Release of endothelium-derived relaxing factor from pig cultured aortic endothelial cells, as assessed by changes in endothelial cell cyclic GMP content, is inhibited by a phorbol ester.

1. Cultured aortic endothelial cells of the pig respond to the endothelium-derived relaxing factor (EDRF) they release with an increase in cyclic GMP content. This response is inhibited by haemoglobin or by L-NG-monomethyl-arginine (L-NMMA), and has been used to investigate the effects of phorbol esters on EDRF release. 2. Pretreatment with phorbol-12,13-dibutyrate (PDB) but not the inactive 4 alpha-phorbol-12,13,-didecanoate (PDD), inhibited increases in cyclic GMP induced by substance P (10(-8) M) in a time and concentration-dependent manner. PDB did not affect basal cyclic GMP levels. 3. PDB (3 x 10(-7) M), but not PDD (3 x 10(-7) M), also inhibited ATP (10(-5) M)-induced increases in cyclic GMP, but did not affect those induced by bradykinin (10(-7) M). 4. Increases in cyclic GMP induced by low (10(-7) M) but not high (10(-6) M) concentrations of the calcium ionophore A23187 were inhibited by PDB (3 x 10(-7) M). This inhibitory effect was due to enhanced destruction of EDRF by superoxide anions rather than inhibition of EDRF release, as the inhibition was abolished in the presence of superoxide dismutase (SOD, 30 mu ml-1) and catalase (CAT, 100 mu ml-1). 5. SOD and CAT did not affect the inhibitory action of PDB on substance P or ATP-induced increases in cyclic GMP. 6. Increases in endothelial cell cyclic GMP content induced by sodium nitroprusside (10(-5) M) were unaffected by PDB pretreatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Protein kinase C-mediated contractile responses of arteries from diabetic rats.

1. The role of protein kinase C (PKC) in mediating enhanced contractile responses of aortae and mesenteric arteries from male rats with 12-14 week streptozotocin-induced diabetes to noradrenaline (NA) was investigated using the PKC activator, phorbol 12,13-dibutyrate (PDB), and the PKC inhibitor, staurosporine. 2. Maximum contractile responses of aortae and mesenteric arteries from diabetic rats to NA were significantly enhanced compared with responses of arteries from age-matched control animals. The maximum NA responses were increased by 59.6 +/- 7.9% in aortae and by 54.9 +/- 7.4% in mesenteric arteries from diabetic animals, compared to their respective controls. 3. Pretreatment of aortae and mesenteric arteries from both control and diabetic animals with staurosporine (5 x 10(-8) M) caused marked inhibition of contractile responses to a maximum concentration of NA (10(-5) M in aortae; 3 x 10(-5) M in mesenteric arteries). In the presence of staurosporine, no difference was observed in the magnitude of contractile responses of arteries from control and diabetic rats to NA. 4. Maximum contractile responses of mesenteric arteries from diabetic rats to PDB were significantly increased (by 45.0 +/- 4.9%) compared to responses of arteries from control animals. In contrast, no significant difference was found in the magnitude of contractile responses or aortae from control and diabetic rats to PDB. 5. Staurosporine (5 x 10(-8) M caused marked attenuation of contractile responses of arteries from control and diabetic rats to a maximum concentration of PDB (3 x 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)     

Possible Involvement of Protein Kinase C in the Modulation of Inotropic and Chronotropic Effects Induced by Ouabain in Rat Right Atrial Muscles

