Pervanadate stimulates amylase release and protein tyrosine phosphorylation of paxillin and p125(FAK) in differentiated AR4-2J pancreatic acinar cells

We have studied the role of protein tyrosine phosphorylation in amylase secretion from differentiated AR4-2J cells. The secretagogue bombesin, the protein kinase C activator phorbol 12-myristate 13-acetate (PMA), and the protein-tyrosine phosphatase inhibitor pervanadate induced tyrosine phosphorylation of different proteins, including paxillin and p125(FAK), which was reduced or blocked by the tyrosine kinase inhibitors genistein and tyrphostin B56, respectively. Both PMA and pervanadate continuously increased amylase secretion with a similar time course, reaching the level of bombesin-induced amylase release after 60 min. Their effects were not additive and could be inhibited by preincubation of AR4-2J cells with genistein or tyrphostin B56, respectively. Inhibition of protein kinase C with Ro 31-8220 nearly abolished the effects of PMA, but had no effect on either pervanadate-induced protein tyrosine phosphorylation or amylase secretion. An increase in cytosolic free Ca2+ concentration by thapsigargin or A23187 caused a rapid increase in amylase release within the initial 5 min. In the presence of PMA or pervanadate, amylase secretion was further stimulated to levels comparable to those induced by bombesin after 30 min of stimulation. Inhibition of PMA-induced amylase secretion by Ro 31-8220 was less at elevated cytosolic free Ca2+ concentrations than without Ca2+. Furthermore, an increase in cytosolic free Ca2+ concentration had no effect on protein tyrosine phosphorylation in either the absence or presence of PMA or pervanadate. We therefore conclude that in the cascade of events that lead to bombesin-induced protein secretion from AR4-2J cells, protein tyrosine phosphorylation occurs downstream of protein kinase C activation. A further step in secretion that is Ca2+-dependent occurs distal to protein tyrosine phosphorylation.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

Sodium, PMA and calcium play an important role on intracellular pH modulation in rat mast cells

In a series of experiments aimed to understand the signaling pathways that regulate intracellular pH (pHi) in rat mast cells, the effect of different intracellular mechanisms on the activity of the Na+/H+ exchanger was studied. After promoting an artificial acidification with sodium propionate we determined the variations on pHi rate recovery. pHi was measured with the dye 2, 7-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester. We studied the effect that the inhibition of some cellular exchangers with different drugs induced on pHi. When the Na+/H+ exchanger was inhibited in the presence of amiloride, the recovery rate constant was twofold smaller than the control value. After the recovery, the final pH was lower than the initial value when the cells were treated either with amiloride or with 4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid (an anionic antiport inhibitor). No effect was observed when the Na+/K+-ATPase or the Na+/Ca2+ exchanger were inhibited. The suppression of intracellular and extracellular calcium did not induced any change in pHi. The addition of thapsigargin, an activator of capacitative calcium influx, or the phorbol esther 12-O-tetradecanoylphorbol-13-acetate (PMA), a protein kinase C (PKC) activator, increased the activity of the antiporter. Both effects were abrogated by inhibition of the Na+/K+-ATPase with ouabain. The increase in cAMP levels did not affect the effect of PMA on pHi recovery, but it blocked the effect of thapsigargin. Our results indicate that rat mast cells regulate pHi by the combination of some anionic exchanger and the Na+/H+ antiporter. And also that the modulation of this exchanger is the consequence of the connection between different intracellular mechanisms, Na+/K+-ATPase-PKC-calcium, among which cAMP seems not to have a direct role.     

Physical health impairment and depression among older adults

We studied the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1), and interferon-gamma (IFN-gamma) on the function of thyroid cells and pituitary thyrotrophs. In FRTL-5 rat thyroid cells, both human and murine TNF-alpha inhibited basal and TSH-stimulated [125I]iodide transport. IL-1 shared this action with TNF-alpha, but was less potent. IL-1 and IFN-gamma did not cause a further reduction of TNF-alpha-induced inhibition of [125I]iodide transport. TNF-alpha, phorbol ester 12-myristate 13-acetate (PMA), and calcium ionophore (CI) A23817 all inhibited [125I]iodide transport, but high doses of PMA and CI also blocked the inhibitory action of TNF-alpha on [125I]iodide transport. Inhibition of protein kinase A and protein kinase C by H7 or HA inhibited TSH-stimulated iodide transport, but did not block the TNF-alpha action, suggesting that the mechanism of TNF-alpha action on thyroid cells is independent of protein kinase A and C. In pituitary cells, both human and murine TNF-alpha did not affect basal TSH secretion, but TNF-alpha reduced TRH-stimulated TSH secretion. This study provides further in vitro evidence that TNF-alpha inhibits the function of the hypothalamus-pituitary-thyroid axis acting directly on both the pituitary and thyroid glands.     

