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At least 19 recordsLinked to original sources
PubMed1992

Protein kinase C isoenzymes display differential affinity for phorbol esters. Analysis of phorbol ester receptors in B cell differentiation.

Protein kinase C (PKC) comprises a family of distinct isoenzymes that are involved in signal transduction pathways linking the cell to triggers perceived via membrane receptors. These isoenzymes differ in their tissue distribution, activation requirements, and substrate specificity. One common denominator among different PKC subspecies is their activation by phorbol esters. We have developed a sensitive method permitting the measurement of phorbol ester binding sites, their quantitation, as well as their dissociation kinetics, by performing cytofluorometric analyses on intact cells or on isolated PKC associated to phosphatidylserine vesicles incubated in the presence of fluorochrome-labeled phorbol ester. Both PKC isozymes beta I/beta II and alpha from brain and spleen after incorporation into phosphatidylserine vesicles, display affinities with apparent Kd of 120 and 50 nM, respectively; although PKC gamma from brain exhibits a Kd of 210 nM. In addition to these receptors, on PKC isozymes from spleen, an intermediate affinity phorbol ester receptor (Kd of 3 nM) and an additional high affinity phorbol ester binding site with a Kd of 0.1 to 0.5 nM were also detected. This latter receptor comigrates with high m.w. PKC isoforms. In different cell lines, the phorbol ester binding patterns, as well as the expression of individual PKC isoenzymes, could be positively correlated.

Animals↗
PubMed1992

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.

Binding Sites↗
PubMedDate not supplied

A comparison of the priming effect of phorbol myristate acetate and phorbol dibutyrate on fMet-Leu-Phe-induced oxidative burst in human neutrophils.

Phorbol esters such as phorbol myristate acetate (PMA) and phorbol dibutyrate (PDBU) are generally considered to have similar effects through a similar mechanism, i.e. protein kinase C (PKC) activation. We recently suggested that this was not the case in human neutrophils. To identify further differences between the two phorbol esters, we compared their priming effects on fMet-Leu-Phe-induced superoxide anion (O2-) production, cytosolic PKC activity and binding of fMet-Leu-Phe. Priming could be initiated with a low (0.2 nM) concentration of both PDBU and PMA. Their effects on the pattern of fMet-Leu-Phe-induced superoxide production were similar in both Ca2(+)-containing and Ca2(+)-free medium. PDBU, like PMA, abolished the Ca2+ dependency of fMet-Leu-Phe-induced O2- production in a dose-dependent manner. In cytochalasin B-treated cells and in the presence of Ca2+, priming with PDBU or PMA did not alter the enhancing effect of cytochalasin B on fMet-Leu-Phe-induced O2- production. In Ca2(+)-free medium, priming abolished the Ca2+ dependency of fMet-Leu-Phe stimulation in cytochalasin B-treated cells. Cytochalasin B, however, enhanced the effect of PMA but not that of PDBU. Priming with PDBU was not associated under any experimental conditions with a decrease in cytosolic PKC activity, or an increase in PKM activity before or after fMet-Leu-Phe stimulation. Furthermore, priming effects were abolished by cell washing but not by H-7 or staurosporine, which are potent PKC inhibitors. PDBU, in contrast to PMA, increased fMet-Leu-Phe binding to PMNs through a decrease in the dissociation constant and induced degranulation of specific granules as measured by the release of vitamin B12 binding protein. These findings show that the priming effects of PDBU differ in certain respects from those of PMA, namely with regard to its synergism with cytochalasin B and the expression of fMet-Leu-Phe receptors. In addition, priming concentrations of PDBU, like PMA, did not alter cytosolic PKC activity in fMet-Leu-Phe-stimulated neutrophils.

Cells, Cultured↗
PubMed1992

The protein kinase C activators, phorbol 12-myristate,13-acetate and phorbol 12,13-dibutyrate, are convulsant in the pico-nanomolar range in mice.

Administration of phorbol 12-myristate,13-acetate (PMA, 10 fmol-10 nmol) or phorbol 12,13-dibutyrate (PDB, 0.2-495 nmol) (i.c.v.) to mice induced: hindlimb scratching, tremor, myoclonic jerks, hyperlocomotion, clonic seizure, followed by death or recovery. CD50 values for clonic seizures for PMA and PDB were 1.0 pmol and 1.2 nmol. 4-alpha-Phorbol (68-686 nmol) was inactive. The effects of PDB (24-247 nmol) were reduced by pretreatment with staurosporine (30 nmol, i.c.v.). Protein kinase C activators are potent convulsants in vivo.

