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.     

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.     

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.     

Protein kinase C: regulation of serotonin release from rat brain cortical slices

The effects of phorbol esters on serotonin release were examined in an attempt to investigate the role of protein kinase C in the regulation of serotonin release. Rat brain parietal cortical slices were incubated with [3H]5-HT in the presence of pargyline in order to label the serotonin stores. Potassium stimulated (30 s) release and spontaneous [3H]5-HT efflux were examined in slices during superfusion with Krebs-Ringer solution containing chlorimipramine. Repeated K+ stimulations elicited reproducible responses with release ratios of approximately 1.0. Introduction of phorbol 12-myristate, 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu) 20 min prior to S2, or S3 resulted in dose-related increases in [3H]5-HT or [3H]NE release. PMA was slightly more potent (93% increase) than PDBu in potentiating K+-stimulated [3H]5-HT release. Phorbol and 4 alpha-phorbol 12,13-didecanoate (4 alpha PDD) which do not activate protein kinase C did not alter serotonin release. In contrast, basal [3H]5-HT and [3H]NE release were altered to a far lesser extent which was not always dose related. The response to the phorbol esters was reversible, Ca2+-dependent and reached maximal effect after 20 min of superfusion. The putative protein kinase C inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibited K+-induced [3H]5-HT release significantly (11%) but did not alter basal efflux. The PMA facilitation of serotonin release was, however, markedly prevented by the enzyme inhibitor. The effect of PMA on release was found not to be directly mediated through the prejunctional serotonin autoreceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

The modulatory effects of phorbol esters on histamine release from mast cells induced by a polyethylenimine and a polyallylamine.

1. Histamine release from rat peritoneal mast cells induced by polyethylenimine (mol. wt = 600) was inhibited by phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu), but not by 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD). 2. Histamine release induced by polyallylamine (mol. wt = 10,000) was enhanced by PMA and PDBu, but not by 4 alpha-PDD. 3. K-252a reduced the inhibitory effect of PMA on histamine release induced by polyethylenimine and its enhancing effect on histamine release by polyallylamine.     

Phorbol esters inhibit smooth muscle contractions through activation of Na(+)-K(+)-ATPase.

1. The role of protein kinase C (PKC) in agonist-induced contractions of guinea-pig ileum longitudinal smooth muscle has been investigated. 2. The phorbol esters, phorbol 12,13-dibutyrate (PDBu), phorbol 12,13-diacetate (PDA) and phorbol 12-myristate 13-acetate (PMA), relaxed tissues precontracted by submaximal concentrations of carbachol, histamine or substance P. 3. This inhibitory action of the phorbol esters was reversed following the application of ouabain, a specific inhibitor of Na(+)-K(+)-ATPase. Similarly, pretreatment with ouabain inhibited the ability of phorbol esters to relax tissues precontracted by the above agonists. 4. The slow relaxation of the tonic component of contraction induced by submaximal concentrations of carbachol and histamine, and all concentrations of substance P, was abolished in the presence of ouabain. 5. In Na(+)-loaded tissues, PDBu and carbachol caused a concentration-dependent increase of Na(+)-K(+)-ATPase activity, assessed by ouabain-sensitive 86Rb(+)-uptake. Extrusion of Na+, assessed by the cellular content of the ion, was also stimulated by PDBu (the effect of carbachol was not investigated). 6. We conclude that phorbol esters inhibit the tonic component of contractions induced by submaximal concentrations of these agonists through activation of Na(+)-K(+)-ATPase. We suggest that PKC may exert feedback control over the tonic component of agonist contractions through stimulation of the pump.     

Protein kinase C and alpha 2-adrenoceptor-mediated inhibition of noradrenaline release from the rat tail artery.

In isolated rat tail arteries preincubated with [3H]noradrenaline, electrical field stimulation evoked the overflow of tritium. Phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activating phorbol ester, time-dependently increased the overflow at 1 mumol/L but not at 0.1 mumol/L. In contrast, the overflow was not altered by phorbol 13-acetate (PA, 1 mumol/L), which does not influence the activity of PKC. Polymyxin B (70 mumol/L), an inhibitor of PKC, depressed the overflow when given alone and, in addition, attenuated the effect of PMA, 1 mumol/L. The selective alpha 2-adrenoceptor agonist B-HT 933 depressed the overflow; PMA, 1 mumol/L, did not interfere with the effect of B-HT 933, 10 mumol/L. The results provide evidence for the participation of prejunctionally located PKC in the release of noradrenaline. However, PKC does not seem to be involved in the alpha 2-adrenoceptor-agonist-mediated inhibition of noradrenaline release.     

