Dissimilarities in superoxide anion production by human neutrophils stimulated by phorbol myristate acetate or phorbol dibutyrate

Phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBU) are known to translocate protein kinase C (PKC) and to induce superoxide anion (O2-.) production in human neutrophils. They are thus currently used to probe the role of PKC in O2-. production. We show here that under certain conditions, O2-. production induced by PMA is not associated with a decrease in cytosolic PKC activity, whereas these two events are associated after PDBU stimulation. (1) In the presence of extracellular calcium (1 mM), O2-. production was related to the concentration of PMA. PMA induced O2-. production at all the concentrations studied, but this was not associated with a decrease in cytosolic PKC levels up to 5 ng/ml PMA (50% maximum O2-. production). (2) Under PDBU stimulation, even at very low O2-. production levels, cytosolic PKC decreased and the decrease as well as the O2-. production were related to the concentration of PDBU. (3) For a given decrease in cytosolic PKC, O2-. production induced by PMA was much greater than that induced by PDBU. (4) In calcium-free medium, O2-. production induced by low concentrations of PMA (up to 5 ng/ml) was lower than that observed in the presence of 1 mM calcium, whereas modifications of cytosolic PKC activity were similar. (5) Cytochalasin B had no effect on PMA-induced O2-. production, regardless of the calcium content of the medium, and had no effect on the decrease in cytosolic PKC. On the contrary, following PDBU stimulation, cytochalasin B increased O2-. production, regardless of the medium, but induced a larger decrease in cytosolic PKC when Ca2+ was present. (6) Preincubation of PMN with 100 microM H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) before stimulation with PMA or PDBU led to similar inhibition of O2-. production whatever the degree of decrease in cytosolic PKC activity. These findings show that, in contrast to PDBU, O2-. production induced by PMA is not always related to cytosolic PKC activity.     

Protein kinase C modulates the release of [3H]5-hydroxytryptamine in the spinal cord of the rat: the role of L-type voltage-dependent calcium channels.

The present studies examined the relationship between protein kinase C (PKC) and L-type voltage-dependent calcium channels in modulating the release of neurotransmitter from K(+)-depolarized rat spinal cord synaptosomes. Activators of PKC, such as phorbol 12-myristate 13-acetate (PMA), mezerein and oleoyl acetylglycerol produced a concentration-dependent potentiation of K(+)-induced release of [3H]5-hydroxytryptamine ([3H]5-HT). Enhanced release was dependent on the concentration of both Ca2+ and K+ in the superfusion medium. Calcium-independent release of [3H]5-HT or release induced by the Ca2+ ionophore were unaffected by PKC activators. Calcium-dependent release of [3H]5-HT, evoked by K+, was enhanced under similar conditions by the L-type Ca2+ channel agonists Bay K 8644 and (+)-SDZ 202-791. Nimodipine, an L-type Ca2+ channel antagonist, while having no independent effect on K(+)-induced release of [3H]5-HT, abolished the potentiative effects of Bay K 8644 and PMA. Similarly, the PKC inhibitors, polymyxin B and staurosporine, blocked effects of both PMA and Bay K 8644 on K(+)-stimulated release of [3H]5-HT. Neither PMA nor Bay K 8644 altered the uptake of [3H]5-HT. These results suggest that PKC-dependent mechanisms utilize calcium influx, via the L-type calcium channel, to modulate release of neurotransmitter and indicate a possible functional link between PKC and L-type voltage-dependent calcium channels in the spinal cord.     

Central 5-hydroxytryptamine receptor-linked protein kinase C translocation: a functional postsynaptic signal transduction system

The effect of 5-hydroxytryptamine (5-HT) receptor stimulation on protein kinase C (PKC) activity and translocation was assessed in slices or synaptosomes obtained from rat brain. Serotonin (0.5-100 microM) and the specific 5-HT2 receptor agonist 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) (0.01-10 microM) but not the 5-HT1A or 5-HT1B agonists elicited time- and dose-related translocations in cortical slices. The maximal translocation elicited by 5-HT (10-100 microM, 15 min) or DOI (1 microM, 10 min) was similar to that achievable by the phorbol ester phorbol myristate acetate (PMA) (162 nM). In synaptosomes, short exposures to depolarizing concentrations of K+ (45-65 mM) resulted in PKC translocation. In addition, PMA but not serotonin induced enzyme translocation in synaptosomes. In slices, serotonin-stimulated PKC translocation was prevented by 5-HT2 antagonists but not by dopamine or alpha-adrenergic antagonists. PKC translocation induced by serotonin but not by PMA was inhibited by incubation of slices in a Ca2+-free medium. However, addition of 0.5 mM ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid to the incubation mixture abolished the effects of both serotonin and PMA. These results indicate that, in cortical slices, serotonin operating via a 5-HT2 postsynaptic receptor can induce the translocation of PKC from cytosol to membrane. This action of the neurotransmitter appears to be dependent on extracellular Ca2+.     

