Further evidence for abnormal protein kinase C regulation of macromolecule secretion in fibroblasts from cystic fibrosis patients

In comparison to skin fibroblasts from normal subjects, those from patients with cystic fibrosis (CF): (1) bound [20-³H] phorbol 12,13-dibutyrate (PDBu) with a higher affinity (K_d=25.8 vs 12.8 nM respectively) but expressed a similar number of total phorbol ester binding sites (about 2.5 pmol PDBu bound/mg of protein); (2) exhibited a faster and higher response to 4-phorbol 12-myristate 13-acetate (PMA) for the stimulation of [³⁵S]-labelled glycoconjutate release, but were equally sensitive to the synergistic effect of A23187 on this process; and (3) secreted glycoconjugates with similar [³⁵S]-sulfate and [¹⁴C]-leucine to [¹⁴C]-glucosamine labelling ratios. Taken together, these results provide further evidence for abnormal protein kinase C (PKC) regulation of macromolecule secretion in CF disease.Abbreviations BSA Bovine serum albumin - DBcAMP Dibutyryl cyclic AMP - DMEM Dulbecco's modified Eagle's medium - DMSO Dimethylsulfoxide - LDH Lactate dehydrogenase - PBS Phosphate-buffered saline - PDBu 4-phorbol 12,13-dibutyrate - 4-PDD 4-phorbol 12,13-didecanoate - PMA 4-phorbol 12-myristate 13-acetate - TCA Trichloroacetic acid     

Specific binding of phorbol ester tumor promoters to mouse skin

The tumor promoter 20-³H-phorbol 12,13-dibutyrate bound in a specific manner to particulate preparations from both whole mouse skin and mouse epidermis. The binding, which was comparable in both whole skin and epidermal preparations, occurred rapidly, was reversible upon addition of non-radioactive ligand and showed high affinity (K_D = 2.4 × 10^?8 M). The potencies of phorbol esters for inhibiting binding of ³H-PDBu corresponded to their biological and tumor-promoting activities: phorbol 12-myristate 13-acetate, K_I = 0.74 nM; phorbol 12,13-didecanoate, K_I = 16 nM; phorbol 12,13-dibenzoate, K_I = 82 nM; mezerein, K_I = 98 nM; phorbol 12,13-diacetate, K_I = 3 μM; phorbol 12,13,20-triacetate, K_I = 5.6 μM; phorbol 13-acetate, K_I = 64 μM. The biologically inactive derivatives phorbol (0.88 mM) and 4α-phorbol 12,13-didecanoate (15 μM) did not inhibit binding. Likewise, ³H-PDBu binding was only weakly inhibited by phorbol-related diterpenes which are highly inflammatory but nonpromoting. These structure-activity relationships suggest that the ³H-PDBu binding activity mediates phorbol ester tumor promotion. ³H-PDBu binding was not inhibited by the nonphorbol promoters examined. Similarly, it was not blocked by compounds which antagonize (dexamethasone acetate, 2 μM; retinoic acid, 10 μM) or mimic (epidermal growth factor, 100 ng/ml; melittin, 25 μg/ml; PGE₂, 1 μM) some of the effects of the phorbol esters in vivo or in vitro.     

Vinculin phosphorylation in response to calcium and phorbol esters in intact cells

Vinculin phosphorylation in both chick embryo fibroblasts and Swiss 3T3 cells was increased by either calcium or biologically active phorbol esters. Increased phosphorylation of vinculin was noted as early as 10 min following phorbol 12-myristate 13-acetate treatment and was maximal at about 1 h. Maximal increases in phosphorylation were noted at approximately 100 nM phorbol 12-myristate 13-acetate. Phorbol 12,13-dibutyrate (80 nM), a less potent phorbol ester, resulted in smaller increases in vinculin phosphorylation than phorbol 12-myristate 13-acetate at equimolar concentrations. Phorbol, dibutyryl cAMP, and dibutyryl cGMP had no significant effect on phosphorylation. No correlation was found between vinculin phosphorylation and the morphological changes induced by phorbol esters. Tryptic peptide analysis of vinculin revealed multisite phosphorylation. Phosphorylation of only three of the peptides was significantly increased following phorbol 12-myristate 13-acetate treatment. Phosphoamino acid analysis revealed increases at both serine and threonine residues. The low level of phosphotyrosine present in control cells was not significantly increased by phorbol 12-myristate 13-acetate treatment. These findings combined with studies of vinculin phosphorylation by purified protein kinase C (Werth, D. K., Niedel, J. E., and Pastan I. (1983) J. Biol. Chem. 258, 11423-11426) suggest the hypothesis that protein kinase C may be involved in regulation of phosphorylation of vinculin, a cytoskeletal protein.     

