Osmotic activation of the Na+/H+ antiport in protein kinase C-depleted lymphocytes

The Na+/H+ antiport of rat thymic lymphocytes is activated when protein kinase C is stimulated by phorbol esters. A similar activation of the antiport is obtained when the cells are treated with hypertonic solutions. We tested the possibility that protein kinase C also mediates the osmotic activation of Na+/H+ exchange. Protein kinase C was depleted by preincubation of thymocytes for 24 hr in the presence of high concentrations of phorbol ester. Disappearance of the enzyme was assessed by direct measurement of phosphotransferase activity, and by the loss of biological responses to phorbol esters. The Na+/H+ antiport in protein kinase C-depleted cells was not stimulated by addition of phorbol ester, but responded normally to hypertonic treatment. The results indicate that the osmotic activation of countertransport does not require stimulation of protein kinase C.     

Sphingosine reverses growth inhibition caused by activation of protein kinase C in vascular smooth muscle cells.

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.     

Phorbol Esters Enhance Neurotransmitter-Stimulated Cyclic AMP Production in Rat Brain Slices

The effect of phorbol esters on cyclic AMP production in rat CNS tissue was examined. Using a prelabeling technique for measuring cyclic AMP accumulation in brain slices, it was found that phorbol 12-myristate, 13-acetate (PMA) enhanced the cyclic AMP response to forskolin and a variety of neurotransmitter receptor stimulants while having no effect on second messenger accumulation itself. A short (15-min) preincubation period with PMA was required to obtain maximal enhancement, whereas the augmentation was lessened by prolonged exposure (3 h) to the phorbol. The response to PMA was concentration dependent (EC50 = 1 microM) and regionally selective, being most apparent in forebrain, and was not influenced by removal of extracellular calcium or by inhibition of phosphodiesterase or phospholipase A2. Only those phorbols known to stimulate protein kinase C augmented the accumulation of cyclic AMP. Moreover, the membrane substrates phosphorylated by endogenous C kinase and by a partially purified preparation of this enzyme were similar. The results suggest that phorbol esters, by activating protein kinase C, modify the cyclic AMP response to brain neurotransmitter receptor stimulation in brain by influencing a component of the adenylate cyclase system beyond the transmitter recognition site.     

Phosphatidylcholine hydrolysis stimulated by phorbol myristate acetate is mediated principally by phospholipase D in endothelial cells

The mechanism of phosphatidylcholine (PC) degradation stimulated by phorbol myristate acetate (PMA) was investigated in bovine pulmonary artery endothelial cells prelabeled with [methyl-3H]choline ([3H]choline) or [9,10-3H]myristic acid ([3H]myristic acid). Both labels were selectively incorporated into PC, and addition of PMA stimulated comparable losses of 3H from PC in cells prelabeled with [3H]choline or [3H]myristate. In cells prelabeled with [3H]choline, the loss of 3H from PC correlated with a rapid increase in intracellular free [3H]choline. The increase in intracellular [3H]choline stimulated by PMA was not preceded by an increase in any other 3H-labeled PC degradation product. PMA did not stimulate the formation of PC deacylation products in cells prelabeled with [3H]choline. In permeabilized cells prelabeled with [3H]choline, PMA stimulated the formation of [3H]choline but not [3H]phosphocholine. In intact cells prelabeled with [3H]myristate, the loss of 3H from PC induced by PMA correlated with the formation of [3H]phosphatidic acid ([3H]PA) and [3H]diacylglycerol. In the presence of ethanol, PMA stimulated the formation of [3H]phosphatidylethanol ([3H]PEt) at the expense of [3H]PA. The time-course of [3H]PEt formation was similar to the time-course of intracellular [3H]choline formation in cells stimulated with PMA. These data taken together support the notion that PC degradation in endothelial cells stimulated with PMA is mediated principally by phospholipase D. PC breakdown via phospholipase D was not observed in cells treated with phorbol esters incapable of interacting with protein kinase C. Activation of phospholipase D by phorbol esters was inhibited by long-term pretreatment of cells with PMA to down-regulate protein kinase C and by pretreatment of the cells with staurosporine. These data support the notion that activation of phospholipase D by phorbol esters is dependent upon protein kinase C.     

