Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.     

Stimulation of trehalose efflux from cockroach (Periplaneta americana) fat body by hypertrehalosemic hormone is dependent on protein kinase C and calmodulin

Protein kinase C and calmodulin play key roles in cockroach fat body during activation of phosphorylase and trehalose efflux by HTH-II. The data support the view that an increase in cytosolic Ca2+ is prerequisite for enhanced activity of protein kinase C and calmodulin. Chelation of Ca2+ (i) with BAPTA blocks HTH-II-induced trehalose efflux from the fat body whereas thapsigargin, which raises [Ca2+]i to the same level as HTH-II, produces only a small, yet significant increase in trehalose efflux. Sphingosine, an inhibitor of protein kinase C, inhibits HTH-II-induced trehalose efflux in a concentration-dependent manner. Trehalose efflux is not activated by the protein kinase C activators OAG or PMA alone but in the presence of thapsigargin both agents increase trehalose efflux to a level comparable to that obtained with HTH-II. Thapsigargin has only a moderate activating effect on phosphorylase but in combination with OAG produces an activation indistinguishable from that provoked by HTH-II. Each of the structurally different calmodulin inhibitors, trifluoperazine, W-7, and calmidazolium, blocks completely the action of HTH-II on trehalose efflux, thus confirming the importance of calmodulin in HTH-II initiated trehalose efflux.     

Ca2+.Calmodulin-dependent release of arachidonic acid for renal medullary prostaglandin synthesis. Evidence for involvement of phospholipases A2 and C

The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C.     

Calmodulin and protein kinase C antagonists also inhibit the Ca2+-dependent protein protease, calpain I

The calmodulin and C-kinase antagonists melittin, calmidazolium, N-(6-aminohexyl)-5-chloro-1-napthalenesulfonamide (W7), and trifluoperazine (TFP) also inhibit the activity of the human erythrocyte Ca2+-dependent protease, calpain I. W-5, the nonchlorinated derivative of W-7, was ineffective as an inhibitor of calpain I just as it is for calmodulin and protein kinase C. Dose response studies provided the following IC50 values: melittin, 2.6 microM; calmidazolium, 6.2 microM; trifluoperazine, 130 microM; W-7, 251 microM. These IC50 values indicate that the compounds have affinities 10 to 600 fold less for calpain I than for calmodulin; however, the affinities of the inhibitory compounds are comparable for calpain I and protein kinase C. Kinetic analysis indicates that the compounds are competitive inhibitors of calpain I with respect to substrate.     

Calcium ion regulates the release of lipase of Fusarium oxysporum.

The lipase production of a plant pathogenic fungus, Fusarium oxysporum f. sp. lini SUF 402, was induced by fat as the carbon source, and its release was stimulated by the infusion of intracellular free calcium ion with a calcium ionophore, A23187. N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7, a calmodulin inhibitor) and 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl- L-tyrosyl]-4-phenylpiperazine (KN-62, a Ca2+/calmodulin dependent protein kinase II inhibitor) reduced the extracellular release of lipase in vivo. 1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7, a protein kinase C inhibitor) did not have this ability. After K2H32PO4 had been incorporated into the cells, they were treated with W-7 or KN-62 and stimulated by Ca2+ ionophore. On SDS-PAGE of intracellular proteins followed by autoradiography, W-7- and KN-62-treated cells showed inhibition of the incorporation of 32Pi into the 20 kDa protein resulting from Ca2+ stimulation. F. oxysporum had calmodulin (CaM)-dependent protein kinase activity in the cytoplasmic fraction and had the ability to phosphorylate of syntide 2, a specific substrate of CaM kinase II. The partially purified CaM-dependent protein kinase was inhibited by 10 microM KN-62 in vitro. Increase of the intracellular Ca2+ concentration of F. oxysporum activated CaM and CaM-dependent protein kinase, resulting in the extracellular lipase release. These results suggest the existence of a Ca2+ signalling system in F. oxysporum like those observed in higher eucaryotes.     

