Human T cell activation by phorbol esters and diacylglycerol analogues

Activation of protein kinase C (PKC), by the phorbol ester PMA, or the membrane-permeable diacylglycerol 1-oleoyl 2-acetylglycerol (OAG), had different effects on the proliferation-associated responses of a more than 99% pure population of human T cells. Treatment with PMA or OAG caused down-regulation of the TCR-CD3 complex, but only PMA, in combination with ionomycin, was capable of stimulating IL-2R expression and proliferation. Immunocytochemical staining with antisera specific for the PKC subspecies alpha, beta I, beta II, and gamma showed that untreated resting T cells normally coexpress alpha, beta I, and beta II PKC subspecies, which are distributed diffusely throughout the cell, with some localization around the periphery of the nucleus. There was no difference between the responses of these PKC subspecies to OAG and PMA, redistributing, after 10 min of treatment, to a discrete focal area within the cell. Treatment with OAG resulted in transient redistribution of PKC, maximal at 10 min, while in PMA-stimulated cells, the PKC redistribution was prolonged, persisting for at least 24 h. The results suggest that the difference in cellular response to treatment with PMA and OAG is not a consequence of differential activation of various PKC subspecies.     

Protein kinase C enhances growth hormone releasing factor (1-40)-stimulated cyclic AMP levels in anterior pituitary. Actions of somatostatin and pertussis toxin

The brain peptide human growth hormone releasing factor (1-40) (GRF), which stimulates adenylate cyclase activity in the anterior pituitary, is the predominant hormone signal for pituitary growth hormone (GH) release. Activators of protein kinase C such as teleocidin and 4 beta-phorbol 12-myristate 13-acetate (PMA) double the cyclic AMP accumulation induced by GRF, with no apparent effect on GRF potency; an inactive 4-alpha-PMA has no such action in cultured anterior pituitary cells. This PMA potentiation can be measured as early as 60 s, is maximal by 15 min, and wanes such that by 3-4 h there is no such amplifying effect of PMA. PMA, phorbol 12,13-dibutyrate, and teleocidin ED50 values for potentiating GRF activity are similar to those obtained for direct protein kinase C activation. The major inhibitory peptide somatostatin reduced both GRF- and GRF + PMA-stimulated cyclic AMP accumulation. Pertussis toxin totally blocked this somatostatin action without affecting the degree of maximal GRF potentiation achieved with PMA. Thus, the pertussis toxin target(s) are required for somatostatin inhibition of the cyclic AMP generating system, but may not be involved in the PMA potentiation of GRF-stimulated cyclic AMP accumulation.     

Coupling of antagonistic signalling pathways in modulation of neutrophil function

Modulation of neutrophil activation by catecholamines reflects a fine-tuning by coupling inhibitory and stimulatory receptor pathways. The catecholamine isoproterenol (ISO) binds to beta-adrenergic cell surface receptors and thereby inhibits cell responses such as O2- production stimulated by formyl peptides. However, ISO did not inhibit O2- generation activated by 1 microM ionophore A23187, the protein kinase C activators phorbol ester (PMA, 100 ng/ml) and oleoylacetylglycerol (OAG, 50 microM), and the G-protein activator NaF (40 mM). Furthermore, the overall kinetics of oxidant production in the presence of ISO were unchanged when cells were stimulated with PMA, OAG, A23187, and NaF. These results would imply that neither intracellular calcium, the activation of protein kinase C, nor the activation of G-protein are the primary target of the inhibitory pathway. Accordingly, pertussis toxin did not block PMA or NaF-stimulated superoxide generation. In contrast, formyl peptide-dependent GTPase activity is inhibited by ISO in sonicated cell preparations. Since ISO increases the cAMP concentration in the cell, the possibility is raised that a cAMP-dependent kinase inhibits signal transduction in part by blocking the interaction of this receptor with its G-protein.     

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.     

