Concanavalin a binding and Ca fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca²⁺ content of the cells. ⁴⁵Ca²⁺ fluxes were measured under conditions where artifacts due to Ca²⁺ binding to concanavalin A could be excluded. Both ⁴⁵Ca²⁺ influx into and efflux from these cells are significantly activated by the lectin. If ⁴⁵Ca²⁺ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in ⁴⁵Ca²⁺ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 μg/ml) no significant increase in ⁴⁵Ca²⁺ influx occurs but an increase in ⁴⁵Ca²⁺ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca²⁺ influx into the cells and that activation of Ca²⁺ efflux occurs as a response to an increase in the cytosolic Ca²⁺ activity. Thus, Ca²⁺ may well play a role in triggering lymphocyte activation.     

Concanavalin A binding and Ca2+ fluxes in rat spleen cells

Addition of the mitogenic lectin concanavalin A to rat spleen cells results in a small increase in the steady-state Ca2+ content of the cells. 45Ca2+ fluxes were measured under conditions where artifacts due to Ca2+ binding to concanavalin A could be excluded. Both 45Ca2+ influx into and efflux from these cells are significantly activated by the lectin. If 45Ca2+ is added 30 min after concanavalin A the rate of influx is further enhanced. The increase in 45Ca2+ influx correlates well with binding of concanavalin A to the cells. At low concentrations (optimal mitogenic) of the lectin (1 and 3 micrograms/ml) no significant increase in 45Ca2+ influx occurs but an increase in 45Ca2+ efflux is still observed. The results suggest that concanavalin A binding to the cell surface causes an increase in Ca2+ influx into the cells and that activation of Ca2+ efflux occurs as a response to an increase in the cytosolic Ca2+ activity. Thus, Ca2+ may well play a role in triggering lymphocyte activation.     

Concanavalin A-stimulated Ca2+ uptake in rat splenocytes

Summary Commercially available concanavalin A binds Ca²⁺ with high apparent affinity. In order to dissociate concanavalin A stimulated Ca²⁺ uptake (defined as an increased association of ⁴⁵Ca²⁺ with cells) in rat splenocytes and Ca²⁺ binding to cell-bound concanavalin A, conditions were developed to remove more than 75% of the bound concanavalin A. Under these conditions concanavalin A treated cells showed a considerable increase in ⁴⁵Ca²⁺ uptake over control. The concanavalin A stimulated uptake of ⁴⁵Ca²⁺ occurred within minutes, and required concentrations of concanavalin A which promoted [³H]thymidine uptake into these cells. Succinyl concanavalin A was less potent in promoting Ca²⁺ uptake than concanavalin A. Sodium periodate inhibited Ca²⁺ uptake at concentrations which promoted ³H-thymidine incorporation into splenocytes.It is concluded that con canavalin A promotes Ca²⁺ uptake which is not due to binding of ⁴⁵Ca²⁺ to concanavalin A. Although the concanavalin A-promoted Ca²⁺ uptake occurs at lectin concentrations that cause lymphocyte proliferation as measured by ³H-thymidine incorporation, the role of Ca²⁺ in this event remains unclear.     

Intracellular calcium mobilization is triggered by clustering of membrane glycoproteins in concanavalin A-stimulated platelets

Stimulation of human platelets with concanavalin A resulted in a significant increase in the concentration of cytoplasmic free Ca²⁺. This effect was due to two different processes: Ca²⁺ mobilization from internal stores and Ca²⁺ influx from the extracellular medium. Kinetic analysis revealed that the release of Ca²⁺ from internal storage sites occurred sooner than the opening of plasma membrane Ca²⁺ channels. The ability of concanavalin A to induce a sustained increase in cytoplasmic Ca²⁺ concentration was antagonized and reversed by methyl ∝-D -mannopyranoside, demonstrating that it was promoted by the interaction of the lectin with cell surface glycoproteins. Succinyl–concanavalin A, a dimeric derivative of the lectin, that does not promote patching/capping of the receptor, was able to bind to the platelet surface, and antagonized the effects of native concanavalin A. In addition, succinyl–concanavalin A, per se, was unable to induce Ca²⁺ mobilization in human platelets. Therefore, the action of the native concanavalin A was mediated by receptor clustering events. Concanavalin A mobilized Ca²⁺ from the same internal stores from which Ca²⁺ was mobilized in response to strong platelet agonists, such as thrombin and arachidonic acid. However, while thrombin was ineffective in inducing Ca²⁺ release after stimulation of platelets with Con A, Con A was able to cause a full discharge of Ca²⁺ from internal stores even in platelets previously stimulated with thrombin. These results demonstrate for the first time that the clustering of specific membrane glycoproteins can trigger platelet activation. The physiological implications during platelet aggregation are discussed.     

