phorbol-12-13-didecanoate and phorbol

The activation of protein kinase C (PKC) has been implicated in the pathogenesis of gram-negative sepsis. The effects of PKC modulation on hepatic flow and metabolism were studied using isolated liver perfusion. The liver was isolated from well-fed or overnight-fasted, male Sprague-Dawley rats weighing 250-310 g, and perfused at a constant pressure of 12 cmH2O using a recirculating system. Phorbol 12-myristate 13-acetate (PMA), a potent activator of PKC, decreased hepatic flow and oxygen consumption, and increased net lactate production. It enhanced net glucose production in fed animals. Neither 4 alpha-phorbol didecanoate, an inactive phorbol ester for PKC nor 4 alpha-phorbol, an inactive phorbol had any significant effect. The effects of PMA were augmented by increasing calcium concentration in the medium. PMA at an initial concentration of 4 x 10(-8) M stimulated net lactate and/or glucose production more than a reduction of perfusion pressure from 12 to 6 cmH2O. Staurosporine, a potent PKC inhibitor, significantly attenuated the PMA-induced alterations of hepatic flow and oxygen consumption. These results indicate that modulation of PKC exerts significant effects on hepatic flow and metabolism, which are dependent on extracellular calcium concentrations and feeding conditions, and that the effect of PMA on carbohydrate metabolism is not merely attributed to decreases in hepatic flow and oxygen consumption. It is suggested that PKC activation may be involved in the alterations of hepatic flow and metabolism during severe sepsis.

Topics: Alkaloids; Animals; Enzyme Activation; Fasting; Food; Hemodynamics; In Vitro Techniques; Liver; Liver Circulation; Male; Organ Size; Perfusion; Phorbol Esters; Phorbols; Protein Kinase C; Rats; Rats, Sprague-Dawley; Staurosporine; Tetradecanoylphorbol Acetate

Using a paced Lagendorff-perfused rabbit heart paradigm, we investigated the role of protein kinase C (PKC) in the development of ventricular fibrillation (VF) in hearts subjected to hypoxia (12 min) and re-oxygenation (40 min). We studied the effect of putative activators and inhibitors of PKC on the incidence of VF. Hearts exposed to 4 beta-phorbol,12,13-dibutyrate (PDBu), isophorbol or the membrane permeant diacylglycerol analog, 1-oleoyl-2-acetyl-rac-glycerol (OAG), during the prehypoxic phase had an increased incidence of VF during the hypoxic and reoxygenation periods. The incidence of VF was 90%, 83% and 75% in hearts exposed to PDBu, isophorbol and OAG, respectively (P < 0.05 vs control). Perfusion of hearts with PDBu was associated with a significant increase in the membrane fraction of cardiac PKC activity. In the presence of the inactive phorbol ester 4 alpha-phorbol didecanoate, the incidence of VF was 17% (P > 0.05 vs control). PKC activators were profibrillatory at concentrations that did not affect cardiac function: neither left ventricular developed pressure nor coronary perfusion pressure were affected. The effect of PDBu was antagonized by staurosporine: the incidence of VF was 17% in PDBu+staurosporine treated hearts (P < 0.05 vs control). To further study the profibrillatory effect of PDBu, hearts were exposed to PDBu in the presence of the ATP-dependent potassium channel antagonist glibenclamide. The latter prevented PDBu-induced VF. The results show that under the conditions employed, PDBu-induced activation of PKC induces redistribution of PKC activity and is associated with the development of VF.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Diglycerides; Glyburide; Guanidines; Isoquinolines; Perfusion; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Pinacidil; Piperazines; Protein Kinase C; Rabbits; Staurosporine; Ventricular Fibrillation

We have recently demonstrated that binding by monoclonal antibody (mAb) 8A2 to regenerating retinal ganglion cell axons in goldfish explants specifically induces a sustained, actin-based retraction response that is similar in most respects to a spontaneous retraction (S.G. Finnegan, V. Lemmon, and E. Koenig, Cell Motil. Cytoskeleton, 1992). Experiments were conducted to evaluate potential signal transduction pathways that may play a role in mediating retraction, using the mAb 8A2 retraction model system. Potential roles of cAMP, elevated intracellular calcium, or calmodulin-dependent processes were probed and the results did not appear to implicate them in either the induction or the maintenance of the axon retraction response. In contrast, treatment with phorbol 12-myristate 13-acetate, but not with inactive phorbol esters, induced a retraction response, although the response was more variable and less robust than that produced by mAb 8A2. However, both forms of induction were blocked by staurosporine, a nonspecific kinase inhibitor. Okadaic acid, a potent serine/threonine phosphatase inhibitor produced a very robust retraction response, and subthreshold doses significantly potentiated the retraction response induced by mAb 8A2. Genistein inhibited the mAb 8A2-induced retraction response at concentrations selective for tyrosine kinase activity in a dose-dependent manner. These findings are consistent with the hypothesis that an augmented phosphorylation state of one or more axonal proteins, perhaps catalyzed in part by protein kinase C, produces a sustained physiological retraction. In addition, tyrosine kinase may be involved in transducing surface-mediated interactions that trigger retraction, including the binding reaction signal of mAb 8A2.