Characterization of fMet-Leu-Phe receptor-mediated Ca2+ influx across the plasma membrane of human neutrophils

N-Formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) stimulation of human neutrophils leads to a rapid increase of the cytosolic free Ca2+ concentration, [Ca2+]i, which is significantly reduced by removal of extracellular calcium. In the present study we show that fMet-Leu-Phe-induced [Ca2+]i increases are, in part, mediated by an increase of the plasma membrane permeability to Ca2+. This conclusion is based on the following evidence. In the presence of extracellular calcium, addition of La3+ reduced the fMet-Leu-Phe-induced [Ca2+]i increase to approximately the same level as that observed in the absence of extracellular calcium. A net increase of the plasma membrane permeability for Mn2+ could be observed after fMet-Leu-Phe stimulation, as revealed by intracellular quenching of the quin2 signal. The influx of Mn2+, like that of Ca2+, was inhibited by La3+ and was more pronounced in the absence of extracellular Ca2+, suggesting competition for the same pathway. Temporal dissociation of intracellular Ca2+ release from stores and Ca2+ influx from the medium could be demonstrated by readdition of calcium to cells stimulated in the absence of this cation. This second [Ca2+]i increase could be abolished either by giving the specific chemotactic peptide receptor antagonist, BOC-Met-Leu-Phe, or Co2+. We could also show that the fMet-Leu-Phe-dependent Ca2+ influx was not due to the activation of voltage-dependent calcium channels since depolarization either by K+ or gramicidin D did not affect the resting [Ca2+]i, nor did it affect a subsequent [Ca2+]i increase induced by fMet-Leu-Phe. Furthermore, nifedipine and verapamil, at concentrations known to block classical voltage-dependent calcium channels, had no significant effects on the Ca2+ influx induced by fMet-Leu-Phe. We suggest that fMet-Leu-Phe promotes influx of Ca2+ ions across the plasma membrane of human neutrophils by opening of receptor-dependent calcium channels.     

The effects of phorbol esters on choline phospholipid hydrolysis in heart and brain

The efflux of choline was determined in rat striatal slices, incubated chicken atria and perfused chicken hearts. 4 beta-Phorbol-12 beta,13 alpha-dibutyrate (PDB) and 4 beta-phorbol-12 beta-myristate, 13 alpha-acetate (PMA) were used to stimulate protein kinase C. The other phorbol esters, 4 beta-phorbol-13 alpha-acetate (PAc) and 4 alpha-phorbol-12 beta,13 alpha-didecanoate (4 alpha PDD), known to be inactive, were tested to evaluate the specificity of the responses. PDB markedly enhanced the efflux of choline in all of the three preparations. The PDB-evoked efflux of choline in incubated chicken atria was equal to the net production of choline and, therefore, was not caused by translocation of intracellular free choline. After inhibition of the cholinesterase activity, PDB linearly increased the efflux of choline in rat striatal slices, but failed to alter the spontaneous efflux of acetylcholine. Thus acetylcholine did not serve as the source of the PDB-evoked efflux of choline. PMA was as effective as PDB, whereas PAc and 4 alpha PDD failed to alter the choline efflux in the perfused heart. Both infusion of a Ca2(+)-free EGTA-containing Tyrode solution and mepacrine reduced the spontaneous efflux of choline by about 40% and blocked the PDB-evoked efflux of choline. In contrast, a Ca2(+)-free solution without EGTA failed to alter the spontaneous and the PDB-evoked choline efflux. It is concluded that phorbol esters stimulate the hydrolysis of choline-containing phospholipids in heart and brain via activation of protein kinase C.     

Calphostin C-sensitive enhancements of force by lysophosphatidylinositol and diacylglycerols in mesenteric arteries from the rat.