Modulations of the inotropic and chronotropic effects of ouabain and protein kinase C (PKC) stimulation with phorbol esters in rat right atria were examined. Cumulative administration of ouabain (3–30 μm ) caused a positive inotropic effect in a concentration-dependent manner, but did not produce a chronotropic effect. A single administration of ouabain (30 μm ) also had similar effects: +744 ± 84% (n = 23, P < 0.01) in the contractile force and ?0.7 ± 1.3% (n = 23, P > 0.05) in the sinus rate. Addition of phorbol esters reinforced the ouabain-evoked positive inotropic effect: 26.5 ± 8.9% (n = 6, P < 0.05) with 100 μm 4-β-phorbol-12,13-dibutyrate (PDB), and 6.4 ± 3.3% (n = 6, P > 0.05) with 100 μm 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Simultaneously, the mixture of ouabain and phorbol ester raised the resting tension. Phorbol esters alone caused a positive inotropic effect (by about 21–27%). Non-PKC activating phorbol ester, 4-α-phorbol-12,13-didecanoate (PDD, 100 μm ), did not have any effect. Pretreatment with the PKC inhibitor (staurosporine 100 μm ) significantly decreased the ouabain-induced positive inotropic effect and caused a negative chronotropic effect, but H-7 (1-(5-isoquinolinylsulphonyl)-2-methylpiperazine dihydrochloride) (5 μm ) had no effect. These results suggest that PKC stimulation may be involved in the ouabain-evoked responses in the right atria of rat as seen by increased cellular Ca²⁺ concentration (and Ca²⁺-sensitivity); thus the positive inotropic effect may not be due only to modulation of Na⁺/K⁺ pump activity.     

Characterization of phorbol esters activity on individual mammalian protein kinase C isoforms, using the yeast phenotypic assay

An alternative in vivo assay, based on growth inhibition of yeast expressing an individual mammalian protein kinase C (PKC) isoform (proportional to the degree of PKC activation), was used to characterize the activities of phorbol-12-myristate-13-acetate (PMA) and its analogues on classical (alpha and betaI), novel (delta and eta) and atypical (zeta) PKC isoforms. Effects of PMA, 4alpha-PMA, phorbol-12-myristate-13-acetate-4-O-methyl-ether (MPMA), phorbol-12-monomyristate (PMM), phorbol-12,13-diacetate (PDA), phorbol-13-monoacetate (PA), phorbol-12,13-dibutyrate (PDB), phorbol-12,13-didecanoate (PDD) and 12-deoxyphorbol-13-phenylacetate-20-acetate (dPPA), on growth of yeast expressing individual PKC isoforms was determined. PMA-induced growth inhibition on all isoforms tested (except on PKC-zeta). PDD and PDB presented an efficacy similar to PMA; the other PMA-analogues presented lower efficacies. MPMA and 4alpha-PMA stimulated growth of yeast expressing classical PKCs and reduced the PMA-induced growth inhibition, effects similar to those exhibited by the PKC inhibitors chelerythrine and R-2,6-diamino-N-[[1-(1-oxotridecyl)-2-piperidinyl]methyl]-hexanamide dihydrochloride (NPC 15437). This study reveals that phorbol esters differ on their potency to activate a given PKC isoform, and presents their isoform-selectivity. Furthermore, MPMA and 4alpha-PMA caused effects similar to those expected from PKC inhibition.     

On electrophysiological responses to phorbol esters which stimulate protein kinase C in rabbit sino-atrial node cells

Summary Effects of phorbol esters on spontaneously beating rabbit sino-atrial (SA) node cells were investigated by means of voltage clamp technique. In a small SA node specimen, 12-O-tetradecanoylphorbol-13-acetate (TPA) 10^–7 mol/l lengthened the cycle length (CL) and at over 3 × 10^–7 mol/l prolonged the action potential duration (APD). Action potential amplitude (APA), maximum diastolic potential (MDP) and maximum rate of rise (V _max) were unaffected. Amiloride 10^–3 mol/1, an inhibitor of Na⁺-H⁺ exchange, did not reverse the phorbol ester-induced effects. In voltage-clamp experiments, TPA 1-10 × 10^–7 mol/l slightly increased the slow inward current (I _si) and the time-dependent inward current (I _h) which activates during hyperpolarization. The outward current and the tail current were reduced, although the activation curve was not shifted along the voltage axis. In the presence of 10^–7 mol/l isoprenaline, TPA produced dysrhythmia and a transient inward current in voltage-clamp experiments. In the presence of 5 × 10^–5 mol/l phenylephrine or 2 × 10^–6 mol/l acetylcholine, TPA also elicited dysrhythmia. 4-betaphorbol-12,13-dibutyrate (PBD) induced similar electrophysiological effects as TPA, but 4-alpha-phorbol-12,13-didecanoate (PDD) never did so even in the presence of isoprenaline. These results suggest that TPA and PDB might mobilize intracellular Ca²⁺ via protein kinase C activation in the presence of isoprenaline, phenylephrine or acetylcholine, resulting in dysrhythmia due to delayed afterdepolarization.Send offprint requests to Hiroyasu Satoh     