Topics: Alkaloids; Animals; Antibodies, Monoclonal; Axons; Calcium; Cyclic AMP; Ethers, Cyclic; Goldfish; Nerve Regeneration; Okadaic Acid; Organ Culture Techniques; Phorbol Esters; Phorbols; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Retinal Ganglion Cells; Staurosporine; Tetradecanoylphorbol Acetate

1. The electrophysiological properties of the sensory neurons that mediate withdrawal reflexes in Aplysia are modulated by a number of second messengers. For example, the second messengers adenosine 3',5'-cyclic monophosphate (cAMP) and arachidonic acid modulate the S-K+ current (IK,S) and the calcium-activated K+ current (IK,Ca). Recent evidence suggests that protein kinase C (PKC) may also be an important regulator of cellular plasticity. In the present study we examined the possibility that IK,Ca was modulated by the activation of PKC in the pleural sensory neurons. 2. In voltage-clamped sensory neurons the application of phorbol esters, such as phorbol dibutyrate (PDBu), phorbol myristate (PMA), and phorbol diacetate (PDAc), which activate PKC, caused a dose-dependent increase in a voltage-dependent current with properties that resembled IK,Ca. The inactive isomer of phorbol ester, 4 alpha-phorbol, was without effect. 3. This phorbol ester-sensitive current had the kinetics and pharmacological sensitivity of IK,Ca. The current developed slowly during step depolarizations, showed little inactivation, and was activated at membrane potentials greater than approximately 0 mV. In addition, the current modulated by phorbol esters was blocked by a concentration of tetraethylammonium (TEA) that blocks a component of IK,Ca in the sensory neurons. 4. IK,Ca, which was activated directly by the iontophoretic injection of Ca2+, was also enhanced by PDBu. Moreover, the enhancement of Ca(2+)-elicited responses by PDBu persisted after Ca2+ influx was blocked by cobalt. These results indicate that at least one component of the modulation of IK,Ca by PDBu was independent of the modulation of voltage-dependent Ca2+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Aplysia; Calcium; Dose-Response Relationship, Drug; Enzyme Activation; Ganglia; In Vitro Techniques; Isoquinolines; Kinetics; Membrane Potentials; Neurons, Afferent; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Piperazines; Potassium Channels; Protein Kinase C; Protein Kinase Inhibitors; Second Messenger Systems; Tetradecanoylphorbol Acetate; Tetraethylammonium; Tetraethylammonium Compounds

Angiotensin II increased PGE2 release from superfused glomeruli, and stimulated labeled inositol phosphate production. 12-O-Tetradecanoyl phorbol -13-acetate (TPA, 10(-7) M), which stimulates protein kinase C activity in soluble fractions of glomerular homogenates, suppressed angiotensin II actions on inositol phosphate production and PGE2. By contrast, 4a phorbol 12,13 di-decanoate and phorbol had no effect on protein kinase C activity or angiotensin II induced increases in inositol phosphate or PGE2. 1-(5-Isoquinolinyl)-2-methylpiperazine (H-7), which inhibits protein kinase C activity in soluble fractions of glomerular homogenates, prevented TPA induced suppression of angiotensin II actions on inositol phosphate production and PGE2. Moreover H-7 prolonged the time course of angiotensin II induced inositol phosphate production and enhanced angiotensin II actions on glomerular PGE2 production. The results support a role for inositol phospholipid hydrolysis through the phospholipase C pathway in the mediation of angiotensin II actions on PGE2 in glomeruli and are consistent with negative modulation of these actions by protein kinase C.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Angiotensin II; Animals; Dinoprostone; Female; Isoquinolines; Kidney Glomerulus; Phorbol Esters; Phorbols; Piperazines; Prostaglandins E; Protein Kinase C; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

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.

Topics: Breast Neoplasms; Cell Division; Cell Line; Dose-Response Relationship, Drug; Female; Humans; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Protein Kinase C; Tetradecanoylphorbol Acetate

In cultured human 1321N1 astrocytoma cells, muscarinic receptor stimulation leads to phosphoinositide hydrolysis, formation of inositol phosphates, and mobilization of intracellular Ca2+. Treatment of these cells with 1 microM 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) completely blocks the carbachol-stimulated formation of [3H]inositol mono-, bis-, and trisphosphate ( [3H]InsP, [3H]InsP2, and [3H]InsP3). The concentrations of PMA that give half-maximal and 100% inhibition of carbachol-induced [3H]InsP formation are 3 nM and 0.5 microM, respectively. Inactive phorbol esters (4 alpha-phorbol 12,13-didecanoate and 4 beta-phorbol), at 1 microM, do not inhibit carbachol-stimulated [3H]InsP formation. The KD of the muscarinic receptor for [3H]N-methyl scopolamine is unchanged by PMA treatment, while the IC50 for carbachol is modestly increased. PMA treatment also abolishes carbachol-induced 45Ca2+ efflux from 1321N1 cells. The concomitant loss of InsP3 formation and Ca2+ mobilization is strong evidence in support of a causal relationship between these two responses. In addition, our finding that PMA blocks hormone-stimulated phosphoinositide turnover suggests that there may be feedback regulation of phosphoinositide metabolism through the Ca2+- and phospholipid-dependent protein kinase.