Stimulation of Ca efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10⁻¹⁰ M to 10⁻⁷ M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Stimulation of Ca2+ efflux by N-formyl chemotactic peptides in guinea-pig peritoneal macrophages

Effects of N-formyl chemotactic peptides on the Ca²⁺ influx and efflux were investigated in guinea-pig peritoneal macrophages using an isotope tracer. fMet-Leu-Phe did not enhance the influx of ⁴⁵Ca²⁺ into macrophages, whereas it stimulated the efflux of ⁴⁵Ca²⁺ from macrophages at concentrations ranging from 10^?10 M to 10^?7 M. fMet-Met-Met and fMet-Leu also stimulated the ⁴⁵Ca²⁺ efflux, albeit at much higher concentrations, while there was no stimulation with fMet. The mitochondrial inhibitors, oligomycin and NaN₃, did not modify the ⁴⁵Ca²⁺ efflux induced by the chemoattractants, yet they did induce the release of ⁴⁵Ca²⁺ from the mitochondria. On the other hand, higher concentrations of the calmodulin antagonists, chlorpromazine and trifluoperazine, induced the release of ⁴⁵Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site and mimicked the enhancement of the ⁴⁵Ca²⁺ efflux by N-formyl chemotactic peptides. Thus, N-formyl chemotactic peptides appear to increase the levels of intracellular free Ca²⁺ in guinea-pig peritoneal macrophages, probably by inducing the release of Ca²⁺ from the NaN₃-insensitive Ca²⁺ store site.     

Properties of AMPA Receptors Expressed in Rat Cerebellar Granule Cell Cultures: Ca2+ Influx Studies

Abstract: Cultured cerebellar granule cells become vulnerable to excitatory amino acids, especially to NMDA and kainate, by 9 days in vitro. In the same time, the sensitivity of cells to (RS)-α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), in terms of AMPA-induced toxicity or ⁴⁵Ca²⁺ uptake, was very low. The low AMPA responsiveness was due to receptor desensitization, because agents known to block desensitization, cyclothiazide and the lectins concanavalin A and wheat germ agglutinin, rendered granule cells vulnerable to AMPA and produced a pronounced stimulation of ⁴⁵Ca²⁺ accumulation. ⁴⁵Ca²⁺ influx was induced specifically by AMPA-receptor stimulation, because it was blocked virtually completely by 2,3-dihydroxy-6-nitro-7-sulfamoylbenzoquinoxaline (NBQX) and the benzodiazepine GYKI 52466 (selective non-NMDA receptor antagonists). Nevertheless, indirect routes activated by cellular responses to AMPA-receptor stimulation contributed significantly to the overall ⁴⁵Ca²⁺ influx. These included Ca²⁺ uptake through NMDA-receptor channels, voltage-sensitive Ca²⁺ channels, and via Na⁺/Ca²⁺ exchange. However, nearly one-fifth of the total ⁴⁵Ca²⁺ influx remained unaccounted for and this estimate was similar to ⁴⁵Ca²⁺ influx observed under Na⁺-free conditions. This observation suggested that a significant proportion of the Ca²⁺ flux passes through the AMPA-receptor channel proper, a view supported by Co²⁺ uptake into nearly all granule cells on exposure to AMPA in the presence of cyclothiazide. Results are discussed in light of the reported AMPA receptor-subunit composition of cerebellar granule cells in vitro.     