The inhibition of phosphoenolpyruvate carboxykinase (guanosine triphosphate) gene expression by insulin is not mediated by protein kinase C

The role protein kinase C plays in the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin and phorbol esters was studied in H4IIE hepatoma cells (ATCC CRL 1548). The combined effects of phorbol 12-myristate 13-acetate (PMA) and insulin on the suppression of mRNA coding for PEPCK (mRNAPEPCK) synthesis were additive. A potent inhibitor of both cyclic nucleotide-dependent protein kinases and protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, inhibited the cAMP and PMA-mediated regulation of mRNAPEPCK synthesis, but did not affect the action of insulin. Desensitization of the protein kinase C pathway by exposure to PMA for 16 h abolished the subsequent action of the phorbol ester, but did not affect insulin- or cAMP-mediated regulation of PEPCK gene expression. We conclude that insulin suppresses PEPCK gene expression independently from the protein kinase C-mediated pathway used by phorbol esters.     

Calcium transients in human platelets monitored by aequorin, fura-2 and quin-2: effects of protein kinase C activation and inhibition

Tumour-promoting phorbol esters and 1,2-dioctanoyl-sn-glycerol both induce calcium transients in platelets. However, these can only be detected in platelets loaded with aequorin, but not in those loaded with the fluorescent probes quin-2 and fura-2 presumably because of intracellular calcium buffering. Several effects induced by phorbol esters and diacylglycerols, including the rise in (Ca2+)i, the stimulation of Na+/H+ transporter and the inhibition of the effects of thrombin alone on (Ca2+)i are potently antagonised by staurosporine, a compound known to inhibit protein kinase C. Higher concentrations of staurosporine themselves inhibit the thrombin-induced calcium transient. Staurosporine inhibits the effects of phorbol esters and dioctanoyl glycerol with equal potency although the latter does not cause enzyme translocation of cytosolic protein kinase C to membranes. These results therefore suggest that some, if not all, the effects of protein kinase C activation can occur without translocation of the enzyme.     

Inhibition of receptor-mediated stimulation of cyclic AMP accumulation in neuroblastoma-hybrid NCB-20 cells by a phorbol ester

Activation of protein kinase C by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), modulates responsiveness of the cyclase system in many cell types. In the neuroblastoma-hybrid cell line NCB-20, PMA causes a reduction in receptor-mediated accumulation of cyclic AMP. The reduction in receptor responses by PMA occurs within 3 min and is still apparent at 40 min. This occurs in a concentration-dependent manner with an EC50 for PMA of approx. 30 nM. Accumulations of cyclic AMP that are elicited by prostaglandin E2, vasoactive intestinal peptide or 2-chloroadenosine are decreased in the presence of PMA. Accumulations of cyclic AMP that are elicited by forskolin in the absence of a receptor agonist are unaffected by the presence of PMA. Inhibition of cyclic AMP generation by dopamine is not diminished by PMA suggesting the receptor input through the inhibitory Ni-guanyl nucleotide binding protein is still functional after PMA treatment. The generalized inhibition of receptor-mediated responses by PMA could be due to a protein kinase C-mediated phosphorylation of the stimulatory Ns-guanyl nucleotide binding protein, but other mechanisms are possible.     

