Molecular events in B cell activation. I. Signals required to stimulate G0 to G1 transition of resting B lymphocytes

Anti-immunoglobulin antibodies (anti-Ig) can stimulate a majority of resting B cells via their receptor Ig. Evidence suggests that the signals generated after this ligand-receptor interaction may be transduced via hydrolysis of inositol phospholipids. In other systems, the ability of inositol phospholipid hydrolysis to link receptor-ligand interactions to subsequent activational events has been suggested to relate to the ability of metabolic intermediates of this hydrolytic process to facilitate activation of protein kinase C and mobilization of Ca+2. In this study, we investigated the importance of protein kinase C and Ca+2 mobilization in the signaling mechanism by which anti-Ig drives B cells to undergo G0 to G1 transition. Our results show that pharmacologic inhibition of either protein kinase C activity or channel-mediated Ca+2 influx completely abrogates the increase in RNA synthesis associated with B cell activation after stimulation by anti-Ig. This suggests that pathways leading to both protein kinase C activation and elevation of intracellular Ca+2 are critical for receptor Ig-mediated G0 to G1 transition. Furthermore, studies in which anti-Ig-induced signaling could be bypassed by directly facilitating Ca+2 mobilization and protein kinase C activation using Ca+2 ionophore and phorbol diester show that these events are sufficient to drive the majority of resting B cells into G1 in the absence of additional signaling from accessory cells or extra-cellular factors. However, like anti-Ig-induced stimulation, Ca+2 ionophore and phorbol diester are relatively inefficient in driving B cells that have entered G1 into S phase. We discuss the relevance of these results towards the transduction mechanism linking B cell membrane-associated Ig-generated signals with subsequent activation events.     

Bimodal effect of phorbol ester on B cell activation. Implication for the role of protein kinase C.

The role of protein kinase C PKC in B cell activation is controversial. These studies were undertaken to determine whether protein kinase C has a stimulatory or inhibitory role in B cell activation. We found that treatment of B cells for a short period of time (30 min) with the PKC activator phorbol 12,13-dibutyrate (PDBU) primed the cells for enhanced proliferative responses to anti-immunoglobulin (anti-Ig) antibody whereas treatment for a longer period of time (3 h or more) resulted in suppression of proliferation. The enhanced proliferative response to treatment of B cells with PDBU for short periods of time was associated with inhibition of anti-Ig-stimulated increases in phosphatidyl 4,5-bisphosphate (PIP2) hydrolysis and inhibition of increases in [Ca2+]i, indicating that activation of PKC per se might be sufficient for enhancing B cell activation. The time-dependent effect of phorbol esters on the inhibition of B cell proliferation was found to be closely correlated with the kinetics of disappearance of PKC as measured by Western blot and by enzymatic activity but not with inhibition of [Ca2+]i and PIP2. These data demonstrate a bimodal time-dependent effect of PDBU on B cell activation and suggest that (a) the inhibitory effect of phorbol ester on anti-Ig-induced proliferation may be due to the disappearance of PKC rather than to the inhibition of PIP2 and Ca2+; and (b) the early activation of PKC is a stimulatory rather than an inhibitory signal in the induction of B lymphocyte proliferation by anti-Ig.     

Ethanol-induced phospholipase C activation is inhibited by phorbol esters in isolated hepatocytes.