Characterization of phorbol diester binding to isolated cardiac myocytes

There are specified and saturable binding sites for [20-3H]phorbol-12,13-dibutyrate on enzymatically dissociated rat cardiac myocytes. At 37 degrees C, maximal binding occurs within 20 min, with a KD of 3.9 nM and Bmax of 0.275 pmol/mg. [3H]Phorbol dibutyrate binding is blocked by 12-O-tetradecanoyl phorbol-13-acetate but not by 4 alpha-phorbol or 4 alpha-phorbol-12,13-dibutyrate. Dibucaine, tetracaine, chlorpromazine, and phospholipase C lowered phorbol binding through a competitive mechanism. Similarly, unsaturated (but not saturated) diacylglycerols competed with [3H]phorbol dibutyrate for the binding site. There was a progressive decline in specific binding of phorbol diesters to cardiac myocytes which occurred primarily during the first 3 weeks of postnatal life. Cardiac phorbol diester receptors may mediate protein kinase C-dependent effects on important cellular functions such as Ca2+ transport.     

Phorbol ester-induced activation of protein kinase C leads to increased formation of diacylglycerol in human neutrophils

Human neutrophils stimulated with a phorbol ester (phorbol 12-myristrate 13-acetate or phorbol 12,13-dibutyrate) responded with an increase in diacylglycerol, considered the natural activator of protein kinase C. The amounts of diacylglycerol formed were considerable, reaching 700-900% of basal after 20 min. In contrast, 4-alpha-phorbol 12-myristate 13-acetate did not induce any detectable formation of diacylglycerol. Simultaneously, phorbol 12-myristate 13-acetate exposure caused increased breakdown of both phosphatidylcholine and phosphatidylinositol 4,5-bisphosphate. These results suggest that once activated, protein kinase C can positively modulate its own activity by inducing additional formation of diacylglycerol from at least two different sources.     

Inhibition of insect juvenile hormone synthesis by phorbol 12-myristate 13-acetate

The synthesis of insect juvenile hormone III (JH III) by isolated corpora allata of the cockroach Diploptera punctata incubated in vitro is inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and 1-oleyl-2-acetylglycerol. 4 alpha-Phorbol 12,13-didecanoate and diolein are inactive. The inhibitory effect of phorbol 12-myristate 13-acetate is fully reversed by 2E,6E-farnesol or by 2E,6E-farnesoic acid. It is highest in corpora allata that are past their peak in secretory activity or that have been inhibited by injections of 20-hydroxyecdysone. This effect of phorbol esters implicates protein kinase C in the regulation of insect corpus allatum activity.     

Phorbol ester-induced stimulation of prostaglandin biosynthesis in human amnion cells

Both phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (10(-8)-10(-6) M) induced concentration-dependent increases in prostaglandin E2 (PGE2) production by human amnion cells, with maximum stimulations of 10.8-fold and 5.9-fold, respectively. 4 alpha-Phorbol 12,13-didecanoate, an inactive phorbol ester analogue, had little or no effect on PGE2 production by amnion cells. PMA and phorbol 12,13-dibutyrate (10(-7) M) induced a maximal increase in the rate of PGE2 biosynthesis within 15 min of treatment. These results suggest that there is an active protein kinase C present in amnion cells that is linked to arachidonic acid release and/or metabolism.     

Alteration of the GTP-dependent inhibitory pathway of rat striatal adenylate cyclase by phorbol esters

In membranes of rat striatum, phorbol 12-myristate 13-acetate (PMA), a potent activator of Ca²⁺/phospholipid-dependent protein kinase, enhanced adenylate cyclase activity by counteracting the inhibition elicited by GTP. Exposure to pertussis toxin caused a similar alteration of the GTP-regulation of the enzyme activity and largely prevented the PMA effects. PMA treatment increased by threefold the GTP requirement of acetylcholine-induced inhibition of adenylate cyclase activity but did not affect the GTP-dependence of the enzyme stimulation by dopamine. The hydrolysis of GTP by membrane-bound high affinity GTPase was significantly inhibited by PMA (IC 50 10 nM) in a Ca²⁺-dependent manner. Like PMA, phorbol 12, 13-dibutyrate inhibited the GTPase activity, whereas the biologically inactive 4- phorbol 13-acetate and 4- phorbol were without effect. These results suggest that activation of Ca²⁺/phospholipid-dependent protein kinase by PMA stimulates adenylate cyclase activity by impairing the activity of the GTP-dependent inhibitory protein, possibly through a reduction of the GTP-GDP exchange.     