Alkaloids↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--IV. Lipophilic binding and molecular orientation of spin-labeled phorbol-12,13-diesters in human erythrocyte membrane.

In human erythrocyte membranes, membrane binding of spin-labeled TPA-analogous phorbol (doxyl)esters [(n,m)PA] was investigated during measurement of the kinetics of the decay of their electron paramagnetic resonance signal by ascorbate reduction. In membrane-bound (n,m)PA the reduction rate was dependent of the position of doxyl in the aliphatic chain of their 12-O-acyl moiety. To describe quantitatively the reaction kinetics observed, two hypotheses (models) were developed and used. Model 1 is based on the assumption that ascorbate reduction takes place in the extracellular space. In this case the experimental data could be fitted by the partition and permeability coefficients of (n,m)PA determining model 1 only, if non-realistic values of these parameters were used. The more refined model 2, corresponding to a bilayer membrane structure, assumes the reduction to take place in the hydrophilic region of the membrane. Assuming a finite probability of finding the doxyl group within the hydrophilic membrane region, model 2 describes quantitatively the dependence of the reduction rate on the position of the doxyl in the aliphatic chain of the (n,m)PA used. From the validity of this model it may be postulated that the molecular orientation of TPA-analogous (n,m)PA in the bilayer membrane is determined by an anchoring of their lipophilic ester moiety in the lipophilic region of the membrane bilayer, thus locating the hydrophilic phorbol moiety within the hydrophilic region of the membrane. With regard to the well-known categories of non-specific versus specific binding of bioactive phorbol esters to protein kinase C/membrane complexes it is deduced that anchoring of (n,m)PA (and hence TPA) in the hydrophobic interior of the membrane structure may be the molecular equivalent of their non-specific binding.

Ascorbic Acid↗
PubMedDate not supplied

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.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Prostratin, a nonpromoting phorbol ester, inhibits induction by phorbol 12-myristate 13-acetate of ornithine decarboxylase, edema, and hyperplasia in CD-1 mouse skin.

Pretreatment of CD-1 mouse skin with prostratin (12-deoxyphorbol 13-acetate) inhibited biological response to phorbol 12-myristate 13-acetate. The three responses examined were hyperplasia, induction of ornithine decarboxylase, and edema; the characteristics of inhibition depended on the specific response. Hyperplasia is the best short-term correlate of tumor promotion. Two or more pretreatments with 2.56 mumol (1 mg) prostratin, administered at intervals of 1-4 days, almost completely blocked the hyperplasia induced by phorbol 12-myristate 13-acetate applied 15 min to 6 h after the last pretreatment. Inducibility of hyperplasia was partially restored at 2 days and recovered by 4 days. Prostratin was more potent for inhibition of ornithine decarboxylase induction (50% inhibitory dose = 25.6 nmol) than it was for hyperplasia: the inhibition was largely attained by the first application, and the recovery from inhibition was slower (8 days). Edema was partially inhibited by prostratin (dose giving 50% of maximal inhibition = 512 nmol). We have previously demonstrated that prostratin is a protein kinase C activator. Our present results show that prostratin is a functional antagonist for a class of protein kinase C mediated responses. The findings emphasize the diversity of biological outcome for protein kinase C activators, presumably driven by the extensive heterogeneity in the protein kinase C pathway.

Animals↗
PubMed1991

Effects of the phorbol ester phorbol 12-myristate 13-acetate (PMA) on islet-cell responsiveness.