Protein kinase C modulates effects of prostanoids on cyclic adenosine monophosphate in guinea pig chief cells

In the course of examining the role of protein kinase C in signal transduction in dispersed chief cells from guinea pig stomach, we observed that phorbol esters inhibit prostaglandin (PG)-stimulated increases in cyclic adenosine monophosphate (cAMP). Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, decreased maximal levels of PGE2-stimulated cAMP by 40%. This dose-dependent effect was observed within 30 sec and was maximal by 1 min of incubation at 37 degrees C. Phorbols that do not activate protein kinase C did not have this effect. Adding H7, a protein kinase C inhibitor, abolished the inhibitory effects of PMA, indicating that these effects are not caused by activation of cyclic nucleotide phosphodiesterases. PMA did not alter the increase in cellular cAMP caused by cholera toxin, forskolin, secretin, or vasoactive intestinal peptide. Hence the site of these prostanoid-specific actions of protein kinase C does not appear to be stimulatory or inhibitory guanine nucleotide binding proteins or the catalytic component of the adenylyl cyclase system. In dispersed chief cells, activation of protein kinase C may inhibit prostanoid-induced stimulation of the adenylyl cyclase system by a direct effect on prostaglandin receptors.     

Neutrophil beta-adrenergic receptor responses are potentiated by acute exposure to phorbol ester without changes in receptor distribution or coupling.

Exposure to the phorbol ester, phorbol 12-myristate, 13-acetate (PMA, 100nM) for 10 minutes enhanced cyclic AMP accumulation in human neutrophils under basal conditions and in response to the beta-adrenergic receptor agonist isoproterenol (ISO), 1 microM) and the adenylate cyclase activator forskolin (FSK, 10mM). Potentiation of responses to ISO by PMA was dose-dependent between 0.1 and 100nM PMA. The diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG) (50 microM) also elevated beta-receptor responses, but 4 beta-phorbol (100nM), lacking the capacity to activate PMA, was ineffective. Short-term exposure (12 seconds) to the peptide n-formylmethionine leucyl-phenylalanine (FMLP, 1 microM) also elevated neutrophil cyclic AMP accumulation. All potentiating effects of PMA on cyclic AMP production were inhibited by the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7). Elevation of cyclic AMP by FMLP was insensitive to H7. PMA had no apparent effect on beta-receptor agonist-affinity, distribution between cell-surface and internalised compartments, or the capacity of ISO to induce beta-receptor internalisation. Responses to FSK or ISO in terms of fold-stimulation of basal cyclic AMP accumulation in the presence of PMA were not elevated by PMA. These findings indicate that PMA exerts a potentiating effect on neutrophil adenylate cyclase responses through protein kinase C activation. FMLP elevation of neutrophil cyclic AMP in the absence of other stimuli, appears however, to be insensitive to protein kinase inhibition.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Antagonistic effects of phorbol esters on lymphocyte activation. Evidence that protein kinase C provides an early signal associated with lytic function

There is increasing evidence that protein kinase C plays a role in the transduction of an activation signal in lymphocytes. The bulk of this evidence is based on pharmacological experiments involving the tumor promoter phorbol myristate acetate (PMA) as a protein kinase C agonist. However, in cytotoxic T lymphocytes, PMA has been shown to both stimulate and inhibit lytic function. By examining the effects of a series of phorbol esters on protein kinase C activity in lymphocytes, we will demonstrate that these antagonistic effects of PMA on cytotoxic T lymphocyte function are related to multiple effects of PMA on protein kinase C activity.     

Transforming growth factor beta (TGF-beta) reverses phorbol diester resistance of a breast adenocarcinoma (MCF-7) subline

We investigated the effects of TGF-beta on a MCF-7 subline (MCF-7:RPh-4) which is resistant to phorbol diesters with respect to growth inhibition and estrogen receptor content modulation. This biological unresponsiveness of MCF-7:RPh-4 cells to phorbol esters seems to be unrelated to activation of protein kinase C. In the presence of 80 nM PMA (12-O-tetradecanoylphorbol-13-acetate), TGF-beta induced a dose-dependent inhibition of MCF-7:RPh-4 cell proliferation. MCF-7:RPh-4 cells grown in PMA-free medium for at least 28 days remained insensitive to PMA but lost sensitivity to TGF-beta. Under these conditions, addition of 80 nM PMA restored sensitivity to TGF-beta. In the presence of a fixed concentration of TGF-beta, the dose-dependent inhibition of proliferation and the decrease in estrogen receptor content induced by PMA were comparable to those observed in PMA-treated parental MCF-7 cells. These observations indicate that TGF-beta reverses PMA resistance in MCF-7:RPh-4 cells. In addition, TGF-beta does not modify the basal or PMA-stimulated phosphorylation of Mr 28,000 endogenous protein. These results suggest that TGF-beta interferes with the protein kinase C pathway independently of enzyme activation.     

