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PubMed1989

Analysis of calcium homeostasis in activated human polymorphonuclear leukocytes. Evidence for two distinct mechanisms for lowering cytosolic calcium.

The stimulation of polymorphonuclear leukocytes (PMNs) by chemoattractants triggers a rapid rise in cytosolic free calcium concentration(s) ([Ca2+]i), which quickly returns to base line, suggesting a role for calcium removal in the homeostasis of activated PMNs. To investigate cytosolic calcium homeostasis, PMNs were treated with a fluoroprobe and ionomycin to induce a sustained elevation of [Ca2+]i. The cells were then stimulated, and attenuation of the fluorescence signal was measured as an indication of calcium loss from the cytosol. The formyl peptide chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP), phorbol myristate acetate (PMA), and 1,2-dioctanoyl-sn-glycerol, but not the inactive phorbol ester 4 alpha-phorbol didecanoate, induced a dose-dependent decrease in [Ca2+]i in ionomycin-pretreated cells. However, the decline in [Ca2+]i caused by PMA was sustained and occurred following a lag time, whereas the response to fMLP was immediate, lasted approximately 2 min, and then was followed by a return of [Ca2+]i to its initial level. The restoration of [Ca2+]i required extracellular calcium. Varying the ionomycin concentration allowed studies at different initial [Ca2+]i, which in untreated PMNs was approximately 135 nM. In contrast to fMLP, PMA did not lower calcium at concentrations below 200 nM. The decline in [Ca2+]i induced by fMLP, but not PMA, was blocked by pertussis toxin. In contrast, the decrease in [Ca2+]i caused by PMA and 1,2-dioctanoyl-sn-glycerol, but not fMLP, was inhibited by the protein kinase C antagonists staurosporine, H-7, and sphingosine. These results suggest that formyl peptide chemoattractants transiently stimulate an activity which lowers [Ca2+]i to normal intracellular levels. Activation of this process appears to be independent of protein kinase C. An additional cytosolic calcium lowering activity, dependent on protein kinase C, operates at [Ca2+]i above 200 nM. Thus, activated PMNs can use at least two processes for attentuation of elevated cytosolic calcium levels.

Alkaloids↗
PubMed1987

Transmembrane signaling during interleukin 1-dependent T cell activation. Interactions of signal 1- and signal 2-type mediators with the phosphoinositide-dependent signal transduction mechanism.

The murine T lymphoma line, LBRM-33 1A5, requires synergistic signals delivered by phytohemagglutinin (PHA) and interleukin 1 (IL1) for activation to high level interleukin 2 production. The activation signals provided by PHA and IL1 were replaced by the Ca2+ ionophore, ionomycin, and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), respectively. These observations supported a two-signal model for T cell activation involving increases in intracellular Ca2+ concentration ([Ca2+]i) (signal 1) and activation of protein kinase C (signal 2) as necessary and sufficient events. However, biochemical analyses revealed that additional signals were involved in the activation of LBRM-33 cells by both receptor-dependent and -independent mediators. Both signal 1-type mediators, PHA and ionomycin, exerted pleiotropic effects at the concentrations required for synergy with signal 2-type mediators (IL1, TPA). Within 1-2 min of addition, PHA stimulated phospholipid turnover, including hydrolysis of phosphatidylinositol 4,5-bisphosphate, Ca2+ mobilization, and protein kinase C activation. The [Ca2+]i increase induced by PHA was due to influx from both intracellular and extracellular Ca2+ pools. Similarly, ionomycin increased phospholipid turnover, [Ca2+]i, and directly affected protein kinase C activity in LBRM-33 cells. In contrast, the signal 2-type mediators, TPA and IL1, appeared to act by distinct intracellular mechanisms. TPA induced a protracted association of cellular protein kinase C with the plasma membrane, consistent with the two-signal activation model. Furthermore, acute TPA treatment inhibited PHA-stimulated inositol phosphate release and Ca2+ mobilization, suggesting that this mediator partially antagonized signal 1 delivery. IL1, in contrast, neither activated protein kinase C directly nor did it positively modulate the coupling of signal 1-type mediators to [Ca2+]i or protein kinase C via the phosphoinositide pathway. The intracellular signal delivered by IL1 is, therefore, generated through a mechanism distinct from or distal to the activation of protein kinase C. These studies indicate that the two-signal hypothesis, in its simplest form, is inadequate to explain the signals required for the initiation of IL1-dependent T cell activation.