Topics: Astrocytoma; Calcium; Carbachol; Cells, Cultured; Humans; Hydrolysis; Phorbol Esters; Phorbols; Phosphatidylinositols; Receptors, Muscarinic; Tetradecanoylphorbol Acetate

Preincubation of frog erythrocyte lysates with tumor-promoting phorbol diesters leads to an increase in adenylate cyclase activity. This stimulatory effect of phorbol diesters was specific. Incubation with 12-O-tetradecanoylphorbol 13-acetate led to increases in basal (38%) and isoproterenol- (40%), fluoride- (25%), and Mn-stimulated (68%) adenylate cyclase activities compared with control. The inactive phorbol diesters (4 alpha-phorbol 12,13-didecanoate and beta-phorbol) were ineffective in promoting increases in adenylate cyclase activity. The effect of active phorbol diesters was also observed on isolated frog erythrocyte membranes in the absence of cell supernatant, although to a much lesser extent than in the whole lysates. Addition of the cell supernatant or of purified protein kinase C to the membranes maximized the sensitization by the phorbol diesters. These data are consistent with the notion that some component(s) of the adenylate cyclase system is (are) phosphorylated by protein kinase C, resulting in an enhancement of enzyme activity.

Topics: Adenylyl Cyclases; Animals; Carcinogens; Erythrocyte Membrane; Erythrocytes; Fluorides; Isoproterenol; Kinetics; Manganese; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phorbols; Rana pipiens; Structure-Activity Relationship; Tetradecanoylphorbol Acetate

In hepatocytes isolated from meal-fed rats, phorbol 12-myristate 13-acetate as well as phorbol 12,13-didecanoate stimulated de novo fatty acid synthesis in a dose-dependent manner. Moreover, phorbol 12-myristate 13-acetate inhibited ketogenesis from exogenous oleate, but slightly enhanced oleate esterification. The stimulation of esterification was more pronounced with endogenously synthesized fatty acids. In hepatocytes from 24h-starved rats a moderate stimulation of gluconeogenesis and ureogenesis was observed with glutamine as substrate. It is concluded that tumor-promoting phorbol esters mimic the short-term effects of insulin on hepatic fatty acid metabolism.

Topics: Animals; Fatty Acids; Food; Gluconeogenesis; Glutamine; Ketone Bodies; Liver; Male; Phorbol Esters; Phorbols; Rats; Rats, Inbred Strains; Starvation; Tetradecanoylphorbol Acetate

When human transformed RSa cells were treated simultaneously with potent tumor promoter 12-O-tetradecanoyl-phorbol-13 acetate (TPA) and interferon (IFN) (20-100 IU/ml) at the concentration which suppresses the multiplication of cells slightly, distinct synergistic suppression of cell growth was observed. Synthesis of DNA in these cells was reduced in proportion to the concentration of IFN and it was more strongly reduced by combined treatment with IFN and TPA. When IFN was pretreated with anti-IFN serum, such synergistic effect was not observed. Highly purified IFN (specific activity, 2 X 10(8) U/ml) together with TPA elicited almost the same synergistic effect on RSa cells. Similar concentration of phorbol-12,13-didecanoate (PDD) also produced synergistic effects with IFN on RSa cells. In contrast, phorbol (PHR), which lacks tumor promoting activity, did not produce such synergistic effects even at a concentration of as high as 500 ng/ml. In the case of HeLa cells and IFN-resistant IFr cells, only an additive effect was observed. Effect of TPA on the antiviral action of IFN against VSV was tested and there was almost no influence of TPA on IFN action. In accordance with these results, 2-5A synthetase activity was not induced by TPA treatment and the level of enzyme action did not change significantly by the combined treatment with TPA and IFN.

Topics: 2',5'-Oligoadenylate Synthetase; Cell Division; Cell Line; Cell Transformation, Viral; Drug Resistance; Drug Synergism; Humans; Interferon Type I; Phorbol Esters; Phorbols; Tetradecanoylphorbol Acetate

Topics: Animals; Antibody Formation; B-Lymphocytes; Cytotoxicity, Immunologic; Female; Macrophages; Mice; Neoplasms, Experimental; Phorbol Esters; Phorbols; Structure-Activity Relationship; Tetradecanoylphorbol Acetate