1. A pharmacological characterization was made of the effects of lysophosphatidyl-inositol (lysoPI) and -ethanolamine (lysoPE) on the Ca(2+)-sensitivity of contraction in alpha-toxin permeabilized rat mesenteric arteries. The effect of GTP gamma S (G-protein activator), diacylglycerols (DAGs, dioctanoyl glycerol (diC8) and 1-stearoyl-2-arachidonoyl-sn-glycerol) and phorbol myristate acetate (PMA, protein kinase C (PKC) activator) on Ca(2+)-sensitivity was also assessed. 2. LysoPI increased the Ca(2+)-sensitivity, demonstrated by both an increase in tension induced by 1 microM [Ca2+]free and an increase in the Ca(2+)-sensitivity of Ca2+ concentration-tension curves. LysoPE did not enhance force or Ca(2+)-sensitivity. 3. GTP gamma S enhanced force at constant Ca2+, increased the Ca(2+)-sensitivity, and increased force under Ca(2+)-free conditions. PMA also increased force at constant Ca2+ and increased Ca(2+)-sensitivity, but caused no force development under Ca(2+)-free conditions. 4. DAGs, both diC8 and the more physiological relevant DAG, 1-stearoyl-2-arachidonoyl-sn-glycerol, enhanced force at constant Ca2+ and increased the Ca(2+)-sensitivity. DiC8, in contrast to 1-stearoyl-2-arachidonoyl-sn-glycerol, caused force development under Ca(2+)-free conditions and substantially enhanced force at maximal Ca(2+)-induced contraction. GDP-beta-S abolished the increased Ca(2+)-sensitization induced by noradrenaline, but not that by DAGs. 5. The PKC inhibitor calphostin C completely abolished Ca(2+)-sensitization induced by all of the Ca(2+)-sensitizing agents. 6. These results show that lysoPI can increase the Ca(2+)-sensitivity of smooth muscle contraction, and the Ca(2+)-sensitization induced by DAGs was not completely G-protein mediated, because it was not inhibited by GDP-beta-S. A central role for PKC in regulation of Ca(2+)-sensitization in rat mesenteric small arteries was indicated by the abolishment of Ca(2+)-sensitization by calphostin C.     

Modulation of mitochondrial metabolic function by phorbol 12-myristate 13-acetate through increased mitochondrial translocation of protein kinase Calpha in C2C12 myocytes

Protein kinase C (PKC) agonists including phorbol 12-myristate 13-acetate (PMA) not only induce the redistribution of cytosolic PKC to various subcellular compartments but also activate the kinase domain of the protein. In the present study we have investigated the nature of mitochondrial PKC pool and its effects on mitochondrial function in cells treated with PMA. Treatment of C2C12 myoblasts, C6 glioma and COS7 cells with PMA resulted in a dramatic redistribution of intracellular PKCalpha pool, with large fraction of the protein pool sequestered in the mitochondrial compartment. We also observed mitochondrial PKCdelta accumulation in a cell restricted manner. The intramitochondrial localization was ascertained by using a combination of protection against protease treatment of isolated mitochondria and immunofluorescence microscopy. PMA-induced mitochondrial localization of PKCalpha was accompanied by increased mitochondrial PKC activity, altered cell morphology, disruption of mitochondrial membrane potential, decreased complex I and pyruvate dehydrogenase activities, and increased mitochondrial ROS production. All of these changes could be retarded by treatment with PKC inhibitors. These results show a direct role for PMA-mediated PKCalpha translocation to mitochondria in inducing mitochondrial toxicity.     

Discrimination of histamine H1 and muscarinic receptor-mediated signalling pathways by phorbol ester in human astrocytoma cells

1. Histamine H1 receptor-mediated signalling was compared with muscarinic receptor-mediated signalling in 1321N1 human astrocytoma cells. 2. Short-term (2 min) treatment of cells with phorbol 12-myristate 13-acetate (PMA) resulted in a reduction of increases in intracellular Ca2+ ([Ca2+]i) induced by carbachol or histamine. 3. Carbachol-induced increases in [Ca2+]i were 10-fold more sensitive to PMA than the histamine-induced increases. 4. When cells were treated with PMA for 48 or 72 h (long-term treatment), protein kinase C (PKC) was down-regulated and PMA did not inhibit carbachol-induced increases in [Ca2+]i. 5. Histamine-induced increases in [Ca2+]i were significantly reduced by long-term treatment with PMA. 6. These findings suggest that the signalling pathways mediated by histamine H1 and muscarinic receptors can be distinguished by using PKC in 1321N1 human astrocytoma cells.     

Molsidomine inhibits the chemoattractant-induced respiratory burst in human neutrophils via a no-independent mechanism.

