Continuous phosphorylation of both the 47 and the 49 kDa proteins occurs during superoxide production by neutrophils

Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approx. 47 and 49 kDa. Treatment of unstimulated cells with antagonists of protein kinase C (e.g., staurosporine; 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) is known to inhibit both of these phenomena upon stimulation. These antagonists of PKC also cause a rapid cessation of O2- release when added to cells that are already stimulated. In this paper, we report that the addition of staurosporine or H-7 to stimulated neutrophils resulted in a rapid loss of 32P from both the 47 and the 49 kDa phosphoprotein bands, as detected by autoradiography. This suggests that these two proteins may be regulated by a continual cycle of phosphorylation and dephosphorylation in the stimulated cell, with the phosphorylation reactions predominating, or undergo a rapid degradation subsequent to phosphorylation. Either explanation is consistent with the view that protein kinase C activity is necessary to both initiate and maintain O2- production in neutrophils stimulated with tumor promoters.     

Paradoxical effects of retinal in neutrophil stimulation

Retinal stimulates the activity of phospholipase C and superoxide (O2-) release in neutrophils. The latter response is comparable in magnitude to that observed when phorbol 12-myristate 13-acetate (PMA) is the stimulating agent. Cells treated with retinal, however, do not undergo degranulation, nor do they exhibit the formation of intracellular vesicles, as is commonly observed with other agents (e.g. Lochner, J. E., Badwey, J. A., Horn, W., and Karnovsky, M. L. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7673-7677). Retinal promotes redistribution of the activity of protein kinase C from a soluble to a particulate fraction in neutrophils, and this redistribution precedes O2- release. Superoxide release stimulated with retinal, however, is largely insensitive to inhibitors of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7); staurosporine). These compounds substantially block both O2- release and the phosphorylation of two proteins with molecular masses of about 47 and 49 kDa when the stimulus is PMA. The data indicate that retinal and PMA elicit the formation of active protein kinase C complexes of different natures, or that the mechanism of stimulation of O2- release by retinal does not involve this kinase. The significance of these observations to the common use of retinoids as inhibitors of protein kinase C is discussed.     

Protein phosphorylation associated with synergistic stimulation of neutrophils

Neutrophils treated with optimal amounts of tumor promoters that activate protein kinase C (e.g. mezerein) release large quantities of superoxide (O2-) and exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. These cells can also be stimulated synergistically to release a comparable amount of O2-. This involves treatment with a suboptimal amount of a tumor promoter and an agent capable of elevating cellular Ca2+. Neutrophils treated in the former fashion exhibit a redistribution of the activity of protein kinase C from a soluble to a particulate fraction that is stable in the presence of Ca2+ chelators, whereas cells stimulated synergistically do not do so to an appreciable extent (Badwey, J. A., Robinson, J. M., Horn, W., Soberman, R. J., Karnovsky, M. J., and Karnovsky, M. L. (1988) J. Biol. Chem. 263, 2779-2786). In this paper, we report that neutrophils stimulated synergistically do exhibit a significant incorporation of 32P into the 47-kDa protein, but with little labeling of the 49-kDa species. This labeling of the 47-kDa protein was greater than the sum of those observed with each agent added separately but was less than that observed in cells stimulated with optimal amounts of tumor promoters alone. An inhibitor of protein kinase C (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) blocked O2- release and the phosphorylation of the 47-kDa protein under all conditions of stimulation mentioned, whereas an inhibitor of cyclic nucleotide-dependent kinases had no effect on these phenomena. Thus, labeling of the 47-kDa protein can occur in the absence of a "tight" translocation of protein kinase C to membrane and was always observed during synergy. The data support a role for protein kinase C and the 47-kDa phosphoprotein in the synergistic stimulation of neutrophils.     

Effects of antagonists of protein phosphatases on superoxide release by neutrophils.