Thyroid hormone regulation of cell migration and oxidative metabolism in polymorphonuclear leukocytes: clinical evidence in thyroidectomized subjects on thyroxine replacement therapy

Migration and superoxide anion (O2-) generation were studied in polymorphonuclear leukocytes (PMNs) from 14 athyreotic patients, previously treated by total thyroidectomy and radioiodine therapy for differentiated thyroid carcinoma, and from age- and sex-matched euthyroid healthy controls. Patients were studied twice: in hypothyroidism (visit 1) and after TSH-suppressive L-T4 replacement therapy (visit 2). Random migration and N-formyl-Met-Leu-Phe (fMLP) 0.1-microM induced chemotaxis were similar in cells from patients at both visit 1 and visit 2 and from healthy controls. On the contrary, resting O2- generation in cells from patients was significantly lower than control values, both at visit 1 and 2. At visit 1, fMLP 0.1 muM-induced O2- generation was significantly lower than control values, while phorbol-myristate acetate (PMA) 100-ng/ml induced O2- generation was similar in cells from patients and from controls. At visit 2 both responses increased, resulting in fMLP-induced O2- generation superimposable to control values and PMA-induced O2- generation significantly higher with respect to both visit 1 and cells from controls. In vitro exposure of PMNs from healthy subjects to L-T4 did not affect O2- generation in resting cells, and significantly increased that induced by fMLP or PMA only at high, supra-physiological concentrations. Neither TSH nor T3 had significant effects at any of the concentrations tested. The present results document the existence of a correlation between thyroid status and oxidative metabolism of human PMNs, which is however unlikely to depend upon a direct action of thyroid hormones on these cells.     

Induction of histidine decarboxylase of rat basophilic leukemia (2H3) cells stimulated by higher oligomeric IgE or phorbol myristate acetate

When rat basophilic leukemia (2H3) cells were stimulated by higher oligomer, the chemically cross-linked oligomers of IgE, in the presence of calcium the activity of histidine decarboxylase (HDC, L-histidine carboxylase, E.C.4.1.1.22), a histamine-forming enzyme, was increased by 1 hr, reaching maximum activity by 2 hr, and returning to the original level by 8 hr. A similar increase in enzyme activity was observed in cells treated with phorbol myristate acetate (PMA) or oleoyl-acetylglycerol (OAG), which are known activators of protein kinase C. Removal of calcium from medium abolished the increase in HDC activity in response to higher oligomer but not that induced by PMA or OAG, suggesting that the increase in HDC activity may be mediated by protein kinase C. The increase in the HDC activity probably required induction of enzyme synthesis, because it was prevented by cycloheximide.     

Augmentation of c-fos mRNA expression by activators of protein kinase C in fresh, terminally differentiated resting macrophages.

Expression of c-fos mRNA was investigated in fresh, normal peritoneal macrophages (M phi), which are terminally differentiated, nonproliferating cells. The levels of c-fos mRNA were dramatically increased by stimulation with phorbol myristate acetate (PMA), calcium ionophore, or 1-oleoyl-2-acetoyl glycerol (OAG). Induction of c-fos mRNA by all the above agents followed similar kinetics, with a peak of mRNA 30 min after stimulation. These results demonstrate that c-fos mRNA can be augmented in fresh, terminally differentiated cells. Since the stimuli increasing c-fos mRNA are direct or indirect activators of protein kinase C, our data suggest that in M phi c-fos mRNA is controlled by protein kinase C activation. PMA, calcium ionophore, and OAG were biologically active in M phi. PMA and calcium ionophore induced respiratory burst and tumoricidal activity, respectively, whereas OAG and PMA were chemotactic for M phi. Interferons beta and gamma, potent M phi activators eliciting tumoricidal activity, did not alter the levels of c-fos mRNA. These results indicate that c-fos mRNA augmentation is a stimulus-specific rather than a function-specific response connected to activation of protein kinase C.     