Animals↗
PubMed1986

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells.

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.

Aminoquinolines↗
PubMed1986

Distinct signals are required for proliferation and lymphokine gene expression in murine T cell clones.

Experiments were performed to assess the capacity of lectin (Con A), ionomycin, phorbol ester (PMA), and recombinant IL 2 to mediate proliferation as well as the expression of cell surface IL 2 receptors, two lymphokine genes, IL 2 and IFN-gamma, and the c-myc proto-oncogene in cloned T cell populations. Stimulation of T cell clones with recombinant IL 2 resulted in proliferation and sustained expression of the c-myc cellular proto-oncogene, but did not induce the expression of mRNA for the lymphokines IFN-gamma and IL 2. In contrast, stimulation of cloned T cells with lectin alone induced significant IFN-gamma and IL 2 mRNA expression, up-regulation of the number of cell surface IL 2 receptors, and transient c-myc expression. Ionomycin alone was not a sufficient signal for lymphokine mRNA induction. The phorbol ester PMA alone induced neither proliferation nor lymphokine gene expression but potentiated lectin and ionomycin-mediated signals. We also performed experiments to examine whether the T cell response to extracellular stimuli was a function of the activation state of the cell. Reexposure of 48-hr antigen-activated cloned cells to identical stimuli revealed several differences. Low but significant levels of IFN-gamma mRNA were now also reinduced in activated clones cells in response to IL 2 or PMA alone. Activated cells were refractory to reinduction of IL 2 mRNA by any stimulus, which may reflect a physiologic mechanism to limit clonal expansion after antigenic stimulation. This could be partially reversed by restimulation with lectin in the presence of cycloheximide, suggesting a role for a labile protein repressor in the down-regulation of IL 2 mRNA expression. PMA alone induced an IL 2-independent proliferative response. We demonstrate that distinct signals are required for lymphokine gene expression vs cellular proliferation in cloned T lymphocyte populations, and that the capacity of extracellular stimuli to reinduce expression of lymphokine genes or to mediate cell proliferation is altered by prior activation.

Animals↗
PubMed1987

One-signal requirement for interferon-gamma production by human large granular lymphocytes.

This laboratory has been investigating IFN-gamma gene expression by highly purified human large granular lymphocytes (LGL) and T cells. We report here that within 1 hr after interleukin 2 (IL 2) treatment of freshly isolated human LGL, IFN-gamma mRNA can be detected, with IFN-gamma protein in the culture medium within 4 to 6 hr of treatment. CD3- Leu-11+ LGL require only a single signal for IFN-gamma production because phytohemagglutinin (PHA), phorbol myristate acetate (PMA), IL 2, or ionomycin can each independently induce IFN-gamma production. In addition, PHA and ionomycin (but not IL 2) show significant synergy with PMA as a stimulus to LGL. In contrast, CD3+ T cells require two stimuli for high levels of IFN-gamma production, and not only are PMA plus ionomycin or PHA synergistic, but in addition, IL 2 and PHA demonstrate some synergy. Furthermore, we have found by fractionation of peripheral blood lymphocytes that IL 2-induced IFN-gamma production is associated with the LGL population and not T cells. These results indicate that with certain stimuli, LGL may be the predominant source of IFN-gamma from peripheral blood lymphocytes.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Requirements for the simultaneous presence of phorbol esters and calcium ionophores in the expression of human T lymphocyte proliferation-related genes.

