Inhibitory Effect Of Phorbol 12,13-dibutyrate Research Articles

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Bidirectional regulation of renal cortical Na+,K+-ATPase by protein kinase C.

We examined the role of protein kinase C (PKC) in the regulation of Na+,K+- ATPase activity in the renal cortex. Male Wistar rats were anaesthetized and the investigated reagents were infused into the abdominal aorta proximally to the renal arteries. A PKC-activating phorbol ester, phorbol 12,13-dibutyrate (PDBu), had a dose-dependent effect on cortical Na+,K+-ATPase activity. Low dose of PDBu (10(-11) mol/kg per min) increased cortical Na+,K+-ATPase activity by 34.2%, whereas high doses (10(-9) and 10(-8) mol/kg per min) reduced this activity by 22.7% and 35.0%, respectively. PDBu administration caused changes in Na+,K+-ATPase Vmax without affecting K(0.5) for Na+, K+ and ATP as well as Ki for ouabain. The effects of PDBu were abolished by PKC inhibitors, staurosporine, GF109203X, and Gö 6976. The inhibitory effect of PDBu was reversed by pretreatment with inhibitors of cytochrome P450-dependent arachidonate metabolism, ethoxyresorufin and 17-octadecynoic acid, inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, and by actin depolymerizing agents, cytochalasin D and latrunculin B. These results suggest that PKC may either stimulate or inhibit renal cortical Na+,K+-ATPase. The inhibitory effect is mediated by cytochrome P450-dependent arachidonate metabolites and PI3K, and is caused by redistribution of the sodium pump from the plasma membrane to the inactive intracellular pool.

Protein kinase C may regulate the tonic component of intestinal smooth muscle contraction in response to substance P

(1) The study investigated a possible involvement of protein kinase C (PKC) in the substance P-induced contraction of the longitudinal muscle of the guinea-pig isolated ileum. (2) The predominant effect of the PKC activator, phorbol-12,13-dibutyrate (PDB), was to change the time course of the response to substance P. While the initial peak contraction was hardly influenced by PDB, the fading of the contraction was accelerated to an extent that any tonic contraction which normally followed the initial peak response was prevented. This inhibitory effect of PDB on the tonic contraction was immediate in onset and related to its concentration (20-200 nM); responses to half-maximally (2-7 nM) or maximally effective (0.74 microM) concentrations of substance P were affected in the same manner. Tetrodotoxin (0.6 microM) did not alter the effect of PDB. Phorbol-13-monoacetate (2 microM), a phorbol ester which does not stimulate PKC, failed to change the time course of the substance P-induced contraction. (3) The tonic component of half-maximal contractile responses to histamine (0.2-0.4 microM) was also depressed by PDB (0.2 microM) whereas the tonic component of maximal responses to histamine (9 microM) was enhanced. (4) PDB (0.2 microM) reduced desensitization to substance P as judged by the reduction of the peak response to substance P (2-7 nM) following a 10-min exposure to a high concentration of the peptide (0.74 microM). (5) The PKC inhibitor, polymyxin B (0.1-0.3 mM), reduced the peak contractile response to substance P, slowed the fading of the contraction, and antagonized the inhibitory effect of PDB on the tonic contraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of cAMP accumulation by kappa-receptor activation in isolated iris-ciliary bodies: role of phosphodiesterase and protein kinase C.