Effects of adenosine diphosphate on Ca2+ fluxes and Ca2+ accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Simultaneous Preparation of a DNA Ligase and Restriction Endonucleases from Bacillus amyloliquefaciens N

An N-acetylgalactosamine (GalNAc)-specific Ca²⁺-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 μg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 μg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 μg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.     

Adriamycin-induced changes in the surface membrane of sarcoma 180 ascites cells

Adriamycin increases (a) the rate of agglutination of Sarcoma 180 cells by concanavalin A after brief exposure of 2–3 h and (b) membrane fluidity as measured by ESR within 30 min of exposure at concentrations of the anthracycline of 10^?7–10^?5 M. The effect of adriamycin on agglutination is not due to an increase in the number of surface receptors for concanavalin A, since the extent of binding of the lectin is not altered by adriamycin and no change occurs in the rate of occupancy of the concanavalin A binding sites by the lectin in cells treated with the antibiotic. The order parameter, a measurement of membrane fluidity, decreases in cells exposed to adriamycin and is dose-related. The results indicate that adriamycin can induce changes in the surface membrane of Sarcoma 180 cells within a brief period of exposure to a low but cytotoxic level of this agent.     

Biochemical studies of the excitable membrane of Paramecium aurelia. I. 45Ca2+ fluxes across resting and excited membrane

The characteristics of Ca²⁺ transport across the excitable membrane of Paramecium aurelia were studied by measuring ⁴⁵Ca²⁺ influx and efflux. The intracellular concentration of free Ca²⁺ in resting P. aurelia was at least ten times less than the extracellular concentration. Ca²⁺ influx was easily measurable at 0°C, but not at 23°C. The influx of ⁴⁵Ca²⁺ was stimulated by the same conditions which cause membrane depolarization and ciliary reversal. Addition of Na⁺ and K⁺ (which stimulate ciliary reversal) resulted in a 10-fold increase in the rate of Ca²⁺ influx. An externally applied, pulsed, electric field (1–2 mA/cm² of electrode surface), caused the rate of Ca²⁺ influx to increase 3–5 times, with the extent of stimulation dependent on the current density and the pulse width Ca²⁺ influx had the characteristics of a passive transport system and was associated with the chemically or electrically triggered Ca²⁺ “gating” mechanism, which has been studied electrophysiologically. In contrast, Ca²⁺ efflux appeared to be catalyzed by an active transport system. With cells previously loaded at 0°C with ⁴⁵Ca²⁺, Ca²⁺ efflux was rapid at 23°C, but did not occur at 0°C. This active Ca²⁺ efflux mechanism is probably responsible for maintaining the low internal Ca²⁺ levels in unstimulated cells.     

Effects of adenosine diphosphate on Ca fluxes and Ca accumulation of sarcoplasmic reticulum

