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1.
Inhibitors of protein kinase C block activation of B lymphocytes by bacterial lipopolysaccharide 总被引:1,自引:0,他引:1
Activation of murine splenic B lymphocytes (B cells) by bacterial lipopolysaccharide (LPS) was found to be markedly inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), two potent inhibitors of protein kinases. The higher sensitivity of DNA synthesis, RNA synthesis and protein N-glycosylation activity to H-7, relative to H-8, strongly supports the proposal that protein kinase C plays a critical role in the activation of B cells. A kinetic study on the time of addition of H-7 indicated that protein kinase C promoted the activation process continuously after the addition of LPS. 相似文献
2.
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. 相似文献
3.
Resting murine splenic B lymphocytes (B cells) can be stimulated to proliferate by exposure to a variety of polyclonal activators. To investigate changes in glycoprotein synthesis that occur during the activation process, N-glycosylation activity was assessed by following the incorporation of [2-3H]mannose into dolichol-linked oligosaccharide intermediates and glycoprotein after B cells were exposed to anti-immunoglobulin M (anti-mu). Stimulation of B cells by anti-mu resulted in a dramatic induction of N-glycosylation activity. The incorporation of radiolabeled mannose into oligosaccharide-lipid increased 9-fold while the rate of labeling of glycoprotein increased 27-fold between 18 and 38 h after exposure to anti-mu. Maximal stimulation of N-glycosylation activity was observed at an anti-mu concentration of 20-50 micrograms/ml. Similar results were obtained when B cells were activated by bacterial lipopolysaccharide (LPS), another polyclonal activating agent. The major dolichol-bound oligosaccharide labeled during the induction period was determined to be Glc3Man9GlcNAc2 by HPLC analysis. Nearly full induction of oligosaccharide-lipid synthesis and protein N-glycosylation was also seen when DNA synthesis was suppressed by activating B cells with anti-mu in a serum-free medium, or by activating with anti-mu or LPS in the presence of hydroxyurea. The results suggest that the N-glycosylation pathway is induced during the G0 to G1 transition or during the G1 period, and that entry into S phase is not required. These studies describe a striking developmental increase in N-glycosylation activity and extend the information on biochemical changes occurring during the activation of B cells. 相似文献
4.
The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation 总被引:3,自引:0,他引:3
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. 相似文献
5.
Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst 总被引:2,自引:0,他引:2
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. 相似文献
6.
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. 相似文献
7.
In primary cultured rat hepatocytes, DNA synthesis was markedly induced 48 h after plating by epidermal growth factor (EGF) and insulin added at 24 h, but not by 12-O-tetradecanoyl-phorbol-13-acetate (TPA). When EGF and insulin were added at 6 h, DNA synthesis at 30 h was 7% of DNA synthesis seen at 48 h, but became 27% by pretreatment with TPA. The similar pretreatment effect was also seen with vasopressin. Such induction at 30 h was inhibited by rat liver plasma membrane added at 2 h even in the presence of TPA or vasopressin, and also by 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine more extensively than N-(2-guanidinoethyl)-5-isoquinolinesulfonamide. These results suggest that DNA synthesis induction by EGF and insulin may require a priming period related to protein kinase C activation in primary cultured rat hepatocytes, which is inhibited by plasma membrane. 相似文献
8.
Regulation of T lymphocyte apoptosis. Signals for the antagonism between activation- and glucocorticoid-induced death. 总被引:7,自引:0,他引:7
Apoptotic death can be induced in T cell hybridomas by glucocorticoids or the stimulation via the TCR/CD3 complex. The two apoptotic processes are mutually antagonistic. We have previously proposed that positive selection of thymocytes for the formation of the T cell repertoire might be based on a similar mechanism. We analyzed the TCR/CD3-mediated signals essential for the regulation of apoptosis in T cell hybridomas. We suggest that both an increase in the intracellular Ca2+ level and an activation of protein kinase C are essential for the TCR/CD3-mediated apoptosis, because we obtained the following results: 1) either reduction of extracellular Ca2+ concentration or addition of a protein kinase inhibitor, 1-(5-isoquinolkinelsulfonyl)-2-methylpiperazine or N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide, inhibited anti-CD3-induced but not dexamethasone-induced DNA fragmentation. 2) The combination of ionomycin and PMA, but neither one alone nor the combination of ionomycin and cyclic nucleotide analogs, induced DNA fragmentation. On the contrary, we suggest that only an increase in the intracellular Ca2+ level is essential for the inhibition of glucocorticoid-induced apoptosis, because ionomycin alone as well as the combination of ionomycin and PMA inhibited dexamethasone- but not anti-CD3-induced DNA fragmentation. 相似文献
9.