Neutrophils stimulated with 4 beta-phorbol 12-myristate 13-acetate (PMA) release large quantities of superoxide (O2-) and exhibit phosphorylation of two proteins with molecular masses of 47(p47) and 49 kDa (p49). Addition of inhibitors of protein kinases (e.g. 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7)) to these cells after stimulation with PMA results in the loss of 32P from these proteins and a rapid cessation of O2- release (e.g. Heyworth, P. G., and Badwey, J. A. (1990) Biochim. Biophys. Acta 1052, 299-305). In this paper we report that antagonists of type 1 and 2A protein phosphatases (okadaic acid, calyculin A) prevented both the loss of 32P from p47 and the termination of O2- release in stimulated neutrophils treated with H-7. Calyculin A also caused a remarkable hyperphosphorylation of a number of proteins in neutrophils and increased O2- release from these cells in response to a suboptimal amount of PMA. Enzymes present in both the soluble and particulate fractions of neutrophils catalyzed the near complete dephosphorylation of 32P-labeled p47 and p49 bound to Immobilon-P membranes. Dephosphorylation of these blotted phosphoproteins occurred at physiological rates and was inhibited by okadaic acid and calyculin A. These data strongly suggest that p47 undergoes a continual cycle of phosphorylation and dephosphorylation throughout the period of O2- release when PMA is the stimulus. Moreover, we show that antagonists of type 1 and 2A protein phosphatases block dephosphorylation of p47 both in vivo and in vitro, indicating that these enzymes may modulate O2- release under certain circumstances.     

Interleukin-1 stimulation of arachidonic acid release from human synovial fibroblasts; blockade by inhibitors of protein kinases and protein synthesis

Addition of IL-1 (interleukin-1) to human synovial fibroblasts radiolabelled with [3H]arachidonic acid caused a linear dose-dependent increase in arachidonic acid release and a transient rise in labelled diacylglycerol. Protein kinase C activators PMA 4-phorbol 12-myristate 13-acetate and DiC8 (1,2-dioctanoyl-sn-glycerol) also increased arachidonic acid release, but the time course observed with PMA was different from that of IL-1. When cultures were treated with PMA for 16-24 h to down regulate protein kinase C, the ability of IL-1 to increase arachidonic acid release persisted to the same extent as in nontreated cultures. In contrast, PMA pretreatment prevented the eight-fold stimulation of arachidonic acid release in response to PMA observed in cultures not previously exposed to PMA. To examine the role of other kinases in IL-1 stimulated arachidonic acid release, cultures were treated with H-7 (1-(5-isoquinolinesulphonyl)-2-methylpiperazine dichloride), H-8 (N-[2-(methylamino) ethyl]-5-isoquinolinesulphonamide dichloride), HA1004 (N-(2-guanidoinoethyl)-5-isoquinolinesulphonamide hydrochloride), and staurosporine. IL-1 stimulation of arachidonic acid release was blocked by H-7, H-8 and staurosporine. H-7 was a more potent inhibitor than H-8, suggesting that cAMP dependent kinase did not mediate IL-1 action. Addition of H-7 at various times following IL-1 decreased IL-1 stimulated arachidonic acid release, suggesting that continued protein kinase activity was necessary for IL-1 action. Cycloheximide and actinomycin D inhibited the stimulation of arachidonic acid release by IL-1, PMA or DiC8. The addition of cycloheximide or actinomycin D 15-45 min after IL-1 also inhibited IL-1 stimulated arachidonic acid release, indicating that continued protein synthesis was required for IL-1 action. These results suggest that IL-1 stimulation of acylhydrolyase activity in human synovial cells occurs by a mechanism requiring continued protein synthesis and protein kinase activity and that neither protein kinase C nor cAMP dependent protein kinase is involved.     

Arachidonate activation of the neutrophil NADPH-oxidase. Synergistic effects of protein phosphatase inhibitors compared with protein kinase activators.