Ethanol causes a transient activation of the phosphoinositide-specific phospholipase C in intact hepatocytes and mimics the action of receptor-mediated agonists [Hoek, Thomas, Rubin & Rubin (1987) J. Biol. Chem. 262, 682-691]. Preincubation of the hepatocytes with phorbol esters which activate protein kinase C prevented this effect of ethanol: phorbol ester treatment inhibited the ethanol-induced phosphorylase activation, the increase in intracellular free Ca2+ concentrations measured in quin 2-loaded hepatocytes, and the changes in concentrations of inositol phosphates, phosphoinositides and phosphatidic acid. Several lines of evidence indicate that these effects were mediated by protein kinase C. Phorbol esters acted in a concentration range where they activate protein kinase C; phorbol esters that do not activate protein kinase C were not effective in inhibiting the effects of ethanol. The permeant diacylglycerol oleoyl-acetylglycerol also inhibited the effects of ethanol, but other diacylglycerols were not effective in the intact cells. The inhibition of ethanol-induced Ca2+ mobilization by phorbol esters was prevented by preincubating the cells with the protein kinase C inhibitors 1-(5-isoquinolinesulphonyl)-2-methylpiperazine (H7) and sphingosine. H7 also enhanced the Ca2+ mobilization induced by ethanol in cells that were not pretreated with phorbol esters, indicating that the transient nature of the ethanol-induced Ca2+ mobilization may be due to an activation of protein kinase C caused by the accumulation of diacylglycerol. These data support a model whereby ethanol activates the phosphoinositide-specific phospholipase C, possibly by affecting receptor-G-protein-phospholipase C interactions in the membrane.     

Induction of competence and progression signals in human T lymphocytes by phorbol esters and calcium ionophores

We have investigated the induction of competence (IL-2 responsiveness) and progression in human T lymphocyte proliferation triggered by phorbol ester and calcium ionophore. The degree of proliferation induced with the phorbol ester, phorbol 12,13-dibutyrate (PDB) and the calcium ionophore ionomycin was dependent on the duration of exposure to these agents, with more than 6 h required for obtaining maximum proliferation. Following brief exposure to both agents for 30 min, which did not cause significant proliferation, T cells became competent to proliferate in response to exogenous interleukin 2 (IL-2). These competent T cells also progressed to DNA synthesis following incubation with PDB in the absence of ionomycin. Induction of competence to proliferate in response to either PDB or IL-2 was blocked by EGTA, suggesting that transmembrane Ca2+ flux was obligatory at this stage. Since other phorbol esters and synthetic diacylglycerols also stimulated DNA synthesis in competent cells, it is likely that progression was triggered by activation of protein kinase C. Following a brief exposure to PDB and ionomycin, subsequent incubation with PDB induced gene expression and secretion of IL-2 and augmented the expression of IL-2 receptors in the competent cells. Thus, we have demonstrated that Ca2+ mobilization is required for rendering T cells competent to express functional IL-2 receptors, to produce IL-2 in response to subsequent incubation with PDB, and that sustained activation of protein kinase C seems necessary for IL-2 production and subsequent progression of competent T cells to DNA synthesis.     

Early membrane potential and cytoplasmic calcium changes during mitogenic stimulation of WEHI 231 cell line by polyene antibiotics, lipopolysaccharide and anti-immunoglobulin

Changes in free cytosolic calcium concentration and in membrane voltage are thought to be important initiating events in lymphocyte activation. The antifungal agent amphotericin B (AmB) holds interesting immunomodulating properties and its N-thiopropionyl derivative (AmBSH) is a potent polyclonal B-cell activator. These molecules may then exert their stimulating activity through the production of early ionic signals similar to those delivered by the classical activators lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). We addressed this question in a B-cell line (WEHI 231) which has previously been shown to exhibit characteristic response to LPS and anti-Ig. AmBSH protected these cells against anti-Ig-induced cell growth inhibition, providing a LPS-like response. In contrast, the parental compound AmB did not. The two polyene antibiotics did not modify the resting Ca2+i level of the cells, neither did LPS, whereas anti-Ig induced a rapid increase in the cytosolic calcium concentration. On the other hand, polyene antibiotics and LPS promoted membrane depolarization, whereas membrane voltage remained unchanged after anti-Ig treatment. Polyene antibiotics-induced depolarization originated from the increase of membrane permeability to Na+ ions and occurred independently of Ca2+i changes. The relationship between membrane potential and Ca2+i changes in lymphocyte activation are discussed on the basis of these results. Our conclusion was that constitutive Ca2+(-)dependent K+ channels are absent in the WEHI 231 cell line.     