Phorbol esters inhibit the proliferation of MCF-7 cells. Possible implication of protein kinase C

The effect of tumor promoter phorbol esters on cell proliferation was investigated in human breast cancer cell line MCF-7. During a 4-day culture period, the various phorbol ester derivatives TPA, PDD, PDBu, PDBz and PDA inhibited the proliferation of MCF-7 cells in a dose-dependent manner, with respective IC50 of 0.06, 0.75, 2.4, 3.6 and 15 X 10(-9) M. The 4-O-met-TPA, alpha PDD and alph PHR were ineffective at 2 X 10(-7) M, the highest concentration tested. Using a 3H-PDBu probe, we demonstrated the presence of specific, high affinity binding sites in intact cultured cells, with a Kd of about 9 X 10(-9) M. Unlabelled TPA, PDD, PDBU and PDBz competed with 3H-PDBu with respective IC50 of 35, 12.5, 150 and 220 X 10(-9) M. High concentrations of PDA, 4-O-met-TPA and alpha PDD slightly inhibited the 3H PDBu binding, whereas alpha PHR did not until 10(-5) M. The correlation that we observed between the relative potencies of the various phorbol derivatives for inhibiting both PDBu binding and cell proliferation, suggests that tumor promoter phorbol esters may induce growth arrest in MCF-7 cells by the mediation of protein kinase C.     

Effects of protein kinase C activators upon the late stages of the ACTH secretory pathway of AtT-20 cells.

1. The mouse AtT-20/D16-16 anterior pituitary tumour cell line was used as a model system for the study of phorbol 12-myristate 13-acetate (PMA)-mediated enhancement of calcium-evoked adrenocorticotrophin (ACTH) secretion. 2. PMA stimulated ACTH secretion from intact cells in a concentration-dependent manner. Other phorbol esters; phorbol 12,13-dibutyrate (PDBu) and phorbol 12,13-didecanoate (PDD) and diacylglycerol analogues; 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG) also stimulated ACTH release from intact AtT-20 cells. This would suggest that activation of protein kinase C (PKC) stimulates ACTH secretion from AtT-20 cells. 3. Calcium stimulated ACTH secretion from electrically-permeabilized cells over the concentration-range of 10(-7) M to 10(-5) M. PMA (10(-7) M) enhanced the amount of ACTH secreted at every concentration of calcium investigated. The PKC inhibitor, chelerythrine (10(-5) M) blocked the PMA (10(-7) M)-evoked enhancement of calcium (10(-5) M)-stimulated ACTH secretion but did not alter significantly the calcium (10(-5) M)-evoked secretion itself. This suggests that PKC modulates the secretory response to increases in intracellular calcium but does not mediate the effects of calcium. 4. Guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S, 10(-5) M) stimulated ACTH secretion from permeabilized cells in the absence of calcium and was additive with calcium-evoked ACTH secretion up to a maximum value which could be achieved by calcium acting alone. This suggests that a GTP-binding protein mediates the secretory response to increases in the intracellular calcium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vascular protein kinase C in Wistar-Kyoto and spontaneously hypertensive rats.