3-Morpholino-sydnonimine (SIN-1) is a NO-releasing compound which mimics the effects of cGMP through activation of soluble guanylyl cyclase. Its prodrug, molsidomine (SIN-10), does not release NO but does modulate various cell functions. These findings prompted us to study the effects of SIN-10 and SIN-1 on the respiratory burst in human neutrophils. SIN-10 was more effective than SIN-1 in inhibiting superoxide anion (O2-) formation induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) and by C5a. The effects of SIN-1 and SIN-10 on O2- formation were additive or less than additive, indicating the sydnonimines acted through a common mechanism. The sydnonimines showed no effect on O2- formations induced by gamma-hexachlorocyclohexane, arachidonic acid and a phorbol ester. They did not inhibit O2- formation induced by xanthine oxidase, by autoxidation of pyrogallol and in a cell-free system from HL-60 leukemic cells. Neutrophils did not convert SIN-10 to SIN-1 as assessed by O2 consumption which accompanies NO release from SIN-1. The cell-permeant analogue of cGMP, N2,2'-O-dibutyryl guanosine 3':5'-monophosphate (Bt2cGMP), and SIN-10 but not SIN-1 inhibited fMet-Leu-Phe-induced O2 consumption. SIN-1 and SIN-10 slightly enhanced agonist binding to formyl peptide receptors, whereas Bt2cGMP was inhibitory. The sydnonimines did not affect GTP hydrolysis of heterotrimeric regulatory guanine nucleotide-binding proteins in HL-60 membranes. SIN-1 but not SIN-10 stimulated ADP-ribosylation of a 39-kDa protein in the cytosol of HL-60 cells. SIN-10 reduced fMet-Leu-Phe-induced rises in cytosolic Ca2+ concentration in neutrophils. These data suggest that SIN-10 inhibits the respiratory burst via a NO-independent mechanism which may involve inhibition of rises in cytosolic Ca2+ concentration.     

Role of sodium in intracellular calcium elevation and leukotriene B4 formation by receptor-mediated activation of human neutrophils

The role of Na(+) and Na(+) exchangers in intracellular Ca(2+) elevation and leukotriene B(4) (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na(+) with N-methyl-D-glucamine(+) (NMDG(+)) resulted in over 85% inhibition of the LTBs generation observed (from 14.1+/-0.9pmol/10(6) neutrophils to 1.7+/-1.0pmol/10(6) neutrophils at 0.3 microM fMLP). Isotonic substitution of Na(+) with NMDG(+) also induced a significant inhibition of fMLP-induced rise in cytosolic Ca(2+) concentration ([Ca(2+)](i)) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na(+)/Ca(2+) exchanger (benzamil) did not inhibit either [Ca(2+)](i) rise or LTBs production, indicating that the observed effects of extracellular Na(+)-deprivation were unrelated to the Na(+)/Ca(2+) exchanger in receptor-mediated Ca(2+) influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na(+) depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca(2+) influx is required for leukotriene synthesis and that this process is independent of Na(+)-deprivation. Exposure to Na(+)-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na(+)/H(+) exchanger in intracellular Na(+) depletion. Reducing the time of Na(+)-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca(2+)](i) rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na(+) concentration, and, at variance with previously published results, unrelated to the Ca(2+) influx through the Na(+)/Ca(2+) exchanger.     

The structural requirements for phorbol esters to enhance noradrenaline and dopamine release from rat brain cortex.

