okadaic-acid and phorbol-12-13-didecanoate

Treatment of striatal synaptosomes with the protein phosphatase inhibitor okadaic acid significantly decreased gamma-aminobutyric acid (GABA) uptake, indicating that the GABA transporter may be regulated by phosphorylation. Forskolin and 8-bromoadenosine-3,5-cyclic monophosphate (8-br-cAMP) inhibited GABA uptake to the same extent as okadaic acid, suggesting the involvement of protein kinase A in GABA transporter regulation. In contrast, the same treatments did not alter dopamine (DA) uptake into striatal synaptosomal preparations. The results suggest that the structurally related GABA and DA transporters may be subject to different post-translational regulation.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Carrier Proteins; Colforsin; Dopamine; Dopamine Plasma Membrane Transport Proteins; Ethers, Cyclic; GABA Plasma Membrane Transport Proteins; gamma-Aminobutyric Acid; In Vitro Techniques; Male; Membrane Glycoproteins; Membrane Proteins; Membrane Transport Proteins; Neostriatum; Nerve Tissue Proteins; Okadaic Acid; Organic Anion Transporters; Phorbol Esters; Phosphoprotein Phosphatases; Phosphorylation; Rats; Rats, Sprague-Dawley; Signal Transduction; Synaptosomes

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

12-O-Tetradecanoylphorbol-13-acetate (TPA) markedly enhanced the increase in L-histidine decarboxylase (HDC) activity induced by dexamethasone in mouse mastocytoma P-815 cells, even with a concentration of the latter that had the maximal effect, whereas it induced a rapid and transient increase in HDC activity, which peaked after 3 h in the absence of dexamethasone. The synergistic effect of TPA on HDC activity induced by dexamethasone was detected after 4 h, a plateau level being reached by 6 h, which was similar to the time course with dexamethasone alone. TPA enhanced the induction of HDC activity by various glucocorticoids, but had no effect on the induction by dibutyryl cAMP, prostaglandin E2 or sodium butyrate. Both 1-oleoyl-2-acetylglycerol, a protein kinase C activator, and okadaic acid, a protein phosphatase inhibitor, enhanced the increase in HDC activity induced by dexamethasone, but 4 alpha-phorbol-12,13-didecanoate, an inactive derivative of TPA, did not. Protein kinase C inhibitors, such as staurosporin, H-7 and K255a, suppressed the increase in HDC activity induced by TPA with or without dexamethasone. The enhancement of HDC activity by dexamethasone was completely suppressed by cycloheximide or actinomycin D. Furthermore, TPA markedly enhanced the accumulation of HDC mRNA due to dexamethasone (5 to 10-fold, from 6 to 12 h after). TPA did not cause a significant increase in the level of either [3H]dexamethasone binding capacity or preformed HDC activity in cells. These results taken together suggest that dexamethasone-induced de novo synthesis of HDC in mastocytoma P-815 cells is up-regulated by TPA-activated protein kinase C through the mechanism involving an increased rate of transcription.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Carcinogens; Dexamethasone; Diglycerides; Drug Synergism; Enzyme Activation; Ethers, Cyclic; Histidine Decarboxylase; Isoquinolines; Mast-Cell Sarcoma; Mice; Okadaic Acid; Phorbol Esters; Phosphoprotein Phosphatases; Piperazines; Protein Kinase C; RNA, Messenger; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Recently, the ligand-receptor signal transduction mechanism has been implicated in mediating the zona pellucida (ZP)-induced acrosome reaction. Little is known about the role of protein phosphorylation in this specific event. We examine whether modification of protein phosphorylation and dephosphorylation affects the kinetics of the acid-solubilized ZP-induced acrosome reaction of mouse sperm. Mouse epididymal sperm were incubated in modified Krebs-Ringer bicarbonate medium for a period of 90 to 120 min and then treated with 2 acid-solubilized ZP/microliters for an additional 60 min. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. Capacitated sperm were inhibited from undergoing acid-solubilized ZP-induced acrosome reaction in the presence of an inhibitor of cyclic nucleotide-dependent protein kinase, H8; activators of the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C); an inhibitor of phosphatases 1 and 2A, okadaic acid; or an inhibitor of protein tyrosine kinases, genistein. The addition of inhibitors of protein kinase C, such as staurosporine, H7, and protein kinase C [19-36] pseudosubstrate, inhibited the phorbol ester-dependent inhibition of the acid-solubilized ZP-induced acrosome reaction. The present study suggests that protein phosphorylation and dephosphorylation play a regulatory role in the process of the ZP-induced acrosome reaction.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Acrosome; Alkaloids; Animals; Diglycerides; Ethers, Cyclic; Exocytosis; Female; Genistein; Isoflavones; Isoquinolines; Male; Membrane Fusion; Mice; Okadaic Acid; Phorbol Esters; Phosphoprotein Phosphatases; Phosphorylation; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Protein Processing, Post-Translational; Signal Transduction; Sperm-Ovum Interactions; Staurosporine; Tetradecanoylphorbol Acetate; Zona Pellucida

We examined effects of modulators of protein kinases and phosphatases on the kinetics of mouse sperm capacitation. The chlortetracycline fluorescence assay was used to monitor the process of capacitation (in terms of the appearance of the B pattern). The treatment of sperm with dibutyryl cyclic AMP (cAMP) or dibutyryl cGMP resulted in a higher percentage B pattern at various times during capacitation compared with the control. The addition of 100 microM H8 inhibited the cyclic nucleotide-dependent stimulation of capacitation. Tumor promotors, 12-O-tetradecanoyl phorbol 13-acetate (TPA; a stimulator of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A), induced a rapid appearance of the B pattern (15 min after addition) and maintained a percentage B pattern similar to that of the control in the later period of capacitation. An inhibitor of protein kinase C, staurosporine, inhibited the TPA-dependent acceleration of capacitation. Furthermore, the addition of genistein, an inhibitor of protein tyrosine kinases, resulted in a strong inhibition of capacitation. All agents tested did not affect sperm motility. These data suggest that protein phosphorylation and dephosphorylation may play regulatory roles in mediating mouse sperm capacitation.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Animals; Bucladesine; Chlortetracycline; Dibutyryl Cyclic GMP; Diglycerides; Ethers, Cyclic; Fluorescence; Genistein; Isoflavones; Isoquinolines; Male; Mice; Okadaic Acid; Peptide Fragments; Phorbol Esters; Phosphoprotein Phosphatases; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Sperm Capacitation; Staurosporine; Tetradecanoylphorbol Acetate