Myosin light chain phosphorylation in 32P-labeled rabbit aorta stimulated by phorbol 12,13-dibutyrate and phenylephrine

The mechanism(s) of force development in vascular smooth muscle following pharmacological activation of protein kinase C by phorbol esters are not known. In this study, we examined the myosin light chain phosphorylation response following stimulation by phorbol 12,13-dibutyrate (PDB) or phenylephrine in rabbit aorta which had been incubated with 32PO4 in order to label ATP pools. Through tryptic phosphopeptide mapping of myosin light chain from intact tissue and comparison to controls using purified components, we inferred that Ca2+-dependent force stimulated by PDB was associated with small increases in serine-19 phosphorylation, consistent with a contractile mechanism involving indirect activation of myosin light chain kinase. Additional residues, consistent with the in vitro substrate specificity of protein kinase C, were also observed to be phosphorylated in response to PDB and represented proportionately a larger fraction of the total phosphorylated myosin light chain in Ca2+-depleted tissues. Stimulation by an alpha 1-adrenergic agonist (phenylephrine) resulted in phosphorylation of residues which were consistent with an activation mechanism involving myosin light chain kinase only. These results indicate that in rabbit aorta the contractile effects of PDB may be partially mediated by Ca2+-dependent activation of myosin light chain kinase. However, the data do not rule out a component of the PDB-stimulated contractile response which is independent of myosin light chain phosphorylation on the serine-19 residue. In addition, activation by a more physiological stimulus, phenylephrine, does not result in protein kinase C-mediated myosin light chain phosphorylation.     

Effect of anti-Ig on cytosolic Ca2+ in Daudi lymphoblastoid cells

We examined the response in the free intracellular calcium concentration ([Ca2+]i) of Daudi (human lymphoblastoid) cells to antibodies against human immunoglobulins (anti-Ig), and the relationship of [Ca2+]i to anti-Ig-induced capping. At 80 microM intracellular quin-2 (a fluorescent probe for [Ca2+]i), anti-Ig (10 micrograms/ml) caused a rapid increase in [Ca2+]i from 100 to 600 nM; the signal returned to baseline with approximately 1 min. At 450 microM intracellular quin-2, [Ca2+]i rose to only approximately 250 microM, and the signal declined gradually, returning to base line after greater than 7 min. In subsequent experiments, the lower concentrations of quin-2 were employed. Plots of the amplitude of the [Ca2+]i transients and of the binding of 125I-anti-Ig to Daudi cells versus the concentrations of anti-Ig showed similar saturation kinetics, with half-saturation occurring at 2-3 micrograms/ml. Part of the calcium in the transient is derived from the extracellular medium, and part from the nonmitochondrial intracellular stores. Caffeine (4 mM) and 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate HCl (0.5 mM) suppressed the release of calcium from internal stores and the entry of calcium from outside the cells, but permitted capping in more than half of the cells. Phorbol esters (1-2 nM) inhibited both capping and the anti-Ig-induced decrease in [Ca2+]i. None of these agents blocked the binding of anti-Ig to the cells. It appears that receptor capping is not dependent on the anti-Ig-induced transient increase in calcium concentration.     

The mechanism of action of the immunosuppressive drug FK-506

The novel immunosuppressive drug FK-506 inhibits the induction of lymphocyte proliferation in vitro by mitogenic combinations of phorbol esters and calcium ionophores. Early events inducible by phorbol esters alone are unaffected, while changes induced by calcium ionophores alone are completely suppressed by as little as 0.1 nM FK-506. The event in lymphocyte activation inhibited by FK-506 is completed early in the response. Its completion requires the provision of a Ca2+ signal and concurrent activation of protein kinase C and is accelerated as a function of the strength of protein kinase C activation.     

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.     

Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca2+)i, the stimulation of Na+/H+ transporter and the inhibition of the effects of thrombin alone on (Ca2+)i are potently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.     