1. The effects of various protein kinase C (PKC) activators on the stimulation-induced (S-I) release of noradrenaline and dopamine was studied in rat cortical slices pre-incubated with [3H]-noradrenaline or [3H]-dopamine. The aim was to investigate a possible structure-activity relationship for these agents on transmitter release. 2. 4 beta-Phorbol 12,13-dibutyrate (4 beta PDB, 0.1-3.0 microM), enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner whereas the structurally related inactive isomer 4 alpha-phorbol 12, 13-dibutyrate (4 alpha PDB, 0.1-3.0 microM) and phorbol 13-acetate (PA, 0.1-3.0microM) were without effect on noradrednaline release. Another group of phorbol 12, 13-diesters containing a common 13-ester substituent (phorbol 12, 13-diacetate, PDA, 0.1-3.0 microM; phorbol 12-myristate 13-acetate, PMA, 0.1-3.0 microM; phorbol 12-methylaminobenzoate 13-acetate, PMBA, 0.03-3.0 microM) also enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner with PMA being the least potent. 3. The 12-deoxyphorbol 13-substituted monoesters, 12-deoxyphorbol 13-acetate (dPA, 0.1-3.0 microM), 12-deoxyphorbol 13-angelate (dPAng, 0.1-3.0 microM), 12-deoxyphorbol 13-isobutyrate (dPiB, 0.03-3.0 microM) and 12-deoxyphorbol 13-phenylacetate (dPPhen, 0.1-3.0 microM) enhanced S-I noradrenaline and dopamine release in a concentration-dependent manner. In contrast, 12-deoxyphorbol 13-tetradecanoate (dPT, 0.1-3.0 microM) was without effect. 4. The involvement of PKC in mediating the effects of the various phorbol esters was further investigated. PKC was down-regulated by 20 h exposure of the cortical slices to 4 beta-phorbol 12,13-dibutyrate (1 microM). In this case the facilitatory effect of 4 beta PDB and dPA was abolished whilst that of dPAng was significantly attenuated. This indicates that these agents were acting selectively at PKC. In support of this the PKC inhibitors, polymyxin B (21 microM) and bisindolylmaleimide I (3 microM), attenuated the facilitatory effect of 4 beta PDB and dPAng although that of dPA was not significantly altered. 5. The effects of these agents on transmitter release were not correlated with their in vitro affinity and isozyme selectivity for PKC. Short chain substituted mono- and diesters of phorbol were more potent enhancers of action-potential evoked noradrenaline and dopamine release than the long chain esters. Interestingly, these former agents are the least potent or non effective (e.g. dPA, PDA) tumour promoters. We suggest that the reason for the poor effects of lipophilic long chain phorbol esters (PMA, dPT) on transmitter release is that they are sequestered in the plasmalemma and do not access the cell cytoplasm where the PKC may be located.     

Differentiation of renal Na(+)-K(+)-ATPase in control and streptozotocin-induced diabetic mice by G-protein acting toxins and phorbol esters.

The specific activity of Na(+)-K(+)-ATPase in the renal medulla and cortex of 50-day-old streptozotocin (STZ)-induced diabetic mice was increased 58% and 50%, respectively, as compared to controls. Km values of Na+ and K+ for this enzyme were unaltered, while that of ATP was decreased in diabetic mice. The Na(+)-K(+)-ATPase in control medulla and cortex was activated by both cholera and pertussis toxins, while this effect was abolished in diabetics. Since dibutyryl cAMP stimulates cortical Na(+)-K(+)-ATPase activity in control mice, the activation effect of cholera toxin on this enzyme might be due to its interaction with a Gs-protein and the persistent stimulation of adenylate cyclase activity, while the effect of pertussis toxin might be due to its masking of the inhibitory action of a Gi-protein on adenylate cyclase activity. However, the protein kinase C (PKC)-associated Na(+)-K(+)-ATPase might also be quiescent in diabetes, because the stimulating effect of phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA) on this enzyme was abolished in diabetic cortex. In addition, nicardipine and ouabain were found to have differential effects on this enzyme derived from control and diabetic mice.     

Different pathways of calcium sensitization activated by receptor agonists and phorbol esters in vascular smooth muscle.

1. It has been shown that receptor agonists and activators of protein kinase C, phorbol esters, increase Ca2+ sensitivity of contractile elements in vascular smooth muscle. To discover if protein kinase C is involved in the agonist-mediated Ca2+ sensitization, we examined the effects of receptor agonists in the rat isolated aorta in which protein kinase C activity had been diminished by pretreatment with phorbol 12-myristate 13-acetate for 24 h. 2. In the aorta with protein kinase C activity, a high concentration (1 microM) of 12-deoxyphorbol 13-isobutyrate induced contraction and a low concentration (100 nM) potentiated high K(+)-induced contraction. In addition, prostaglandin F2 alpha induced greater contractions than high K+ at a given cytosolic Ca2+ level. The maximally effective concentrations of noradrenaline and endothelin-1 also induced greater contraction than high K+. In the aorta without protein kinase C activity, the contraction induced by 12-deoxyphorbol 13-isobutyrate and its potentiation of the high K(+)-induced contraction were abolished. However, prostaglandin F2 alpha, noradrenaline and endothelin-1 still induced a greater contraction than high K+. 3. In the aorta without protein kinase C activity, noradrenaline, endothelin-1 and prostaglandin F 2 alpha, but not 12-deoxyphorbol 13-isobutyrate, induced contractions in the presence of the Ca2+ channel blocker, verapamil, or in the absence of external Ca2+, by increasing Ca2+ sensitivity. 4. In the permeabilized preparations, inhibition of protein kinase C activity abolished the effect of potentiation of the Ca(2+)-induced contraction by 12-deoxyphorbol 13-isobutyrate although the potentiation of the contraction by prostaglandin F2 alpha did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C-mediated Ca2+ entry in HEK 293 cells transiently expressing human TRPV4