The lipophilicity of phorbol esters as a critical factor in determining the pattern of translocation of protein kinase C delta fused to green fluorescent protein

Our previous study showed differential subcellular localization of protein kinase C (PKC) delta by phorbol esters and related ligands, using a green fluorescent protein-tagged construct in living cells. Here we compared the abilities of a series of symmetrically substituted phorbol 12,13-diesters to translocate PKC delta. In vitro, the derivatives bound to PKC with similar potencies but differed in rate of equilibration. In vivo, the phorbol diesters with short, intermediate, and long chain fatty acids induced distinct patterns of translocation. Phorbol 12,13-dioctanoate and phorbol 12,13-nonanoate, the intermediate derivatives and most potent tumor promoters, showed patterns of translocation typical of phorbol 12-myristate 13-acetate, with plasma membrane and subsequent nuclear membrane translocation. The more hydrophilic compounds (phorbol 12,13-dibutyrate and phorbol 12,13-dihexanoate) induced a patchy distribution in the cytoplasm, more prominent nuclear membrane translocation, and little plasma membrane localization at all concentrations examined (100 nM to 10 microM). The highly lipophilic derivatives, phorbol 12,13-didecanoate and phorbol 12, 13-diundecanoate, at 1 microM caused either plasma membrane translocation only or no translocation at incubation times up to 60 min. Our results indicate that lipophilicity of phorbol esters is a critical factor contributing to differential PKC delta localization and thereby potentially to their different biological activities.     

Phorbol ester receptors and the induction of differentiation in the human T lymphoblastic cell line MOLT-3

To examine the mechanism of induction of differentiation in the human malignant T-lymphoblastic cell line, MOLT-3, by 12-O-tetradecanoylphorbol-13-acetate (TPA), the role of receptors for phorbol esters was investigated. Binding of [20-3H]-phorbol-12,13-dibutyrate to TPA-resistant subclones derived from MOLT-3 was less than 50% of that of the parental MOLT-3. Scatchard analysis showed that the concentration of phorbol ester receptors in a TPA-resistant subclone was about 50% of that in the parental MOLT-3, but affinities of binding were similar, indicating that more than a certain number of phorbol ester receptors is required to induce differentiation by TPA in this human T cell line.     

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling) : Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [³H]phorbol-12,13-dibutyrate ([³H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 10⁵ PDBu molecules were bound to each cell. The binding of [³H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [³H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.     

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.     

Protein Kinase C of Sympathetic Neuronal Membrane Is Activated by Phorbol Ester-Correlation Between Transmitter Release, 45Ca2+ Uptake, and the Enzyme Activity

The effects of phorbol esters [phorbol 12,13-dibutyrate (PDB), 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 13-acetate] were investigated on the release of [3H]norepinephrine, 45Ca2+ accumulation, and protein kinase C activity in cultured sympathetic neurons of the chick embryo. Sympathetic neurons derived from 10-day-old chick embryo were cultured in serum-free medium supplemented with insulin, transferrin, and nerve growth factor. After 3 days, neurons were loaded with [3H]-norepinephrine and the release of [3H]norepinephrine was determined before and after electrical stimulation. Stimulation at 1 Hz for 15 s increased the release of [3H]-norepinephrine over the nonstimulation period. Stimulation-evoked release gradually declined with time during subsequent stimulation periods. Incubation of neurons in Ca2+-free Krebs solution containing 1 mM EGTA completely blocked stimulation-evoked release of [3H]-norepinephrine. Stimulation-evoked release of [3H]-norepinephrine was markedly facilitated by 3 and 10 nM PDB or TPA. The spontaneous release was also enhanced by PDB and TPA. The net accumulation of 45Ca2+ during stimulation of sympathetic neurons was increased by two- to fourfold in the presence of PDB or TPA. PDB at 1-100 nM produced a concentration-dependent increase in the activation of protein kinase C. PDB at 30 nM increased the activity of protein kinase C of the particulate fraction from 0.09 to 0.58 pmol/min/mg protein. There was no significant change in protein kinase C activity of the cytosolic fraction (0.14 pmol/min/mg versus 0.13 pmol/min/mg protein). The ratio of the particulate to cytosolic protein kinase C increased from a control value of 0.62 to 4.39 after treatment with 30 nM PDB. TPA (10 and 30 nM) also increased protein kinase C activity of the particulate fraction by six- to eightfold. Phorbol 13-acetate had no effect on protein kinase C activity, [3H]norepinephrine release, and 45Ca2+ accumulation. These results provide direct evidence that activation of protein kinase C enhances Ca2+ accumulation, which in turn leads to the facilitation of transmitter release in sympathetic neurons.     