Lymphoma models for B-cell activation and tolerance. VII. Pathways in anti-Ig-mediated growth inhibition and its reversal

WEHI-231, CH33, and CH31 are B-cell lymphomas that are inhibited in their growth by crosslinking of surface Ig receptors during early G1. This "negative signaling" process can be prevented or reversed under certain conditions. In the present paper, we use a cell synchronization procedure to demonstrate that activation of protein kinase C (PKC) is not involved in the negative signal for growth, but rather that PKC activation prevents growth inhibition when present early in the cell cycle. Indeed, the prevention of negative signaling is only accomplished by active phorbol esters. Moreover, phorbol esters and a calcium ionophore fail to deliver a negative signal under conditions in which anti-Ig can significantly prevent cell cycle progression into S phase, thereby ruling out synergy between PKC and calcium in growth inhibition. Whether phorbol esters reverse negative signaling by preventing internalization of the immune complex or phosphorylation of a critical intracellular protein is discussed.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Increases in intracellular Ca2+ regulate the binding of [3H]phorbol 12,13-dibutyrate to intact 1321N1 astrocytoma cells

The redistribution of protein kinase C (Ca2+/phospholipid-dependent protein kinase) from a cytosolic or a loosely associated membrane compartment to a more integral membrane compartment is stimulated by Ca2+ in vitro. This event is thought to be necessary for activation of the enzyme. To determine whether such a redistribution of protein kinase C occurs following hormonally stimulated increases in cytoplasmic Ca2+, we measured [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to protein kinase C in intact 1321N1 astrocytoma cells. The muscarinic agonist carbachol causes a 2-fold increase in [3H]PDB binding. This increase is transient, peaking at 1 min and returning toward control levels by 5 min. Scatchard analysis of [3H]PDB binding in the presence of carbachol reveals a 2-fold increase in the Bmax and no change in the KD compared to control values. This increase in Bmax likely represents a redistribution of protein kinase C to the membrane because [3H]PDB binding in intact cells is predominantly to membrane-associated enzyme. The Ca2+ ionophore ionomycin, and two other Ca2+-mobilizing hormones, bradykinin and histamine, mimic the effects of carbachol. Furthermore, when hormone-sensitive Ca2+ stores are depleted by prior agonist treatment, the carbachol-induced increases in intracellular [Ca2+] and [3H]PDB binding are completely blocked. Under these conditions, phosphoinositide hydrolysis and diacylglycerol (DAG) formation are not inhibited. We also examined the time course of DAG accumulation in response to carbachol. DAG is not yet significantly elevated when the increase in [3H]PDB binding is maximal. Furthermore, [3H]PDB binding has returned to control levels when DAG concentrations are maximally elevated. These data suggest that hormone-stimulated increases in cytoplasmic Ca2+ cause a marked and rapid redistribution of protein kinase C which precedes any significant increase in DAG. Our findings also demonstrate that [3H]PDB binding to intact cells may be a useful measure of the ability of Ca2+-mobilizing hormones to affect protein kinase C.     

Mechanism of activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and protein kinase C-independent pathway

Treatment of thymic lymphocytes with the mitogenic lectin concanavalin A (ConA) increases the intracellular free Ca2+ concentration and stimulates phosphoinositide turnover. ConA also induced a rapid, amiloride-sensitive, Na+-dependent increase in cytosolic pH of 0.13 +/- 0.01, indicative of stimulation of the Na+/H+ antiport. To investigate the mechanism underlying activation of Na+/H+ exchange by ConA, the intracellular free Ca2+ concentration changes induced by this lectin were precluded by loading the cells with Ca2+-buffering agents and suspension in Ca2+-free media. Under these conditions, the ConA-induced cytoplasmic alkalinization proceeded normally. Two approaches were used to assess the role of protein kinase C. First, this enzyme was inhibited by the addition of 1-(5-isoquinolinysulfonyl)-2-methylpiperazine. In the presence of this potent antagonist, stimulation of the antiport by 12-O-tetradecanoylphorbol-13-acetate was greatly inhibited. In contrast, stimulation by ConA was unaffected. Second, protein kinase C was depleted by overnight incubation with phorbol esters. Following this treatment, Na+/H+ exchange was no longer activated by 12-O-tetradecanoyl-13-acetate, but was still stimulated by ConA. These data suggest that a Ca2+- and protein kinase C-independent mechanisms mediates the activation of Na+/H+ exchange by ConA. The possible role of GTP-binding proteins in the activation was also studied. The antiport was not stimulated by either fluoroaluminate or vanadate. Moreover, pretreatment with pertussis toxin failed to inhibit the ConA-induced cytoplasmic alkalinization. In contrast, preincubation with cholera toxin partially inhibited activation. Under these conditions, cholera toxin significantly elevated intracellular cAMP levels. Inhibition was also observed in cells treated with forskolin at concentrations that increased [cAMP]. The data suggest that a novel cAMP-sensitive signaling mechanism not involving Ca2+ and protein kinase C is involved in the stimulation of Na+/H+ exchange by mitogens in T lymphocytes.     