Collagenase-isolated rat islets were labelled for 2 h in myo-[2-3H]inositol solution supplemented with 2.75 mM-glucose. The phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.1 or 1 microM) was also present in some experiments. After labelling, islets were washed and then perifused in 2.75 mM-glucose to establish basal [3H]inositol-efflux and insulin-secretory rates. Subsequently, the responses of these islets to stimulation with various agonists were assessed. Inositol phosphate accumulation was measured at the termination of the perifusion. In separate experiments, the cellular location of protein kinase C (PKC) after PMA pretreatment was measured by quantitative immunoblotting of membrane and cytosolic fractions. The following observations were made. (1) Labelling in 0.1-1 microM-PMA had no deleterious effect on total [3H]inositol incorporation during the 2 h labelling period. However, islets labelled for 2 h in 1 microM-PMA were unable to respond, in terms of increases in insulin release, to a 1 microM-PMA stimulus during the subsequent perifusion. (2) As compared with the responses of control islets labelled in 2.75 mM-glucose alone, islets labelled in the additional presence of 1 microM-PMA displayed a significant impairment in phosphoinositide (PI) hydrolysis, but an enhancement of both first-and second-phase insulin secretion, in response to subsequent 20 mM-glucose stimulation. (3) Decreasing extracellular Ca2+ level to 0.1 mM and including the Ca(2+)-channel antagonist nitrendipine (0.5 microM) along with 1 microM-PMA during the [3H]inositol-labelling period did not alter the response of the islets to the subsequent addition of 20 mM-glucose. Glucose-induced PI hydrolysis was still inhibited and 20 mM-glucose-induced insulin release was still enhanced. (4) A markedly amplified and sustained insulin-secretory response to 200 microM-tolbutamide in the presence of 2.75 mM-glucose was also obtained from 1 microM-PMA-pretreated islets. This contrasts sharply with the small and transient response to tolbutamide noted in control islets. (5) When present only during the perifusion phase of the experiments, nitrendipine (0.5 microM) abolished the amplified insulin-secretory responses to both 20 mM-glucose and 200 microM-tolbutamide noted in PMA-pretreated islets. (6) Prior labelling in 1 microM-PMA dramatically amplified the insulinotropic effect of 25 mM-K+ or 5 microM-A23187 stimulation. The amplified insulin-secretory response to K+, but not to A23187, was abolished by inclusion of nitrendipine during the perifusion.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Transcriptional activation of human (2'-5')oligoadenylate synthetase gene expression by the phorbol ester 12-O-tetradecanoyl-phorbol 13-acetate in type-I-interferon-treated HL-60 and HeLa cells.

(2'-5')Oligoadenylate [(2'-5')(A)n] synthetase is a key enzyme in the interferon-elicited antiviral response whose controlled expression in interferon-treated cells has been only partially elucidated. In this investigation, we have compared the modulation of the (2'-5')(A)n synthetase gene by interferon alone and by the combination of interferon and a second cellular effector, 12-O-tetradecanoyl-phorbol 13-acetate (TPA). Although TPA alone had no effect on (2'-5')(A)n synthetase, it potentiated the induction of (2'-5')(A)n of synthetase by interferon in HL-60 and HeLa cells by increasing content of its mRNA and an immunoreactive 40-kDa isoenzyme. Since TPA activates protein kinase C (PKC), other PKC-activating phorbol-ester analogues were tested and found to be effective, whereas the PKC inhibitor staurosporine reduced the potentiative activity of TPA. By using the (2'-5')(A)n synthetase gene promoter linked to a reporter gene, chloramphenicol acetyltransferase (CAT), TPA and interferon were found to result in a doubling of CAT activity compared to cells treated with interferon alone. Moreover, when nuclear extracts prepared from control cells or cells treated with TPA and interferon (IFN), separately or together, were incubated with radioactively labeled oligodeoxynucleotides containing the interferon-responsive element (IRE), TPA was shown to down-regulate an IFN-inducible IRE/protein complex. These data further suggest that TPA regulates (2'-5')(A)n synthetase gene expression at the level of transcription.

2',5'-Oligoadenylate Synthetase↗
PubMed1991

Phorbol diester-induced alterations in the expression of protein kinase C isozymes and their mRNAs. Analysis in wild-type and phorbol diester-resistant HL-60 cell clones.

In an HL-60 cell subline (PR-17) which was greater than 100-fold resistant to the differentiating and cytostatic activities of phorbol 12-myristate 13-acetate (PMA), the protein kinase C phenotype was found to be nearly identical to that of wild-type HL-60 cells. A measurable decrease (30%) in the specific activities of crude preparations of PR-17 cell protein kinase C was observed when the enzyme was measured with histone as the phosphate acceptor substrate, but other aspects of the protein kinase C phenotype (intracellular concentrations and binding affinities of phorbol diester receptors, translocation of activated enzyme from cytosolic to particulate subcellular fractions, relative expression of the alpha and beta isozyme proteins) were equivalent in both PMA-resistant PR-17 cells and in wild-type HL-60 cells. Direct analysis of the behavior of the alpha and beta isozymes after the exposure of each cell type to 100 nM PMA for 12 h revealed that the activities and intracellular concentrations of both isozymes were downregulated to an equivalent extent in both wild-type and PMA-resistant cells. These results suggest that the cellular basis for the resistance to the effects of PMA was present "down-stream" from the activation and down-regulation of protein kinase C and was perhaps a nuclear component. Among the genes which were likely to be differentially regulated when each of the two cell lines were treated with PMA were those for the protein kinase C isozymes themselves. In wild-type HL-60 cells, the intracellular concentrations of type HL-60 cells, the intracellular concentrations of mRNA for each of the beta isozymes were increased (up to 5-fold) 48 h after the initiation of PMA treatment; further studies indicate that an activator of protein kinase C could influence the expression of HL-60 cell protein kinase C genes in an isozyme-specific manner. Comparable PMA-induced alterations in mRNA levels were not observed in PMA-resistant cells, even under conditions of significant activation and subsequent down-regulation of protein kinase C protein. Taken together, these data suggest that activation and down-regulation of the isozymes of protein kinase C may not represent absolute determinants of the PMA-induced differentiation of HL-60 cells, but that specific alterations in the levels of the mRNA for the beta isozymes of protein kinase C, or of other genes which may be regulated by the activated kinase isozymes, are important to the induction of leukemia cell differentiation by PMA.