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     

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

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

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

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

Effect of phorbol ester and pertussis toxin on the enhancement of noradrenaline release by angiotensin II in mouse atria.

1. Mouse atria were incubated with [3H]-noradrenaline, and the outflow of radioactivity due to electrical field stimulation (5 Hz, 60 s) was used as an index of noradrenaline release. Angiotensin II (0.01 and 0.1 microM) significantly enhanced the stimulation-induced (S-I) outflow of radioactivity. 2. Phorbol 12-myristate 13-acetate (0.001, 0.03, 0.1 and 1.0 microM), a protein kinase C activating phorbol ester, significantly enhanced the S-I outflow of radioactivity. When angiotensin II (0.1 microM) was present with the concentration of phorbol 12-myristate 13-acetate that was maximally effective in increasing the S-I outflow (0.1 microM), the enhancement of S-I outflow produced by angiotensin II was maintained. 3. Polymyxin B (70 microM), an inhibitor of protein kinase C, significantly inhibited the S-I outflow. Polymyxin B also inhibited the enhancement of the S-I outflow produced by angiotensin II (0.1 microM). 4. In another series of experiments mice were injected with pertussis toxin (1.5 micrograms per mouse), 4 days before their atria were removed. The effectiveness of pertussis toxin pretreatment was determined indirectly using carbachol. Carbachol caused a concentration-dependent fall in both the rate and force of beating of isolated spontaneously beating atria from mice pretreated with vehicle. This effect of carbachol was not seen with atria from mice pretreated with pertussis toxin. 5. Pertussis toxin pretreatment did not alter the enhancement of the S-I outflow of radioactivity produced by angiotensin II (0.01 and 0.1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid colorimetric microassay to detect agonists/antagonists of protein kinase C based on adherence of EL-4.IL-2 cells

A rapid, colorimetric, microassay for detection of agents which are known agonists/antagonists of protein kinase C (PKC) was developed, utilizing their effects on adherence of EL-4.IL-2 cells. Cells that were incubated with agents which are known inducers of PKC activation, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate (PDBu), mezerein and indolactam V, readily adhered to wells of 96 well microtiter plates within 1-2 hours, whereas cells incubated with the negative PKC activator, 4 alpha-phorbol 12-myristate 13-acetate (4 alpha-PMA), which is structurally related to PMA (4 beta-PMA), did not adhere. The adherent cells withstood repeated vigorous washings with tissue culture medium. Adherence of EL-4.IL-2 cells in the presence of PMA could be blocked by the addition of two known inhibitors of PKC, 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride and staurosporine. Detection of the presence of adherent cells was accomplished by the addition of a tetrazolium salt to culture wells and determination of the remaining viable cells by scanning using a multiwell spectrophotometer (ELISA reader). The EL-4.IL-2 adherence assay meets several important criteria for use as a primary screen in the detection of potential PKC agonists/antagonists, i.e. its selectivity, simplicity, rapid performance through automation and reproducibility.     

Phorbols: chemical synthesis and chemical biology

The phorbol esters, such as phorbol 12- myristate 13-acetate (PMA), are known to be powerful tumor promoters and activators of protein kinase C (PKC). First discovered by Nishizuka et al., PKC is a phospholipid- and calcium-dependent serine/threonine kinase, phisiologically activated by 1,2-diacyl-sn-glycerol (DAG). PKC is also known to be an important target for other structurally diverse tumor promoters such as ingenols, teleocidins, and aplysiatoxins. Structure-activity analyses of a variety of analogs of DAG and these tumor promoters have been carried out. Although many pharmacophore models have been proposed from molecular modeling, no information about specific amino acid residues that interact with these ligands is available. Moreover it has been shown that the biological activity of 11-demethyl-13-deoxyphorbol esters 1, which were synthesized by our group, was not fully consistent with the pharmacophore models so far. Thus, we are now interested in determining the importance of the 13-acetoxy group in phorbol ester-PKC complexes. This has led us to design new photoaffinity probes 66 and 67 and to carry out previously unprecedented photoaffinity labeling of PKC. Photoaffinity labeling of protein kinase C isozymes by both the probes resulted in specific cross-linking. Although the cross-linking yield is not very high, we suppose that determination of the cross-linking site can be realized by taking advantage of subpicomole order analysis by mass spectrometry and other methodologies to clarify the role of individual cysteine rich domein (CRD) in native PKC. We have also designed a new phorbol ester-phosphatidylserine hybrid molecule 69. Because phosphatidylserines in phospholipid membranes are known to have specific interactions with phorbol ester-PKC complexes, such a hybrid molecule can be expected to act as a specific inhibitor of PKC by preventing PKC from interacting with phospholipid membranes. The hybrid molecule was synthesized and preliminary biological activities were examined to inhibit PKC. A catalytic asymmetric synthesis of phorbol PMA is also currently under investigation. Progress is discussed.     