Arachidonate activation of the NADPH-oxidase in intact neutrophils and in a cell-free O2- generation system was compared to synergistic activation in response to arachidonate and agents that effect protein phosphorylation. In intact neutrophils, suboptimal doses of retinal which increase protein phosphorylation, or 4B-phorbol 12-myristate 13-acetate (PMA) an activator of protein kinase C, induced minimal O2- release, but primed neutrophils to release enhanced amounts of O2- in response to 2.5 microM arachidonate. In contrast to retinal or PMA, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, did not induce any release of O2-, but significantly increased the maximal rate and duration of O2- release in response to arachidonate. In the cell-free system, only arachidonate induced O2- generation. Consistent with previous findings, activation of the cell-free system was dependent of the presence of light membranes, cytosol, NADPH, Mg2+, and 82 microM arachidonate. Pretreatment of neutrophils with suboptimal doses of PMA or retinal had little effect on the arachidonate-stimulated release of O2- in cell-free preparations of these cells. However, cytosol (but not light membranes) from PMA or retinal-primed neutrophils was more effective in completing resting membrane NADPH-oxidase activity when compared to cytosol from resting cells. The addition of protein kinase C inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine decreased the effectiveness of PMA-primed cytosol to complete the cell-free system, but had little effect on cytosol obtained from cells primed with retinal. The addition of protein phosphatase inhibitors, p-nitrophenyl phosphate or okadaic acid to neutrophil cavitates increased 3-fold the release of O2- in cell-free preparations of these cells. Okadaic acid and p-nitrophenyl phosphate also increased the effectiveness of both cytosol and light membranes to complete the cell-free system when combined with cytosol or light membranes from resting neutrophils, respectively, indicating that both fractions are affected by the inhibition of protein phosphatase activity. These data indicate that increases in protein phosphorylation alone do not lead to the activation of the NADPH-oxidase, but in addition to the requirement of an anionic amphiphile, the release of O2- from intact neutrophils or in the cell-free system is increased by stimulus activation of protein kinase C or more impressively by inhibition of protein phosphatase activity.     

Phorbol myristate acetate and the calcium ionophore A23187 synergistically induce release of LTB4 by human neutrophils: involvement of protein kinase C activation in regulation of the 5-lipoxygenase pathway

Phorbol myristate acetate (PMA), a tumor-promoting phorbol ester, and the calcium ionophore A23187 synergistically induced the noncytotoxic release of leukotriene B4 (LTB4) and other 5-lipoxygenase products of arachidonic acid metabolism from human neutrophils. Whereas neutrophils incubated with either A23187 (0.4 microM) or PMA (1.6 microM) alone failed to release any 5-lipoxygenase arachidonate products, neutrophils incubated with both stimuli together for 5 min at 37 degrees C released LTB4 as well as 20-COOH-LTB4, 20-OH-LTB4, 5-(S),12-(R)-6-trans-LTB4, 5-(S),12-(S)-6-trans-LTB4, and 5-hydroxyeicosatetraenoic acid, as determined by high pressure liquid chromatography. This synergistic response exhibited concentration dependence on both PMA and A23187. PMA induced 5-lipoxygenase product release at a concentration causing a half-maximal effect of approximately 5 nM in the presence of A23187 (0.4 microM). Competition binding experiments showed that PMA inhibited the specific binding of [3H]phorbol dibutyrate ([3H]PDBu) to intact neutrophils with a 50% inhibitory concentration (IC50) of approximately 8 nM. 1-oleoyl-2-acetyl-glycerol (OAG) also acted synergistically with A23187 to induce the release of 5-lipoxygenase products. 4 alpha-phorbol didecanoate (PDD), an inactive phorbol ester, did not affect the amount of lipoxygenase products released in response to A23187 or compete for specific [3H]PDBu binding. PMA and A23187 acted synergistically to increase arachidonate release from neutrophils prelabeled with [3H]arachidonic acid but did not affect the release of the cyclooxygenase product prostaglandin E2. Both PMA and OAG, but not PDD, induced the redistribution of protein kinase C activity from the cytosol to the membrane fraction of neutrophils, a characteristic of protein kinase C activation. Thus, activation of protein kinase C may play a physiologic role in releasing free arachidonate substrate from membrane phospholipids and/or in modulating 5-lipoxygenase activity in stimulated human neutrophils.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.     

sn-1,2-Dioctanoylglycerol. A cell-permeable diacylglycerol that mimics phorbol diester action on the epidermal growth factor receptor and mitogenesis