We have investigated the synergistic effects of phorbol ester and calcium ionophore on human T lymphocyte proliferation and the expression of the proliferation-related genes, c-myc, c-fos, interleukin 2 receptors (IL-2R) and interleukin 2 (IL-2). Incubation of T lymphocytes with both the phorbol ester, phorbol 12,13-dibutyrate (PDB), and the calcium ionophore, ionomycin, leads to the expression of a series of proliferation-related genes, followed by T cell proliferation. In contrast, stimulation of T cells sequentially with PDB and then ionomycin did not induce mitogenesis, demonstrating that simultaneous exposure to both agents is necessary for proliferation. Exposure of T cells to both agents together for different time periods resulted in a proliferative response in proportion to the duration of the exposure, with more than 6 hr required for maximum proliferation. In contrast, a 1-hr exposure to both drugs was sufficient for maximum expression of c-fos or c-myc proto-oncogene mRNA. The expression of IL-2R and the production of IL-2 were also dependent on the duration of simultaneous exposure to both phorbol ester and calcium ionophore. Levels of IL-2 mRNA became detectable at 1 hr and peaked at 3 hr after stimulation. The induction of IL-2 mRNA occurred only in the presence of both agents and became undetectable within 2 hr after the drugs were removed. In contrast, the expression of IL-2R mRNA became detectable at 1 hr, but was maintained even after the drugs were removed and reached a peak at 24 hr. Both IL-2 and IL-2R mRNA accumulated in proportion to the duration of the exposure. Augmentation of cell proliferation by exogenous IL-2 was observed in T cells exposed to the drugs for less than 3 hr. These data demonstrated that the induction of maximum expression of the nuclear proto-oncogenes c-myc and c-fos was not sufficient for PDB-ionomycin-induced T cell proliferation. The level of IL-2 mRNA accumulation and resultant IL-2 secretion is one of the limiting factors for proliferation of T cells exposed to the drugs for less than 3 hr, but not for longer exposures. Additional events such as accumulation of IL-2R mRNA and protein triggered by a long exposure to the drugs were obligatory for obtaining maximum proliferation.

Cell Division↗
PubMed1988

Regulation of intestinal apolipoprotein B synthesis and secretion by Caco-2 cells. Lack of fatty acid effects and control by intracellular calcium ion.

To investigate the mechanism of control of intestinal apolipoprotein B (apoB) secretion, we studied the effects of fatty acids and calcium ionophores on the human intestinal model cell line Caco-2. Although treatment with various fatty acids (18:1w9, 18:2w6, and 20:5w3) complexed to bovine serum albumin resulted in a dramatic redistribution of apoB-100 from the low density and high density lipoproteins to the very low density lipoprotein fraction, there was no effect of any of the fatty acids on the overall rate of total apoB (apoB-100 and apoB-48) secretion. Treatment of differentiated monolayers with calcium ionophores A23187 or ionomycin caused dose-specific increases (125% at 1 microM) in the accumulation of total apoB, but not apoA-I, in conditioned medium as measured by specific immunoassays. Incubation studies with 35S-labeled Caco-2 apoB,E-containing low density lipoprotein particles revealed that treatment with ionomycin over a broad concentration range had no effect on the reuptake of secreted apoB-100. The effect on A23187 on total apoB secretion was blocked by prior chelation of medium calcium and was significantly enhanced by the addition of calcium (up to 50 mM) to the medium. The effect of A23187 was significantly blunted by treatment with the calmodulin antagonist trifluoperazine (10 microM). The time course of A23187 action on Caco-2 apoB secretion required at least 6 h to occur. In contrast to the concentration of apoB in the medium, cellular apoB content was not influenced by treatment with ionophore. Pulse-chase experiments demonstrated a significant reduction in the synthesis-secretion interval for apoB-100 and apoB-48 after 24 h of exposure to ionomycin. Neither fatty acid treatment nor stimulation with ionophore affected the ratio of apoB-100 to apoB-48 produced by the cells. These findings with calcium ionophores implicate the involvement of calcium ion in the mechanism of intestinal apoB secretion. A role for calcium-dependent processes in apoB production raises the possibility that, rather than fatty acid flux, calcium-evoked or calcium-dependent hormones may be important regulators of apoB secretion.

Adenocarcinoma↗
PubMed1988

Calcium dependence of myosin light chain phosphorylation in smooth muscle cells.

Smooth muscle cells grown in culture may provide a model system for studying the Ca2+ dependence of myosin light chain phosphorylation. Tracheal smooth muscle cells in culture had 60% of the myosin content of tracheal tissue. Western analysis with appropriate antibodies demonstrated one 20-kDa light chain and the presence of a 150-kDa myosin light chain kinase in both tracheal smooth muscle tissue and cells. Moreover, tracheal cells contained 74% of the myosin light chain kinase activity measured in tissue. Similar types of analyses of nonmuscle cells showed a much lower myosin and myosin light chain kinase content. Carbachol (10 microM) or ionomycin (10 microM) stimulation of fura-2-containing cells resulted in a rapid increase in cytosolic free Ca2+ concentration and in the extent of myosin light chain phosphorylation. Maximal increases in Ca2+ concentrations were greater with ionomycin than with carbachol (4400 versus 492 nM). Light chain phosphorylation increased after the Ca2+ concentration exceeded 200 nM from control values of 165 nM. Half-maximal phosphorylation (33%) occurred at 260 nM Ca2+. There was a similar relationship between free cytosolic Ca2+ concentrations and the extent of myosin light chain phosphorylation in carbachol- and ionomycin-stimulated cells. This relationship had a Hill coefficient of 2.7. These observations indicate that small changes in Ca2+ concentrations stimulate myosin light chain phosphorylation and thus presumably contraction in smooth muscle cells.