Phorbol esters which activate protein kinase C (PKC) produced concentration-related force development in aorta from spontaneously hypertensive rat (SHR) and Wistar-Kyoto rat (WKY); all were 2-7 x more potent in SHR. However, total PKC activity in aortas, as well as carotid, caudal and renal arteries, was not different, when SHR was compared with WKY. Binding of phorbol dibutyrate to particulate aortic PKC was similar in SHR and WKY (same apparent Kd and Bmax values), as was potency for displacement of phorbol dibutyrate by phorbol myristate acetate. Furthermore, there was no difference in potency with staurosporine, H-7, and calmidazolium in inhibiting SHR and WKY aortic PKC. These data demonstrate enhanced contractile sensitivity to PKC-activating phorbol esters in SHR aortic smooth muscle that is not related to activity, phorbol ester binding, or sensitivity to inhibitors when SHR PKC is compared with WKY PKC. Thus, signal transduction events distal to PKC activation may be responsible for enhanced vascular contractile sensitivity to phorbol esters in SHR.     

Activation of protein kinase C potentiates postsynaptic acetylcholine response at developing neuromuscular synapses.

1. Phorbol 12-myristate 13-acetate (TPA, 1 microM) and phorbol 12,13-dibutyrate (PDBu, 2 microM), activators of protein kinase C (PKC), increased the mean amplitude and decay time of the spontaneous synaptic currents of Xenopus nerve-muscle coculture, whereas, 4 alpha-phorbol (2 microM) which is an inactive phorbol analogue had no effect. 2. Staurosporine (0.5 microM) and H-7 (10 microM), inhibitors of PKC, inhibited the potentiation effects of TPA on the spontaneous synaptic currents. 3. Effects of TPA on the postsynaptic acetylcholine (ACh) sensitivity were examined by iontophoresis of ACh to the surface of embryonic muscle cells of 1-day-old Xenopus cultures. TPA increased both the amplitude and decay time of ACh-induced whole-cell currents in isolated myocytes. 4. TPA concentration-dependently increased the mean open time of low-conductance ACh channels but did not affect those of high-conductance ACh channels. PDBu but not 4 alpha-phorbol exhibited similar effects to TPA. Staurosporine and H-7 inhibited the increasing effects of TPA. 5. These results suggest that activation of PKC might be involved in synaptogenesis at developing neuromuscular synapses by the postsynaptic potentiation of ACh sensitivity.     

Regulation of cyclic AMP levels by phorbol esters in bovine adrenal medullary cells

Cyclic AMP responses to phorbol esters were studied in cultured bovine adrenal medullary cells. Phorbol esters that activate protein kinase C (PKC: phorbol 12,13-dibutyrate, phorbol 12,13-didecanoate) increased cellular cyclic AMP levels by up to 100% over 5 min, and this was maintained for up to 3 h. The effect was mimicked by 1,2-dioctanoyl-sn-glycerol but not by inactive phorbol esters. The effect of active phorbol esters was concentration dependent over the range 50–500 nM, and was abolished by the PKC inhibitor, Ro 31–8220 (10μM). The response was enhanced by 3-isobutyl-1-methylxanthine (1 mM) and by forskolin (0.3 μM), was enhanced following pertussis toxin pretreatment (100 ng/ml, 7.5 h) and was unaffected by removing extracellular Ca²⁺. The phorbol ester cyclic AMP response was additive with that to K⁺ depolarisation, and synergised with those to prostaglandin E₁ and dimaprit. The results indicate PKC activation increases cyclic AMP formation in bovine adrenal medullary cells, probably by a direct action on adenylate cyclase or G_s.     