Norepinephrine stimulates mitogen-activated protein kinase activity in GT1-1 gonadotropin-releasing hormone neuronal cell lines

The GT1-1 GnRH neuronal cell lines exhibit highly differentiated properties of GnRH neurons. We have used GT1-1 cells to study the roles of norepinephrine (NE), membrane depolarization, calcium influx, and phorbol esters in the regulation of mitogen-activated protein (MAP) kinase. NE, which is known to stimulate the release of GnRH, induced MAP kinase activity, the tyrosine phosphorylation of MAP kinase, and MAP kinase kinase activity. Forskolin led to activation of MAP kinase comparable with that induced by NE, and a selective inhibitor of cAMP-dependent protein kinase, H8, attenuated the NE-induced activation of MAP kinase. On the other hand, elimination of extracellular calcium by EGTA completely blocked NE-induced tyrosine phosphorylation of MAP kinase, and a selective inhibitor of calcium/calmodulin-dependent protein kinase, KN-62, attenuated the NE-induced activation of MAP kinase. Furthermore, depolarization of GT1-1 cells with 75 mM KCl, 10 microM BayK 8644, or 1 microM calcium ionophore (A23187) induced rapid tyrosine phosphorylation of MAP kinase. The omission of calcium from the extracellular medium completely abolished these effects of tyrosine phosphorylation of MAP kinase. Phorbol 12-myristate 13-acetate (PMA) also induced MAP kinase activity, but pretreatment of the cultured cells with PMA to down-regulate protein kinase C did not abolish the activation of MAP kinase by NE. In addition, although phosphorylation of Raf-1 kinase was stimulated by PMA, this phosphorylation was not induced by either NE or A23187. These results demonstrate that NE activates MAP kinase directly in GT1-1 cells, and that the effect of NE is mediated by increase in the cAMP level and by calcium influx, but not by PMA-sensitive protein kinase C or Raf-1 kinase.     

DNA damage induced by m-phenylenediamine and its derivative in the presence of copper ion

We have previously reported that long-term priming of human polymorphonuclear neutrophilic granulocytes (PMN) with interferon-gamma (IFN-gamma) increased the fMLP-stimulated calcium influx. We now show that also after short-term incubation with IFN-gamma, PMN calcium metabolism is modulated. Single adherent cells in three different calcium-containing buffers (high, normal, and low [Ca2+]) were stimulated with the bacterial peptide fMLP or the Ca-ATPase inhibitor thapsigargin (Tg) after about 5 min preincubation with IFN-gamma. The results of this protocol indicated that IFN-gamma increases both calcium influx and calcium sequestration. Store dependent Ca2+ influx, directly measured on readdition of calcium to Tg-treated cells incubated in EGTA buffer, was significantly enhanced in IFN-gamma-treated cells. This effect of IFN-gamma was enhanced by the tyrosine kinase inhibitor herbimycin A. Strikingly, in low extracellular calcium concentrations, IFN-gamma induced calcium transients in 20%-60% of the cells. The proportion of PMN responding with Ca2+ transients increased with decreasing extracellular calcium concentration. Average lagtime from addition of IFN-gamma to a response that could be measured was 7.3 sec, and average increase in [Ca2+] above the basal level was 790 nM. These IFN-gamma-induced transients could not be depressed by herbimycin A. Thus, IFN-gamma can increase capacitative calcium influx, induce calcium transients, and possibly affect calcium sequestration in human PMN.     