Animals↗
PubMed1988

Heterogeneity of immature (Lyt-2-/L3T4-) thymocytes. Identification of four major phenotypically distinct subsets differing in cell cycle status and in vitro activation requirements.

Thymocytes that bear neither Lyt-2 nor L3T4 differentiation Ag (2-4- thymocytes) contain the precursors of mature Lyt-2+ and L3T4+ T cells. In the present study, we have identified four major subpopulations of 2-4- cells in adult C57BL/6 mice that differ in surface phenotype and in situ proliferative status. Two-color immunofluorescence analysis with RL-73 (a mAb recognizing an as yet unidentified activation Ag) and PC-61 (an anti-IL-2R mAb) revealed three distinct subsets of 2-4-thymocytes: RL-73+ IL-2R- (30%), RL-73+/-IL-2R+ (45%), and RL-73- IL-2R- (25%). The RL-73+ IL-2R- subset had the highest percentage of large blasts and cycling cells, whereas the RL-73+/- IL-2R+ and RL-73- IL-2R- subsets had intermediate and low percentages, respectively, indicating that in situ proliferation correlated better with RL-73 intensity than with IL-2R expression. An additional marker, heat-stable Ag (HSA), was found to further subdivide the RL-73- population into RL-73- HSA- (10% of total 2-4-) and RL-73- HSA+ (15%) fractions. The two latter (RL-73-) subsets appeared to be more "mature" than the former since they expressed high levels of Lyt-1 and appeared later during fetal thymus ontogeny. In parallel with the phenotypic analysis, we compared the in vitro activation requirements of each of the four purified 2-4- subsets. All four populations proliferated well to the combination of phorbol ester (PMA), ionomycin, and IL-2. In response to PMA and ionomycin (without added IL-2), only RL-73- HSA-cells proliferated and this proliferation was correlated with IL-2 production. However, if IL-1 was included with PMA and ionomycin then all four populations responded. Finally, a proliferative response to Con A or mitogenic anti-Thy-1 mAb was observed only for RL-73- HSA+ and (to a lesser extent) RL-73- HSA-cells. These data indicate that each of the four phenotypically distinct subpopulations of immature thymocytes can also be distinguished on the basis of their in vitro activation requirements.

Animals↗
PubMed1988

Intracellular activation signal requirements for the induction of IL-2 responsiveness in resting T cell subsets in humans.

Activation signal requirements for the induction of the IL-2 responsiveness in purified subsets of human resting T cells, T4+ or T8+, have been investigated under the monocyte-depleted conditions. Substantial levels of IL-2 responsiveness were induced in T8+ cells by lectin, Con A, mAb directed against the CD3 Ag, OKT3, Ca2+ ionophore, ionomycin or phorbol ester, PMA. In contrast, none of these stimuli was by itself sufficient for the induction of IL-2 responsiveness in the T4+ subset. The latter cells could, however, be induced to respond to IL-2 by combinations of PMA plus either of Con A, OKT3, or ionomycin (but not any combination of Con A, ionomycin, and OKT3). These data indicate that induction of IL-2 responsiveness in the resting T4+ subset is more complex, possibly requiring two intracellular activation signals, increase in the concentration of intracellular Ca2+ and activation of protein kinase C, whereas either signal may directly trigger IL-2 responsiveness in the resting T8+ cells. The data further suggest that under optimal conditions, growth of both resting T4+ and T8+ subsets may be independent of monocytes.

Antibodies, Monoclonal↗
PubMed1986

Signals involved in T cell activation. II. Distinct roles of intact accessory cells, phorbol esters, and interleukin 1 in activation and cell cycle progression of resting T lymphocytes.