Animals↗
PubMed1992

Spin-labeled phorbol esters and their interactions with cellular membranes--V. Electron paramagnetic resonance of spin-labeled phorbol-12,13-diesters bound to their receptors in mouse brain particulate fraction.

The relatively small concentrations required for in vivo bioactivity of diterpene ester skin irritants and promoters (approximately 10 nmol per animal; approximately 10 nM in cell cultures) has discouraged studies of EPR spectra of bioactive, TPA-analogous, spin-labeled phorbol-12,13-diesters [(n,m)PA] bound to their membrane receptors, protein kinases C (PKC). To meet the requirements of present EPR spectrometers, particulate fraction from mouse brain containing at least 25 x 10(-12) mol of receptors/mg protein (PKC species) were employed together with certain (n,m)PA selected to give an optimal ratio of specific to non-specific binding. For selection and optimization of experimental conditions, a theoretical model was developed that considers all characteristic parameters of the system. By fitting the model calculations to the experimental data of competitive agonist displacement from the particulate fraction of tritium-labeled TPA, the dissociation constants Kd for four selected (n,m)PA used as antagonists were determined. Optimal experimental conditions are met by (5,6)PA and by (5,8)PA, in that for both compounds the relative amount of displaced (n,m)PA is in accordance with the predictions derived from the model. Moreover, the model turned out also to be reliable for samples containing either small or large amounts of membranes. To obtain an EPR spectrum of an agonist bound to brain particulate fraction, the (5,6)PA was used. It shows a broad EPR spectrum typical for an immobilized molecule. The spectrum changes if an excess of TPA is added to the system; the slight differences in shape are due to displacement of (5,6)PA from specific receptor sites by non-labeled TPA and show up as a decreased central peak amplitude. This is the first time that the agonist/receptor interaction of a diterpene ester type irritant and tumor promoter has been demonstrated by direct spectroscopic measurement.

Animals↗
PubMed1992

Staurosporine induces a neuronal phenotype in SH-SY5Y human neuroblastoma cells that resembles that induced by the phorbol ester 12-O-tetradecanoyl phorbol-13 acetate (TPA).

Treatment of SH-SY5Y human neuroblastoma cells with the protein kinase inhibitor staurosporine, induced both morphological and functional differentiation in these cells. The effects of staurosporine were comparable to those induced by the protein kinase C (PKC) activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA), with respect to induction of neuronal differentiation, i.e. neurite outgrowth, inhibition of DNA synthesis, induction and down-regulation of c-myc protein expression, induction of mRNA for both neuropeptide Y (NPY) and growth associated protein 43 (GAP-43) and stimulation of tyrosine hydroxylase expression. Staurosporine failed to translocate PKC to the membrane fraction or to stimulate phosphorylation of the endogenous PKC substrate M(r) 80,000 (p80). Instead, staurosporine inhibited TPA-induced phosphorylation of p80.

Alkaloids↗
PubMed1991

Phorbol-ester-mediated expression of the collagen type I pro-alpha 2 gene in mouse 3T3-L1 cells and its absence in a phorbol 12-myristate 13-acetate-non-responsive variant.