Ca²⁺ transport by sarcoplasmic reticulum vesicles was examined by incubating sarcoplasmic reticulum vesicles (0.15 mg/ml) at 37°C in, either normal medium that contained 0.15 M sucrose, 0.1 M KCl, 60 μM CaCl₂, 2.5 mM ATP and 30 mM Tes at pH 6.8, or a modified medium for elimination of ADP formed from ATP hydrolysis by including, in addition, 3.6 mM phosphocreatine and 33 U/ml of creatine phosphokinase. In normal medium, Ca²⁺ uptake of sarcoplasmic reticulum vesicles reached a plateau of about 100 nmol/mg. In modified medium, after this phase of Ca²⁺ uptake, a second phase of Ca²⁺ accumulation was initiated and reached a plateau of about 300 nmol/mg. The second phase of Ca²⁺ accumulation was accompanied by phosphate uptake and could be inhibited by ADP. Since, under these experimental conditions, there was no significant difference of the rates of ATP hydrolysis in normal medium and modified medium, extra Ca²⁺ uptake in modified medium but not in normal medium could not be explained by different phosphate accumulation in the two media. Unidirectional Ca²⁺ influx of sarcoplasmic reticulum near steady state of Ca²⁺ uptake was measured by pulse labeling with ⁴⁵Ca²⁺. The Ca²⁺ efflux rate was then determined by subtracting the net uptake from the influx rate. At the first plateau of Ca²⁺ uptake in normal medium, Ca²⁺ influx was balanced by Ca²⁺ efflux with an exchange rate of 240 nmol/mg per min. This exchange rate was maintained relatively constant at the plateau phase. In modified medium, the Ca²⁺ exchange rate at the first plateau of Ca²⁺ uptake was about half of that in normal medium. When the second phase of Ca²⁺ uptake was initiated, both the influx and efflux rates started to increase and reached a similar exchange rate as observed in normal medium. Also, during the second phase of Ca²⁺ uptake, the difference between the influx and efflux rates continued to increase until the second plateau phase was approached. In conditions where the formation of ADP and inorganic phosphate was minimized by using a low concentration of sarcoplasmic (7.5 μg/ml) and/or using acetyl phosphate instead of ATP, the second phase of Ca²⁺ uptake was also observed. These data suggest that the Ca²⁺ load attained by sarcoplasmic reticulum vesicles during active transport is modulated by ADP accumulated from ATP hydrolysis. ADP probably exerts its effect by facilitating Ca²⁺ efflux, which subsequently stimulates Ca²⁺ exchange.     

Mechanistic distinction between activation and inhibition of (Na+K)-ATPase-mediated Ca influx in cardiomyocytes

(Na⁺+K⁺)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na⁺/Ca²⁺-exchanger (NCX) plays a critical role in increasing intracellular Ca²⁺ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on ⁴⁵Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced ⁴⁵Ca influx, suggesting that the Ca²⁺ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca²⁺ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced ⁴⁵Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca²⁺. In contrast, the inhibition of NKA by ouabain induces 4.7-fold ⁴⁵Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced ⁴⁵Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca²⁺ and that the NCX reverse-mode is the major source for the ⁴⁵Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca²⁺ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca²⁺ influx path ways in cardiomyocytes.     

Metal ion binding to concanavalin A

Metal ion activation of saccharide binding has been studied for concana-valin A near pH 7.0. Although two metal ions, a transition metal ion and a Ca²⁺ ion, can bind, both are not required. Ca²⁺ alone, Mn²⁺ alone, or Ca²⁺ with other transition metal ions can activate this lectin. Only one Ca²⁺ ion per subunit or only one Mn²⁺ per subunit is sufficient. Metal ion binding was studied by magnetic resonance techniques and direct binding assays. Saccharide binding activity was monitored by following the fluorescence of 4-methylumbelliferyl a-D-mannopyranoside. When Ca²⁺ binds to demetalized concanavalin A, the transition metal ion site is hindered. When Mn²⁺ alone binds to demetalized concanavalin A, saccharide binding activity is induced. A subsequent conformational change, not necessary for carbohydrate binding activity, covers the Mn²⁺.     

Effects of Cd2+ upon Ca2+ fluxes and proliferation in concanavalin A-stimulated lymphocytes

The mitogenic response of human peripheral blood lymphocytes to the lectin concanavalin A (conA) is inhibited by micromolar concentrations of CdCl2. This inhibition is partially relieved by an increase in the external Ca2+ concentration (from 0.6 to 2.2 mM). The initial rate of conA-induced 45Ca2+ influx is unaltered by CdCl2, although the level of 45Ca2+ accumulation increases. The basal rate of 45Ca2+ entry is not measurably disturbed by CdCl2 (100 microM). The steady-state efflux of 45Ca2+ and the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of erythrocyte ghosts are inhibited by CdCl2 (10 microM). Thus, the mechanism behind the Cd2+-induced suppression of the mitogenic response to conA is not due to alteration of mitogen-stimulated Ca2+ influx. We suggest that Cd2+ competes with Ca2+ for intracellular Ca2+-binding molecules, such as calmodulin, essential for the induction of cell proliferation.     