The signals involved in the initiation of mitogen-induced activation of resting guinea pig T cells were examined. The combination of phytohemagglutinin (PHA) and 4 beta-phorbol 12-myristate 13-acetate (PMA) stimulated DNA synthesis by accessory cell (AC)-depleted T cells cultured at high density, but the use of low density cultures indicated that intact AC were absolutely necessary for PHA-stimulated T cell DNA synthesis even in the presence of PMA, interleukin 1 (IL 1), or interleukin 2 (IL 2). In contrast, AC-depleted T cells were able to respond to the combination of the calcium ionophore, ionomycin, and PMA regardless of the cell density at which they were cultured. Cell cycle analysis by acridine orange staining indicated that neither PHA nor ionomycin, in the absence of AC, activated resting T cells. PMA in the absence of all AC, supported cell cycle entry and progression to the DNA synthetic phase of the majority of ionomycin-stimulated T cells, but permitted only a small number of PHA-triggered T cells to enter the initial stage of the cell cycle (G1a) characterized by a modest increase in cellular RNA content. Although PMA permitted some PHA-stimulated T cells to enter the cell cycle, most required intact AC to enter G1, and all required intact AC to progress through G1 and synthesize maximal amounts of RNA. No PHA-stimulated cells reached the S phase without intact AC. In PHA-stimulated cultures containing intact AC, PMA increased the number of cells entering the cell cycle and increased the rate of their progress to the DNA synthetic phase. IL 1 also augmented PHA-stimulated AC-dependent T cell DNA synthesis in the presence or absence of PMA, but appeared to be most active during the later stage of the first cell cycle, augmenting the number of activated cells that entered the S phase of the cell cycle. These results support the conclusion that intact AC, IL 1, and a PMA-like signal play distinct roles in the progression of mitogen-stimulated T cells through the first round of the cell cycle.

Animals↗
PubMed1986

Immunodeficiency of aging: restorative effects of phorbol ester combined with calcium ionophore.

Current models for lectin-induced T cell proliferation suggest that activation of protein kinase C (PK-C) and elevation of cytoplasmic Ca2+ may both play important roles in the earliest phases of signal transduction. To learn more about the relative inability of T cells from old mice to proliferate in response to mitogenic stimuli, we attempted to stimulate T cells by the synergistic effects of a PK-C activator, phorbol myristate acetate (PMA), and the calcium ionophore ionomycin. T cells from young mice respond as well to optimal combinations of these agents as they do to the strong polyclonal activator Con A, but T cells from old mice respond much better to PMA plus ionomycin than they do to Con A. This result suggests that an inability to transduce the signal supplied by extracellular ligands into the intracellular signals represented by Ca2+ and PK-C activators may underlie the age-associated loss of T cell reactivity. We also found evidence for a second defect in old T cells related to their response to elevated intracellular Ca2+: old T cells, compared with young, required higher levels of ionomycin for maximal proliferation.

Aging↗
PubMed1987

Signal requirement for interleukin-1-dependent interleukin 2 production by a human leukemia-derived HSB.2 subclone.

We have previously established subclones from human leukemia-derived HSB.2 cell line that produced high levels of interleukin (IL) 2 when stimulated with phytohemagglutinin (PHA) and IL-1. Herein, we investigated the signal requirement for IL-2 production, particularly concerning the role of IL-1 in this system. PHA but not IL-1 rendered marked protein kinase C (PKC) activation and IL-2 production induced by PHA plus IL-1 was totally abrogated by a potent PKC inhibitor, H-7. Concomitantly, PHA alone caused marked Ca2+ influx, whereas IL-1 neither induced Ca2+ influx nor augmented PHA-induced Ca2+ influx. As expected, a signal delivered by PHA could be substituted by phorbol 12-myristate 13-acetate (PMA) and ionomycin while IL-1 was still indispensable, indicating that at least three signals, i.e., those delivered by IL-1 as well as PKC activation and Ca2+ influx were required for optimal IL-2 production. Kinetic study indicated that while PMA and ionomycin should be added at the initiation of culture, delayed addition of IL-1 up to 4 hr later induced even higher levels of IL-2 production, demonstrating the requirement for IL-1 after PKC activation and Ca2+ influx. In this system, it was revealed that IL-1 was not involved in PKC activation, Ca2+ influx, and breakdown of phosphatidylinositols. Whereas PMA, ionomycin, and IL-1 stimulated high levels of IL-2 production, those combinations of signals did not induce breakdown of phosphatidylinositols. It should be noted that IL-2 production induced by these three signals seemed to bypass hydrolysis of phosphatidylinositols in contrast to PHA plus IL-1 stimulation that was accompanied with a marked breakdown of phosphatidylinositols.