Adhesion-dependent degranulation of neutrophils requires the Src family kinases Fgr and Hck

Polymorphonuclear neutrophils (PMN) adherent to integrin ligands respond to inflammatory mediators by reorganizing their cytoskeleton and releasing reactive oxygen intermediates. As Src family tyrosine kinases are implicated in these responses, we investigated their possible role in regulating degranulation. Human PMN incubated on fibrinogen released lactoferrin in response to TNF-alpha and this response was inhibited by PP1, a Src family tyrosine kinase inhibitor. This drug had no effect on lactoferrin secretion induced by PMA, an adhesion-independent agonist of PMN degranulation. However, PP1 blocked secretion in PMN plated on plain tissue culture plastic, a surface inducing PMN spreading in the absence of any stimulus. Double knockout hck-/- fgr-/- PMN adherent to collagen or fibrinogen failed to release lactoferrin in response to TNF-alpha but responded to PMA as wild-type PMN. Degranulation induced by spreading over tissue culture plastic was also defective in hck-/- fgr-/- PMN. Defective adhesion-dependent degranulation required the absence of both kinases, because single knockout fgr-/- or hck-/- PMN responded as wild-type cells. Analysis of lactoferrin secretion in hck-/- fgr-/- or PP1-treated, suspended PMN showed that Src kinases are not implicated in degranulation dependent on activation of protein kinase C or increase in intracellular free Ca2+ but may play a role in the response to FMLP of cytochalasin B-treated PMN. These findings identify a role for Src family kinases in a signaling pathway leading to granule-plasma membrane fusion and suggest that Fgr and Hck would be targets for pharmacological control of adhesion-dependent degranulation in the inflammatory site.     

Purinergic receptor-induced changes in paracellular resistance across cultures of human cervical cells are mediated by two distinct cytosolic calcium-related mechanisms

In human cervical (CaSki) cells, extracellular adenosine triphosphate (ATP) induces an acute decrease in the resistance of the lateral intercellular space (RLIS), phase I response, followed by an increase in tight junctional resistance (RTJ), phase II response. ATP also stimulates release of calcium from intracellular stores, followed by augmented calcium influx, and both effects have similar sensitivities to ATP (EC50 of 6 microM). The objective of the study was to determine the degree to which the changes in [Ca2+]i mediate the responses to ATP. 1,2-bis (2-aminophenoxy) ethane-N,N,N1,N1-tetraacetic acid (BAPTA) abrogated calcium mobilization and phase I response; in contrast, nifedipine and verapamil inhibited calcium influx and attenuated phase II response. Barium, La3+, and Mn2+ attenuated phase I response and attenuated and shortened the ionomycin-induced phase I-like decrease in RLIS, suggesting that store depletion-activated calcium entry was inhibited. Barium and La3+ also inhibited the ATP-induced phase II response, but Mn2+ had no effect on phase II response, and in the presence of low extracellular calcium it partly restored the increase in RTJ. KCl-induced membrane depolarization stimulated an acute decrease in RLIS and a late increase in RTJ similar to ATP, but only the latter was inhibited by nifedipine. KCl also induced a nifedipine-sensitive calcium influx, suggesting that acute increases in [Ca2+]i, regardless of mobilization or influx, mediate phase I response. Phase II-like increases in RTJ could be induced by treatment with diC8, and were not affected by nifedipine. Biphasic, ATP-like changes in RTE could be induced by treating the cells with ionomycin plus diC8. We conclude that calcium mobilization mediates the early decrease in RLIS, and calcium influx via calcium channels activates protein kinase C and mediates the late increase in RTJ.     

Expression and phosphorylation of a MARCKS-like protein in gastric chief cells: further evidence for modulation of pepsinogen secretion by interaction of Ca2+/calmodulin with protein kinase C