Effects of methylprednisolone on the energy metabolism of quiescent and conA-stimulated thymocytes of the rat

The short-term effects of high concentrations of Methylprednisolone (MP) on the energy metabolism of quiescent and Concanavalin A-stimulated rat thymocytes were investigated in vitro. Concanavalin A (ConA) stimulated the respiration rate of quiescent thymocytes by 35%. Addition of more than 0.15 mg MP/10⁷ cells to ConA-stimulated cells reversed this respiratory stimulation; in addition, higher concentrations of MP caused a similar progressive decrease in the rate of respiration of both quiescent and ConA-stimulated cells. Similarly, the stimulation of respiration by ConA was greatly reduced in MP-treated cells. MP addition lowered cytoplasmic [Ca²⁺] and, at high concentrations, abolished the ability of ConA to increase [Ca²⁺]. Thus MP both reverses and prevents the immediate stimulation of thymocytes by ConA.In quiescent thymocytes, MP strongly inhibited that part of the oxygen consumption used to drive the cycle of Na⁺ influx across the plasma membrane and Na⁺ efflux on the Na⁺K⁺-ATPase, but did not inhibit oxygen consumption used to drive protein synthesis. In ConA-stimulated thymocytes MP had the same effects and also strongly inhibited oxygen consumption dependent on the cycle of Ca²⁺ influx across the plasma membrane and Ca²⁺ efflux on the Ca²⁺-ATPase, but had little effect on oxygen consumption used to drive RNA and DNA synthesis.These results show that MP prevents cation cycling in thymocytes (either by preventing cation influx or by inhibiting cation pumps) and prevents mitogenic stimulation of the cells. The high MP concentration required and the speed of onset of the effect (lless than 30s) provide strong evidence that these effects of MP are not mediated by glucocorticoid receptors and subsequent activation of gene expression. They may be caused by direct effects of MP on the properties of the plasma membrane. These effects are considered to be, at least partially, responsible for the beneficial results that currently have been obtained using MP megadoses in various clinical situations.     

α-Adrenergic stimulation of trans-sarcolemma electron efflux in perfused rat heart. Possible regulation of Ca2+-channels by a sarcolemma redox system

The role of trans-sarcolemma membrane electron efflux in the α-adrenergic control of Ca²⁺ influx in perfused rat heart was examined. Electron efflux was measured by monitoring the rate of reduction of extracellular ferricyanide and compared with changes in contractility, as an indirect assessment of changes in cytoplasmic Ca²⁺ concentration. Methoxamine and phenylephrine each increased the rate of ferricyanide reduction from 80 to approx. 114 nmol/min per g wet wt. of heart, with half-maximal activation occurring at 10 μM for each agonist. Activation of the rate of ferricyanide reduction by both 10 μM methoxamine and 10 μM phenylephrine was blocked by the α-adrenergic antagonist, phenoxybenzamine, but not by the β-antagonist, propranolol. Stimulation of the rate of ferricyanide reduction by the α-agonist coincided with the increase in contractility, each reaching maximum values at approx. 80 s. Removal of the α-agonists led to parallel decreases in contractility and the rate of reduction, each returning to pre-stimulation values in approx. 400 s. In addition, the relationship between Ca²⁺ and ferricyanide reduction was examined. Perfusion of the heart with medium containing 6 mM CaCl₂ significantly increased contractility and the rate of ferricyanide reduction. Perfusion of the heart with low Ca²⁺ diminished contractility, did not affect the rate of ferricyanide reduction, but amplified the stimulatory effect of methoxamine on this rate. The increase in ferricyanide reduction by α-adrenergic agonists resulted from a change in the apparent V_max, indicative of an increase in electron efflux sites in the plasma membrane. It is concluded that α-adrenergic control of electron efflux closely parallels changes in contractility and therefore changes in the cytoplasmic concentration of Ca²⁺. The data suggest that α-agonist-mediated changes in electron efflux may lead to Ca²⁺ influx.     

