The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.     

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.     

Extracellular signal-regulated kinases regulate leukotriene C4 generation, but not histamine release or IL-4 production from human basophils

Human basophils secrete histamine and leukotriene C4 (LTC4) in response to various stimuli, such as Ag and the bacterial product, FMLP. IgE-mediated stimulation also results in IL-4 secretion. However, the mechanisms of these three classes of secretion are unknown in human basophils. The activation of extracellular signal-regulated kinases (ERKs; ERK-1 and ERK-2) during IgE- and FMLP-mediated stimulation of human basophils was examined. Following FMLP stimulation, histamine release preceded phosphorylation of ERKs, whereas phosphorylation of cytosolic phospholipase A2 (cPLA2), and arachidonic acid (AA) and LTC4 release followed phosphorylation of ERKs. The phosphorylation of ERKs was transient, decreasing to baseline levels after 15 min. PD98059 (MEK inhibitor) inhibited the phosphorylation of ERKs and cPLA2 without inhibition of several other tyrosine phosphorylation events, including phosphorylation of p38 MAPK. PD98059 also inhibited LTC4 generation (IC50 = approximately 2 microM), but not histamine release. Stimulation with anti-IgE Ab resulted in the phosphorylation of ERKs, which was kinetically similar to both histamine and LTC4 release and decreased toward resting levels by 30 min. Similar to FMLP, PD98059 inhibited anti-IgE-mediated LTC4 release (IC50, approximately 2 microM), with only a modest effect on histamine release and IL-4 production at higher concentrations. Taken together, these results suggest that ERKs might selectively regulate the pathway leading to LTC4 generation by phosphorylating cPLA2, but not histamine release or IL-4 production, in human basophils.     

1,2-dioctanoyl-sn-glycerol can stimulate neutrophils by different mechanisms. Evidence for a pathway that does not involve phosphorylation of the 47-kDa protein

Neutrophils treated with 1,2-dioctanoyl-sn-glycerol (DiC8) are known to release large quantities of superoxide (O2-) and to exhibit an intense phosphorylation of two proteins with molecular masses of approximately 47 and 49 kDa. In this paper, we report that O2- release from guinea pig cells stimulated with a near optimal amount of DiC8 (2.0 microM) is markedly inhibited (greater than or equal to 70%) by antagonists of protein kinase C (i.e. 150 nM staurosporine; 200 microM 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7], whereas that from cells stimulated with an optimal amount of DiC8 (7.8 microM) is not (approximately 25% inhibition). However, staurosporine (150 nM) effectively reduced the level of phosphorylation of the 47- and the 49-kDa proteins to that observed in unstimulated cells when either amount of DiC8 (i.e. 2.0 or 7.8 microM) was utilized. Thus, neutrophils stimulated with 7.8 microM DiC8 in the presence of staurosporine release large quantities of O2- without an enhanced phosphorylation of the 47- and the 49-kDa proteins. In contrast, these antagonists of protein kinase C effectively blocked O2- release from neutrophils stimulated with an optimal amount of phorbol 12-myristate 13-acetate (PMA), and the percentage of inhibition was not affected by increasing the concentration of PMA 160-fold. These data show that DiC8 and PMA, both activators of protein kinase C, can have distinct effects on O2- release by neutrophils. Moreover, they suggest that DiC8 (or a metabolite) under certain circumstances may function in a stimulatory pathway for O2- release that is independent of protein kinase C. Differences in the morphology of neutrophils stimulated with PMA and DiC8 are presented. Ancillary data on human neutrophils are also provided.     

Effects of the protein kinase C inhibitor H-7 and calmodulin antagonist W-7 on superoxide production in growing and resting human histiocytic leukemia cells (U937)

Serum, phorbol 12,13-didecanoate (PDD) and 1-oleoyl-2-acetoy-sn-glycerol (OAG) stimulated O2- release in human histiocytic leukemia U937 cells. The kinetics of O2- release caused by PDD but not by serum or OAG in growing cells differed from those in resting cells. Both the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl) 2-methylpiperidine (H-7) and calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) reduced the superoxide generation induced by these stimuli. H-7 inhibited the O2- release either from growing or resting cells but the effect of W-7 varied according to the growth phase. From these results, it is suggested that activation of protein kinase C and calmodulin-dependent process has an important role in O2(-)-release induced by serum, OAG and PDD, and that the mechanism for PDD-induced O2(-)-release is different in growing and resting cells.     