In gastric chief cells, agents that activate protein kinase C (PKC) stimulate pepsinogen secretion and phosphorylation of an acidic 72-kDa protein. The isoelectric point and molecular mass of this protein are similar to those for a common PKC substrate; the MARCKS (for Myristoylated Alanine-Rich C Kinase Substrate) protein. We examined expression and phosphorylation of the MARCKS-like protein in a nearly homogeneous suspension of chief cells from guinea pig stomach. Western blotting of fractions from chief cell lysates with a specific MARCKS antibody resulted in staining of a myristoylated 72-kDA protein (pp72), associated predominantly with the membrane fraction. Using permeabilized chief cells, we examined the effect of PKC activation (with the phorbol ester PMA), in the presence of basal (100 nM) or elevated cellular calcium (1 microM), on pepsinogen secretion and phosphorylation of the 72-KDa MARCKS-like protein. Secretion was increased 2.3-, 2.6-, and 4.5-fold by incubation with 100 nM PMA, 1 microM calcium, and PMA plus calcium, respectively. A PKC inhibitor (1 microM CGP 41 251) abolished PMA-induced secretion, but did not alter calcium-induced secretion. This indicates that calcium-induced secretion is independent of PKC activation. Chief cell proteins were labeled with 32P-orthophosphate and phosphorylation of pp72 was detected by autoradiography of 2-dimensional polyacrylamide gels. In the presence of basal calcium, PMA (100 nM) caused a > two-fold increase in phosphorylation of pp72. Without PMA, calcium did not alter phosphorylation of pp72. However, 1 microM calcium caused an approx. 50% attenuation of PMA-induced phosphorylation of pp72. Experiments with a MARCKS "phosphorylation/calmodulin binding domain peptide" indicated that calcium/calmodulin inhibits phosphorylation of pp72 by binding to the phosphorylation/calmodulin binding domain and not by inhibiting PKC activity. These observations support the hypothesis that, in gastric chief cells, interplay between calcium/calmodulin binding and phosphorylation of a common domain on the 72-kDa MARCKS-like protein plays a role in modulating pepsinogen secretion.     

Relative maxillary retrusion as a natural consequence of aging: combining skeletal and soft-tissue changes into an integrated model of midfacial aging

Depletion of intracellular calcium stores by agonist stimulation is coupled to calcium influx across the plasma membrane, a process termed capacitative calcium entry. Capacitative calcium entry was examined in cultured guinea pig enteric glial cells exposed to endothelin 3. Endothelin 3 (10 nM) caused mobilization of intracellular calcium stores followed by influx of extracellular calcium. This capacitative calcium influx was inhibited by Ni2+ (89 +/- 2%) and by La3+ (78 +/- 2%) but was not affected by L-, N-, or P-type calcium channel blockers. Chelerythrine, a specific antagonist of protein kinase C, dose-dependently inhibited capacitative calcium entry. The nitric oxide synthase inhibitor NG-nitro-L-arginine decreased calcium influx in a dose-dependent manner. The combination of chelerythrine and NG-nitro-L-arginine produced synergistic inhibitory effects. Capacitative calcium entry occurs in enteric glial cells via lanthanum-inhibitable channels through a process regulated by protein kinase C and nitric oxide.     

Phorbol ester and calcium regulation of corticotrophin-releasing factor receptor 1 expression in a neuronal cell line

We have previously demonstrated that corticotrophin-releasing factor receptor 1 (CRF-R1) mRNA levels can be down-regulated via activation of the cyclic AMP pathway in CATH.a cells, a neuronal cell line. In this study, we show evidence for down-regulation of CRF-R1 mRNA levels via activation of the protein kinase C (PKC) and calcium second messenger pathways. Incubation of CATH.a cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, resulted in a time- and concentration-dependent down-regulation of CRF-R1 mRNA levels. Pretreatment with the inactive phorbol ester 4alpha-phorbol failed to influence significantly CRF-R1 mRNA levels. Incubation with carbachol, a cholinergic agonist known to activate PKC and increase intracellular calcium levels via phosphatidylinositol breakdown, also down-regulated CRF-R1 mRNA levels. Intracellular calcium levels were directly increased using A23187, a calcium ionophore, and thapsigargin, a calcium-ATPase inhibitor. Elevation of intracellular calcium content using either A23187 or thapsigargin significantly down-regulated levels of CRF-R1 mRNA. Furthermore, chelation of calcium with EGTA or blockade of voltage-dependent calcium channels with nifedipine inhibited agonist-mediated down-regulation of CRF-R1 mRNA levels. These results indicate that activation of PKC or calcium signal transduction pathways is sufficient to cause down-regulation of CRF-R1 mRNA levels and that calcium is required for agonist-mediated down-regulation of this receptor.     