Inhibition of phorbol ester stimulated superoxide production by 1-oleoyl-2-acetyl-sn-glycerol (OAG); fact or artefact?

OAG-stimulated superoxide (O2) production by HL-60 granulocytes showed enantiomeric specificity but reached a maximum of only 5% of that produced by either phorbol myristate acetate (PMA) or phorbol dibutyrate (PDBu). At 10-100 microM, OAG displaced specifically-bound [3H]PDBu from intact HL-60 cells by only 25%, suggesting limited cell penetration. OAG (10-100 microM) also inhibited PDBu-stimulated O2 production by 25%; this inhibition was enantiomerically specific. However, at a lower concentration (3 microM), both enantiomers of OAG fully blocked O2 production stimulated by PMA (0.5 microM). This inhibition is probably artefactual, due to the hydrophobic PMA physically associating with OAG in the extracellular fluid.     

Effect of protein kinase C activation on agonist-mediated phosphositide metabolism in rabbit retinal cells

4β-Phorbol 12-myrisate 13-acetate (PMA), a tumour-promoting phorbol ester, and 1-oleoly-2-acetylglycerol (OAG), a synthetic diacylglycerol, induced an inhibition of muscarinic and ₁-adrenergic receptor-mediated stimulation of PIP₂ breakdown and IPs accumulation in both rabbit retinal slices and primary retinal cultures. Furthermore, an increase in [Ca²⁺]_i, mediated by activation of these receptors in 3–5 and 25–30 day old rabbit retinal cultures, was also inhibited by PMA. Neither PMA nor OAG had an effect on the serotonin-mediated PIP₂ breakdown, IPs accumulation or Ca²⁺ mobilization. Although A23187 also stimulated IPs formation by acting directly on phospholipase C, PMA had no effect. Maximal inhibition of the carbachol- and noradrenaline-mediated responses was achieved with a 15 min preincubation with PMA at concentrations of 0.1 and 0.01 μM in retinal slices and primary retinal cultures, respectively. Neither PMA nor OAG influenced the basal levels of phosphoinositides, IPs or [Ca²]_i. In addition, the inactive phorbol ester, 4-phorbol 12,13-didecanoate, had no effect on any of the agonist-induced responses. Staurosporine, a potent inhibitor of protein kinase C, significantly attenuated the inhibitory effects exerted by PMA and OAG. These results suggest that calcium- and phospholipid-dependent protein kinase, which is activated by either PMA or OAG, exert inhibitory effects on muscarinic and ₁-adrenergic responses. This modulatory feedback “down regulation” role by PKC does not, however, affect serotonergic mediated responses, and thus exhibits a certain selectivity about the site of action. The possible mechanism(s) by which PKC induces its actions are discussed.     

Phorbol esters stimulate cyclic adenosine 3', 5'-monophosphate accumulation in hamster spermatozoa during in vitro capacitation

Phorbol ester, phorbol 12-myristate 13-acetate (PMA), induced a 20- to 50-fold increase (ED50: 2 microM) in cyclic adenosine 3', 5'-monophosphate (cAMP) levels in spermatozoa incubated in capacitation medium for short periods of time (30 min). Similar results were obtained with 1-oleoyl 2-acetylglycerol (OAG), whereas 1, 2 diolein, 1-oleoyl glycerol, or 4 alpha-phorbol 12, 13-didecanoate had no effect. When extracellular Ca2+ was complexed by [ethylenebis(oxyethyleneitrilo)] tetraacetic acid (EGTA), a 50% reduction of maximal stimulation was observed, and 90% inhibition was seen after chelation of both extra- and intracellular Ca2+ with EGTA and 2-[[2-[bis [(carbonyl) methyl] amino]-5-methylphenoxy] methyl]-6-methoxy-8-[bis[(carbonyl) methyl] amino] quinoline acetoxy methyl (Quin 2). The acrosome reaction was not affected by similar concentration of PMA or OAG at different periods of incubation. These results suggest the involvement of protein kinase C activity in the regulation of cAMP levels in sperm during capacitation. This stimulation is dependent on intracellular Ca2+ and probably is not linked to the process of the acrosome reaction.     