Inhibitors of IL-2 production and IL-2 receptor expression in human leukemic T-cell line, Jurkat

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H-7), a protein kinase inhibitor, suppressed interleukin 2 (IL-2) production and IL-2 receptor (IL-2R) expression of the human leukemic T-cell line, Jurkat, induced by 12-O-tetradecanoyl-phorbol-13-acetate and phytohemagglutinin-P. This effect was significant at 5 microM H-7 without loss of cell viability. Such activity was not observed with N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), a potent inhibitor of cGMP- and cAMP-dependent kinases, and a weak inhibitor of Ca2+-phospholipid-dependent protein kinase (protein kinase C). These findings suggest that protein kinase C is more closely associated with IL-2 receptor expression and IL-2 production of T cells than cGMP- or cAMP-dependent kinases. In addition, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin inhibitor, suppressed both IL-2 production and IL-2R expression. Cycrosporin A (Cy A), a potent immunosuppressive drug, markedly inhibited IL-2 production of Jurkat cells whereas it did not affect the IL-2R expression. Thus, the mechanism of action of Cy A appears to differ from that of the protein kinase inhibitor, H-7, and the calmodulin inhibitor, W-7.     

Comparative study on the stimulation of superoxide production in guinea-pig eosinophils by the calcium ionophore A23187

The effect of calcium and/or magnesium on O2- production by guinea-pig eosinophils stimulated by the calcium ionophore A23187 was studied in comparison to neutrophils. In the absence of calcium, A23187 did not stimulate O2- production in resting eosinophils and neutrophils, regardless of the presence of extracellular magnesium. The A23187-induced O2- production by both cells increased linearly with extracellular Ca2+ concentrations. However, the concentration of Ca2+ required for maximum O2- production in eosinophils was about 10-times lower than that required of neutrophils. The addition of Mg2+ strongly inhibited O2- production, especially in eosinophils at low Ca2+ concentrations. The intracellular Ca2+ concentration was lower in eosinophils than in neutrophils in the resting state, and the enhancement of the intracellular Ca2+ concentration in response to A23187 was much lower in eosinophils than in neutrophils. The activation of the NADPH-dependent O2(-)-forming enzyme (NADPH oxidase) in eosinophils depended on extracellular calcium, as observed in O2- production. However, the NADPH oxidase activity in the particulate fraction from A23187-stimulated eosinophils was only slightly affected by the addition of divalent cations or EDTA. The compound W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride), a calmodulin antagonist, significantly inhibited O2- production by both cells. On the other hand, the compound H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride), a protein kinase C antagonist, was less effective on O2- production than was W-7. H-7 had little effect on O2- production of eosinophils. These findings suggest that NADPH oxidase might be activated by a smaller Ca2+ concentration through the calmodulin-dependent reaction. This was not observed with protein kinase C, at least in eosinophils.     

Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae

The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM. H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca2+ channel blocker. Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 mM of EGTA and by 1 to 100 microM of H-7. While it was not inhibited by N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotide-dependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist, and verapamil.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mechanism of polymorphonuclear leukocyte activation by myristate. Involvement of calcium ion and protein kinase C

The stimulative effects of myristate on the superoxide generation and depolarization of membrane potential of polymorphonuclear leukocytes (PMN) are particularly strong, yet myristate does not affect the intracellular free Ca2+ level ([Ca2+]i) in the presence of 1 microM free calcium in calcium-EGTA buffer. The half maximum concentration of myristate was 10 microM. Myristate inhibited the transitory changes in [Ca2+]i induced by formylmethionyl-leucyl-phenylalanine (FMLP), but stimulated further the FMLP-induced superoxide generation; these effects are similar to those of phorbol myristate acetate (PMA). The myristate-induced superoxide generation was partially inhibited by H-7, a specific inhibitor of protein kinase C. Myristate stimulated the activity of Ca2+- and phospholipid-dependent protein kinase (protein kinase C) in a concentration-dependent manner in the presence of 10(-6) M Ca2+. The Ka was 100 microM. These results suggested that there is no relation between the superoxide generation and the [Ca2+]i change in PMNs and that the effects of myristate are similar to those of PMA against PMN.     