Differential regulation of IL-4 and IL-13 secretion by human basophils: their relationship to histamine release in mixed leukocyte cultures

Human basophils are an important source of IL-4, a cytokine that is central to the pathogenesis of allergic inflammation. Recent reports have indicated that these cells also generate IL-13, which shares a number of biologic properties with IL-4. We found basophils to be the major source of IL-13 produced in mixed leukocyte cultures following 20-h activation with a variety of stimuli. While the magnitude of IL-4 protein generated correlated with the percent histamine secreted (r = 0.8; p = 0.007), there was no relationship between the levels of IL-13 detected and the amount of either IL-4 or histamine in cultures activated with IL-3/anti-IgE. The induction of IL-13 secretion also occurred in response to IL-3 alone, without concomitant secretion of either IL-4 or histamine. Although previously shown to inhibit IL-4 secretion, the phorbol ester PMA was a potent stimulus for IL-13 generation from basophils, and this secretion was sensitive to the protein kinase C inhibitor, bisindolylmaleimide. In contrast, bisindolylmaleimide did not prevent cytokine secretion induced by either anti-IgE or IL-3. The immunosuppressant, FK506, while strikingly inhibiting the accumulation of IL-4 mRNA and the secretion of protein in response to IL-3/anti-IgE, had no effect on the generation of IL-13 in these cultures; the resistance was attributed to the IL-3-dependent signaling. Similarly, FK506 had no effect on the secretion of IL-13 in basophil cultures stimulated with PMA. This study suggests that multiple intracellular mechanisms control the generation of IL-13 in basophils, some of which are distinct from those regulating IL-4.     

Protein kinase C activation stimulates calcium transport in adrenal zona glomerulosa cells

Adrenal zona glomerulosa (ZG) cells produce aldosterone in response to angiotensin II and extracellular potassium through different mechanisms which involve changes in cytosolic free calcium (Cai). Protein kinase C (PKC) activation is part of the angiotensin II signalling cascade but its effects on Cai are unknown. PKC activation with 1 microM phorbol 12-myristate 13-acetate (PMA) and 8 mM Ko significantly increased the rate of calcium influx (P < 0.001). Both the PKC- and the Ko-induced calcium influx occurred via a nifedipine-sensitive pathway. When both were combined, PKC activation and 8 mM Ko were not additive over either agent alone. PKC activation and 8 mM Ko also stimulated calcium efflux (P < 0.01). When combined together PKC activation and 8 mM Ko had additive effects on calcium efflux (P < 0.05). PKC activation did not increase Cai nor the exchangeable calcium pool in contrast to 8 mM Ko which significantly increased both (P < 0.001). Thus, PKC activation in ZG cells induces a pattern of calcium transport characterized by accelerated calcium recycling across the cell membrane without increasing cell calcium content.     

Protein kinase C modulates cytosolic free calcium by stimulating calcium pump activity in Jurkat T cells

Although protein kinase C (PKC) activation has been shown to inhibit Ca2+ influx in T lymphocytes, the role of PKC on Ca2+ sequestration or extrusion processes has not been fully explored. We examined the effect of CD3 stimulation and PKC activators on cytosolic Ca2+ (Ca2+i) extrusion and 45Ca2+ efflux in human leukemic Jurkat T cells. Treatment of Fura-2 loaded cells with phorbol 12-myristate 13-acetate (PMA) or thymeleatoxin (THYM) resulted in a decrease in Ca2+i both in the presence and absence of extracellular Ca2+, whereas inactive phorbol esters had no effect. PKC activators added at the peak of a Ca2+i transient induced by anti-CD3 mAb, ionomycin or thapsigargin (TG) stimulated the rate and extent of return of Ca2+i to basal levels by 17-53%. PKC stimulation of the Ca2+i decline was not enhanced by the presence of Na+, indicating that PKC activators increase Ca2+ pump activity rather than a Na+/Ca2+ exchange mechanism. As CD3 receptor activation enhanced the Ca2+i decline in TG-treated cells, antigen-mediated activation of phospholipase C (PLC) signaling includes enhanced Ca2+ extrusion at the plasma membrane. The effect of PKC activators on parameters of Ca2+i extrusion were further explored. PMA significantly increased the rate of Ca2+ extrusion in TG-treated cells from 0.28 +/- 0.02 to 0.35 +/- 0.03 s-1 (mean +/- SEM) and stimulated the initial rate of 45Ca2+ efflux by 69% compared to inactive phorbol ester treated cells. The effects of PKC activation on the Ca2+i decline were eliminated by PKC inhibitors, PKC down regulation (24 h PMA pretreatment), ATP-depletion and conditions that inhibited the Ca2+ pump. In contrast, pretreatment of cells with okadaic acid enhanced the PMA-stimulated response. We suggest that Jurkat T cells contain a PKC-sensitive Ca2+ extrusion mechanism likely to be the Ca2+ pump. In lymphocytes, receptor/PLC-linked PKC activation modulates Ca2+i not only by inhibiting Ca2+ influx but also by stimulating plasma membrane Ca2+i extrusion.     