Regulation of Na+/K+/Cl- cotransport and [3H]bumetanide binding site density by phorbol esters in HT29 cells

The involvement of protein kinase C in the regulation of Na+/K+/Cl- cotransport was investigated in cultured HT29 human colonic adenocarcinoma cells. We have demonstrated previously the presence of a Na+/K+/Cl- cotransport pathway in HT29 cells (Kim, H.D., Tsai, Y-S., Franklin, C.C., and Turner, J.T. (1989) Biochim. Biophys. Acta 946, 397-404). Treatment of cells with the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu) caused an increase in membrane-associated protein kinase C activity that was accompanied by a concomitant decrease in cytosolic protein kinase C activity. PMA also produced a rapid transient increase in cotransport to 137% of control values by 5 min followed by a progressive decrease to 19% of control values by 2 h. To determine the underlying mechanism for the reduction in Na+/K+/Cl- cotransport, changes in cotransporter number and/or affinity were determined in radioligand binding studies using [3H]bumetanide. PMA and PDBu produced essentially identical time- and dose-dependent decreases in specific [3H]bumetanide binding that were similar to the observed decreases in cotransport. Analysis of saturation and competition binding data indicated that the decrease in binding was due to a lowered Bmax with no change in affinity. Both the decrease in binding and the changes in cotransport elicited by PMA were prevented by the protein kinase inhibitor H7. These findings suggest that phorbol esters cause a decrease in the number of cotransporters in HT29 cells, resulting in a reduction in Na+/K+/Cl- cotransport activity.     

Multiple regulation of proenkephalin gene expression by protein kinase C

In the present study we investigated the role of protein kinase C (Ca2+/phospholipid-dependent enzyme)-mediated processes in the regulation of proenkephalin gene expression in primary cultures of bovine adrenal chromaffin cells. Activators of protein kinase C such as 1-oleoyl-2-acetylglycerol, mezerein, and the phorbol esters phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate induced a time-dependent increase in proenkephalin mRNA levels, whereas the inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate had no effect. The increase in phorbol ester-induced proenkephalin mRNA was potentiated by low concentrations of the Ca2+ ionophore A23187, suggesting an interaction between protein kinase- and Ca2+-mediated processes in the regulation of proenkephalin mRNA. The phorbol ester-induced stimulation does not appear to be mediated by an interaction with the cAMP-generating system or increases in Ca2+ uptake. However, when proenkephalin mRNA levels were stimulated by KCl (10 mM) and the dihydropyridine BayK8644, PMA exhibited an inhibitory effect on proenkephalin mRNA, which was detectable at a 10-fold lower concentration of PMA than the stimulatory effect. This inhibitory effect appears to be mediated by an inhibition of Ca2+ entry through voltage-dependent Ca2+ channels, as suggested by 45Ca2+ uptake experiments. Thus, the net effect of PMA depends on and varies with the state of voltage-dependent Ca2+ channel activity. A third mode of action by protein kinase C to modulate proenkephalin gene expression is by interaction with the phosphatidylinositol second messenger system. Stimulation of phosphoinositide hydrolysis and proenkephalin mRNA by histaminic H1-receptor activation was inhibited by low concentrations of PMA. We suggest that protein kinase C may act as a positive and negative regulator of proenkephalin gene expression by interacting with at least three receptor-coupled second messenger systems.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Inhibition by phorbol esters of antiimmunoglobulin-induced calcium signalling and B-cell activation