Dissimilar effects of the protein kinase C inhibitors, staurosporine and H-7, on cholecystokinin-induced enzyme secretion from rabbit pancreatic acini.

The effects of two putative inhibitors of protein kinase C activity, staurosporine and H-7, on partially purified protein kinase C and amylase secretion from isolated rabbit pancreatic acini were investigated. Staurosporine dose-dependently inhibited amylase release stimulated by an optimal concentration of cholecystokinin C-terminal octapeptide. At a concentration of 100 nM, the drug inhibited the secretory response to the secretagogue by approximately 50%. At the same concentration, staurosporine inhibited 12-O-tetradecanoylphorbol 13-acetate-stimulated enzyme secretion by 90%. Moreover, the potentiating effect of this phorbol ester on cholecystokinin-induced amylase release was completely abolished in the presence of staurosporine. Interestingly, amylase release was decreased to the level observed with the combination of cholecystokinin and staurosporine. In contrast, H-7, potentiated rather than inhibited cholecystokinin-stimulated enzyme secretion, whereas the secretory response to 12-O-tetradecanoylphorbol 13-acetate was not affected by the drug. Both staurosporine and H-7, however, inhibited protein kinase C purified from exocrine pancreatic tissue. Kinetic analysis revealed that both compounds inhibited protein kinase C competitively with respect to ATP. The Ki value for staurosporine was 0.55 nM and for H-7 13.5 microM. Our results obtained with staurosporine are in line with a stimulatory role of protein kinase C in cholecystokinin-induced enzyme secretion from the exocrine pancreas. The results obtained with H-7 emphasize that care has to be taken in interpreting the biological effects of this drug.     

Effect of lysophosphatidylserine on immunological histamine release

The effect of lysophosphatidylserine on immunological histamine release has been studied in rat peritoneal mast cells actively sensitized with horse serum and in human basophils challenged with anti-IgE. In contrast to other lysophospholipids, lysophosphatidylserine enhances the immunological histamine release in rat mast cells. The effect shows the kinetics of a saturable process with an apparent Km for lysophosphatidylserine of 0.26 microM. A similar Km value (0.21 microM) is found when measuring the non-immunological histamine release activated by lysophosphatidylserine plus nerve growth factor. A comparison with phosphatidylserine shows that a half-maximal response to lysophosphatidylserine occurs at a concentration 4-times lower. In addition, the magnitude of the response is higher. At variance with rat mast cells, lysophosphatidylserine does not influence the histamine release elicited by immunological and non-immunological stimuli in human basophils. The histamine secretion in these cells is instead affected by a calcium ionophore or tetradecanoylphorbolacetate, a compound producing activation of protein kinase C.     

Monoclonal antibodies to the leukocyte common antigen (CD45) inhibit IgE-mediated histamine release from human basophils.

mAb were selected that inhibited IgE-mediated histamine release from human basophils. The two mAb, HB 9AB6 and HB 10AB2, are of the IgG1 subclass and have a 50% inhibitory concentration of 0.16 to 1.1 micrograms/ml. The mAb required several hours of incubation with the basophils at 37 degrees C to induce maximum inhibition. Neither mAb directly released histamine from human basophils nor did they inhibit release induced by formylmethionine tripeptide, calcium ionophore A23187, or PMA. There was little inhibition of IgE-mediated release when the cells were preincubated with the mAb at 4 degrees C. By FACS analysis the 2 mAb bound to all peripheral blood leukocytes and immunoprecipitated a approximately 200-kDa protein from peripheral blood leukocytes and several cell lines of human origin. In binding studies and by sequential immunoprecipitation the 2 mAb and a known anti-CD45 mAb bound to the same protein. However, the mAb recognized different epitopes. Therefore, mAb to the CD45 surface Ag, a membrane protein tyrosine phosphatase, inhibits IgE-receptor mediated histamine release from human basophils. The data suggest a link between protein tyrosine phosphorylation and high affinity IgE receptor-mediated signal transduction in human basophils.     

Cyclosporin A rapidly inhibits mediator release from human basophils presumably by interacting with cyclophilin.