Calcium↗
PubMed1985

Distinction between antigen receptor and IL 2 receptor triggering events in the activation of alloreactive T cell clones with calcium ionophore and phorbol ester.

A previous study indicated that Ca++ ionophores in conjunction with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) could induce normal T lymphocytes to express receptors for the T cell growth factor, interleukin 2 (IL 2), to secrete IL 2, and to proliferate (1). Here we used long-term alloreactive Lyt-2+ cytotoxic or T4+ "helper" T cell clones. In response to their specific alloantigen, all of the clones secreted IFN-gamma but only the T4+ clone secreted IL 2 and proliferated in response to the appropriate alloantigen in the absence of exogenous IL 2. The Ca++ ionophore ionomycin and TPA, used in conjunction, mimicked the effect of specific alloantigen on these T cell clones, i.e., they induced the secretion of IFN-gamma in all clones and the secretion of IL 2 in the T4+ clone. In the absence of exogenous IL 2, a proliferative response was induced only for the IL 2 secreting clone. Increased sensitivity to exogenous IL 2 for some T cell clones was also observed after either alloantigen or ionomycin and TPA treatment; this could be correlated with an increase in the expression of IL 2 receptors 6 hr after a pulse with ionomycin and TPA. These results suggest that, for a given T cell clone, activation of the Ca++ -dependent protein kinase c can replace the antigen-receptor triggering events leading to interleukin secretion and increased expression of IL 2 receptors but cannot substitute for the IL 2 dependent triggering of the IL 2 receptor.

Animals↗
PubMed1982

On the relative roles of Ca2+ and Mg2+ in regulating the endogenous K+/H+ exchanger of rat liver mitochondria.

1. A23187 and ionomycin cause the release of Ca2+, Mg2+, and K+ from the mitochondrial matrix. The electroneutral K+ efflux does not reflect direct transport by these ionophores but, rather, results from release of the endogenous K+/H+ exchanger from inhibition by divalent cations. 2. A23187 and ionomycin differ in their affinities for CA2+ and Mg2+, having relative affinity ratios (Ca2+/Mg2+) in respiring mitochondria of 3 and 35, respectively. 3. This difference in Ca2+/Mg2+ affinities was exploited in dose-response studies designed to determine which cation exerts primary control over K+/H+ exchange activity. The results of such studies demonstrate that removal of Ca2+ has no effect on K+ efflux, and that K+ efflux follows Mg2+ efflux whether induced by ionomycin or A23187. 4. K+ steady states, in which K+ uniport equals K+/H+ antiport, were induced by adding low levels of valinomycin to respiring mitochondria. The addition of Ca2+ perturbed the steady state in the direction of increased K+/H+ exchange. 5. Addition of Ca2+ to respiring mitochondria resulted in electroneutral K+ efflux which was strongly affected by the anion composition of the medium. We propose that Ca2+ turns on the K+/H+ exchanger indirectly, through a decrease in matrix [Mg2+] secondary to swelling and uptake of chelating anions. This phenomenon may be of physiological importance as a temporary protection against excessive swelling due to Ca2+ uptake. 6. These studies support the contention that Mg2+, not Ca2+, is the inhibitory cation responsible for the physiological regulation of mitochondrial K+/H+ exchange.

Animals↗
PubMed1980

Mechanisms of ionophore-induced catecholamine secretion.