In this study, we report that the potent and selective inhibitor of cyclic AMP-dependent protein kinase, N-[2-bromocinnamyl(amino)ethyl]-5-isoquinolinesulfonamide (H-89) interferes with the incorporation of choline into phosphatidylcholine in HeLa cells. Treatment of cells with 10 microM H-89 for 1 h decreases the phosphatidylcholine biosynthesis by 50%. This inhibition is prevented by simultaneous addition of 10 microM forskolin, while the choline uptake itself is not affected by H-89. 相似文献
10.
Sn-1,2-diacylglycerols (DAG) and ionized-free calcium can act as intracellular second messengers for cell activation. Traditionally, T-lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T-lymphocytes demonstrate rapid enhancement of A-(alanine-favoring) system amino acid uptake when treated with DAG or ionomycin. A 30-40% increase in the initial velocity of uptake (vi) of the synthetic A-system specific amino acid, methylamino-isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 mumol/liter cell water per min after 30 min exposure of T-cells to optimal concentrations of dioctanoylglycerol (30 microM), oleoylacetylglycerol (30 microM), or ionomycin (5 microM) (P less than .01 for each agent). A 50-fold excess of non-radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier-mediated on treatment with these agents. Cycloheximide, 100 micrograms/ml, inhibited protein synthesis but did not block the A-system amino acid transport enhancement induced by DAG or ionomycin. The DAG-induced increase in the vi was blocked 40% with 100 microM H-7, an inhibitor of protein kinase C. H-7 treatment did not inhibit the ionomycin-induced A-system enhancement. A marked increase in cytoplasmic free calcium was measured when T-lymphocytes were exposed to ionomycin but not on DAG exposure, and the A-system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A-system amino acid uptake in T-lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A-system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium. 相似文献
11.
H Kido N Fukusen N Katunuma 《Biochemical and biophysical research communications》1987,144(1):152-159
Induction of tyrosine aminotransferase by glucocorticoid in rat hepatocytes was inhibited concentration-dependently by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, but not by N- [2-(methyl-amino)-ethyl]-5-isoquinolinesulfonamide dihydrochloride, an inhibitor of cyclic nucleotide dependent protein kinases. H-7 also inhibited the accumulation of glucocorticoid-receptor complexes in the nuclear fraction with associated accumulation of these complexes in the cytoplasmic fraction, but did not affect incorporation of glucocorticoid into hepatocytes. These results indicate that protein kinase C may be essential in translocation of glucocorticoid-receptor complexes to the nuclei. 相似文献
12.
Phorbol 12-myristate 13-acetate (PMA) induces the cytolytic T lymphocyte (CTL) clone 4D (H-2b anti-H-2d) to promiscuously kill the inappropriate target EL-4 (H-2b). The protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) inhibited the PMA-induced promiscuous lympholysis. The concentration of H-7 that inhibited PMA-induced lympholysis by 50% (IC50) was calculated to be 4 microM, which closely approximates the reported IC50 of H-7 of 6 microM for PKC activity in vitro. In striking contrast, specific cytolysis of appropriate P815 (H-2d) target cell by CTL clone 4D was not inhibited by concentrations of H-7 which inhibited PMA-induced promiscuous lympholysis. These results indicate that PMA-induced promiscuous lympholysis of inappropriate target cell is triggered via activation of PKC, whereas PKC activation is not obligatory in triggering CTL clone 4D to specifically kill appropriate target cells. Thus, these data suggest that cloned CTL have two or more triggering mechanisms than may initiate one or more cytolytic pathways. 相似文献
13.