The cell-permeable diacylglycerol, sn-1,2-dioctanoylglycerol (DiC8), is shown to mimic the effect of tumor promoting phorbol diesters on epidermal growth factor (EGF) binding and action in intact cells. DiC8 inhibited the binding of [3H]phorbol dibutyrate to A431 cell monolayers indicating that the diacylglycerol interacts with the phorbol diester receptor. At 0.3 microM, DiC8 half-maximally inhibited the high affinity binding of 125I-EGF to A431 human epidermoid carcinoma cells. Scatchard analysis indicated that the inhibition of 125I-EGF binding was very similar to that observed in the presence of 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). DiC8 also mimicked the action of PMA to increase the phosphorylation state of the EGF receptor in 32P-labeled cells. Phosphoamino acid analysis demonstrated that DiC8 and PMA caused an increase in the level of EGF-receptor phosphoserine and phosphothreonine, whereas EGF caused an increase in the level of phosphoserine, phosphothreonine, and phosphotyrosine. Phosphopeptide mapping of the EGF receptor showed that DiC8 and PMA enhanced the phosphorylation of the same tryptic peptides. DiC8 inhibited the EGF-dependent tyrosine phosphorylation of the EGF receptor in A431 cells in a similar manner to that observed with PMA. In further experiments with quiescent Swiss 3T3 fibroblasts, DiC8 mimicked the ability of PMA to stimulate the incorporation of [methyl-3H]thymidine synergistically with low concentrations of EGF. This result indicates that DiC8 will mimic the long-term effects of PMA to regulate mitogenesis and raises the possibility that it may be active in two stage carcinogenesis. As both DiC8 and PMA stimulate the Ca2+- and phospholipid-dependent protein kinase (C-kinase) in vitro, the results support the hypothesis that the activation of C-kinase is a critical component of phorbol diester action on EGF receptor modulation and cell proliferation.     

Comparison of phorbol-12-myristate-13-acetate and dioctanoyl-sn-glycerol in the activation of EL4/6.1 thymoma cells

The present study compared the role of two protein kinase C (PK-C) activating agents, the phorbol ester phorbol-12-acetate-13-myristate (PMA) and the membrane-permeating diacylglycerol dioctanoyl-sn-glycerol (DiC8) in the activation of EL4/6.1 thymoma cells. These cells have been shown to express interleukin-2 receptors (IL-2R) upon stimulation with optimal amounts of PMA (10 ng/ml); also, suboptimal amounts of PMA (1 ng/ml) synergized with the Ca2+ ionophore ionomycin and recombinant interleukin-1 (rIL-1) (Lowenthal et al., 1986). Comparing PMA and DiC8 led to the following results: PMA at 10 ng/ml induced IL-2R; in contrast, DiC8 (30-3 micrograms/ml) alone was unable to induce IL-2R, although it did synergize with ionomycin (0.5 micrograms/ml) and rIL-1. Bihourly additions of DiC8 did not change this pattern. The addition of DiC8 together with rIL-2 also resulted in no IL-2R expression. Furthermore, DiC8 (10 micrograms/ml) effectively translocated PK-C. Therefore, the differences observed between PMA and DiC8 do not seem to be due to differences in metabolism or to an inability to translocate PK-C. Analysis of messenger (m) RNA produced in stimulated EL4/6.1 cells revealed that DiC8 was also unable to induce mRNA for IL-2R. Our data suggest that PMA, especially at "optimal" concentrations, might have effects that cannot be mimicked by diacylglycerol. Furthermore, it seems that the deficient activity of diacylglycerols can be compensated for by a Ca2+ ionophore and, depending on the cellular system, by further signals such as IL-1.     

Stimulation of respiratory burst by cyclocommunin in rat neutrophils is associated with the increase in cellular Ca2+ and protein kinase C activity

In this study, the underlying mechanisms of stimulation by cyclocommunin, a natural pyranoflavonoid, of respiratory burst in rat neutrophils was investigated. Cyclocommunin evoked a concentration-dependent stimulation of superoxide anion (O2*-) generation with a slow onset and long lasting profile. The maximum response (16.4+/-2.3 nmol O2*-/10 min per 10(6) cells) was observed at 3-10 microM cyclocommunin. Cyclocommunin did not activate NADPH oxidase in a cell-free system. Cells pretreated with pertussis toxin or n-butanol did not affect the cyclocommunin-induced O2*- generation. However, a protein kinase inhibitor staurosporine and EGTA greatly reduced the O2*-generation caused by cyclocommunin. Treatment of neutrophils with phorbol 12-myristate 13-acetate (PMA), but not with formylmethionyl-leucyl-phenylalanine (fMLP), for 20 min significantly reduced the O2*- generation following the subsequent stimulation of cells with cyclocommunin. Cyclocommunin did not affect the cellular mass of phosphatidic acid (PA). Neither the tyrosine kinase inhibitor, genistein, nor the p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, affected cyclocommunin-induced O2*- generation. The enzyme activities of neutrophil cytosolic and membrane-associated protein kinase C (PKC) were both increased significantly with 100 microM cyclocommunin. The membrane-associated PKC-theta and PKC-beta were increased following the stimulation of neutrophils with 30 and 100 microM cyclocommunin, respectively. Cyclocommunin reduced the [3H]phorbol 12,13-dibutyrate ([3H]PDB) binding to cytosolic PKC in a concentration-dependent manner. Cyclocommunin (> or =3 microM) significantly evoked a slow and long lasting [Ca2+]i elevation in neutrophils, and a phospholipase C (PLC) inhibitor U73122 greatly inhibited these Ca2+ responses. Moreover, the increase in cellular inositol bis- and trisphosphate (IP2 and IP3) levels were observed in neutrophils stimulated with 30 microM cyclocommunin for 3 min. Collectively, these results indicate that the stimulation of respiratory burst by cyclocommunin is probably mediated by the synergism of PKC activation and [Ca2+]i elevation in rat neutrophils.     