A dual role for calcium in regulation of superoxide generation by stimulated rat alveolar macrophages

Superoxide production in alveolar macrophages is stimulated by agonists which act through Ca2+-mediated (concanavalin A) and/or protein kinase C (phorbol ester or diacylglycerol analogues) -mediated events. Simultaneous addition of saturating concentrations of concanavalin A and a protein kinase C activator (either phorbol 12-myristate-13-acetate or 1-oleoyl-2-acetyl-sn-glycerol) caused a supra-additive enhancement of the initial rate of O2-. production. This synergism closely correlated with the known time-course of Ca2+ mobilization induced by concanavalin A; however, it occurred under conditions in which protein kinase C activation is reportedly not Ca2+ dependent. Phorbol ester-induced O2-. production was partially inhibited by the Ca2+ ionophore, A23187. Although phorbol ester-stimulated O2-. production initially was enhanced by concanavalin A, the duration of this O2-. production was reduced in comparison to that induced by phorbol ester alone. These results suggest a dual role for intracellular Ca2+ in both stimulatory and inhibitory regulation of O2-. production.     

Activation of a Ca2+-inhibitable protein kinase that phosphorylates microtubule-associated protein 2 in vitro by growth factors, phorbol esters, and serum in quiescent cultured human fibroblasts

Treatment of quiescent human embryonic lung fibroblastic cells (TIG-3) with 10 nM epidermal growth factor (EGF) resulted in 4-6-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein 2 (MAP2) on serine and threonine residues in vitro. The half-maximal activation of the kinase activity occurred within 5 min after EGF treatment, and the maximal level was attained at 15 min. Casein and histone were very poor substrates for this EGF-stimulated MAP2 kinase activity. The activation of the kinase activity persisted after brief dialysis. Interestingly, the EGF-stimulated MAP2 kinase activity was sensitive to micromolar concentrations of free Ca2+; it was inhibited 50% by 0.5 microM Ca2+ and almost totally inhibited by 2 microM Ca2+. The activated MAP2 kinase activity was recovered in flow-through fractions on phosphocellulose column chromatography, while kinase activities that phosphorylate 40 S ribosomal protein S6 (S6 kinase activities) were mostly retained on the column and eluted at 0.5 M NaCl. Platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, phorbol esters (12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate), and fresh fetal calf serum also induced activation of the MAP2 kinase in the quiescent TIG-3 cells. The activated MAP2 kinase activity in cells stimulated by platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor-I, insulin, 12-O-tetradecanoylphorbol 13-acetate, phorbol 12,13-dibutyrate, or fetal calf serum was almost completely inhibited by 2 microM Ca2+, like the EGF-stimulated kinase. In addition, MAP2 phosphorylated by the kinase activated by different stimuli gave very similar phosphopeptide mapping patterns. These results suggest that several growth factors, phorbol esters, and serum activate a common, Ca2+-inhibitable protein kinase which is distinct from S6 kinase in quiescent human fibroblasts.     