Primary 24-hour cultures of hepatocytes on slides in a serum-free medium were studied. Circahoralian rhythm of protein synthesis served as a marker of cell cooperation. Stimulation of protein kinase activities by phorbol 12-myristate 13-acetate at 0.5 or 1.0 microM or forskolin at 10 microM led to visualization of the protein synthesis rhythm in sparse cultures, which were asynchronous in the control and with linear kinetics of protein synthesis. Inhibitors of protein kinase activities H7 (1-(5-isoquinolinylsulfonyl)-5-methylpiperasine dihydrochloride) at 40 microM or H8 (N-(2-[methylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride) at 25 microM eliminated the protein synthesis rhythm in dense cultures, which are normally synchronous with oscillatory kinetics of protein synthesis. After inhibition of the protein kinase activities, gangliosides or phenylephrine did not synchronize the protein synthesis rhythm. Phorbol 12-myristate 13-acetate modulated the protein synthesis rhythm, shifted the rhythm phase, i.e., stimulation of the protein kinase activities, and, correspondingly, protein phosphorylation may be a factor of synchronization of synthesis oscillations in individual cells and of population rhythm formation. Thus, a cascade of processes leading to self-organization of hepatocytes during formation of summarized protein synthesis was revealed in a series of studies: signal of gangliosides or other calcium agonists-->changes in the level of calcium ions in cytoplasm-->increased protein kinase activities-->protein phosphorylation-->modulation of individual oscillations in the intensity of protein synthesis and their coordination in a summarized rhythm. cAMP-dependent protein kinases also affect the protein synthesis rhythm. Protein phosphorylation is a key process. The mechanisms of cell self-organization are similar in vitro and in vivo, specifically in the liver in situ. 相似文献
14.
Primary cultures of rat hepatocytes grown on slides were studied in serum-free medium. Ultradian protein synthesis rhythm was used as a marker of synchronization of individual oscillations, resulting in the formation of a common rhythm of the cell population, i.e. cell-cell self-organization. Dense synchronous and sparse non-synchronous cultures were used to estimate effect of protein kinase activity on the kinetics of protein synthesis. Treatment of dense cultures with the inhibitors H7 (40 microM) or H8 (25 microM) resulted in a loss of the protein synthesis rhythm, a suppression of the cell-cell self-organization. Stimulation of protein kinase activity with either 0.5 or 1.0 microM phorbol 12-miristate-13-acetate (PMA) or 10 microM forskolin caused the appearance of the synthetic rhythm in non-synchronous sparse cultures under otherwise normal conditions. Inhibition of protein kinase activity with H7 resulted in signal factors, such as gangliosides and phenylephrine, failing to initiate this rhythm in sparse cultures. Activation of protein kinase activity with PMA shifted the phase pattern of the protein synthesis rhythm. Thus, according to our previous and the new data, protein kinase activity and consequently protein phosphorylation is the crucial step of sequence of processes resulting in synchronization during self-organization of cells in producing a common rhythm in the population. The general pathway can be presented as follows: signaling of gangliosides or other calcium agonists-->efflux of calcium ion from intracellular stores, with elevation of calcium concentration in the cytoplasm-->activation of protein kinases-->protein phosphorylation-->synchronization of individual oscillations in protein synthesis rates-->induction of a common rhythm throughout this population. The data have been discussed concerning similarity of the direct cell-cell communication and the cell self-organization in cultures and in organism. 相似文献
15.
Specific aspects of the prolactin stimulation of RNA, DNA and protein synthesis in the Nb2 node lymphoma cell line were determined. In time sequence studies the onset of the prolactin stimulation of the incorporation of radiolabeled precursors into these macromolecules was found to be 0.5-1 h for [3H]uridine incorporation into RNA, 1-2 h for [3H]leucine incorporation into protein, and 4-8 h for [3H]thymidine incorporation into DNA. The total DNA content of the cell cultures was increased by 12-18 hours after addition of prolactin. Amiloride, an inhibitor of the plasma-membrane-bound Na+/H+ antiporter, was found to inhibit the mitogenic effects of prolactin. Amiloride was also found to inhibit the prolactin stimulation of DNA, RNA and protein synthesis, thus suggesting that the initial regulation of the Na+/H+ antiporter may initiate these responses as well as the mitogenic effect of prolactin. In contrast, H-7, a drug which inhibits protein kinase C, had no effect on the magnitude of the prolactin stimulation of DNA, RNA or protein synthesis at a drug concentration (100 muM) that abolished the mitogenic effect of prolactin. The early effects of prolactin on RNA, DNA and protein synthesis would therefore appear not to involve an activation of protein kinase C. 相似文献
16.