Effects of phorbol ester on carbachol-induced contraction in bovine ciliary muscle: possible involvement of protein kinase C

The aim of the research was to characterize muscarinic receptors of bovine ciliary muscle and to investigate the desensitization process. The role of protein kinase C was analyzed. The results show that muscarinic receptors of bovine ciliary muscle have the pharmacological characteristics of the M₃ subtype. Acute exposure to phorbol esters (1 μM phorbol 12,13-dibutyrate, PDB, or 0.1 μM phorbol 12-myristate 13-acetate, PMA, for 15 and 5 min, respectively) resulted in antagonism of muscarinic receptor-mediated contraction. Long-term pretreatment (18 h) with PMA to down-regulate protein kinase C resulted in potentiation of carbachol-induced contraction, reduction of agonist-induced desensitization and loss of phorbol ester-induced desensitization. Staurosporine (3 μM) and H₇ [1-(5-isoquinolinesulfonyl)-2-methyl-piperazine] (1 μM), protein kinase C inhibitors, produced a significant potentiation of the contractile effect of carbachol, reduced the desensitization produced by repeated addition of carbachol and suppressed that induced by phorbol esters. In vitro incubation with carbachol, PDB or PMA did not cause any modification of the binding of labeled [³H]quinuclidinyl benzilate. In vitro incubation with PDB and PMA produced, as expected, a significant translocation of protein kinase C from the cytosol to the membrane. The incubation of the ciliary muscle with carbachol, using the protocol of exposure that induced maximal desensitization of contractile responses, produced a significant redistribution of the enzyme from the cytosol to the membrane. These findings suggest that agonist-induced modulation of functional cholinergic sensitivity in ciliary muscle is correlated, at least partially, to the translocation of protein kinase C from the cytosol to the membrane. The desensitization by phorbol esters is completely due to protein kinase C activation; during the desensitization process, direct modification of the density and affinity of muscarinic receptors is not involved.     

Polymyxin B,a selective inhibitor of protein kinase C,diminishes the release of noradrenaline and the enhancement of release caused by phorbol 12,13-dibutyrate

Summary Slices of the rabbit hippocampus were labelled with ³H-noradrenaline, superfused continuously with a modified Krebs-Henseleit medium containing the uptake inhibitor cocaine and stimulated electrically (2 ms, 3 Hz, 24 mA, 5 V/cm). Phorbol 12,13-dibutyrate (PDB), a potent activator of protein kinase C (PKC), strongly enhanced the electrically-evoked overflow of tritium. In contrast, polymyxin B, a relatively selective inhibitor of PKC, diminished the evoked tritium overflow in a time-and concentration-dependent manner. The enhancement of the evoked overflow of tritium caused by PDB was strongly reduced in the presence of polymyxin B (100 mol/l). These results suggest 1. that PKC may be involved in the physiological mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals and 2. that the PDB-induced enhancement of noradrenaline release may be due to a direct activation of PKC.Abbreviations PKC protein kinase C - PDB phorbol 12,13-dibutyrate - TPA 12-O-tetradecanoyl 13-acetate     

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.     

Effects of the two enantiomers, S-16257-2 and S-16260-2, of a new bradycardic agent on guinea-pig isolated cardiac preparations.

1. The electromechanical effects of two enantiomers, S-16257-2 (S57) and S-16260-2 (R60), were studied and compared in guinea-pig isolated atria and ventricular papillary muscles. The possible stereoselectivity of the interaction on the cardiac Na+ channel was analysed by comparing the effects of the two enantiomers on the onset and recovery kinetics of the frequency-dependent Vmax block. 2. In spontaneously beating right atria, S57 and R60 (10(-8)M-10(-4M) exerted a negative chronotropic effect (pIC50 = 5.07 +/- 0.19 and 4.76 +/- 0.18, respectively) and prolonged the sinus node recovery time, this effect being more marked with S57. In electrically driven left atria, S57 decreased (P < 0.05) contractile force only at 10(-4M) and R60 at concentrations > or = 5 x 10(-5M), whereas in papillary muscles the negative inotropic effect appeared at concentrations > 10(-5M). 3. In papillary muscles driven at 1 Hz, S57 and R60 at concentrations higher than 5 x 10(-6M) produced a concentration-dependent decrease in the maximum upstroke velocity (Vmax) and amplitude of the cardiac action potential without altering the resting membrane potential or the action potential duration. S57 and R60 had no effect on the characteristics of the slow action potentials elicited by isoprenaline in ventricular muscle fibres depolarized in high K+ (27 mM) solution. 4. At 5 x 10(-5M), S57 and R60 produced a small tonic Vmax block. However, in muscles driven at rates between 0.5 and 3 Hz both enantiomers produced an exponential decline in Vmax (frequency-dependent Vmax block) which augmented at higher rates of stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological effects of platelet-activating factor (PAF-acether) in guinea-pig papillary muscles