Diacylglycerols and PMA are particularly effective stimulators of fluid pinocytosis in human neutrophils

Diacylglycerols (OAG, diC8) and PMA were found to stimulate fluid pinocytosis (net uptake of FITC-dextran) to a far greater extent than other neutrophil activators, such as the chemotactic agents fNLPNTL and LTB4, the microtubule disassembling agents colchicine and nocodazole, the kinase inhibitor H-7, or D2O. OAG and diC8 produce a dose-dependent increase in the uptake of FITC-dextran, which is up to about 25- to 30-fold the control value of unstimulated neutrophils. The protein kinase inhibitor H-7 alone had a small stimulating effect on the net uptake, and it failed to inhibit stimulation of fluid pinocytosis by PMA, OAG, and diC8. Also, the protein kinase inhibitor staurosporine failed to inhibit fluid pinocytosis stimulated by OAG, diC8, and PMA. Stimulated fluid pinocytosis and vacuolization in response to PMA or diacylglycerols is associated with surface ruffling of neutrophils. Pinocytosis as well as surface ruffling stimulated by PMA, OAG, diC8, or diC10 are suppressed in the presence of cytochalasin D. The results suggest that diacylglycerols may be instrumental in transducing the signal for stimulated pinocytosis and that the surface movements induced by diacylglycerols, and PMA may be instrumental in fluid pinocytosis.     

Further evidence for the involvement of a phosphoprotein in the respiratory burst oxidase of human neutrophils.

Phosphorylation of a 47 kDa protein in human neutrophils is induced by phorbol 12-myristate 13-acetate (PMA), opsonized latex beads, fMet-Leu-Phe, calcium ionophore A23187 and fluoride. All of these stimuli activate the specialized microbicidal respiratory burst of neutrophils, and in each case the kinetics of activation correspond with the kinetics of phosphorylation of the 47 kDa protein. Trifluoperazine (50 microM) and chlorpromazine (100 microM), inhibitors of calmodulin and protein kinase C, abolish the increase in oxygen consumption and selectively prevent phosphorylation of the 47 kDa protein after PMA stimulation. Treatment of neutrophils with pertussis toxin totally inhibits both superoxide production and phosphorylation of this protein in response to fMet-Leu-Phe, but not in response to PMA, indicating that a GTP-binding protein modulates the fMet-Leu-Phe receptor signal. Phosphorylation of the 47 kDa protein, a phenomenon absent from the neutrophils of subjects with autosomal recessive chronic granulomatous disease, which lack the respiratory burst, appears to be the common trigger for activation of the burst in normal neutrophils.     

A diacylglycerol kinase inhibitor, R 59 022, potentiates superoxide anion production and 46-kDa protein phosphorylation in guinea pig polymorphonuclear leukocytes

A diacylglycerol (DG) kinase inhibitor, R 59 022, potentiated superoxide anion (O2-) production in guinea pig polymorphonuclear leukocytes (PMNL) induced by N-formyl-methionyl-leucyl-phenylalanine (FMLP). R 59 022 also potentiated O2- production induced by 1-oleoyl-2-acetylglycerol, a permeable DG. However, the production induced by phorbol 12-myristate 13-acetate (PMA), a direct activator for protein kinase C, was not potentiated by R 59 022. R 59 022 by itself had no significant effects on unstimulated O2- production. The potentiation of FMLP-induced O2- production by R 59 022 was correlated closely with increased formation of DG and decreased formation of phosphatidic acid, a product of DG kinase. R 59 022 had no effect on the breakdown of phosphoinositides. Phosphorylation of 46-kDa protein(s) by protein kinase C was also examined in relation to O2- production in PMNL. In coincidence with the increase in O2- production, the phosphorylation was potentiated by R 59 022 in the response to FMLP, but not in the response to PMA. In addition, staurosporine, a protein kinase C inhibitor, inhibited increases in both O2- production and phosphorylation of the 46-kDa protein(s) after PMA stimulation. Similar inhibitory effects of staurosporine were also observed upon stimulation with FMLP, irrespective of the presence of R 59 022. These results indicate that retention of DG as a result of the inhibition of further metabolism induces marked stimulation of O2- production via protein kinase C activation in PMNL. These results also provide further evidence for the close relationship between 46-kDa protein phosphorylation by protein kinase C and stimulation of O2- production in PMNL.     