Treatment of the human promyelocytic leukemia cell line HL-60, with 12-o-tetradecanoylphorbol acetate (TPA) results in the differentiation into macrophage-like cell. A potent inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine(H-7), suppressed the proliferation of HL-60 cells and also inhibited TPA-induced cell differentiation of these cells. N-(2-guanidinoethyl)-5-isoquinolinesulfonamide(HA-1004), a weaker analog of H-7, failed to inhibit this TPA-induced cell differentiation. H-7 also inhibited TPA-induced protein phosphorylation in these cells. Thus, protein kinase C-mediated phosphorylation may be involved in the process of TPA-induced HL-60 cell differentiation. 相似文献
17.
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) 相似文献
18.
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. 相似文献
19.
Phorbol diesters and dioctanoylglycerol stimulate accumulation of both diacylglycerols and alkylacylglycerols in human neutrophils 总被引:5,自引:0,他引:5
L G Rider R W Dougherty J E Niedel 《Journal of immunology (Baltimore, Md. : 1950)》1988,140(1):200-207
Human neutrophils treated with phorbol 12-myristate 13-acetate (PMA) or dioctanoylglycerol exhibited a large (10-fold), sustained accumulation of the mass of diradylglycerol, beginning 1 min after stimulation and continuing for 30 to 60 min. Phorbol dibutyrate was less potent than PMA in stimulating diradylglycerol accumulation, whereas the 4-alpha analogs of PMA and phorbol dibutyrate were inactive. Submaximal concentrations of PMA (0.5 to 2.5 nM) plus the calcium ionophore, ionomycin (15 to 60 nM), led to synergistic accumulation of diradylglycerols. Chlorpromazine and sphingosine, inhibitors of protein kinase C, blocked PMA-stimulated accumulation of diradylglycerol with IC50 concentrations of 32 and 9 microM, respectively, paralleling their inhibition of PMA-stimulated O2- production. These compounds also inhibited the ionomycin-stimulated accumulation of diradylglycerols. A third protein kinase C inhibitor, H-7, was less effective, inhibiting PMA-stimulated accumulation of diradylglycerol by 25% at 100 microM. Differential sensitivity to alkaline hydrolysis suggests that diradylglycerols that accumulate in response to PMA or ionomycin stimulation are composed of a mixture of two distinct diglyceride species, diacylglycerols and alkylacylglycerols. Whereas diacylglycerol may activate cellular protein kinase C, the importance of the production of alkylacylglycerols is uncertain. 相似文献
20.
Axokinin phosphorylation by cAMP-dependent protein kinase is sufficient for activation of sperm flagellar motility 总被引:4,自引:3,他引:1
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Using a selective inhibitor of cAMP-dependent protein kinase, N-[2(methylamino)ethyl]-5-isoquinolinesulfonamide (H-8), the requirement for cAMP-dependent phosphoproteins in the initiation of dog sperm flagellar motility was examined. H-8 inhibited motility of live as well as reactivated sperm in a dose-dependent manner. The half-maximal inhibition of reactivated motility (32 microM) paralleled the inhibition of pure catalytic subunit of cAMP-dependent protein kinase (50 microM) measured under the same conditions. H-8 inhibited protein phosphorylation both in whole models and in isolated Nonidet P-40 (NP-40) extracts of sperm. Axokinin, the heat-stable NP-40-soluble protein whose phosphorylation is required for flagellar reactivation, represented 97% of the de novo phosphate incorporation in the NP-40 extract after stimulation by cAMP. 500 microM H-8 inhibited axokinin phosphorylation by 87%. When sperm were reactivated in the presence of up to 5 mM H-8 with NP-40 extract that had been prephosphorylated with cAMP-dependent protein kinase, then neither cAMP nor cAMP-dependent protein kinase activity was required for full flagellar reactivation. If sperm were rendered completely immotile by pretreatment with H-8, then the resulting model remained immotile in the continued presence of H-8 unless prephosphorylated axokinin was added. These results suggest that phosphorylated axokinin is not only required for flagellar reactivation but is sufficient as well. 相似文献