Properties of the basal calcium influx in human red blood cells

The basal ⁴⁵Ca²⁺ influx in human red blood cells (RBC) into intact RBC was measured. ⁴⁵Ca²⁺ was equilibrated with cells with t_1/2=15-20 s and the influx reached the steady state value in about 90-100 s and the steady state level was 1.5±0.2 μmol/l_{packed cells} (n=6) at 37 °C. The average value of the Ca²⁺ influx rate was 43.2±8.9 μmol/l_{packed cells} hour. The rate of the basal influx was pH-dependent with a pH optimum at pH 7.0 and on the temperature with the temperature optimum at 25 °C. The basal Ca²⁺ influx was saturable with Ca²⁺ up to 5 mmol/l but at higher extracellular Ca²⁺ concentrations caused further increase of basal Ca²⁺ influx. The ⁴⁵Ca²⁺ influx was stimulated by addition of submicromolar concentrations of phorbol esters (phorbol 12-myristate-13-acetate (PMA) and phorbol-12,13-dibutyrate (PDBu)) and forskolin. Uncoupler (3,3′,4′,5-tetrachloro-salicylanilide (TCS) 10⁻⁶-10⁻⁵ mol/l) inhibited in part the Ca²⁺ influx. The results show that the basal Ca²⁺ influx is mediated by a carrier and is under control of intracellular regulatory circuits. The effect of uncoupler shows that the Ca²⁺ influx is in part driven by the proton-motive force and indicates that the influx and efflux of Ca²⁺ are coupled via the RBC H⁺ homeostasis.     

On the significance of the influx of calcium ions into stimulated human blood platelets

Blood platelets, upon stimulation with various substances, take up calcium ions from the suspending medium. This influx occurs simultaneously with the release reaction, i.e. the specific secretion of a variety of substances from storage organelles and the second wave of aggregation. Various inhibitors of the release reaction inhibit this Ca²⁺ influx. Platelets previously loaded with ⁴⁵Ca show an increased efflux of the cation upon stimulation by thrombin. These results suggest that the plasma membrane acquires an increased permeability to Ca²⁺ only in a later phase of platelet activation, in most cases after the earlier release of Ca²⁺ into the cytoplasm from Ca-storing organelles. Rapid shape change and release proceed independently of external calcium, whereas clot retraction depends upon a prolonged increased permeability of the plasma membrane to this cation.     

The Ca2+ permeability of sarcoplasmic reticulum vesicles II. Ca2+ efflux in the energized state of the calcium pump

Ca²⁺ efflux from sarcoplasmic reticulum vesicles was studied by measurements of net Ca²⁺ uptake, ⁴⁵Ca²⁺ flux and hydrolysis of energy-rich phosphate. The maximal Ca²⁺ uptake capacity (150–200 nmol/mg protein at pH 6.7, 10 mM MgCl₂ and μ=0.26) was independent of the nature and concentration of the energy-donating substrate (ATP or carbamyl phosphate) and of temperature (15–35°C), suggesting coupling between influx and efflux of Ca²⁺. In the presence of high concentrations of ATP, this efflux of Ca²⁺ was much higher than the passive Ca²⁺ permeation, measured after ATP or Ca²⁺ depletion of the reaction medium. Ca²⁺ efflux was imperceptible at vesicle filling levels below 35–40 nmol Ca²⁺/mg protein, and uncorrelated to the inhibition of the Ca²⁺-ATPase by high intravesicular Ca²⁺ concentrations. Analysis of the data indicated that Ca²⁺ efflux under our conditions probably is associated with one of the Ca²⁺-ATPase partial reactions occurring after dephosphorylation, rather than with a reversal of the Ca²⁺ translocation step in the phosphorylated state of the enzyme. Furthermore, passive Ca²⁺ permeation may be concurrently reduced during the enzymatically active state. It is proposed that both Ca²⁺ efflux and passive Ca²⁺ permeation (Ca²⁺ outflow) proceed via the same channels which are closed (occluded) during part of the Ca²⁺-ATPase reaction cycle.