We have previously isolated a phorbol 12-myristate 13-acetate (PMA)-non-responsive variant line (VT-1) from mouse 3T3-L1 cells [Shimizu, Fujiki, Sugimura & Shimizu (1986) Cancer Res. 46, 4027-4031]. Differential hybridization of cDNAs obtained from PMA-treated 3T3-L1 and VT-1 cells resulted in the isolation of a number of unique cDNA clones, including one with a high degree of sequence similarity to the type I pro-alpha 2 collagen gene (COL1A2) [Amagai, Inokuchi, Nishikawa, Shimizu & Shimizu (1989) Somat. Cell Mol. Genet. 15, 153-158]. Here we examined the expression of type I collagen pro-alpha 2 [pro-alpha 2(I)] mRNA and production of type I collagen in these two cell lines. In quiescent cells, the pro-alpha 2(I) steady-state mRNA levels were four times greater in 3T3-L1 cells than in VT-1 cells. PMA addition caused the steady-state levels of pro-alpha 2(I) mRNA to be six times greater in 3T3-L1 cells than in VT-1 cells, with a maximum at 30-60 min. The pro-alpha 2(I) protein levels in the extracellular matrix or culture media of 3T3-L1 cells were substantially elevated by PMA treatment, but no significant increase was detected in VT-1 cells. The correlation of collagen expression with a PMA-mediated mitogenic response suggests a new role for collagen as an early component of mitogenic signal transduction.

Animals↗
PubMed1990

Phorbol esters and d-tubocurarine up-regulate alpha-bungarotoxin sites in chromaffin cells in culture via distinct mechanisms.

Previous work had shown that nicotinic antagonists resulted in a marked up-regulation of alpha-bungarotoxin sites in chromaffin cells in culture. The present experiments were done to determine the intracellular mechanism(s) whereby nicotinic antagonists might mediate their effects on these receptors. Chromaffin cells were cultured for three days with various concentrations of 4 beta-phorbol 12-myristate 13-acetate, an agent which affects protein kinase C by mimicking the actions of diacylglycerol. The phorbol ester resulted in a dose-dependent increase in alpha-bungarotoxin binding which was maximal with 100 nM 4 beta-phorbol 12-myristate 13-acetate. This increase in binding appeared to be due to an increase in the maximal number of alpha-bungarotoxin sites. Time dependence studies showed that the effect of the phorbol was undetectable with incubations of 24 h or less and appeared to plateau by 72-96 h. A similar increase in toxin binding was also observed with 4 beta-phorbol 12,13-dibutyrate. On the other hand, an inactive analog of 4 beta-phorbol 12-myristate 13-acetate had no significant effect on binding. D-Sphingosine, an inhibitor of protein kinase C, was able to partially block the phorbol ester-induced increase in toxin binding while polymyxin B, another protein kinase C inhibitor, completely prevented the up-regulation of the alpha-bungarotoxin sites. Carbachol and nicotine prevented this enhancement of toxin binding in the presence of 4 beta-phorbol 12-myristate 13-acetate. Although the phorbol ester resulted in an increase in toxin binding, acetylcholine-evoked catecholamine secretion from chromaffin cells in culture was decreased, indicating a dissociation between the functional nicotinic acetylcholine receptor population and the alpha-bungarotoxin sites. To determine whether agents which affect protein kinase C can alter the up-regulation of alpha-bungarotoxin sites by d-tubocurarine, 4 beta-phorbol 12-myristate 13-acetate was added to the cells in combination with the nicotinic antagonist. The up-regulation of toxin binding sites induced by d-tubocurarine was additive with that induced by the phorbol and was not affected by polymyxin B. Thus, the results would suggest that there are at least two mechanisms by which alpha-bungarotoxin binding sites can be regulated. One is mediated via an interaction at nicotinic receptors, while the other occurs in response to phorbol esters and thus may be mediated by protein kinase C. Interestingly, although the molecular mechanisms resulting in alpha-bungarotoxin receptor up-regulation differ, both the d-tubocurarine- and the phorbol ester-induced increases were prevented by nicotinic receptor ligands.

Adrenal Medulla↗
PubMed1992

Positive and negative inotropic effects of phorbol 12-myristate 13-acetate: relationship to PKC-dependence and changes in [Ca2+]i.