Activity of protein kinase C modulates the apoptosis induced by polychlorinated biphenyls in human leukemic HL-60 cells

Polychlorinated biphenyls (PCBs) induce apoptotic cell death of HL-60 cells. In the present study, we examined the possible involvement of protein kinase C (PKC) in PCB-induced apoptosis of HL-60 cells. Treatment of cells with phorbol 12-myristate 13-acetate (PMA), an activator of PKC, suppressed DNA fragmentation induced by PCBs in HL-60 cells. Treatment with another active phorbol ester, phorbol-12,13-dibutyrate (PDBu), also suppressed PCB-induced DNA fragmentation, whereas 4alpha-phorbol-12,13-didecanoate (4alphaPDD), an inactive phorbol ester, did not affect PCB-induced apoptosis of HL-60 cell. Moreover, 1-oleoyl-2-acetyl-sn-glycerol (OAG), an activator of PKC that is not a phorbol ester, also suppressed PCB-induced DNA fragmentation. However, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of PKC, increased DNA fragmentation induced by PCBs. These results demonstrate that the activation of PKC is responsible for the suppression of PCB-induced apoptosis of HL-60 cells. Furthermore, inhibition of PKC promotes DNA fragmentation of HL-60 cells treated with PCBs, thereby suggesting the involvement of PKC activity in PCB-induced apoptosis of HL-60 cells.     

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

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

Increased cyclic AMP response to forskolin in Epstein-Barr virus-transformed human B-lymphocytes derived from schizophrenics

Phorbol 12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, elevated basal cyclic AMP levels and enhanced isoproterenol-, prostaglandin E₁- (PGE₁), forskolin- and cholera toxin-stimulated cyclic AMP accumulation in Epstein-Barr virus (EBV)-transformed human B-lymphocytes. Staurosporine, a PKC inhibitor, significantly antagonized the increase in cyclic AMP accumulation produced by PMA, whereas the inactive phorbol ester, 4α-phorbol 12,13-didecanoate (4αPDD), had no effect. Basal levels of cyclic AMP and the accumulation of cyclic AMP produced by PMA, isoproterenol, PGE₁, cholera toxin and the combination of these compounds with PMA were not significantly different between schizophrenics and controls. The cyclic AMP response to forskolin in the presence and absence of PMA was significantly greater in EBV-transformed human B-lymphocytes from schizophrenics. These results suggest that activation of adenylyl cyclase by forskolin is elevated in EBV-transformed B-lymphocytes derived from schizophrenics and that this elevation is further enhanced through a PKC-dependent phosphorylation mechanism. Received: 20 May 1996/Final version: 6 November 1996     

Involvement Of B-50 (GAP-43) Phosphorylation In The Modulation Of Transmitter Release By Protein Kinase C

1. Protein kinase C (PKC) is a family of enzymes that is activated by diacylglycerol (DAG) following phospholipase (PL) C activation. Protein kinase C may also be activated by metabolites and arachidonic acid generated by breakdown of membrane phospholipids by PLD and PLA2, respectively. Subsequent to PKC activation, key protein substrates are phosphorylated, resulting in the facilitation of transmitter release. 2. Phorbol esters are compounds that mimic the actions of DAG on PKC and have been shown to facilitate stimulation-induced (S-I) transmitter release in rat brain. However, some phorbol esters that have a high affinity for PKC have no effect on transmitter release, whereas others with a lower affinity for PKC markedly elevate S-I transmitter release. 3. The structure and, more importantly, the lipophilicity of the phorbol esters determines their ability to access and activate the intraneuronal pools of PKC that are involved with transmitter release. In studies in which cell membranes were intact, phorbol esters did not display the characteristics expected based on their affinities for PKC in contrast with studies in disrupted synaptosomes. This supports the hypothesis that the membrane plays a critical role in determining the effects of phorbol esters on PKC. 4. B-50, a PKC substrate thought to be involved in transmitter release, also appears to be differentially phosphorylated by various phorbol esters. The effects on B-50 phosphorylation in intact synaptosomes, but not disrupted synaptosomes, are well correlated with the effects of phorbol esters on S-I transmitter release. 5. B-50 is colocalized with actin, which has also been suggested to play an important role in facilitating the movement of reserve pools of transmitter vesicles to the readily releasable state. Therefore, it is possible that the phosphorylation status of B-50 directly influences the organization of actin filaments, thereby allowing transmitter output to be sustained under high levels of stimulation.     

