okadaic-acid and phorbol

Adaptive regulation and modulation by phosphorylation are mechanisms by which some cells control taurine transport. Goldfish and rat retinal cells were incubated with the activator of protein kinase C, phorbol 12,13-dibutyrate (PDBu), or the inhibitor of protein phosphatases, okadaic acid (OKA). OKA, 1 nM, inhibited the uptake of taurine at short period of incubation in goldfish retinal cells, and at low concentrations in rat retinal cells incubated with the inhibitor for 1 h. PDBu treatment did not produce significant effects. Isolated Müller cells from the goldfish retina presented a clear adaptive regulation and a decrease of taurine uptake by increasing phosphorylation either by the stimulation of PKC with PDBu or the inhibition of phosphatases with OKA.

Topics: Animals; Biological Transport; Cells, Cultured; Enzyme Activation; Enzyme Inhibitors; Goldfish; Okadaic Acid; Phorbol 12,13-Dibutyrate; Phorbols; Phosphoprotein Phosphatases; Protein Kinase C; Rats; Retina; Taurine

1. The present work was aimed to study the effect of PKC activation and protein-serine/threonine phosphatase (PP1/PP2 A) inhibition on P-glycoprotein (P-gp) mediated transport of L-DOPA in LLC-GA5 Col300 cells, a renal cell line expressing the human P-glycoprotein in the apical membrane. 2. L-DOPA accumulation was a time-and concentration-dependent process with the following kinetic characteristics: kin, 57.3 +/- 1.2 pmol mg protein(-1) min(-1); k(out), 3.3 +/- 0.1 pmol mg(-1) protein min(-1); Amax, 10.6 +/- 0.8; Kn, 198 +/- 64 microM; Vmax, 5.2 +/- 0.7 nmol mg protein(-1). 3. Verapamil (25 microM), a P-glycoprotein inhibitor, markedly increased (approximately 40% increase) the accumulation of a non-saturating concentration of L-DOPA (2.5 microM) at both initial rate of uptake (IRU, 6 min incubation) and at steady-state (SS, 30 min incubation). 4. PKC activation with phorbol 12,13-dibutyrate (PDBu, 1, 3 and 10 nM) produced a concentration-dependent decrease in L-DOPA accumulation at SS, but not at IRU. The inactive phorbol ester, 4alpha-phorbol 12,13-didecanoate (100 nM), produced no change in L-DOPA accumulation. The effect of PDBu was completely reverted by staurosporine (100 nM). The phosphatase inhibitor okadaic acid (100 nM) reduced by 20% the accumulation of L-DOPA at IRU, but not at SS. 5. It is suggested that P-glycoprotein plays a role in regulation of intracellular availability of L-DOPA in renal epithelial cells, and phosphorylation/dephosphorylation of P-glycoprotein may be involved in the regulation of the transporter.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cells, Cultured; Dose-Response Relationship, Drug; Drug Interactions; Epithelium; Humans; Kidney; Levodopa; Okadaic Acid; Phorbols; Phosphoric Monoester Hydrolases; Phosphorylation; Protein Kinase C; Staurosporine; Time Factors; Verapamil

Cross-regulation from the stimulatory phospholipase C to the adenylyl cyclase pathways was explored in neuroblastoma-glioma NG-108-15 cells in culture. Activation of protein kinase C by phorbol myristic acid resulted in a markedly attenuated activation of the inhibitory adenylyl cyclase response to delta-opiate agonists and epinephrine but not to the muscarinic agonist carbachol. The ability of okadaic acid to mimic the effects of phorbol myristic acid on the inhibitory response suggested a role for protein phosphorylation. Adenylyl cyclase activity from cells in which protein kinase C had been activated demonstrated a loss in the inhibitory adenylyl cyclase response at the level of the G-protein. Activation of protein kinase C prompted a 2-4-fold increase in phosphorylation of G1 alpha 2 in cells metabolically labeled with [32P]orthophosphate. The phosphate content of Gi alpha 2 was determined to be approximately 0.5 mol/mol subunit in the unstimulated cells and approximately 1.5 mol/mol subunit for cells in which protein kinase C was activated. The effects of okadaic acid, 4-alpha-phorbol, and calphostin C on inhibition of adenylyl cyclase in cells treated with phorbol myristic acid correlate with the effects of these agents on phosphorylation of Gi alpha 2. The time courses for attenuation of inhibitory adenylyl cyclase and that for phosphorylation of Gi alpha 2 were similar in cells challenged with phorbol myristic acid. These data argue for cross-regulation from the stimulatory protein kinase C to inhibitory adenylyl cyclase pathways at the level of Gi alpha 2 via protein phosphorylation.

Topics: Adenylyl Cyclases; Enkephalin, Leucine-2-Alanine; Enzyme Activation; Ethers, Cyclic; Glioma; GTP-Binding Proteins; Hybrid Cells; Naphthalenes; Neuroblastoma; Okadaic Acid; Phorbols; Phosphorylation; Polycyclic Compounds; Protein Kinase C; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

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