We have examined the effects of cyclosporin A (CsA) and a series of CsA analogs that bind with decreasing affinity to cyclophilin, to evaluate the involvement of this protein in the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4; LTC4) mediators of inflammatory reactions from human basophils. CsA (8 to 800 nM) concentration-dependently inhibited (5 to 60%) histamine release from peripheral blood basophils challenged with anti-IgE. CsA was more potent (92.6 +/- 1.8 vs 59.1 +/- 4.5%; p less than 0.001) and, at low concentrations, more effective when the channel-operated influx of Ca2+ was bypassed by the ionophore A23187 (IC40 = 24.1 +/- 3.9 vs 105.5 +/- 22.2 nM; p less than 0.05). CsA had no effect on the release of histamine caused by phorbol myristate and bryostatin 1 that activate different isoforms of protein kinase C. Inhibition of histamine release from basophils challenged with anti-IgE was not abolished by washing (three times) the cells before anti-IgE challenge. CsA also inhibited the de novo synthesis of LTC4 from basophils challenged with anti-IgE. The inhibitory effect of CsA was very rapid, and the drug, added from 1 to 10 min during the reaction, inhibited the ongoing release of histamine caused by anti-IgE and by A23187. The experiments with CsA analogs (CsG, CsC, CsD, and CsH) showed that CsH, which has an extremely low affinity for cyclophilin, has no effect on basophil mediator release. In addition, there is a significant correlation between the concentrations of CsA, G, C, and D that inhibited by 30% the histamine release induced by anti-IgE (r = 0.99; p less than 0.001) and by A23187 (r = 0.87; p less than 0.001) and their affinity for cyclophilin.     

Inhibitory effect of H-7, H-8 and polymyxin B on liver protein kinase C-induced phosphorylation of endogenous substrates

Inhibitory actions of 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H-7), N-[2-(methylamine)ethyl]-5-isoquinolinesulfonamide [H-8] and polymyxin B on the calcium-activated, phospholipid-dependent protein kinase (protein kinase C) of rat liver were compared. Using a partially purified liver protein kinase C and an exogenous substrate histone-III S, polymyxin B showed maximum inhibition (IC50, 9.5 microM) followed by H-7 (IC50, 25 microM) and H-8 (IC50, 36 microM). These inhibitors also inhibited protein kinase C-induced phosphorylation of endogenous cytosolic and particulate proteins in a dose-dependent manner though polymyxin B was relatively less effective with the particulate fraction. With the aid of protein kinase-C activators and these inhibitors, seven proteins in cytosolic (Mr 170K, 150K, 43K, 34K, 30K, 25K and 19K daltons) and six proteins in particulate (Mr 150K, 43K, 34K, 25K, 19K and 16K daltons) fractions were identified as probable substrates for protein kinase C in liver. The identity of these proteins remains to be determined.     

FK-506, a potent novel inhibitor of the release of proinflammatory mediators from human Fc epsilon RI+ cells.

FK-506, a macrolide that binds with high affinity to a specific binding protein, and the structurally related macrolide rapamycin (RAP) were compared to cyclosporin A (CsA) for their effects on the release of preformed (histamine) and de novo synthesized (peptide leukotriene C4) inflammatory mediators from human basophils. FK-506 (1 to 300 nM) concentration dependently inhibited histamine release from basophils activated by Der p I Ag, anti-IgE, or compound A23187. FK-506 was more potent than CsA when basophils were challenged with Ag (IC50 = 25.5 +/- 9.5 vs 834.3 +/- 79.8 nM; p less than 0.001), anti-IgE (IC50 = 9.4 +/- 1.7 vs 441.3 +/- 106.7 nM; p less than 0.001), and A23187 (IC50 = 4.1 +/- 0.9 vs 36.7 +/- 3.8 nM; p less than 0.001). The maximal inhibitory effect of FK-506 was higher than that caused by CsA when basophils were activated by Der p I (80.0 +/- 3.6 vs 49.5 +/- 4.7%; p less than 0.001) and anti-IgE (90.4 +/- 1.8 vs 62.3 +/- 2.9%; p less than 0.001). FK-506 had little or no effect on the release of histamine caused by f-met peptide, phorbol myristate (12-tetradecanoyloxy-13-acetoxy-phorbol), and bryostatin 1. RAP (30 to 1000 nM) selectively inhibited only IgE-mediated histamine release from basophils, although it had no effect on mediator release caused by f-met peptide, A23187, 12-tetradecanoyloxy-13-acetoxy-phorbol, and bryostatin 1. FK-506 also inhibited the de novo synthesis of sulfidopeptide leukotriene C4 from basophils challenged with anti-IgE. Low concentrations of FK-506 and CsA synergistically inhibited the release of mediators from basophils induced by anti-IgE or compound A23187. IL-3 (3 and 10 ng/ml), but not IL-1 beta (10 and 100 ng/ml), reversed the inhibitory effect of both FK-506 and CsA on basophils challenged with anti-IgE or A23187. RAP was a competitive antagonist of the inhibitory effect of FK-506 on A23187-induced histamine release from basophils with a dissociation constant of about 30 nM. In contrast, RAP did not modify the inhibitory effect of CsA on A23187-induced histamine release. These data indicate that FK-506 is a potent antiinflammatory agent that acts on human basophils presumably by binding to a receptor site (i.e., FK-506 binding protein).     