The inhibitory effect of phorbol dibutyrate (PDB) on B-cell stimulation was evaluated using a model in which activation is induced by modest doses of antiimmunoglobulin antibody (anti-Ig) and progression to DNA synthesis is induced by cytochalasin. PDB preferentially inhibited anti-Ig-induced activation and did so during brief (2 hr) preincubation with anti-Ig. Activation was inhibited whether PDB was added before or shortly after anti-Ig. Since activation for cytochalasin responsiveness appears to be mediated by Ca2+, the effect of PDB on the anti-Ig-induced rise in intracellular Ca2+ was evaluated. PDB (and other phorbol esters that activate protein kinase C) inhibited the rise in Ca2+ normally associated with anti-Ig treatment; moreover, PDB reversed an established anti-Ig-induced Ca2+ response. These data suggest that phorbol esters inhibit B-cell activation by interfering with the elevated levels of intracellular Ca2+ produced by cross-linking of surface immunoglobulin by anti-Ig. This could represent a "feedback inhibition" type of response, but it remains to be seen if this occurs under physiological conditions of protein kinase C activation.     

Agonist-induced desensitization of cholinergically stimulated phosphoinositide breakdown is independent of endogenously activated protein kinase C in HT-29 human colon carcinoma cells.

Activation of M3 muscarinic receptors in HT-29 cells by carbachol rapidly increases polyphosphoinositide breakdown. Pretreatment of these cells with carbachol (0.1 mM) for 5 h completely inhibits the subsequent ability of carbachol to increase [3H]inositol monophosphate ([3H]InsP) accumulation, paralleled by a total loss of muscarinic binding sites. In contrast, protein kinase C (PK-C)-mediated desensitization by incubation with phorbol esters [PMA (phorbol 12-myristate 13-acetate)], leading to a time- and dose-dependent inhibition of cholinergically stimulated InsP release (95% inhibition after 4 h with 0.1 microM-PMA), is accompanied by only a 40% decrease in muscarinic receptor binding, which suggests an additional mechanism of negative-feedback control. Neither carbachol nor PMA pretreatment had any effect on receptor affinity. Incubation with carbachol for 15 min caused a small increase of membrane-associated PK-C activity (15% increase, P less than 0.05) as compared with the potency of phorbol esters (PMA) (3-4-fold increase, P less than 0.01). Long-term incubation (4-24 h) with PMA resulted in a complete down-regulation of cytosolic and particulate PK-C activity. Stimulation of InsP release by NaF (20 mM) was not affected after a pretreatment with phorbol esters or carbachol, demonstrating an intact function of G-protein and phospholipase-C (PL-C) at the effector side. Determination of PL-C activity in a liposomal system with [3H]PtdInsP2 as substrate, showed no change in PL-C activity after carbachol (13 h) and short-term PMA (2.5 h) pretreatment, whereas long-term preincubation with phorbol esters (13 h) caused a small but significant decrease in PL-C activity (19%, P less than 0.05). Our results indicate that agonist-induced desensitization of phosphoinositide turnover occurs predominantly at the receptor level, with a rapid loss of muscarinic receptors. Exogenous activation of PK-C by phorbol esters seems to dissociate the interaction between receptor and G-protein/PL-C, without major effects on total cellular PL-C activity.     

Inhibition of stimulated cell responses by phorbol esters and other activators of protein kinase C: sites of action

The addition of the protein kinase C activator phorbol esters to cell suspension a few minutes prior to stimulation inhibits the agonists-induced biochemical changes and cell responses. This inhibition is prevented by protein kinase C inhibitors. Activation of protein kinase C down regulates the stimulated responses by affecting one or more of the steps in the exitation-response coupling. This includes the receptors, the quanine-nucleotide-binding protein, the activity or distribution of phospholipase C, and other steps.