Priming of the respiratory burst of neutrophils by diacylglycerol. Independence from activation or translocation of protein kinase C

Preincubation of neutrophils with certain agonists may "prime" the cells to cause increased responses to a second stimulus ("primed stimulation"). We used two approaches to examine the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in priming and stimulation by 1-oleoyl-2-acetylglycerol (OAG), phorbol 12-myristate 13-acetate (PMA), and N-formyl-Met-Leu-Phe (fMLP): inhibition of protein kinase C by 1-(5-isoquinolinesulfonyl)-piperazine (C-I) and measurement of protein kinase C translocation induced by priming and stimulatory concentrations of OAG. C-I had little effect on stimulation or primed stimulation by fMLP, suggesting that fMLP invokes events independent of protein kinase C. C-I equally inhibited stimulation and primed stimulation by PMA. Direct stimulation by OAG was inhibited, but priming and primed stimulation by OAG was unaltered by C-I. OAG concentrations greater than or equal to 100 microM caused translocation of protein kinase C, in correlation with direct stimulation of the respiratory burst. Lower OAG concentrations (10-30 microM) primed to stimulation by fMLP and, conversely, stimulated neutrophils primed with fMLP, yet did not cause translocation of protein kinase C. The data are compatible with previous assumptions that PMA and OAG directly stimulate polymorphonuclear neutrophil leukocytes by translocation and activation of protein kinase C. However, priming and primed stimulation by OAG apparently invoke distinct transduction mechanisms other than protein kinase C translocation.     

The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester.

Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.     

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.     

Role and mechanism of PKC in ischemic preconditioning of pig skeletal muscle against infarction

Protein kinase C (PKC) inhibitors, chelerythrine (Chel, 0.6 mg) and polymyxin B (Poly B, 1.0 mg), and PKC activators, phorbol 12-myristate 13-acetate (PMA, 0.05 mg) and 1-oleoyl-2-acetyl glycerol (OAG, 0.1 mg), were used as probes to investigate the role of PKC in mediation of ischemic preconditioning (IPC) of noncontracting pig latissimus dorsi (LD) muscles against infarction in vivo. These drugs were delivered to each LD muscle flap (8 x 12 cm) by 10 min of local intra-arterial infusion. It was observed that LD muscle flaps sustained 43 +/- 5% infarction when subjected to 4 h of global ischemia and 24 h of reperfusion. IPC with three cycles of 10 min ischemia-reperfusion reduced muscle infarction to 25 +/- 3% (P < 0.05). This anti-infarction effect of IPC was blocked by Chel (42 +/- 7%) and Poly B (37 +/- 2%) and mimicked by PMA (19 +/- 10%) and OAG (14 +/- 5%) treatments (P < 0.05), given 10 min before 4 h of ischemia. In addition, the ATP-sensitive K(+) (K(ATP)) channel antagonist sodium 5-hydroxydecanoate attenuated (P < 0.05) the anti-infarction effect of IPC (37 +/- 2%), PMA (44 +/- 17%), and OAG (46 +/- 9%). IPC, OAG, and Chel treatment alone did not affect mean arterial blood pressure or muscle blood flow assessed by 15-microm radioactive microspheres. Western blot analysis of muscle biopsies obtained before (baseline) and after IPC demonstrated seven cytosol-associated isoforms, with nPKCepsilon alone demonstrating progressive cytosol-to-membrane translocation within 10 min after the final ischemia period of IPC. Using differential fractionation, it was observed that nPKCepsilon translocated to a membrane compartment other than the sarcolemma and/or sarcoplasmic reticulum. Furthermore, IPC and preischemic OAG but not postischemic OAG treatment reduced (P < 0.05) muscle myeloperoxidase activity compared with time-matched ischemic controls during 16 h of reperfusion after 4 h of ischemia. Taken together, these observations indicate that PKC plays a central role in the anti-infarction effect of IPC in pig LD muscles, most likely through a PKC-K(ATP) channel-linked signal-transduction pathway.     