Decline in c-myc mRNA expression but not the induction of c-fos mRNA expression is associated with differentiation of SH-SY5Y human neuroblastoma cells

The induction of differentiation in SH-SY5Y human neuroblastoma cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is accompanied by a rapid and a transient expression of c-fos mRNA and a down-regulation of c-myc mRNA. The TPA-induced expression of c-fos mRNA was inhibited by H-7, a specific inhibitor of protein kinase C (PK-C). Dioctanoylglycerol (DiC8) failed to induce differentiation of SH-SY5Y cells or to down-regulate c-myc mRNA but it did induce the expression of c-fos mRNA. Treatment of IMR-32 human neuroblastoma cells with TPA did not cause differentiation although c-fos mRNA was induced. Since PK-C in SH-SY5Y cells was activated by both TPA and DiC8 it is suggested that the activation of PK-C alone is not sufficient to induce differentiation in SH-SY5Y cells. The down-regulation of c-myc mRNA rather than the induction of c-fos mRNA seems to be associated with differentiation process in SH-SY5Y cells.     

Thrombin- and histamine-induced signal transduction in human endothelial cells. Stimulation and agonist-dependent desensitization of protein phosphorylation

Treatment of human endothelial cells with thrombin, histamine, or dioctanoylglycerol (DiC8), a synthetic diacylglycerol, resulted in the rapid and transient phosphorylation of a Mr = 29,000 protein (P29) in a dose-dependent manner. Various tumor promoters also promoted P29 phosphorylation while the adenylate cyclase activator, forskolin, did not. The level of phosphorylation with all three agonists was similar (2.5-4 fold), and analysis of P29 by two-dimensional gel electrophoresis revealed identical patterns in each case. Receptor specificity was demonstrated for the histamine-stimulated changes; pyrilamine (10(-6) M; H1) but not cimetidine (10(-4); H2) blocked the response. The thrombin effect was active site-dependent. Phosphorylation induced by thrombin and histamine occurred within 1 min, peaked between 5 and 10 min, and returned to control levels by 1 h. DiC8-induced phosphorylation occurred more slowly but was also reduced by 1 h while phorbol ester treatment prolonged phosphorylation for at least 4 h. Treatment of these cells with thrombin or histamine for 1 h desensitized P29 to further phosphorylation by the homologous agonist although secondary phosphorylation could occur with heterologous compounds. However, if the primary agonist was removed following the onset of a desensitized state, secondary phosphorylation of P29 could be stimulated by the same compound. These same results were observed with two other phosphoproteins Mr = 18,000 (P18) and 80,000 (P80) which became more highly phosphorylated in response to thrombin treatment and with histamine/thrombin-stimulated prostaglandin I2 production. In contrast, homologous down-regulation of P29 phosphorylation was not observed with DiC8-treated cells, and the decline in phosphorylated P29 was associated with the loss of functional DiC8. The protein kinase inhibitors staurosporine and H-7 blocked P18 and P80 phosphorylation by thrombin but had no effect on P29 phosphorylation by histamine, thrombin, or DiC8 suggesting distinct pathways leading to the phosphorylation of these different proteins. These data suggest that multiple and independent thrombin/histamine-induced events are susceptible to receptor occupancy-dependent homologous down-regulation.     

IL-8 inhibits histamine release from human basophils induced by histamine-releasing factors, connective tissue activating peptide III, and IL-3.