The effects of PAF-acether (10(-11) to 10(-7) M) were studied on the electrical and mechanical activity of guinea-pig papillary muscles. At 10(-11) M PAF-acether did not modify the amplitude and Vmax of the upstroke or the resting membrane potential. At higher concentrations PAF-acether produced a dose-dependent increase in the amplitude and Vmax of the upstroke, shortened the action potential duration and hyperpolarized the resting membrane potential. These effects were accompanied by a biphasic effect on ventricular contractile force. The shortening of the APD was inhibited in muscles pretreated with tetraethylammonium or verapamil. In papillary muscles depolarized by 27 mM K Tyrode solution PAF-acether induced slow action potentials which were blocked by verapamil. PAF-acether produced a dose-dependent increase in amplitude and Vmax of the upstroke on the slow action potentials elicited by isoproterenol, prolonged the action potential duration and hyperpolarized the resting membrane potential. These results suggest that in guinea-pig papillary muscles PAF-acether increased Ca influx via the slow inward current.     

Inhibition in L-type Ca2+ channel by stimulation of protein kinase C in isolated guinea pig ventricular cardiomyocytes.

1. Electrophysiological effects of phorbol esters on the L-type Ca2+ current (ICa(L)) in isolated single ventricular cells from guinea pig hearts were investigated. 2. In whole-cell voltage-clamped myocytes, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) at 10(-7) M inhibited ICa(L). An antagonist of protein kinase C (PK-C), H-7, at 10(-5) M did not modify the TPA-induced inhibition. The time-course of inactivation process for ICa(L) was greatly slowed. 3. In cell-attached patch-clamp experiments, TPA (10(-7) M) also markedly decreased the opening of L-type Ca2+ channels. The conductance was unaffected. 4. Even H-7 (10(-5) M) alone inhibited the opening of the channels. Addition of TPA (10(-7)-10(-8) M) caused further decrease in the opening. 5. On the other hand, 4-alpha-phorbol-12,13-didecanoate (not a PK-C activator) had no effect on the Ca2+ channels. 6. These results indicate that the PK-C activation induced by TPA greatly depresses the opening of L-type Ca2+ channels in ventricular cell membranes.     

Identification of protein disulfide isomerase as a phorbol ester-binding protein

A specific binding protein for 12-O-tetradecanoylphorbol 13-acetate (TPA), different from protein kinase C (PKC) and histone H1, was purified from HeLa cell extract by the use of affinity gel pendanted with phorbol ester (TPA-GEL). The purified binding protein was identified as protein disulfide isomerase (PDI, EC 5.4.3.1) by peptide sequence analysis. The dissociation constants (Kd's) of TPA to PDI, histone H1 and PKCalpha were determined to be 1.03 x 10(-6) M, 5.70 x 10(-7) M, and 4.00 x 10(-7) m, respectively, by the surface plasmon resonance (SPR) method. TPA moderately inhibited PDI activity assessed in terms of reactivation of denatured RNase A.     