Effect of n-3 and n-6 polyunsaturated fatty acids and their ethylesters on stimuli-dependent superoxide generation in neutrophils

Polymorphonuclear leukocytes from healthy volunteers (HPMN) generated superoxide (O2*-) following treatment with various stimuli, such as phorbol myristate acetate (PMA), opsonized zymozan (OZ) and arachidonic acid (AA). Other types of n-3 polyunsaturated fatty acids (PUFAS), such as docosahexaenoic acid (DHA), docosapentaenoic acid (DPA), and eicosapentaenoic acid (EPA), also stimulated O2*- generation. The free form of DHA enhanced the generation of O2*- induced by PMA but inhibited that induced by OZ. In contrast, the ethylester of DHA (DHA-E) inhibited O2*- generation induced by PMA but stimulated that induced by OZ. Similar effects were also observed with ethylesters of EPA (EPA-E), DPA (DPA-E) and AA (AA-E). High concentrations of DHA-E reduced the PMA-induced formation of superoxide without affecting the cellular activity of protein kinase C (PKC). Similar phenomena were also observed with oral neutrophils from healthy volunteers (OPMN). These results indicate that PUFAS and their esters affect 02*- generation in human PMN via different pathways, thereby modulating inflammatory reactions.     

Activation of human TRPC6 channels by receptor stimulation

The human TRPC6 channel was expressed in human embryonic kidney (HEK) cells, and activity was monitored using the giga-seal technique. Whole cell membrane currents with distinctive inward and outward rectification were activated by carbachol (CCh) in TRPC6-expressing cells, but not in lacZ-transfected controls. The effect of CCh was steeply dose-dependent with a K(0.5) of approximately 10 microm and a Hill coefficient of 3-4. A steep concentration-response relationship was also observed when TRPC6 activity was measured using a fluorescence-based imaging plate reader (FLIPR) assay for membrane depolarization. Ionomycin, thapsigargin, and dialysis of the cell with inositol 1,4,5-trisphosphate via the patch pipette had no effect on TRPC6 currents, but exogenous application of 1-oleoyl acetyl-sn-glycerol (OAG, 30-300 microm) produced a slow increase in channel activity. The PKC activator, phorbol 12-myristate 13-acetate (PMA, 0.5 microm) had no significant acute effect on TRPC6, or on the subsequent response to OAG. In contrast, the response to CCh was blocked >90% by PMA pretreatment. To further explore the role of DAG in receptor stimulation, TRPC6 currents were monitored following the sequential addition of CCh and OAG. Surprisingly, concentrations of CCh that produced little or no response in the absence of OAG, produced increases in TRPC6 currents in the presence of OAG that were larger than the sum of either agent alone. Likewise, the response to OAG was superadditive following prior stimulation of the cells with near threshold concentrations of CCh. Overall, these results suggest that generation of DAG alone may not fully account for activation of TRPC6, and that other receptor-mediated events act synergistically with DAG to stimulate channel activity. This synergy may explain, at least in part, the steep dose-response relationship observed for CCh-induced TRPC6 currents expressed in HEK cells.     