Role of Ca2+-ATPase inhibitors in activation of cytosolic phospholipase A2 in human polymorphonuclear neutrophils

In the present study, we investigated the involvement of Ca2+-signaling and protein kinases in the effect of Ca2+-ATPase inhibitors on the activation of cytosolic phospholipase A2 (cPLA2) in human polymorphonuclear neutrophils. We found that activity and mobility on electrophoresis gels of the cPLA2 protein were significantly increased by f-Met-Leu-Phe (fMLP), 12-myristate 13-acetate (PMA) and the Ca2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid. This effect was completely suppressed by staurosporine. Calphostin C partially inhibited the fMLP- and PMA-induced cPLA 2 activation, but had no influence on thapsigargin- and cyclopiazonic acid-treated cells. Thapsigargin and cyclopiazonic acid also showed no effect on protein kinase C activity. However, the thapsigargin- and cyclopiazonic acid-induced cPLA2 activation was completely inhibited by the tyrosine kinase inhibitor, erbstatin, and Ca2+ chelator, EGTA. In addition, the cPLA2 activity was reduced after pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059. The arachidonic acid release was significantly reduced in cells pretreated with the cPLA2 inhibitor, AACOCF3. Furthermore, we found that the human neutrophil cPLA2 cDNA contain a Ca2+-dependent-lipid binding domain which shares homology to several other enzymes such as protein kinase C and phospholipase C. Our results suggest that tyrosine kinases and the MAP kinase cascade are involved in Ca2+-ATPase inhibitor-induced activation and phosphorylation of cPLA2. Protein kinase C is not required in this event.     

Calcium- and calmodulin-dependent PMA-activation of the CD44 adhesion molecule

The ability of the CD44 adhesion molecule to interact with its ligand hyaluronic acid (HA) is tightly regulated. CD44-positive mouse LB lymphoma cells are unable to bind HA unless activated by the tumor promoter phorbol 12-myristate 13-acetate (PMA). PMA causes a dose-dependent increase in both CD44 expression level and HA-binding capacity, with the binding of HA observed only above a threshold amount of CD44 molecules. This induction of HA-binding as well as the increase in CD44 expression are prevented by cycloheximide, suggesting a requirement for new additional CD44 molecules on the cell surface and/or cooperating proteins. In the present study, we have investigated which of the signal transduction pathways activated by PMA leads to the increased CD44 expression with subsequent acquisition of HA-binding capacity. By comparing the influence of each inhibitory agent on PMA-activated LB lymphoma cells versus that on a constitutive HA-binder cell line derived from LB cells (designated HA9 cells), we could distinguish between an effect on the PMA-activation phase and a one on the HA-binding phase. Our data show that the PMA-induced HA-binding could not be blocked by agents inhibiting protein kinase C (PKC) (staurosporine, sphingosine, polymyxin B, quercetin) or genestein, an inhibitor of tyrosine protein kinases. However, this PMA response was strongly inhibited by calmodulin antagonists (chlorpromazine, trifluoperazine, W-7) and the calcium blocker verapamil. The calmodulin antagonists inhibited the PMA-induced increase in CD44 expression on LB cells, but had no influence on the ability of the constitutive HA-binder HA9 cell line to interact with HA, indicating an effect on the PMA induction phase rather than on the binding itself. Verapamil also blocked the PMA-induced increase in CD44 expression on LB cells, but in addition it slightly reduced the ability of the HA9 cells to bind HA without affecting their CD44 expression level. In conclusion, our data suggest that CD44 activation by PMA is calcium and calmodulin dependent, rather than mediated by protein kinase C.     

Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells

To separate the role of changes in parathyroid diacylglycerol (DG) from other effects of extracellular calcium, we studied the effect of inhibition of DG metabolism on PTH secretion and cellular DG content in acutely dispersed bovine parathyroid cells. R 59 022, an inhibitor of DG kinase, increased cellular DG, but significantly decreased PTH secretion. Particulate protein kinase C (PKC) activity decreased in bovine parathyroid cells incubated at high extracellular calcium or in the presence of R 59 022, which is the opposite of what was observed in the presence of the phorbol ester, phorbol myristate acetate. Sphinganine, a normal cellular product that is a known inhibitor of PKC, significantly inhibited PTH secretion at low extracellular calcium, but had no significant effect at normal or high extracellular calcium. We then measured sphingosine in bovine parathyroid cells incubated with high extracellular calcium or R 59 022. Both conditions were associated with significant elevations of cellular sphingosine. These studies suggest that inhibition of PTH secretion and PKC activity by enhanced cellular DG may result from the activation of an inhibitory second messenger pathway involving the sphingoid lipids.     

Tyrphostin A9 inhibits calcium release-dependent phosphorylations and calcium entry via calcium release-activated channel in Jurkat T cells

The mechanism by which calcium-depleted intracellular stores may trigger an external calcium influx through a calcium release-activated channel was investigated by analyzing the effects of several protein tyrosine kinase inhibitors on calcium movements in Jurkat T cells. Tyrphostin A9, an inhibitor of the kinase activity of the platelet-derived growth factor (PDGF) receptor, dramatically impaired the sustained elevation of cytosolic calcium concentration, induced by either CD3 mAbs, thapsigargin, ionomycin at low (10(-7) M) concentration, or passive depletion of intracellular stores; other tested tyrphostins, lavendustin, genistein, and compound 5 lacked significant effect. Tyrphostin A9, added during the plateau phase, was able to return cytosolic calcium to resting concentration. Likewise, it abrogated manganese entry in cells stimulated by CD3 or thapsigargin, measured by the quenching of the fluorescence of Indo-1. However, it did not measurably modify kinetics of intracellular calcium releases monitored in the absence of extracellular calcium, nor did it reverse the inhibition of phosphatidylserine that occurs as a consequence of emptying intracellular stores. Analyses of tyrosine phosphorylations demonstrated that A9 inhibited the phosphorylation of proteins, which occurred every time that internal calcium stores were depleted. These phosphorylations were not impaired by chelation of external Ca2+, nor by La3+ that inhibits calcium release-induced calcium entry. We concluded that their inhibition was not a consequence, but may be a cause, of the blockade of calcium release-activated channel by tyrphostin A9.     

Regulation of alphaIIb beta3 function in human B lymphocytes

We studied the function of the platelet integrin alphaIIb beta3 using a B lymphocyte model in which alphaIIb beta3 can be induced to interact with fibrinogen using phorbol myristate acetate (PMA). To determine whether a G protein-coupled receptor could also activate alphaIIb beta3 in lymphocytes, we coexpressed the human formyl peptide receptor (fPR) and alphaIIb beta3, finding that the fPR agonist formyl Met-Leu-Phe (fMLP)-stimulated lymphocyte adherence to immobilized fibrinogen and binding of soluble fibrinogen to the lymphocyte surface. The response to fMLP, but not PMA, was abrogated by pertussis toxin, indicating that the fPR was coupled to the G-protein Galphai, whereas the protein kinase C inhibitor bisindolylmaleimide I inhibited the response to both fMLP and PMA, indicating that signaling from the fPR included protein kinase C. On the other hand, the tyrosine kinase inhibitor genistein, the Syk inhibitor piceatannol, and the RhoA inhibitor C3 exoenzyme had no effect, implying that neither tyrosine phosphorylation nor the GTPase RhoA were involved. Furthermore, whereas micromolar concentrations of cytochalasin D inhibited the PMA-stimulated interaction of alphaIIb beta3 with fibrinogen, nanomolar concentrations actually induced fibrinogen binding to unstimulated cells. Our studies demonstrate that alphaIIb beta3 expressed in B lymphocytes can be activated by a physiologic agonist and outline an activating pathway that includes Galphai, protein kinase C, and the actin cytoskeleton.