We have previously purified and partially characterized histamine releasing factors (HRF), which were derived from a mixture of human mononuclear cells and platelets. We now report the effect of IL-8 upon HRF-, connective tissue activating peptide III (CTAP III)-, and IL-3-induced histamine release from human basophils. We determined that IL-8 itself, at concentrations between 10(-7) to 10(-11) M, does not release histamine from basophils, although positive results are observed in two of 26 subjects at 10(-7) M. Unfractionated (crude) HRF released histamine in 25 of 26 donors, in the range of 6.7% to 100% of total basophil histamine stores. When basophils were preincubated with IL-8 (10(-7) to 10(-11) M) for 5 min, followed by a 40-min incubation with HRF, histamine release was significantly inhibited in 20 of 25 donors. Inhibition was observed at as little as 10(-11) M IL-8, with maximal inhibition being attained at 10(-9) M. HRF-containing supernatants contain a mixture of different histamine-releasing moieties. To better define which factor(s) may be inhibited by IL-8, fractionated supernatants, purified CTAP III, and IL-3 were studied. Histamine release produced by two different HRF-containing chromatographic fractions (HRFvoid and HRFpeak 2) and purified CTAP-III (5 micrograms/ml) was inhibited by IL-8 in 10 of 12 donors, three of three donors, and seven of 10 donors, respectively. IL-3 (5000 U/ml)-dependent histamine release was inhibited by IL-8 in all subjects tested. In contrast, histamine release by anti-IgE and FMLP was not affected by IL-8. Thus, IL-8 appears to be an inhibitor of cytokine-like molecules that induce histamine release and may represent the previously described 8-kDa histamine release inhibitory factor present in mononuclear cell supernatants.     

The protein kinase C inhibitor H-7, inhibits antigen and IL-2-induced proliferation of murine T cell lines

Activation of protein kinase C has been shown to be involved in the activation pathway of many cell types. Recently, a number of investigations have suggested that protein kinase C plays an essential role in T lymphocyte activation. The recent synthesis of the protein kinase inhibitors, H-7 and HA1004, have now made possible a new approach for testing the relevance of protein kinase C in T cell activation and proliferation. We now report that the antigen-induced and interleukin-2-induced proliferation of murine T cell lines can be consistently inhibited by the protein kinase C inhibitor, H-7. HA1004, a somewhat more potent inhibitor of cyclic nucleotide-dependent protein kinases, but a significantly weaker inhibitor of protein kinase C than H-7, demonstrated no consistent inhibition of these T cell responses. These results represent a further demonstration that protein kinase C plays an essential role in the activation of T cells.     

Possible role of calmodulin in the control of histamine release from human basophil leukocytes

We investigated the possible role of calmodulin (CaM) in the control of histamine release from human basophil leukocytes using several CaM antagonists. Trifluoperazine (TFP) (10(-6)-2 X 10(-5) M), pimozide (10(-6)-1.5 X 10(-5) M), chlorpromazine (CPZ) (10(-5)-10(-4) M) and promethazine (PMZ) (2 X 10(-5)-10(-4) M) inhibited in vitro histamine secretion from human basophils induced by several immunological (antigen, anti-IgE, and formyl-L-methionyl-L-leucyl-L-phenylalanine: f-met peptide) and nonimmunological (Ca2+ ionophore A23187 and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate: TPA) stimuli. Trifluoperazine sulfoxide (TFP-S) and chlorpromazine sulfoxide (CPZ-S), which have very low affinity to CaM, had practically no inhibitory effect on histamine release from human basophils. The inhibitory effect of TFP could be made irreversible by irradiating the cells with UV light. A sulfonamide derivative, the compound N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (2.5 X 10(-5)-2 X 10(-4) M), which selectively binds to CaM, inhibited the release of histamine from basophils. In contrast, the chloride deficient analogue, W-5, which interacts only weakly with CaM, had practically no inhibiting effect. The IC50 for enzyme release by a series of eight CaM antagonists was closely correlated (r = 0.91; p less than 0.001) with the CaM specific binding, supporting the concept that these agents act by binding to CaM and thereby inhibiting histamine release. TFP and W-7 inhibited histamine release in the absence and in the presence of increasing concentrations of extracellular Ca2+. These results emphasize the possible role of CaM in the control of histamine secretion from human basophils.     