Tumor necrosis factor receptors and cytocidal activity are down-regulated by activators of protein kinase C

Human HeLa cells and murine L(S) cells are highly sensitive to the cytocidal activity of tumor necrosis factor (TNF) when simultaneously treated with the inhibitor of protein synthesis cycloheximide. This cytocidal activity of TNF was inhibited up to 90% in both cell lines after a 15-60-min pretreatment with 3-10 ng/ml of phorbol 12-myristate 13-acetate (PMA). This inhibition was long lasting for HeLa cells but transient for L(S) cells. The protection afforded by PMA was most effective when the cells were pretreated with this phorbol ester, but it decreased when PMA was added together with TNF or after TNF addition. This finding suggested that PMA interfered with one of the early steps in the mechanism of action of TNF. A pretreatment with the calcium ionophore A23187 also reduced the cytocidal activity of TNF in both HeLa and L(S) cells to about the same extent. Treatment of these cells with either PMA or A23187 significantly decreased the binding of 125I-TNF to cell surface receptors. This decrease paralleled the time course and dose-response of the inhibition of cytocidal activity. In addition, treatment of HeLa cells with 1-oleyl-2-acetyl-glycerol (OAG) also induced a rapid loss of TNF binding capacity. Since OAG, PMA, and A23187 are all activators of protein kinase C (Ca2+/phospholipid-dependent enzyme), these results suggest that this kinase is involved in modulation of TNF sensitivity. Furthermore, depletion or inhibition of protein kinase C antagonized PMA-induced effects on TNF cytotoxicity and binding to receptors. Internalization of bound TNF was not significantly affected by PMA treatment, and Scatchard analysis of binding data indicated that PMA decreased TNF receptor binding affinity rather than the number of TNF-binding sites. These findings suggest that protein kinase C may have a physiological role in mediating TNF sensitivity.     

Studies on the inactivation of superoxide dismutase activity by nitric oxide from rat peritoneal macrophages

Rat peritoneal macrophages stimulated with lipopolysaccharide (LPS) and Phorbol myristate acetate (PMA) generated increased levels of superoxide anions (O2ú-) by 122% as compared to those stimulated with PMA alone. However, Nitric oxide (NO) synthase inhibitors-n-monomethyl arginine (nMMA) or spermine-HCI lowered the enhanced levels of O2ú- released by LPS treated macrophages. The Superoxide dismutase (SOD) activity in LPS treated macrophages was 51% lower than that observed in resident cells. NO synthase inhibitors prevented the loss of SOD activity in LPS treated cells. Exogenously added SOD during sensitization of cells with LPS also inactivated the enzyme. This inactivation of SOD is inhibited by Nitric oxide synthase inhibitors. PMA alone did not affect SOD activity. NO synthase inhibitors also did not affect PMA activated superoxide anion generation in macrophages. These studies indicate that nitric oxide generated by LPS treated macrophages can inactivate SOD activity.     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous protein kinase C

Single IK(Ca) channels of human erythrocytes were studied with the patch-clamp technique to define their modulation by endogenous protein kinase C (PKC). The perfusion of the cytoplasmic side of freshly excised patches with the PKC activator, phorbol 12-myristate 13-acetate (PMA), inhibited channel activity. This effect was blocked by PKC(19-31), a peptide inhibitor specific for PKC. Similar results were obtained by perfusing the membrane patches with the structurally unrelated PKC activator 1-oleoyl-2-acetylglycerol (OAG). Blocking of this effect was induced by perfusion with PKC(19-31) or chelerythrine. Channel activity was not inhibited by the PMA analog 4alpha-phorbol 12,13-didecanoate (4alphaPDD), which has no effect on PKC. Activation of endogenous cAMP-dependent protein kinase (PKA), which is known to up-modulate IK(Ca) channels, restored channel activity previously inhibited by OAG. The application of OAG induced a reversible reduction of channel activity previously up-modulated by the activation of PKA, indicating that the effects of the two kinases are commutative, and antagonistic. Kinetic analysis showed that down-regulation by PKC mainly changes the opening frequency without significantly affecting mean channel open time and conductance. These results provide evidence that an endogenous PKC down-modulates the activity of native IK(Ca) channels of human erythrocytes. Our results show that PKA and PKC signal transduction pathways integrate their effects, determining the open probability of the IK(Ca) channels.     

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.