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 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.     

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.     

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.     

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.     

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.     

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.     

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.     

Oxygen free radicals and calcium homeostasis in the heart

Many experiments have been done to clarify the effects of oxygen free radicals on Ca²⁺ homeostasis in the hearts. A burst of oxygen free radicals occurs immediately after reperfusion, but we have to be reminded that the exact levels of oxygen free radicals in the hearts are yet unknown in both physiological and pathophysiological conditions. Therefore, we should give careful consideration to this point when we perform the experiments and analyze the results. It is, however, evident that Ca²⁺ overload occurs when the hearts are exposed to an excess amount of oxygen free radicals. Though ATP-independent Ca²⁺ binding is increased, Ca²⁺ influx through Ca²⁺ channel does not increase in the presence of oxygen free radicals. Another possible pathway through which Ca²⁺ can enter the myocytes is Na⁺?Ca²⁺ exchanger. Although, the activities of Na⁺?K⁺ ATPase and Na⁺?H⁺ exchange are inhibited by oxygen free radicals, it is not known whether intracellular Na⁺ level increases under oxidative stress or not. The question has to be solved for the understanding of the importance of Na⁺?Ca²⁺ exchange in Ca²⁺ influx process from extracellular space. Another question is ‘which way does Na⁺?Ca²⁺ exchange work under oxidative stress? Net influx or efflux of Ca^2+?’ Membrane permeability for Ca²⁺ may be maintained in a relatively early phase of free radical injury. Since sarcolemmal Ca²⁺-pump ATPase activity is depressed by oxygen free radicals, Ca²⁺ extrusion from cytosol to extracellular space is considered to be reduced. It has also been shown that oxygen free radicals promote Ca²⁺ release from sarcoplasmic reticulum and inhibit Ca²⁺ sequestration to sarcoplasmic reticulum. Thus, these changes in Ca²⁺ handling systems could cause the Ca²⁺ overload due to oxygen free radicals.     

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.     

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.     

H-mediated coupling of transmembrane Ca fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca concentrations

The adaptation to extreme concentrations of Ca²⁺ and its consequence on the properties of the ⁴⁵Ca²⁺ transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca²⁺]_o did not cause changes in kinetic parameters of the ⁴⁵Ca²⁺ influx but the adaptation to high [Ca²⁺]_o increased the K_M(Ca2+). The V_max of the ⁴⁵Ca²⁺ influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K_M(Ca2+) these changes were prevented in mycelia adapted to high Ca²⁺. High [Ca²⁺]_o decreased the stimulation by the uncoupler, 3, 3′, 4′, 5-tetrachloro salicylanilide (TCS) (30 μM), as compared to the control, whereas the Ca²⁺ chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the ⁴⁵Ca²⁺ influx faded away, in parallel with the activity of the H⁺-ATPase. The ⁴⁵Ca²⁺ efflux from mycelia was affected by TCS in a similar way as the ⁴⁵Ca²⁺ influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca²⁺ homeostasis and ageing are in agreement with a notion that both Ca²⁺-influx and-efflux are coupled by the H⁺-homeostasis at the plasma membrane.     

Systemic signalling in barley through action potentials

Using apoplastic voltage- and ion selective microprobes, in barley leaves action potentials (APs) have been measured, which propagate acropetally as well as basipetally from leaf to leaf or from root to leaf following the application of mild salt stress (e.g. 30–50 mM KCl or NH₄Cl) or amino acids (e.g. 1 mM glutamic acid or 5 mM GABA). Voltage changes were biphasic, followed an ‘all-or-none’ characteristic, and propagated at 20–30 cm min⁻¹ irrespective of the direction. With the salt-induced APs, a strong initial depolarization is the main AP-releasing factor that first causes Ca²⁺ influx and then anion efflux. Ca²⁺ influx coincides with an initial slower depolarization, the rapid anion efflux causes the typical voltage ‘break-through’. Subsequently, K⁺-efflux starts after the depolarizing voltage has passed the K⁺ equilibrium potential (inversion of the K⁺ driving force). Glutamic acid and GABA induce APs not through membrane depolarization, but presumably by binding to a putative receptor or to ligand-gated Ca²⁺-conducting channels, respectively, followed by Ca²⁺ induced activation of anion efflux. APs are accompanied by transient apoplastic pH increase (about 1 unit), and by cytoplasmic pH decrease (about 0.5 units). The apoplastic pH change is interpreted as an indicator of stress, the cytoplasmic pH change as a prerequisite for defence related gene activation. Since APs are released by agents added in a moderate concentration range, it is suggested that they may serve as first and fast systemic signals following attack from pathogens.     

Bradykinin-induced potassium current in cultured bovine aortic endothelial cells

Summary Bovine aortic endothelial cells (BAECs) respond to bradykinin with an increase in cytosolic-free Ca²⁺ concentration, [Ca²⁺] _i , accompanied by an increase in surface membrane K⁺ permeability. In this study, electrophysiological measurement of K⁺ current was combined with⁸⁶Rb⁺ efflux measurements to characterize the K⁺ flux pathway in BAECs. Bradykinin- and Ca²⁺-activated K⁺ currents were identified and shown to be blocked by the alkylammonium compound, tetrabutylammonium chloride and by the scorpion toxin,noxiustoxin, but not by apamin or tetraethylammonium chloride. Whole-cell and single-channel current analysis suggest that the threshold for Ca²⁺ activation is in the range of 10 to 100nm [Ca²⁺] _i . The whole-cell current measurement show voltage sensitivity only at the membrane potentials more positive than 0 mV where significant current decay occurs during a sustained depolarizing pulse. Another K⁺ current present in control conditions, an inwardly rectifying K⁺ current, was blocked by Ba²⁺ and was not affected bynoxiustoxin or tetrabutylammonium chloride. Efflux of⁸⁶Rb^– from BAEC monolayers was stimulated by both bradykinin and ionomycin. Stimulated efflux was blocked by tetrabutyl- and tetrapentyl-ammonium chloride and bynoxiustoxin, but not by apamin or furosemide. Thus,⁸⁶Rb⁺ efflux stimulated by bradykinin and ionomycin has the same pharmacological sensitivity as the bradykinin- and Ca²⁺-activated membrane currents. The results confirm that bradykinin-stimulated⁸⁶Rb⁺ efflux occurs via Ca²⁺-activated K⁺ channels. The blocking agents identified may provide a means for interpreting the role of the Ca²⁺-activated K⁺ current in the response of BAECs to bradykinin.     

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.     

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.