainly by releasing Ca2+ from membrane-bound, intracellular stores. Thus, in order to readily appreciate the effect of cytochalasins on [Ca2+ )i, these cells must be loaded with low intracellular quin2 concentrations. On the other hand, in peripheral blood lymphocytes, splenocytes and thymocytes, the increase in [Ca2+]i is predominantly due to an increased Ca2+ influx from the extracellular medium. In addition, we found that in neutrophils these drugs prolong the increase in [Ca2+]i induced by chemotactic peptides, probably by increasing the cell permeability to Ca2+. Finally, in thymocytes, cytochalasins potentiate the production of inositol phosphates induced by the polyclonal mitogen concanavalin A (conA).     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Mechanism of alterations in isolated rat liver mitochondrial function induced by gold complexes of bidentate phosphines

Au(DPPE)+2 (bis[1,2-bis(diphenylphosphino)ethane] gold(I] is an organo-gold antineoplastic agent that has anti-tumor activity in a variety of in vitro cell lines and in vivo rodent tumor models. Preliminary studies suggested that this compound represented a novel class of inhibitors of mitochondrial function. The purpose of this study was, therefore, to determine the mechanism of mitochondrial dysfunction induced by Au(DPPE)+2. Au(DPPE)+2 induced a rapid, dose-related collapse of the inner mitochondrial membrane potential (EC50 = 28.0 microM) that was not potentiated by Ca2+ preloading. Au(DPPE)+2-induced dissipation of mitochondrial membrane potential was accompanied by an efflux of Ca2+ from mitochondria upon exposure to Au(DPPE)+2. Ca2+ efflux in these experiments was via a reversal of the Ca2+ uniporter as efflux could be inhibited with ruthenium red. Au(DPPE)+2 did not increase the permeability of mitochondria to oxalacetate, indicating that the collapse of membrane potential may not be a result of gross increased inner membrane permeability. However, Au(DPPE)+2 may mediate an increased permeability of the inner membrane to cations and protons. Au(DPPE)+2 caused passive swelling in potassium acetate buffer in the absence of valinomycin, suggesting Au(DPPE)+2 facilitated the exchange of H+ and K+. Ca2+ cycling was not extensive and did not contribute to the decrease in membrane potential. These data suggest that one possible mechanism of Au(DPPE+2-induced uncoupling of mitochondrial oxidative phosphorylation is via increased permeability of the inner mitochondrial membrane to cations. The disruption of mitochondrial function may be a key process leading to hepatocyte cell injury by this drug.