Distinct effects of phorbol esters and exogenous diacylglycerols in the induction of murine thymocyte proliferation.

Murine thymocytes were stimulated with the protein kinase C activating agents Phorbol-12-myristate-13-acetate (PMA) or a more physiological membrane permeant diacylglycerol (dioctanoyl-sn-glycerol, DiC8) in the presence or absence of exogenous lymphokines (rIL-1 beta, rIL-2). Whereas PMA directly induced reactivity to rIL-2, DiC8 did not but had to synergize with the calcium ionophore Ionomycin. Expression of the p55 chain of the IL-2 receptor behaved similarly. In the absence of exogenous rIL-2, thymocytes proliferated in response to a combination of Ionomycin and PMA; however, replacing PMA by a single addition of DiC8 did not result in proliferation. Stimulation with Ionomycin plus repeated addition of DiC8 induced a low level of thymocyte proliferation and further addition of rIL-1 beta resulted in a significant increase. Purified immature (L3T4-Lyt2-) thymocytes behaved similarly, but showed an increased sensitivity to rIL-1 beta. Taken together, the data support the idea that PMA and the more physiological diacylglycerols do not possess totally equivalent activities in lymphocyte stimulation.     

A protein kinase C inhibitor, staurosporine, activates phospholipase D via a pertussis toxin-sensitive GTP-binding protein in rabbit peritoneal neutrophils.

In rabbit peritoneal neutrophils prelabeled with [3H] lyso platelet-activating factor, a protein kinase C inhibitor, staurosporine (> 1 microM), increased [3H]phosphatidylethanol ([3H]PEt) level in the presence of ethanol in a concentration- and time-dependent manner, providing evidence for staurosporine activation of phospholipase D (PLD). The staurosporine activation of the enzyme absolutely required both extracellular calcium and cytochalasin B, and was almost completely inhibited by pretreatment of the cells with pertussis toxin (IAP). In a reconstituted system where the purified Gi1 had been incorporated into phospholipid vesicles, staurosporine activated GTPase activity of Gi1 in a concentration-dependent fashion, with a maximal 4-5-fold effect. ADP-ribosylation by IAP of Gi1 in vesicles significantly suppressed the staurosporine activation. As with the GTPase activity of Gi1, GTPase activities of other purified IAP-sensitive G proteins, such as Gi2 and G(o), were significantly stimulated by staurosporine, but the cholera toxin substrate Gs was appreciably less sensitive to the staurosporine stimulation. The staurosporine activation of GTPase was also observed in rabbit neutrophil membranes from control cells, but not in membranes from IAP-treated neutrophils. From these results, we conclude that the staurosporine activation of PLD in rabbit neutrophils is attributed to the direct activation of an IAP-sensitive G protein in a similar manner to receptors occupied by agonists. By contrast, staurosporine failed to activate phosphoinositide-specific phospholipase C (PI-PLC) under the conditions in which it activated PLD, indicating that there exists a PLD activation pathway independent of PI-PLC. Furthermore, it was found that N-acetyl-beta-glucosaminidase release from the granules of intact neutrophils was evoked by staurosporine to almost the same extent as by fMLP (100 nM), but O2- generation was not affected. These results suggest a possibility that PLD pathway plays an important role in enzyme release, but is not sufficient for O2- generation, in rabbit peritoneal neutrophils.     