Inhibition of denuded mouse oocyte meiotic maturation by tumor-promoting phorbol esters and its reversal by retinoids

Two tumor-promoting phorbol esters, phorbol 12,13-dibutyrate (PDBu) and phorbol 12-myristate 13-acetate (PMA), when added to the culture medium of denuded mouse oocytes prevent their spontaneous meiotic maturation, whereas phorbol 13-acetate, which is inactive as a tumor promoter, does not inhibit this process. Retinoids appear to antagonize this inhibitory action of tumor promoters. However, the inhibitory effect of forskolin on meiotic maturation is not prevented, but is potentiated by retinal. These data indirectly suggest a role for calcium and/or phospholipids in the regulation of meiotic maturation. They also suggest that forskolin and phorbol esters mediate their effects through different pathways.     

Phorbol-ester stimulated lysis of weak and nonspecific target cells by cytotoxic T lymphocytes

We have investigated the effects of short-term incubation of cloned and in vivo-produced cytotoxic T lymphocytes (CTL) with phorbol esters on their lytic activity against weak and nonspecific targets. These experiments demonstrate that 4 beta-phorbol-12-myristate, 13-acetate (PMA), 4 beta-phorbol-12,13-dibutyrate, and 4 beta-phorbol-12,13-didecanoate, but not the 4 alpha-phorbol-12,13 didecanoate esters stimulate the lytic apparatus. The stimulation is specific for the CTL rather than the target and appears to be nearly instantaneous in action. This rapid stimulation of the CTL lytic process is consistent with previously reported effects of phorbol esters associated with T cell activation in other functional assays.     

Effects of phorbol-12,13-diesters on prostaglandin production and phospholipase activity in canine kidney (MDCK) cells

Two tumor promoting phorbol-12,13-diesters, 12-0-tetradecanoyl-phorbol-13-acetate and phorbol-12,13-didecanoate, at concentrations of 10^?9 to 10^?10 M, stimulated prostaglandin production by dog kidney (MDCK) cells cultured in serum-supplemented medium. The non-tumor producing phorbol diester, 4α-phorbol-12,13-didecanoate, at a concentration of 10^?7 M, had no effect. The two biologically active phorbol diesters, but not the non-tumor promoting analog, stimulated deacylation of the cellular phospholipids of MDCK cells radioactively labelled with [³H]arachidonic acid. Most of the arachidonic acid was converted into prostaglandins.     

Schistosoma mansoni: evidence for protein kinase-C-like modulation of muscle activity

The phorbol esters, phorbol-12,13-dibutyrate, phorbol-12-myristate-13-acetate, phorbol-12,13-didecanoate, and phorbol-12,13-diacetate, as well as mezerin at concentrations as low as 10 nM produce a spastic paralysis of the schistosome musculature. The action of these protein kinase-C activators is dependent on the sites of esterification and is stereo-specific since phorbol-13,20-diacetate, phorbol-12,13,20-triacetate, 20-oxo, 20-deoxy-beta-phorbol-12,13-dibutyrate, alpha-phorbol-12,13-didecanoate, and alpha-phorbol are inactive. A phospholipid and phorbol ester-dependent protein kinase is identified. This kinase is stimulated by all of the phorbol esters that increase muscle tone but is not stimulated by phorbol esters that do not affect muscle tone. A high affinity, stereo-specific phorbol ester receptor is identified. Dose-response curves of phorbol-12,13-dibutyrate-induced muscle tension and -stimulated kinase activity and receptor binding indicate that these responses are mediated by the same system. These results indicate that protein kinase-C-like enzyme may play an important role in modulating activity of the schistosome musculature.     