The present study was designed to examine the roles of protein kinase C (PKC) and phosphodiesterase (PDE) in modulating the action of kappa receptor stimulation on cAMP accumulation in isolated iris-ciliary bodies (ICBs) of New Zealand White rabbits. The kappa receptor agonist, (+/-)-1-(3,4-dichlorophenyl)acetyl-2-(1-pyrrolidinyl)methylpiperidine (BRL-52537) (BRL), and the PKC activator, phorbol 12,13-dibutyrate (PDBu), both caused a concentration-dependent inhibition of forskolin-stimulated cAMP production. The inhibitory effect of BRL on cAMP levels was significantly reduced in the presence of the selective kappa receptor antagonist, norbinaltorphimine (10(-6) M), but the effect of PDBu was not, thus supporting the involvement of kappa-opioid receptors in the response to BRL. In the presence of 3-isobutyl-1-methylxanthine or rolipram (10(-5) M), the inhibitory effect of BRL or PDBu (10(-6) M) on cyclic AMP accumulation was abolished. In the presence of the selective PKC antagonist, chelerythrine (10(-6) M), the inhibitory effect of PDBu or BRL (10(-6) M) was significantly reduced. Direct measurement of PDE activity demonstrated the ability of BRL and PDBu (10(-6) M) to augment the activity of these enzymes. Preincubation of ICBs with rolipram (10(-5) M) or chelerythrine (10(-6) M) caused significant reversal of both BRL- and PDBu-induced increases in PDE activity. These results indicate that stimulation of PKC and PDE4 activity is part of the complex mechanism whereby kappa-opioid receptor agonists reduce levels of cAMP in the rabbit ICB. This mechanism of action could contribute to the ability of kappa-opioid agonists to suppress aqueous flow rate and to lower intraocular pressure.

Involvement of protein kinase C in homologous desensitization of histamine-evoked secretory responses in rat chromaffin cells

The secretory responses in rat adrenal chromaffin cells to histamine H 1 receptor stimulation desensitize during repetitive stimulation. The rate of development of this desensitization was slowed by Ro 31-8220, a protein kinase C (PKC) inhibitor. Ro 31-8220 also reversed part of the desensitization which had been induced by earlier histamine stimulation. Phorbol 12,13-dibutyrate (PDBu), an activator of protein kinase C, inhibited histamine-evoked catecholamine (CA) secretion almost completely. The inhibitory effect of PDBu on the H 1-receptor-mediated secretory response was antagonized by Ro 31-8220. Histamine induced [Ca 2+] i increases due to Ca 2+ entry and Ca 2+ release from intracellular Ca 2+ stores in fura-2-loaded adrenal medullary cells. These [Ca 2+] i increases were abolished in PDBu-treated cells. These results suggest that the activation of PKC following histamine H 1 receptor stimulation plays a significant role in the process of homologous desensitization of histamine-evoked secretory responses in rat chromaffin cells, through modulation by PKC of H 1 receptors and/or GTP-binding proteins coupled with H 1 receptors.

Protein kinase C inhibits Na(+)-K(+)-2Cl- cotransporter activity in cultured rabbit nonpigmented ciliary epithelium.

We examined the regulation of Na(+)-K(+)-2Cl- transporter activity by protein kinase C (PKC) in a cell line derived from rabbit nonpigmented ciliary epithelium. Na(+)-K(+)-2Cl- cotransporter activity was measured as the rate of bumetanide-sensitive potassium (86Rb) transport. Phorbol 12,13-dibutyrate (PBDu) was used to activate PKC. PBDu inhibited bumetanide-sensitive potassium (86Rb) uptake, with a half-maximal inhibitory concentration of approximately 0.1 microM. The inhibitory effect of PBDu on potassium uptake by the N(+)-K(+)-2Cl- cotransporter was abolished by PCK downregulation and diminished by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a PKC inhibitor. PBDu inhibited Na(+)-K(+)-2Cl- cotransporter-mediated inward potassium (86Rb) transport by approximately 26% in control cells and by 40% in cells pretreated with ouabain. PKC activation also reduced the rate of bumetanide-sensitive potassium (86Rb) efflux in ouabain-treated cells but not in control (no oubain) cells. PBDu caused little change of intracellular sodium, potassium, or chloride, suggesting that an alteration of cytoplasmic ion composition is not responsible for the observed PBDu-induced changes in the rate of either inward or outward potassium movement mediated by the Na(+)-K(+)-2Cl- cotransporter.

Role of a pertussis toxin sensitive G-protein in mediating the effects of phorbol esters on receptor activated cyclic AMP accumulation in Jurkat cells.