Inhibition of protein kinase C by H-7 potentiates the release of oleic, linoleic and arachidonic acids in A23187-stimulated human neutrophils

This present report describes the effect of H-7, a protein kinase C inhibitor, on the release of oleic, linoleic and arachidonic acids in A23187-stimulated neutrophils. Surprisingly, the inhibitor potentiated the release of all three unsaturated fatty acids in neutrophils stimulated with A23187 alone. In contrast, released oleic acid, linoleic acid and arachidonic acid in phorbol 12-myristate 13-acetate-primed neutrophils were attenuated by 35, 47 and 33%, respectively, in the presence of H-7 (300 microM). Phorbol 12-myristate 13-acetate (PMA) had no effect on A23187-stimulated release of saturated fatty acids. Both PMA and H-7 when used alone had no effect on the release of saturated or unsaturated fatty acids. We, therefore, conclude that H-7 may have effects other than inhibiting PMA-primed responses including superoxide generation, degranulation and arachidonic acid release in human neutrophils.     

Synergistic release of arachidonic acid from platelets by activators of protein kinase C and Ca2+ ionophores. Evidence for the role of protein phosphorylation in the activation of phospholipase A2 and independence from the Na+/H+ exchanger

The protein kinase C activators phorbol myristate acetate (PMA), mezerein, oleoylacetylglycerol, and (-)-indolactam V, although without direct effect on arachidonic acid release, greatly enhance the release of platelet arachidonic acid caused by the Ca2+ ionophores A23187 and ionomycin. In contrast, 4 alpha-phorbol 12,13-didecanoate and (+)-indolactam V, which lack the ability to activate kinase C, do not potentiate arachidonate release. Release of arachidonic acid occurs without activation of phospholipase C and is therefore mediated by phospholipase A2. Synergism between PMA and A23187 is not affected by inactivation of the Na+/H+ exchanger with dimethylamiloride. The time course and dose-response for the effect of PMA at 23 degrees C closely correlate with the phosphorylation of a set of relatively "slowly" phosphorylated proteins (P20, P35, P41, P60), but not the rapidly phosphorylated P47 protein. P20 is myosin light chain, and P41 is probably Gi alpha, but the other proteins have not been positively identified. Depletion of metabolic ATP stores by antimycin A plus 2-deoxyglucose abolishes both protein phorphorylation and the potentiation of arachidonate release by PMA, but does not prevent fatty acid release by the ionophores. Similarly, the kinase C inhibitors H-7 and staurosporine produce, respectively, partial and complete inhibition of PMA-potentiated arachidonic acid release and protein phosphorylation, without affecting the direct response to ionophores. These results indicate that protein phosphorylation, mediated by kinase C, promotes the phospholipase A2 dependent release of arachidonic acid in platelets when intracellular Ca2+ is elevated by Ca2+ ionophores.     

Protein kinase C induces phosphorylation and desensitization of the human 5-HT1A receptor

The effects of short-term phorbol ester treatment of CHO cells that stably express 900 fmol of recombinant human serotonin 5-HT1A receptor/mg of protein on coupling to the inhibition of adenylyl cyclase and on phosphorylation of the receptor were studied. Pretreatment of cell monolayers with phorbol 12-myristate 13-acetate (PMA) caused a dose- and time-dependent shift of the half-maximal dose of serotonin (5-HT) required to inhibit membrane adenylyl cyclase (from IC50 approximately 100 nM to approximately 400 nM). This desensitization (shift in IC50) was rapid, occurring with 5 min of pretreatment and being maximal by 10-15 min; it was also dose-dependent, being half-maximal at approximately 300 nM PMA. Desensitization was also induced by sn-dioctanoylglycerol (DiC8) and blocked by the protein kinase C (PKC) inhibitors sphingosine and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). In detached permeabilized cells, PMA pretreatment caused a rapid phosphorylation of immunoprecipitated 5-HT1A receptors, with an approximately 3-4-fold increase that was maximal after 15 min and persisted for 90 min. The phosphorylation occurred at a similar dose of PMA as that which induced desensitization (half-maximal at approximately 300 nM, maximal at 500 nM to 1 microM), could be reproduced by pretreatment with the PKC activators DiC8 or phorbol 12,13-dibutyrate (PDBu), and could be blocked by the PKC inhibitors sphingosine or H-7. The stoichiometry of the phosphorylation was approximately 2 mol of [32P]ATP/mol of receptor, suggesting the involvement at least two of three putative PKC sites within the 5-HT1A receptor. The close concordance between the PKC-induced desensitization and phosphorylation suggests a potential causative link between these two effects of PKC on the human 5-HT1A receptor.