microM Phorbol Dibutyrate Research Articles

Tyrosine hydroxylase phosphorylation in digitonin-permeabilized bovine adrenal chromaffin cells: the effect of protein kinase and phosphatase inhibitors on Ser19 and Ser40 phosphorylation.

The protein kinases and protein phosphatases that act on tyrosine hydroxylase in vivo have not been established. Bovine adrenal chromaffin cells were permeabilized with digitonin and incubated with [gamma-32P]ATP, in the presence or absence of 10 microM Ca2+, 1 microM cyclic AMP, 1 microM phorbol dibutyrate, or various kinase or phosphatase inhibitors. Ca2+ increased the phosphorylation of Ser19 and Ser40. Cyclic AMP, and phorbol dibutyrate in the presence of Ca2+, increased the phosphorylation of only Ser40. Ser31 and Ser8 were not phosphorylated. The Ca2+-stimulated phosphorylation of Ser19 was incompletely reduced by inhibitors of calcium/calmodulin-stimulated protein kinase II (46% with KN93 and 68% with CaM-PKII 273-302), suggesting that another protein kinase(s) was contributing to the phosphorylation of this site. The Ca2+-stimulated phosphorylation of Ser40 was reduced by specific inhibitors of protein kinase A (56% with H89 and 38% with PKAi 5-22 amide) and protein kinase C (70% with Ro 31-8220 and 54% with PKCi 19-31), suggesting that protein kinases A and C contributed to most of the phosphorylation of this site. Results with okadaic acid and microcystin suggested that Ser19 and Ser40 were dephosphorylated by PP2A.

Functional Uncoupling between the Receptor and G-Protein as the Result of PKC Activation, Observed in Aplysia Neurons.

Application of either acetylcholine (ACh), dopamine (DA), histamine (HA), or Phe-Met-Arg-Phe-NH2 (FMRFamide) induces a K+-current response in the identified neurons of Aplysia under voltage clamp. This type of response is mediated by a pertussis toxin (PTX)-sensitive G-protein, Gi or Go. Extracellular application of 60 microM phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), to these cells markedly depressed all the K+-current responses to ACh, DA, HA, and FMRFamide. The depressing effect of PDBu lasted for at least 60 min despite continuous washing with the normal perfusing medium. Application of PKC inhibitors such as 100 microM H-7 or 10 microM staurosporine and PKCI(19-31) prior to the application of PDBu significantly decreased the depressing effects of PDBu. In contrast, an intracellular injection of okadaic acid (OA), an inhibitor of protein phosphatase 1 and 2A, significantly augmented the blocking effect of PDBu. Intracellular injection of the PKC catalytic subunit induced a similar depressing effect as observed with PDBu. The dose-response curves obtained with different transmitters all shifted downward after the activation of PKC, but the ED50 of each transmitter remained unchanged. Furthermore, the K+-current responses induced by the intracellular application of GTPgammaS were not depressed at all, even after the receptor-induced K+-current responses of the same cell were markedly depressed. These results strongly suggest that PKC phosphorylated a certain coupling site between the receptor and G-protein, and impaired the signal transduction necessary for triggering the K+-channel opening.

Effects of PKC downregulation on norepinephrine- and prostaglandin F2 alpha-induced contraction in rat aorta

The purpose of this study was to investigate, through phorbol ester-induced protein kinase C (PKC) downregulation, the role of PKC in the regulation of alpha-adrenergic agonist- and prostaglandin (PG) F2 alpha-induced contraction in vascular smooth muscle. In rat aorta, long-term phorbol ester exposure (10 microM phorbol dibutyrate for 17 h), a procedure that decreased PKC activity by > 95%, and maximal phorbol myristate acetate (PMA)-induced contraction by approximately 75%, decreased tissue sensitivity to norepinephrine (NE) and PGF2 alpha 2.8- and 4.6-fold, respectively, while maximal contraction was not significantly decreased. In contrast, long-term phorbol ester exposure did not alter tissue sensitivity to KCl, while maximal KCl contraction was decreased by 40%. Long-term phorbol ester exposure, as well as short-term phorbol ester exposure (1 microM PMA for 1 h), abolished the initial transient NE contraction elicited in Ca(2+)-free solution. In contrast, long-term phorbol ester exposure did not alter the plateau NE or PGF 2 alpha contraction elicited in Ca(2+)-free solution. Short-term phorbol ester exposure of PKC-downregulated tissues potentiated the plateau PGF2 alpha contraction elicited in Ca(2+)-free solution, although the magnitude of potentiation was less than that observed in non-PKC downregulated tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of protein kinase C reduces thromboxane receptors in glomeruli and mesangial cells

The potential role of protein kinase C (PKC) in the modulation of thromboxane A2 (TX) receptor density was evaluated in intact glomeruli and cultured renal mesangial cells (MC) from the rat. Incubation of glomeruli with 0.1 microM phorbol dibutyrate (PDBu) or 30 mM glucose for four hours activated PKC as reflected by increased in situ phosphorylation of the 80 kDa MARCKS protein, a specific endogenous substrate for PKC. High affinity binding to TX receptors, as assessed from the binding of the stable TX antagonist [3H]-Sq-29548 (Sq), was decreased 30% in glomeruli exposed to PDBu and 28% in glomeruli incubated in 30 mM D-glucose for four hours. Concurrent incubation with 0.05 microM of the PKC inhibitor staurosporine blocked both MARCKS protein phosphorylation and the decrease in TX receptor sites in response to either PDBu or 30 mM glucose. Neither 30 mM L-glucose nor 30 mM mannitol altered glomerular PKC activity or TX receptor density, thus excluding an osmotic effect of D-glucose, and implicating cellular metabolism of glucose in the expression of these actions. Inhibition of endogenous production of TX with indomethacin during exposure of glomeruli to 30 mM glucose did not prevent the decrease in TX binding. Homologous down-regulation of TX receptors mediated by endogenous TX was therefore not implicated in this action of glucose. The affinity of the glomerular receptor sites for [3H]-Sq was not affected by PKC activation. MC in passages 3 to 7 also demonstrated high affinity sites for [3H]-Sq (Kd, 2.8 nM). Culture of MC with PDBu (0.05 or 0.1 microM) for four hours decreased TX receptor density.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of the M current in NG108-15 neuroblastoma � glioma hybrid cells