In the human T-cell line, Jurkat, the accumulation of cyclic AMP induced by adenosine is enhanced by tumor-promoting phorbol esters, whereas prostaglandin E2 receptor-stimulated cAMP accumulation is antagonized (Nordstedt et al. 1989). In the present study we examine the involvement of pertussis toxin sensitive guanine nucleotide binding proteins (G-proteins) in producing the phorbol ester effects. Pertussis toxin pretreatment of the Jurkat cells invariably caused an ADP ribosylation of two G-proteins that inhibit adenylyl cyclase, tentatively identified as Gi2 and Gi3, using Western blots. Pertussis toxin treatment had little effect on basal cAMP accumulation, but sometimes inhibited, sometimes stimulated agonist and cholera toxin induced cAMP accumulation. The latter effect was not mimicked by the B-oligomer. Irrespective of whether pertussis toxin stimulated or inhibited NECA and cholera toxin-induced cAMP accumulation it could not block the effect of phorbol-12,13-dibutyrate (PDBu). The inhibitory effect of PDBu on prostaglandin E2-induced cAMP accumulation was, however, invariably eliminated by pertussis toxin treatment. In conclusion, activation of protein kinase C by phorbol esters reveals a Gi-mediated prostaglandin E receptor-induced inhibition of adenylate cyclase in addition to the prostaglandin E receptor-mediated stimulation of cAMP accumulation in Jurkat cells. The enhancement of adenosine A2 receptor stimulated cAMP accumulation by PDBu, on the other hand, does not involve a PTX sensitive Gi-protein.

Ontogeny of the regulation of Na+,K(+)-ATPase activity in the renal proximal tubule cell.

This study examines the ontogeny of the regulation of Na+,K(+)-ATPase activity in the proximal tubule (PT) by a first messenger, dopamine (DA), and by direct stimulation of a third messenger, protein kinase C (PKC). PT segments dissected from 10- (PT10), 15-(PT15), 20- (PT20), and 40- (PT40) d-old rats were preincubated with DA 10(-5) M, diacylglycerol (DAG) 10(-5) M (an endogenous activator of PKC), or phorbol 12,13-dibutyrate (PDBu) 10(-6) M (an exogenous activator of PKC). DA inhibited Na+,K(+)-ATPase activity in PT40. In PT20, DA also inhibited Na+,K(+)-ATPase activity, but the inhibitory effect in PT20 was less pronounced than in PT40. In PT15, DA had no effect on Na+,K(+)-ATPase activity. DAG significantly inhibited Na+,K(+)-ATPase activity in PT40. DAG also inhibited Na+,K(+)-ATPase activity in PT20, but the inhibition was slightly less pronounced than in PT40. DAG had no effect on Na+,K(+)-ATPase activity in PT15. Na+,K(+)-ATPase activity in PT40 and PT20 preincubated with PDBu was significantly lower than with vehicle. The inhibitory effect in PT20 was less pronounced than in PT40. When PT40 and PT20 were preincubated with both PDBu and 5 x 10(-5) M sphingosine, an inhibitor of PKC activation, the inhibitory effect of PDBu was abolished. In both PT40 and PT20 incubated with 4-alpha-12,13 phorbol didecanoate 10(-7) M, a phorbol ester that will not activate PKC, Na+,K(+)-ATPase activity was not different from the control. In PT10, Na+,K(+)-ATPase activity was the same after PDBu incubation and after vehicle incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Phorbol-ester-induced alterations of free calcium ion transients in single rat hepatocytes.

The effect of the phorbol esters phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDB) on changes in free Ca2+ concentration ([Ca2+]i) in single rat hepatocytes, microinjected with the photoprotein aequorin, were investigated. [Arg8]vasopressin and phenylephrine induced a series of repetitive [Ca2+]i transients. Phorbol esters inhibited the alpha 1-adrenoceptor-induced response; sub-nanomolar concentrations decreased the transient frequency, and higher concentrations abolished the transients. The inhibitory effect of PDB was readily reversible. Phorbol esters were less effective in decreasing the frequency of [Arg8]-vasopressin-induced transients, and the inhibition could be overcome by high [Arg8]vasopressin concentrations.