The present study examined the concentration-dependent effects of phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, on contractile force and [Ca2+]i in guinea-pig hearts and isolated cardiac myocytes, respectively. Contractile force was measured using isolated Langendorff-perfused hearts while [Ca2+]i was measured independently in isolated cardiac myocytes loaded with fura2-AM. Phorbol 12-myristate 13-acetate, as well as another PKC-activating phorbol, phorbol dibutyrate (PDBu), and two non-PKC-activating phorbols, alpha-phorbol didecanoate (alpha PDD) and 4 alpha-phorbol, exerted time- and concentration-dependent effects on contractility. A significant positive inotropic response was observed with either PMA (10(-12) M; 5-15 min of perfusion) or PDBu (10(-12) M; 5 min of perfusion). In contrast, 10(-10) M PMA caused a significant negative inotropic effect following 30 min of perfusion while 10(-8) M PMA produced a significant negative inotropic effect which occurred earlier (10 min) and was sustained throughout the 30 min perfusion period. A similar negative inotropic effect was seen with 10(-8) M of either PDBu or alpha PDD. In addition, 4 alpha-phorbol (10(-8) M) exerted a modest, but significant negative inotropic effect following 25 and 30 min of perfusion. Both concentration-dependent increases and decreases of +dF/dt and -dF/dt were observed in the presence of PMA. In addition, both PMA and PDBu caused a concentration-dependent increase in coronary perfusion pressure. The positive inotropic responses and coronary perfusion pressure effects elicited by PMA and PDBu were largely prevented by the addition of the PKC inhibitors H7 (6 nM) or HAG (10 nM); however, these drugs were without effect on the negative inotropic response to higher concentrations of both PKC-activating (PMA, PDBu) and non-PKC-activating (alpha PDD, 4 alpha-phorbol) phorbol compounds. The lowest concentration of either PMA or PDBu (10(-12) M) increased the 340/380 fluorescence ratio of isolated cardiac myocytes loaded with fura2-AM on a time scale similar to that at which the positive inotropic response was seen in the whole heart. However, in contrast to results in the isolated heart, PDBu elicited a greater and sustained increase in the fluorescence ratio measured in isolated cardiac myocytes. The higher concentration of either PMA or PDBu (10(-8) M), resulted in a decrease in the 340/380 ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Concentration-dependent effects of protein kinase C-activating and -nonactivating phorbol esters on myocardial contractility, coronary resistance, energy metabolism, prostacyclin synthesis, and ultrastructure in isolated rat hearts. Effects of amiloride.

An extensive investigation of the cardiac actions of phorbol esters and the potential role of the Na(+)-H+ exchanger in those actions was carried out using isolated rat hearts. Sixty minutes of perfusion with 10(-9) M phorbol 12-myristate 13-acetate (PMA) or 10(-8) M phorbol 12,13-dibutyrate (PDBu) produced marked cardiac dysfunction associated with depressed contractility, coronary constriction, and elevated resting tension, the latter being particularly evident with PMA. These effects were also associated with disturbances in tissue levels of energy metabolites manifested primarily by a reduction in ATP and an elevation in lactate. Furthermore, both phorbols produced a sustained stimulation of the release of 6-ketoprostaglandin F1 alpha (6-keto PGF1 alpha), the hydrolysis product of prostacyclin (prostaglandin I2). Amiloride, an inhibitor of the Na(+)-H+ exchanger, significantly attenuated the loss in contractility and elevation in coronary pressure as well as the stimulated release of 6-keto PGF1 alpha but was without effect on elevations in resting tension or on changes in energy metabolites. Increasing concentrations of PMA or PDBu 10-fold resulted in a much more rapid and severe (greater than 80% loss in contractile function after 30 minutes) effect that was nonetheless qualitatively identical to that seen with the lower concentrations of phorbol. However, the effects were not prevented by amiloride. Surprisingly, 4 alpha-phorbol 12,13-didecanoate (alpha-PDD, 10(-6) M), which does not activate protein kinase C, was found to be a potent inhibitor of cardiac function (greater than 80% loss in contractility and 50% increase in resting tension) after 30 minutes of perfusion, although these effects were not associated with changes in levels of energy metabolites or with elevations in coronary pressure. Similarly, none of the actions of this compound were attenuated by amiloride. In contrast to the sustained effects of other phorbols on 6-keto PGF1 alpha release, the effect of alpha-PDD was transient (less than 10 minutes). In all hearts studied, the marked depression in contractile function caused by all phorbol esters occurred in the absence of any ultrastructural changes. 4 alpha-Phorbol (10(-6) M), which does not activate protein kinase C, was without effect on any parameter studied. Our results demonstrate very complex effects of phorbol esters on numerous parameters of cardiac function, including an amiloride-sensitive component that occurs at low concentrations. The latter observation suggests the involvement of Na(+)-H+ exchange activation, possibly occurring as a consequence of protein kinase C stimulation, in mediation of the effects of phorbol esters at low concentrations.(ABSTRACT TRUNCATED AT 400 WORDS)

Amiloride↗