Effect of some psychotropic drugs on luminol-dependent chemiluminescence induced by O2-, *OH,HOCl

We studied antioxidant activity of six neuroleptics (chlorpromazine, levomepromazine, promethazine, trifluoperazine and thioridazine) and two antidepressants (imipramine and amitriptyline) in the range of concentration of 10(-7)-10(-4) M. We applied luminol-dependent chemiluminescence to test the ability of these drugs to scavenge the biologically relevant oxygen-derived species: hydroxyl radical, superoxide radical, hypochlorous acid in vitro. We found that the phenothiazines were powerful scavengers of hydroxyl and superoxide radicals. Chlorprothixene, amitriptyline and imipramine had no scavenge activity to the superoxide radical. All drugs showed a moderate scavenger effect on hypochloric anion.     

Calcium-stimulated, ATP-magnesium-dependent inactivation of pig liver glycogen synthase

Ca2+-stimulated inactivation of liver glycogen synthase was observed when a partially purified liver phosphorylase kinase fraction containing glycogen synthase was incubated with ATP-Mg2+. The Ca2+-stimulated portion of this inactivation was partially counteracted by trifluoperazine and slightly stimulated by exogenously added calmodulin. These results suggest that Ca2+-calmodulin may be involved as one of the factors causing this glycogen synthase inactivation. Although the exact mechanism mediated by Ca2+ has not been clearly determined, the possibility of the participation of some Ca2+-dependent protein kinase is discussed.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.     

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.     

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.     

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.     

Does protein kinase C activation mediate thrombin-induced arachidonate release in human platelets?

Thrombin stimulated rapid formation of diacylglycerol, inositol 1,4,5-trisphosphate (IP3) and thromboxane B2 (TXB2) in human platelets. Formation of diacylglycerol and IP3 appeared to precede that of TXB2. Activation of protein kinase C by diacylglycerol combining with Ca+2 mobilization by IP3 has been implicated in mediating arachidonate release. However, addition of the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) to platelet suspension did not inhibit thrombin-stimulated arachidonate release and TXB2 synthesis, whereas addition of the Ca+2 antagonist, 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8) or the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) abolished arachidonate release. The correlation of IP3 production with arachidonate release on increasing the concentrations of thrombin was further examined. IP3 production reached near maximum at 0.2 U/ml, whereas TXB2 synthesis continued to increase at 1 U/ml. These results suggest that protein kinase C activation may not mediate arachidonate release and that Ca+2 mobilization by IP3 may only partially account for arachidonate release in platelets stimulated with relatively high concentrations of thrombin.     

Distinct mechanisms for two amplification systems of insulin release.

The mechanisms whereby activation of the cyclic AMP-dependent protein kinase A or the Ca2+-phospholipid-dependent protein kinase C amplifies insulin release were studied with mouse islets. Forskolin and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) were used to stimulate adenylate cyclase and protein kinase C respectively. The sulphonylurea tolbutamide was used to initiate insulin release in the presence of 3 mM-glucose. Tolbutamide alone inhibited 86Rb+ efflux, depolarized beta-cell membrane, triggered electrical activity, accelerated 45Ca2+ influx and efflux and stimulated insulin release. Forskolin alone only slightly inhibited 86Rb+ efflux, but markedly increased the effects of tolbutamide on electrical activity, 45Ca2+ influx and efflux, and insulin release. In the absence of Ca2+, only the inhibition of 86Rb+ efflux persisted. TPA (100 nM) alone slightly accelerated 45Ca2+ efflux and insulin release without affecting 45Ca2+ influx or beta-cell membrane potential. It increased the effects of tolbutamide on 45Ca2+ efflux and insulin release without changing 86Rb+ efflux, 45Ca2+ influx or electrical activity. Omission of extracellular Ca2+ suppressed all effects due to the combination of TPA and tolbutamide, but not those of TPA alone. Though ineffective alone, 10 nM-TPA amplified the releasing action of tolbutamide without affecting its ionic and electrical effects. In conclusion, the two amplification systems of insulin release involve at least partially distinct mechanisms. The cyclic AMP but not the protein kinase C system initiating signal (Ca2+ influx) triggered by the primary secretagogue.     