Modes of inhibitory action of 4 beta-phorbol 12-myristate 13-acetate in thrombin-stimulated arachidonic acid release in intact and permeabilized platelets

The tumor-promoting phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited thrombin-stimulated arachidonic acid (AA) release in rabbit and human platelets. PMA was effective over the same concentration range that activates protein kinase C in intact rabbit platelets: IC50 vs thrombin = 0.5 nM, greater than 90% inhibition at 10 nM. Suppression of thrombin-stimulated AA release was evident within 5 min of pretreatment with 1 nM PMA. A non-tumor-promoting phorbol ester, 4-O-methyl PMA, showed a very weak ability to inhibit AA release. Thrombin-stimulated serotonin secretion was progressively inhibited by PMA pretreatment in platelets, while PMA was a stimulus for secretion at higher concentrations. 1-(5-Isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), a selective inhibitor of protein kinase C, blocked PMA-induced inhibition of AA release. Furthermore, H-7 enhanced the effect of thrombin on AA release. PMA pretreatment reduced the inhibitory effect of thrombin on forskolin-stimulated cAMP accumulation, but had no effect on nonstimulated cAMP metabolism in the presence of thrombin. PMA did not inhibit AA release caused by A23187 or melittin. In digitonin-permeabilized platelets, thrombin plus guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)-stimulated AA release, but not GTP gamma S- and AIF4(-)-stimulated AA release, was abolished by PMA pretreatment. These results suggest that activation of protein kinase C may exert negative feedback on the receptor-mediated activation of phospholipase A2. A possible uncoupling of thrombin receptor to GTP-binding protein leading to activation of phospholipase A2 by PMA pretreatment is discussed.     

Xanthine oxidase-catalysed oxidation of paracetamol.

[3H]Arachidonic acid is released after stimulation of rabbit neutrophils with fMet-Leu-Phe or platelet-activating factor (PAF). The release is rapid and dose-dependent, and is inhibited in phorbol 12-myristate 13-acetate (PMA)-treated rabbit neutrophils. The protein kinase C (PKC) inhibitor 1-(5-isoquinoline-sulphonyl)-2-methylpiperazine (H-7) prevents this inhibition. In addition, PMA increases arachidonic acid release in H-7-treated cells stimulated with fMet-Leu-Phe. [3H]Arachidonic acid release, but not the rise in the concentration of intracellular Ca2+, is inhibited in pertussis-toxin-treated neutrophils stimulated with PAF. The diacylglycerol kinase inhibitor R59022 increases the concentration of diacylglycerol and potentiates [3H]arachidonic acid release in neutrophils stimulated with fMet-Leu-Phe. This potentiation is not inhibited by H-7. These results suggest several points. (1) A rise in the intracellular concentration of free Ca2+ is not sufficient for arachidonic acid release in rabbit neutrophils stimulated by physiological stimuli. (2) A functional pertussis-toxin-sensitive guanine nucleotide regulatory protein and/or one or more of the changes produced by phospholipase C activation are necessary for arachidonic acid release produced by physiological stimuli. (3) Agents that stimulate PKC potentiate arachidonic acid release, and this potentiation is not inhibited by H-7. These agents produce their actions in part by direct membrane perturbation.     

Exogeneous diacylglycerols downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes.

In this study, cell permeable diacylglycerols, sn-1,2-dioctanoglycerol (DiC8), and sn-1-oleoyl-2-acetylglycerol (OAG) were found to downregulate the activity of Na(+)-K+ pump in Xenopus laevis oocytes. Both DiC8 and OAG decreased the binding of [3H]ouabain to intact oocytes while phorbol esters did not appreciably influence the same. These diacylglycerols inhibited the amiloride-sensitive 22Na+ influx and ouabain-sensitive 86Rb+ uptake in the oocytes. Furthermore, DiC8 prevented the 22Na+ efflux from the oocytes preloaded with 22Na+. Addition of H-7 to DiC8- and OAG-treated oocytes stimulated the pump activity curtailed by the two latters. The impairment of Na(+)-K+ pump activity by diacylglycerols suggests that protein kinase C activators may stimulate endocytosis of membrane-coupled Na(+)-K+ ATPase.     

Effect of H-7 on the modulation of glucagon actions by activators of protein kinase-C

The stimulations of ureagenesis and cyclic AMP accumulation induced by glucagon were inhibited by 10 nM vasopressin or 100 nM phorbol 12-myristate 13-acetate (PMA). The maximal accumulation of cyclic AMP induced by glucagon was clearly diminished by these agents without change in the EC50 for the peptide hormone suggesting a non-competitive type of inhibition. H-7 blocked the inhibition of glucagon-stimulated ureagenesis induced by PMA and vasopressin and diminished their effect on the accumulation of cyclic AMP induced by glucagon. It is concluded that activation of protein kinase C inhibits the stimulation of ureagenesis and the accumulation of cyclic AMP induced by glucagon in liver cells from hypothyroid rats; H-7 inhibits the effects of protein kinase C activation.