A number of carboxylic ionophores stimulate the secretion of norepinephrine from cell suspensions prepared from a transplantable rat pheochromocytoma. The divalent-cation ionophore ionomycin stimulates catecholamine secretion by a mechanism that is dependent upon the presence of extracellular Ca++. It is likely that ionomycin-induced catecholamine secretion results from the ionophore-mediated entry of Ca++ into the cells. The monovalent-cation ionophore monensin stimulates catecholamine secretion by a mechanism that is independent of extracellular Ca++, but is markedly dependent upon extracellular Na+. Monensin probably transports Na+ into the pheochromocytoma cells and increases the intracellular concentration of Na+ in these cells. This rise in intracellular Na+ may cause the release of Ca++ from some intracellular store. Lasalocid stimulates catecholamine secretion by a mechanism that is independent of extracellular Ca++ and is only slightly dependent upon extracellular Na+. The action of lasalocid, in contrast to the actions of ionomycin and monensin, is potentiated by decreased pH. It is likely that lasalocid enters the cells in its uncharged, protonated form. Once inside the cells, lasalocid may promote the release of intracellular Ca++. Alternatively, lasalocid and monensin may stimulate catecholamine secretion by the process which is independent of Ca++. These experiments show that ionophores can stimulate catecholamine secretion by at least three distinct ionic mechanisms.

Adrenal Gland Neoplasms↗
PubMed1994

A tyrosine phosphorylation requirement for cytotoxic T lymphocyte degranulation.

Phorbol myristate acetate (PMA) plus ionomycin induces the tyrosine phosphorylation of several cytotoxic T lymphocyte (CTL) substrates, including one with an apparent molecular weight of 100,000 (pp100) in cloned murine CTL. cis-Unsaturated fatty acids and low concentrations of phenylarsine oxide specifically inhibit the tyrosine phosphorylation of pp100. Genistein also inhibits tyrosine phosphorylation of pp100, but not with the same specificity as cis-fatty acids or low concentrations of phenylarsine oxide. Degranulation triggered by PMA plus ionomycin is inhibited by cis-fatty acids, low concentrations of phenylarsine oxide, and genistein, under the same conditions that these agents inhibit tyrosine phosphorylation of pp100. Depleting CTL of protein kinase C (PKC) activity by prolonged exposure to PMA eliminates the increase in tyrosine phosphorylation when challenged by PMA plus ionomycin, but not when these PKC-depleted CTL are activated by cognate target cells, immobilized anti-T cell receptor (TCR) antibodies, or concanavalin A. Tyrosine phosphorylation of pp100 triggered by TCR engagement in PKC-depleted cells is inhibited by cis-fatty acids and phenylarsine oxide, indicating that the inhibitory mechanism of the tyrosine phosphorylation of pp100 is independent of PKC. Furthermore, because all three tyrosine phosphorylation inhibitors are unlikely to inhibit PKC, these results suggest that, in addition to PKC activation and a rise in intracellular Ca2+, CTL degranulation requires the tyrosine phosphorylation of a CTL substrate(s), in addition to phospholipase C, and the present results are consistent with pp100 as that substrate. Taken together with previous studies, these results suggest that tyrosine phosphorylation of pp100 may play a central role in CTL function.

Animals↗
PubMed1995

Regulatory effect of the level of free Ca2+ of the host cell on the capacity of Trypanosoma cruzi to invade and multiply intracellularly.

We studied whether modification of the free intracellular Ca2+ level of a mammalian host cell would affect its susceptibility to infection by Trypanosoma cruzi or its capacity to support trypomastigote-->amastigote transformation and amastigote replication. Pretreatment of rat heart myoblasts (RHM) with BAPTA.AM or Quin-2.AM, intracellular Ca2+ chelators, decreased the susceptibility of these cells to infection by untreated trypomastigotes. This was evidenced by a significant drop in both the percentage of infected RHM and the average number of organisms per 100 host cells relative to control values. Similar RHM treatment with the Ca2+ ionophore ionomycin had the opposite effects. The rate of trypomastigote-->amastigote transformation measured in RHM that had been treated with BAPTA.AM, Quin-2.AM, or ionomycin before and after, but not during co-culture with trypomastigotes was not significantly altered. The rate of intracellular amastigote multiplication measured in RHM exposed to the intracellular Ca2+ chelators only after virtually all of the internalized trypomastigotes had transformed into amastigotes was significantly decreased by incubation with BAPTA.AM or Quin-2.AM but was increased by ionomycin. None of the drug treatments affected RHM viability to any significant extent. These results suggest that T. cruzi relies on host cell Ca(2+)-dependent events, utilizes host cell free Ca2+ during invasion, or both, and highlight a requirement for an adequate free Ca2+ level for effective intracellular T. cruzi multiplication but not for trypomastigote-->amastigote transformation.

Aminoquinolines↗