Stimulatory effect of gangliosides on phagocytosis, phagosome–lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome–lysosome (P–L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P–L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P–L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P–L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P–L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P–L fusion stimulated by gangliosides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Involvement of protein kinase C in the phosphorylation of 46 kDa proteins which are phosphorylated in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes

Two proteins (Mr 46,000, pI 6.4 and 7.0), the phosphorylation of which was increased by any of the membrane-perturbing agents in parallel with activation of NADPH oxidase in intact guinea-pig polymorphonuclear leukocytes in our previous study (Okamura, N., Ohashi, S., Nagahisa, N. and Ishibashi, S. (1984) Arch. Biochem. Biophys. 228, 270-277), were also phosphorylated in a cell-free system prepared from the leukocytes. The in vitro phosphorylation of these two proteins was stimulated by the addition of phosphatidylserine in the presence of higher concentrations of Ca2+ (300-500 microM). The phosphorylation was further increased when protein kinase C partially purified from guinea-pig brain was added to the system. At a low concentration of Ca2+ (about 10 microM), stimulation of the phosphorylation was not attained by phosphatidylserine alone but required the addition of diacylglycerol or phorbol myristate acetate. On the other hand, the increase in the phosphorylation was inhibited by H-7, an inhibitor for protein kinase C. These results indicate that protein kinase C is involved in the phosphorylation of the two proteins, which may be related to the superoxide anion production stimulated by various membrane-perturbing agents.     

Stimulation of polyphosphoinositide turnover upon activation of protein kinases in human erythrocytes

Activation of protein kinase C in erythrocytes by 4-beta-phorbol 12-myristate 13-acetate (PMA) resulted in a parallel stimulation (time course and dose response) of the phosphorylation of both membrane proteins (heterodimers of 107 kDa and 97 kDa, protein 4.1 and 4.9, respectively) and of phosphatidylinositol 4-phosphate (PIP) and, to a lesser extent, of phosphatidylinositol 4,5-bisphosphate (PIP2). Evidence that the effect on lipid was mediated by protein kinase C activation and not by a direct action of PMA was provided by (1) the lack of effect of a phorbol ester that did not activate protein kinase C or of PMA addition on isolated membranes from control erythrocytes, (2) the reversal of the effect in the presence of protein kinase C inhibitors (alpha-cobrotoxin, H-7 (1-(5-isoquinolinesulfonyl)-2-methylpiperazine) or trifluoperazine). PMA treatment did not change the specific activity of ATP or the content of PIP2, but increased the content of PIP and decreased that of PI, indicating that the phosphorylation or dephosphorylation reactions linking PI and PIP were the target for the action of PMA. PMA treatment had no effect on the Ca2+-dependent PIP/PIP2 phospholipase C activity measured in isolated membranes. Mezerein, another protein kinase activator, had similar effects on both protein and lipid phosphorylation, when added with alpha-cobrotoxin. Activation of protein kinase A by cAMP also produced increases in phosphorylation, although quantitatively different from those induced by protein kinase C, in proteins and PIP. Simultaneous addition of PMA and cAMP at maximal doses resulted in only a partially additive effect on PIP labelling. These results show that inositol lipid turnover can be modulated by a protein kinase C and protein kinase A-dependent process involving the phosphorylation of a common protein. This could be PI kinase or PIP phosphatase or another protein regulating the activity of these enzymes.