1. We investigated whether protein kinase C (PKC) activation stimulates Ca2+ entry in HEK 293 cells transfected with human TRPV4 cDNA and loaded with fura-2. 2. Phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, increased the intracellular Ca2+ concentration ([Ca2+]i) in a dose-dependent manner, with an EC50 value of 11.7 nm. Exposure to a hypotonic solution (HTS) after PMA further increased [Ca2+]i. Two other PKC-activating phorbol esters, phorbol 12,13-didecanoate (PDD) and phorbol 12,13-dibutyrate, also caused [Ca2+]i to increase. 3. The inactive isomer 4alpha-PMA was less effective and the peak [Ca2+]i increase was significantly smaller than that induced by PMA. In contrast, 4alpha-PDD produced a monophasic or biphasic [Ca2+]i increase with a different latency, while 4alpha-phorbol had no effect. 4. The PMA-induced [Ca2+]i increase was abolished by prior exposure to bisindolylmaleimide (BIM), a PKC-specific inhibitor, and suppressed by the nonspecific PKC inhibitor 1-(5-isoquinolinesulphonyl)-2-methylpiperazine. The [Ca2+]i increase induced by 4alpha-PMA, 4alpha-PDD or HTS was not significantly affected by BIM. 5. These results suggest that both PKC-dependent and -independent mechanisms are involved in the phorbol ester-induced activation of TRPV4, and the PKC-independent pathway is predominant in HTS-induced Ca2+ entry.     

Protein kinase C isoforms in bovine aortic endothelial cells: role in regulation of P2Y- and P2U-purinoceptor-stimulated prostacyclin release.

1. Enhanced synthesis of prostacyclin (PGI2) and inositol polyphosphates in bovine aortic endothelial cells in response to ATP and ADP is mediated by co-existing P2Y- and P2U-purinoceptors. Here we examine the regulation of these responses by isoforms of protein kinase C (PKC). 2. Immunoblots with antisera specific for 8 different PKC isoforms revealed the presence of alpha, epsilon and zeta, while no immunoreactivity was found for beta, gamma, delta, eta and theta isoforms. PKC-alpha was largely cytosolic in unstimulated cells and almost all translocated to the membrane (Triton X-100 soluble) after a 1 min treatment with the PKC activating phorbol myristate acetate (PMA); PKC-epsilon was always in a Triton X-100 insoluble membrane fraction, while PKC-zeta was found in both soluble and membrane bound (Triton X-100 soluble) forms in the unstimulated cells and was unaffected by PMA. 3. Treatment with PMA for 6 h led to a 90% downregulation of PKC-alpha, while the immunoreactivity to the epsilon and zeta isoforms remained largely unchanged. 4. After either 10 min or 6 h exposure to PMA the PGI2 response to activation of both receptors was enhanced, while the inositol 1,4,5-trisphosphate response to P2Y-purinoceptor activation was substantially attenuated and the P2U-purinoceptor response was unchanged. Thus the PGI2 response to PMA under conditions when 90% of the PKC-alpha was lost resembles that seen on acute stimulation of PKC by PMA, and the PGI2 response does not correlate with phospholipase C response. 5. Inhibition of PKC with the isoform non-selective inhibitors, Ro 31-8220 and Go 6850 abolished the PGI2 response to both P2U- and P2Y-purinoceptor stimulation. However, Go 6976, which preferentially inhibits Ca2+ sensitive isoforms (such as PKC-alpha) and not Ca2+ insensitive isoforms (such as PKC-epsilon), had no effect on the PGI2 response. 6. The results show that there is a requirement for PKC in the stimulation of PGI2 production by endothelial P2Y- and P2U-purinoceptors. Both downregulation and inhibition studies show that PKC-alpha is not responsible for the regulation of the response to P2-purinergic stimulation, and imply that the response is mediated by PKC-epsilon (PKC-zeta is unresponsive to PMA), or an as yet uncharacterized PKC isoform.     