Differential sensitivity of agonist- and antagonist-occupied gonadotropin-releasing hormone receptors to protein kinase C activators. A marker for receptor activation

Gonadotropin-releasing hormone (GnRH) stimulates release of pituitary gonadotropins by activating specific plasma membrane receptors. In the present studies, we have used activators of the Ca2+- and phospholipid-dependent protein kinase (protein kinase C) to probe the binding characteristics of agonist- or antagonist-occupied GnRH receptors in intact cell cultures, using a radioligand receptor assay. Specific binding of [125I-Tyr5,D-Ser(tBu)6,Pro9,NHEt]GnRH (Buserelin), a high-affinity GnRH agonist, was increased to 180% of control in the presence of 150 nM phorbol 12-myristate 13-acetate (PMA) or 100 nM phorbol 12,13-dibutyrate (PDB), and to 125% of control in the presence of 200 microM 1,2-dioctanoylglycerol, after 20 min at 23 degrees C. The PMA effects were associated with apparent increases in both binding affinity and number of binding sites. The effects of protein kinase C activators on Buserelin binding were concentration- and time-dependent and were not seen with 4 alpha-PMA or 1,2-dioctanoyl-3-Cl-glycerol, neither of which activate protein kinase C. In contrast, PMA had no measurable effects on specific binding of a GnRH receptor antagonist, Ac[D-pCl-Phe1,2,D-Trp3,125I-Tyr5,D-Lys6,D-Ala10]GnRH. When cell cultures were pretreated with 100 nM PDB in the absence of GnRH and then washed to remove the phorbol ester, no effects of prior protein kinase C activation were detected upon subsequent addition of Buserelin. However, when PDB pretreatment was carried out in the presence of 0.3 microM GnRH, residual enhancement of Buserelin binding, but not antagonist binding, was observed at either 23 or 4 degrees C. The radiolabeled agonist activated, and the antagonist blocked, GnRH receptor-mediated luteinizing hormone release and [3H]inositol phosphate production in cells preloaded with [3H]inositol. These findings suggest that the action of protein kinase C on the GnRH receptor, either direct or indirect, requires the receptor to be in an activated (agonist-occupied) state but does not require receptor internalization. The mechanism of these effects on GnRH agonist binding is not known but may involve sequestration of surface receptors, expression of new receptors, and/or modulation of GnRH receptor affinity.     

Regulation of calcium channels in aortic muscle cells by protein kinase C activators (diacylglycerol and phorbol esters) and by peptides (vasopressin and bombesin) that stimulate phosphoinositide breakdown

Voltage-dependent Ca2+ channels of the aortic cell line A7r5 were studied using 45Ca2+ flux experiments. Ca2+ channels which have been studied belong to the L-type and are very sensitive to inhibitors and activators in the 1,4-dihydropyridine series as well as to (-)desmethoxyverapamil and d-cis-diltiazem. L-type Ca2+ channels in these smooth muscle cells are not affected by cyclic 8-bromo-AMP and dibutyryl cyclic AMP. However, the activity of these channels is strongly depressed after treatment with diacylglycerols (1-oleyl 2-acetylglycerol and 1,2-dioctanoylglycerol). Phorbol esters, which like diacylglycerols are well-known activators of protein kinase C (the Ca2+- and phospholipid-dependent enzyme), inhibit 70% of Ca2+ channel activity (K0.5 = 25 nM for phorbol 12-myristate 13-acetate and K0.5 = 200 nM for phorbol 12,13-dibutyrate). Phorbol esters that are inactive on kinase C are without effect on Ca2+ channel activity. [Arg8]Vasopressin and bombesin, two peptides that are well known for their action on polyphosphoinositide metabolism, inhibit Ca2+ channel activity to the same extent as active phorbol esters (65-70%). Oxytocin has the same type of effect presumably by acting at the V1-receptor. Both effects of [Arg8]vasopressin and oxytocin are suppressed by [1-(beta-mercapto-beta,beta-diethylpropionic acid)4-valine]arginine vasopressin, a specific vasopressin antagonist at the V1-receptor.     

The effects of phorbol ester on anabolism of lipids of cultured human skin fibroblasts

We have investigated the independent effects of phorbol ester (phorbol 12-myristate 13-acetate) on anabolism of the major lipid components in cultured diploid human skin fibroblasts. When we incubated these cells with [3H]acetate in serum-free medium for 18 h in the presence of 16 nM phorbol ester, [3H]acetate incorporation and the cellular content of cholesterol ester increased, and free cholesterol decreased. Enhancement of [3H]acetate incorporation into cholesterol ester was also observed when the cells were incubated with phorbol ester for 5 h in medium containing lipoprotein-deficient serum. Incorporation of [3H]galactose into glycosphingolipids increased many fold upon exposure of the cells either to fetal calf serum or separately to phorbol ester. Therefore, phorbol ester independently affects cholesterol and glycosphingolipid metabolism in a way that may be similar to that reported for serum low-density lipoproteins and unknown other factors in fetal calf serum. We have observed these effects of phorbol ester in the intact living cell. These findings should provide useful means for the study of metabolism of the plasma membrane lipid components in fibroblasts.