The M current, IM, a voltage-dependent non-inactivating K current, was recorded in NG108-15 neuroblastoma x glioma hybrid cells, using the whole-cell mode of the patch-clamp technique. We studied inhibition of the M current by bradykinin, phorbol dibutyrate (PDBu), an activator of protein kinase C (PKC), and methylxanthines. Focal application of 0.1-5 microM bradykinin inhibited IM by about 60%; 5 nM bradykinin inhibited by about 40%. Bath application of 0.1 microM and 1 microM PDBu diminished IM to about half of the control value. Staurosporine, a PKC inhibitor, applied for 35-43 min in a concentration of 0.3 microM significantly reduced the effect of 1 microM PDBu. M current blockage by PDBu could be partly reversed by bath application of H-7 (51-64 microM), another PKC inhibitor. These observations suggest that the PDBu effect is really due to activation of PKC. The findings are compatible with the view [Brown DA, Higashida H (1988) J Physiol (Lond) 397:185-207] that the bradykinin effect on IM is mediated by PKC. However, three further observations suggest that this is only true for part of the bradykinin effect. When the suppression of IM by 1 microM PDBu was fully developed, 0.1 microM bradykinin produced a further inhibition of IM. Down-regulation of PKC by long-term treatment with PDBu reduced the effect of 0.1 microM bradykinin significantly but did not abolish it. Staurosporine (0.3 microM, applied for 31-46 min) failed to reduce the effect of 5 nM bradykinin significantly. The M current could be reversibly blocked by methylxanthines (caffeine, isobutyl-methylxanthine, theophylline) in the millimolar range, probably because of a direct action on the M channels.

Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms.

Protein kinase C interaction with calcium: a phospholipid-dependent process

The calcium-binding properties of calcium- and phospholipid-dependent protein kinase C (PKC) were investigated by equilibrium dialysis in the presence and the absence of phospholipids. Calcium binding to PKC displayed striking and unexpected behavior; the free proteins bound virtually no calcium at intracellular calcium concentrations and bound limited calcium (about 1 mol/mol of PKC) at 200 microM calcium. However, in the presence of membranes containing acidic phospholipids, PKC bound at least eight calcium ions per protein. The presence of 1 microM phorbol dibutyrate (PDBu) in the dialysis buffer had little effect on these calcium-binding properties. Analysis of PKC-calcium binding by gel filtration under equilibrium conditions gave similar results; only membrane-associated PKC bound significant amounts of calcium. Consequently, PKC is a member of what may be a large group of proteins that bind calcium in a phospholipid-dependent manner. The calcium concentrations needed to induce PKC-membrane binding were similar to those needed for calcium binding (about 40 microM calcium at the midpoint). However, the calcium concentration required for PKC-membrane binding was strongly influenced by the phosphatidylserine composition of the membranes. Membranes with higher percentages of phosphatidylserine required lower concentrations of calcium. These properties suggested that the calcium sites may be generated at the interface between PKC and the membrane. Calcium may function as a bridge between PKC and phospholipids. These studies also suggested that calcium-dependent PKC-membrane binding and PKC function could be regulated by a number of factors in addition to calcium levels and diacylglycerol content of the membrane.

Regulation of Protein Kinase C by Nerve Growth Factor, Epidermal Growth Factor, and Phorbol Esters in PC12 Pheochromocytoma Cells

We have used a permeabilized cell assay and a synthetic peptide substrate (KRTLRR) to specifically monitor the activity of protein kinase C in PC12 cells preincubated with nerve growth factor (NGF), epidermal growth factor (EGF), or phorbol esters. Pretreatment of PC12 cells with 1 microM 12-O-tetradecanoylphorbol 13-acetate or 1 microM phorbol dibutyrate stimulated the rate of KRTLRR peptide phosphorylation 4.8- and 2.6-fold, respectively. Furthermore, pretreatment of cells with NGF or EGF transiently increased the KRTLRR peptide kinase activity. Peak stimulations of KRTLRR peptide kinase (1.3-2-fold) were observed after 1-5 min of growth factor treatment and returned to control levels within 15-20 min. The KRTLRR peptide kinase activity fulfilled two criteria of protein kinase C. A synthetic peptide inhibitor of protein kinase C inhibited both growth factor- and phorbol ester-stimulated KRTLRR peptide kinase activity. In addition, growth factors and phorbol esters failed to stimulate KRTLRR peptide kinase activity in cells rendered protein kinase C-deficient by long-term treatment with 1 microM 12-O-tetradecanoylphorbol 13-acetate. In contrast to the transient activation of protein kinase C, ribosomal S6 kinase, assayed with the synthetic peptide RRLSSLRA, was persistently activated by NGF and EGF. The findings indicate that protein kinase C serves an early and transient role in the molecular actions of NGF and EGF in PC12 cells.