Distinct PKC isoforms mediate the activation of cPLA2 and adenylyl cyclase by phorbol ester in RAW264.7 macrophages

1. The modulatory effects of protein kinase C (PKC) on the activation of cytosolic phospholipase A₂ (cPLA₂) and adenylyl cyclase (AC) have recently been described. Since the signalling cascades associated with these events play critical roles in various functions of macrophages, we set out to investigate the crosstalk between PKC and the cPLA₂ and AC pathways in mouse RAW 264.7 macrophages and to determine the involvement of individual PKC isoforms. The cPLA₂ and AC pathways were studied by measuring the potentiation by the phorbol ester PMA of ionomycin-induced arachidonic acid (AA) release and prostagladin E₁ (PGE₁)-stimulated cyclic AMP production, respectively.
2. PMA at 1 μM caused a significant increase in AA release both in the presence (371%) and absence (67%) of ionomycin induction, while exposure of RAW 264.7 cells to PMA increased PGE₁ stimulation of cyclic AMP levels by 208%.
3. Treatment of cells with staurosporine and Ro 31-8220 inhibited the PMA-induced potentiation of both AA release and cyclic AMP accumulation, while Go 6976 (an inhibitor of classical PKC isoforms) and LY 379196 (a specific inhibitor of PKCβ) inhibited the AA response but failed to affect the enhancement of the cyclic AMP response by PMA.
4. Long term pretreatment of cells with PMA abolished the subsequent effect of PMA in potentiating AA release, but only inhibited the cyclic AMP response by 42%.
5. Neither PD 98059, an inhibitor of MEK, nor genistein, an inhibitor of tyrosine kinases, had any effect on the ability of PMA to potentiate AA or cyclic AMP production.
6. The potentiation of AA release, but not of cyclic AMP formation, by PMA was sensitive to inhibition by wortmannin. This effect was unrelated to the inhibition of PKC activation as deduced from the translocation of PKC activity to the cell membrane.
7. Western blot analysis revealed the presence of eight PKC isoforms (α, βI, βII, δ, ε, μ λ and ξ) in RAW 264.7 cells and PMA was shown to induce the translocation of the α, βI, βII, δ, ε and μ isoforms from the cytosol to the cell membrane within 2 min.
8. Pretreatment of cells with PMA for 2–24 h resulted in a time-dependent down-regulation of PKCα, βI, βII, and δ expression, while the levels of the other four PKC isozymes were unchanged after PMA treatment for 24 h. A decrease in the potentiation of AA release by PMA was observed, concomitant with the time-dependent down-regulation of PKC.
9. These results indicate that PKCβ has a crucial role in the mediation of cPLA₂ activation by the phorbol ester PMA, whereas PMA utilizes PKC ε and/or μ to up-regulate AC activity.

     

Divergent effects of co-carcinogenic phorbol esters and a synthetic diacylglycerol on human neutrophil chemokinesis and granular enzyme secretion

The effects of two co-carcinogenic phorbol esters (phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBu] and a synthetic diacylglycerol (OAG, 1-oleoyl-2-acetyl-glycerol), which all stimulate protein kinase C, were compared with two inactive phorbol compounds (4 alpha-phorbol and 4 alpha-phorbol didecanoate (4 alpha-PDD)) on three functional properties of stimulated human polymorphonuclear leukocytes (PMNs): release of granular enzymes lysozyme and beta-glucuronidase, chemokinesis, and changes in cytoplasmic free calcium [Ca2+]i. PMA, PDBu and the diacylglycerol, OAG, all caused a dose-dependent and slow (max by 15 min) release of small amounts of lysozyme with much less beta-glucuronidase and no release of cytoplasmic lactate dehydrogenase. Release was unaffected by removal of extracellular Ca2+. PMA, PDBu and OAG inhibited random movement of the cells, did not cause chemokinesis and induced a slow reduction in the basal [Ca2+]i, as measured by the quin-2 method. PMA, PDBu and OAG increased the capacity of five independently-acting stimulants (N-formyl-Met-Leu-Phe, leukotriene B4, C5a des-Arg, platelet activating factor and A23187) to cause release of lysozyme and beta-glucuronidase but strongly inhibited PMN chemokinesis induced by the same five agents and reduced the stimulant-induced increases in [Ca2+]i. PMA was always more potent than PDBu and much more potent than OAG in eliciting these stimulatory or inhibitory effects on human PMNs. In all tests, 4 alpha-phorbol and 4 alpha-PDD were inactive. The results confirm that stimulation of the diacylglycerol/protein kinase C system in human PMN, either by active phorbol esters or the synthetic diacylglycerol, causes bidirectional effects on human PMN function. In particular, activation of the C-kinase causes inhibition of stimulated neutrophil motility, whereas the secretory functions of the cells are enhanced.     