Ca2+ and phorbol ester activation of protein kinase C at intracellular Ca2+ concentrations and the effect of TMB-8

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from human polymorphonuclear leukocytes and shown to be identical to bovine protein kinase C. The Ca2+ activation of the enzyme was studied and the Ca2+ concentrations required to activate the enzyme were compared to free cytosolic Ca2+ concentrations in resting and activated polymorphonuclear leukocytes. The free calcium concentrations in the cytosol and in the enzyme assay mixture were determined using the calcium indicator quin 2. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range, but was not activated by phosphatidylserine at Ca2+ concentrations corresponding to the intracellular free Ca2+ concentration under resting conditions. However, at similar Ca2+ concentrations (less than 2.5 X 10(-7) M) the enzyme was highly activated by phorbol 12-myristate 13-acetate (PMA) or diolein in the presence of phosphatidylserine. It was demonstrated that PMA stimulation of human polymorphonuclear leukocytes did not induce any increase in the level of the intracellular free calcium concentration. It was concluded that PMA activation of protein kinase C occurred independently of a rise in the intracellular Ca2+ concentration. K0.5 (half-maximal activation) for the PMA activation of purified protein kinase C was shown to be equivalent to the K0.5 for PMA stimulation of superoxide (O-2) production in human polymorphonuclear leukocytes, suggesting that protein kinase C is involved in activation of the NADPH oxidase. The presumed intracellular Ca2+ antagonist TMB-8 inhibited the PMA-induced superoxide production, but neither by an intracellular Ca2+ antagonism nor by a direct inhibition of protein kinase C activity.     

Control of exocytosis from adrenal chromaffin cells

1. Calcium-dependent exocytosis of catecholamines from intact and digitonin-permeabilized bovine adrenal chromaffin cells was investigated. 2. 45Ca2+ uptake and secretion induced by nicotinic stimulation or depolarization in intact cells were closely correlated. The results provide strong support for Ca2+ entry being the trigger for exocytosis. 3. Experiments in which the H+ electrochemical gradient across the intracellular secretory granule (chromaffin granule) membrane was altered indicated that the gradient does not play an important role in exocytosis. 4. Ca2+ entry into the cells is associated with activation of phospholiphase C and a rapid translocation of protein kinase C to membranes. 5. The plasma membrane of chromaffin cells was rendered permeable to Ca2+, ATP, and proteins by the detergent digitonin without disruption of the intracellular secretory granules. In this system in which the intracellular milieu can be controlled, micromolar Ca2+ directly stimulated catecholamine secretion. 6. Treatment of the cells with phorbol esters and diglyceride, which activate protein kinase C, enhanced phosphorylation and subsequent Ca2+-dependent secretion in digitonin-treated cells. 7. Phorbol ester-induced secretion could be specifically inhibited by trypsin. The experiments indicate that protein kinase C modulates but is not necessary for Ca2+-dependent secretion.     

Role of protein kinase C and intracellular calcium mobilization in the induction of macrophage tumoricidal activity by interferon-gamma

These studies were designed to test the hypothesis that changes in intracellular Ca2+ levels and activation of the calcium ion- and phospholipid-dependent protein kinase C were required for the induction of macrophage tumoricidal activity by interferon-gamma (IFN-gamma). Phenothiazines and R24571, known antagonists of calcium-binding proteins and therefore nonspecific inhibitors of protein kinase C, blocked in a dose-dependent manner the induction of macrophage cytocidal activity by either natural or recombinant IFN-gamma. Macrophages depleted of intracellular Ca2+ by chelation with Quin 2, were also unresponsive to IFN-gamma. These treatments effected neither the binding of IFN-gamma to its cell surface receptor nor the normal intracellular processing of IFN-gamma. Activators of protein kinase C (such as phorbol esters) and Ca2+ ionophores when added alone did not effect the activation state of the macrophage population. However, macrophages exposed to both drugs in combination were elevated into the primed activation state such that in the presence of a second signal (lipopolysaccharide or heat killed Listeria monocytogenes), the cells were triggered to express full levels of tumoricidal activity. The capacity of phorbol esters to induce cellular activation correlated with their ability to bind and to activate protein kinase C. No synergistic effect was observed between IFN-gamma and protein kinase C activators and/or Ca2+ ionophores, indicating that the drugs could only prime and could not trigger macrophages for tumor cell killing. These results thus support the concept that protein kinase C activation and mobilization of intracellular Ca2+ are essential steps in the pathway of IFN-gamma-dependent induction of non-specific tumoricidal activity in macrophages.