Differential abilities of phorbol esters in inducing protein kinase C (PKC) down-regulation in noradrenergic neurones

1. The ability of several phorbol ester protein kinase C (PKC) activators (phorbol 12, 13-dibutyrate, PDB; phorbol 12, 13-diacetate, PDA; and 12-deoxyphorbol 13-acetate, dPA) to down-regulate PKC was studied by assessing their effects on electrical stimulation-induced (S-I) noradrenaline release from rat brain cortical slices and phosphorylation of the PKC neural substrate B-50 in rat cortical synaptosomal membranes. 2. In cortical slices which were incubated for 20 h with vehicle, acute application of PDB, PDA and dPA (0.1 - 3.0 microM) enhanced the S-I noradrenaline release in a concentration-dependent manner to between 200 - 250% of control in each case. In slices incubated with PDB (1 microM for 20 h), subsequent acute application of PDB (0.1 - 3.0 microM) failed to enhance S-I release, indicating PKC down-regulation. However, in tissues incubated with PDA or dPA (3 microM) for 20 h, there was no reduction in the facilitatory effect of their respective phorbol esters or PDB (0.1 - 3.0 microM) when acutely applied, indicating that PKC was not down-regulated. This was confirmed using Western blot analysis which showed that PDB (1 microM for 20 h) but not PDA (3 microM for 20 h) caused a significant reduction in PKCalpha. 3. Incubation with PDB for 20 h, followed by acute application of PDB (3 microM) failed to increase phosphorylation of B-50 in synaptosomal membranes, indicating down-regulation. In contrast, tissues incubated with PDA or dPA for 20 h, acute application of their respective phorbol ester (10 microM) or PDB (3 microM) induced a significant increase in B-50 phosphorylation. 4. Acutely all three phorbol esters elevate noradrenaline release to about the same extent, yet PDA and dPA have lower affinities for PKC compared to PDB, suggesting unique neural effects for these agents. This inability to cause functional down-regulation of PKC extends their unusual neural properties. Their neural potency and lack of down-regulation may be related to their decreased lipophilicity compared to other phorbol esters. 5. We suggest that PKC down-regulation appears to be related to binding affinity, where agents with high affinity, irreversibly insert PKC into artificial membrane lipid and generate Ca(2+)-independent kinase activity which degrades and deplete PKC. We suggest that this mechanism may also underlie the ability of PDB to down-regulate PKC in nerve terminals, in contrast to PDA and dPA.     

A phorbol ester inhibits the release of endothelium-derived relaxing factor

The effect of the phorbol ester phorbol-12,13-dibutyrate (PDB), an activator of protein kinase C, on endothelium-dependent relaxation was studied in noradrenaline-constricted isolated aortic ring preparations of the rabbit. Endothelium-dependent relaxation induced by acetylcholine or substance P was inhibited by PDB (greater than or equal to 10(-7) M). Endothelium-dependent relaxation induced by the calcium ionophore A23187 (7.5 X 10(-8) and 10(-7) M) was unaffected by PDB (to 10(-6) M). The mechanical responses to acetylcholine or sodium nitroprusside in endothelium-denuded rings were not altered by PDB (to 10(-6) M). The results suggest a role for protein kinase C in receptor-mediated EDRF release mechanisms.     

Effects of TYB-3823, a new antiarrhythmic drug, on the electrophysiological properties of guinea-pig ventricular muscles

1. The effects of TYB-3823 (B-GYKI 38233), a new antiarrhythmic drug, were electrophysiologically examined in the guinea-pig ventricular muscles. 2. TYB-3823 at concentrations of 1-3 x 10(-7) M significantly prolonged the action potential duration (APD) of the papillary muscle. However, the resting potential, action potential amplitude and maximum rate of depolarization (Vmax) were unaffected by the drug at such concentrations. At a higher concentration (1 x 10(-4) M) TYB-3823 reduced Vmax. 3. Voltage clamp experiments with single ventricular cells revealed that TYB-3823 at concentrations higher than 1 x 10(-7) M reduced the outward potassium currents, especially the time-dependent outward current, but that TYB-3823 failed to affect the calcium current. 4. These results suggested that TYB-3823 at low concentrations reduces the outward potassium current to give rise to a prolongation of APD and that at higher concentrations it additionally inhibits the sodium channels; both the effects may be related to the antiarrhythmic action of this drug.