Different patterns of protein kinase C redistribution mediated by alpha 1-adrenoceptor stimulation and phorbol ester in rat isolated left ventricular papillary muscle.

1. In rat left ventricular papillary muscle, phenylephrine, an alpha 1-adrenoceptor agonist, had a staurosporine-sensitive positive inotropic effect and increased the particulate-associated protein kinase C (PKC) activity without significant changes in total PKC activity or in cytosolic Ca2+/phospholipid-independent kinase (PKI) activity. 2. A PKC stimulant, phorbol 12,13-dibutyrate (PDBu), decreased contractility and slightly increased PKC activity in the particulate fractions, with a marked decrease and increase in total PKC and PKI activities, respectively. 3. The PDBu-induced negative inotropic response was attenuated by two protease inhibitors, leupeptine and a microbial peptide isolated from Aspergillus japonicus (E-64), which are known to inhibit the conversion of particulate-associated PKC to PKI. 4. Such differences in the patterns of PKC redistribution, i.e. marked increases in particulate PKC and cytosolic PKI activities caused by phenylephrine and PDBu, respectively, may account for the opposite inotropic effects of PKC stimulation by an alpha 1-agonist and a phorbol ester.     

Phorbol ester-induced contractility and Ca2+ influx in human cultured prostatic stromal cells

In this study, we investigated the effects of protein kinase C (PKC)-activating phorbol esters upon Ca(2+) influx and contractility in human cultured prostatic stromal cells. Tissue obtained from patients undergoing transurethral resection of the prostate was used to generate explant cultures of prostatic stromal cells. These cells expressed detectable levels of PKCalpha, delta, gamma, lambda, and zeta, but not epsilon, iota, mu, or theta; isoforms and responded to both phorbol 12,13-diacetate (PDA) and 12-deoxyphorbol 13-tetradecanoate (DPT) with concentration-dependent contractions (pEC50+/-SEM 7.07+/-0.41 and 6.39+/-0.27, respectively). The L-type Ca2+ channel blocker nifedipine (3 microM), and the PKC inhibitors G? 6976, G? 6983 (both 100 nM), myristoylated PKC inhibitor 19-27 (20 microM) and bisindolylmaleimide (1 microM) all abolished PDA-stimulated (1 microM) contractions. Neither PDA nor DPT (at 1 microM) caused translocation of any PKC isoform from the cytosolic to the particulate fraction. Nifedipine (3 microM), myristoylated PKC inhibitor 19-27 (20 microM), and bisindolylmaleimide (1 microM) inhibited PDA-stimulated Ca2+ influx into FURA-2 loaded cells. This study indicates that human cultured prostatic stromal cells respond to phorbol esters with contractions that are dependent upon the influx of Ca2+ through L-type Ca2+ channels and that this effect may be independent of the translocation of PKC from cytosolic to particulate fractions.     

Contractile and relaxant effects of phorbol ester in the intestinal smooth muscle of guinea-pig taenia caeci.

1. Effects of phorbol esters on the cytosolic Ca2+ level ([Ca2+]i) and muscle tension in the intestinal smooth muscle of guinea-pig taenia caeci were examined. 2. 12-Deoxyphorbol 13-isobutyrate (DPB, 1 microM) did not change the [Ca2+]i and tension in resting muscle. 3. In high K(+)-stimulated muscle, 1 microM DPB transiently augmented the contraction and decreased [Ca2+]i. 12-Deoxyphorbol 13-isobutyrate 20-acetate (1 microM) and phorbol 12, 13-dibutyrate (1 microM) showed similar effects to DPB whereas phorbol 12-myristate 13-acetate (1 microM) and phorbol 12, 13-didecanoate (1 microM) were ineffective. 4. DPB (1 microM) inhibited both [Ca2+]i and tension stimulated by 300 nM carbachol or 3 microM histamine. In the presence of a higher concentration of carbachol (1 microM), DPB decreased [Ca2+]i and transiently increased muscle tension. 5. In the muscle strips permeabilized with bacterial alpha-toxin, 1 microM DPB shifted the Ca(2+)-tension curve to the left. An inhibitor of protein kinase C, H-7 (30 microM), inhibited the effect of DPB. 6. DPB did not change the high K(+)-induced contraction in the muscle strips pretreated with 3 microM phorbol 12-myristate 13-acetate for 24 h. 7. These results suggest that activation of protein kinase C has dual effects; it augments contraction by increasing the Ca2+ sensitivity of the contractile elements and it inhibits contraction by decreasing [Ca2+]i.