MECHANISMS OF THE EFFECTS OF ENDOTHELIN ON RESPONSES TO NORADRENALINE AND SYMPATHETIC NERVE STIMULATION

1. The effects of endothelin (0.1-1 nmol/L) and the phorbol ester, phorbol 12-myristate 13-acetate (PMA, 0.1-100 nmol/L) have been studied on vasoconstrictor responses to sympathetic nerve stimulation and noradrenaline in the rabbit isolated ear artery. 2. Endothelin (0.1 nmol/L) significantly enhanced the biphasic response of the arteries to a prolonged period of sympathetic nerve stimulation (2 Hz for 60 s). Both the component of the response attributable to mobilization of intracellular calcium (phasic response) and the component due to the influx of extracellular calcium (tonic response) were enhanced. 3. PMA (0.1 and 0.3 nmol/L), which activates protein kinase C, enhanced vasoconstrictor responses to noradrenaline but a higher concentration of PMA (100 nmol/L) inhibited responses to noradrenaline. 4. It is concluded that the enhancement of responses to sympathetic nerve stimulation and noradrenaline produced by endothelin may be due to facilitation of the influx of extracellular calcium and to the activation of protein kinase C.     

Pre- and postjunctional effects of phorbol 12-myristate-13-acetate in the mouse vas deferens.

The protein kinase C stimulator phorbol 12-myristate-13-acetate (PMA) increased the release of noradrenaline from field-stimulated mouse vasa deferentia and antagonized the inhibitory effect of xylazine and FK 33-824. The mechanical response to field stimulation was not modified by PMA in unpretreated vasa, but the contractility was partly restored when the stimulation-response curve had been depressed by xylazine or FK 33-824. In contrast, PMA decreased the contractile response to exogenous noradrenaline and bethanechol. A similar but smaller reduction was obtained with 1,2-dioctanoyl-sn-glycerol. In vasa depolarized by KCl, PMA and verapamil reduced the amplitude of contractions elicited by CaCl2. Only the effect of verapamil could be reversed by the calcium ionophore A 23187, while the effect of PMA was greatly attenuated in muscles pretreated with ouabain. Phorbol 12-myristate-13-acetate-4-O-methylether was ineffective in all experiments. These results suggest that PMA increases noradrenaline release (prejunctional effect) and decreases vasal contractility (postjunctional effect) by activation of protein kinase C.     

Protein kinase C activation and alpha 2-autoreceptor-modulated release of noradrenaline.

1 Effects of phorbol esters on the evoked noradrenaline release were studied in slices of the rabbit hippocampus, labelled with [3H]-noradrenaline, superfused continuously with a medium containing the reuptake inhibitor cocaine and stimulated electrically for 2 min (stimulation parameters: 2 ms, 24 mA, 5 V cm-1, 3 or 0.3 Hz). 2 The electrically-evoked overflow of [3H]-noradrenaline in the slices was increased in a concentration-dependent manner by the protein kinase C (PKC) activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 4 beta-phorbol 12,13-dibutyrate (4 beta-PDB). Phorbol esters, which do not activate PKC, 4-O-methyl-TPA and 4 alpha-PDB, showed no effect on neurotransmitter release. The effect of 4 beta-PDB was abolished in the presence of tetrodotoxin and in the absence of calcium. The PKC inhibitor polymyxin B inhibited the evoked noradrenaline release. 3 In the presence of 4 beta-PDB the inhibitory effects of the alpha 2-adrenoceptor agonist clonidine or the facilitatory effects of the alpha 2-adrenoceptor antagonist yohimbine seemed to be modified only by changes in the concentration of noradrenaline in the synaptic region. At a stimulation frequency of 3 Hz the inhibitory action of clonidine was reduced whereas the facilitatory effect of the yohimbine was even slightly enhanced by the phorbol ester. At 0.3 Hz and in the presence of 4 beta-PDB the effect of clonidine remained and that of yohimbine was strongly enhanced. 4 Pretreatment of the slices with islet-activating protein or N-ethylmaleimide significantly reduced the enhancement of noradrenaline release caused by 4 beta-PDB. It is possible that a regulatory N-protein is involved in steps following PKC activation. 5 These results suggest that PKC participates in the mechanism of action-potential-induced noradrenaline release from noradrenergic nerve terminals of the rabbit hippocampus and that effects on the autoinhibitory feedback system were not responsible for the 4 beta-PDB-induced increase of neurotransmitter release.