Inhibitory Effect Of Phorbol 12-myristate 13-acetate Research Articles

Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A–protein kinase C cross talk

Dopamine cellular signaling via the D1 receptor (D1R) involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D1R-mediated inhibition of NADPH oxidase activity involves cross talk between PKA and a specific PKC isoform(s). In HEK-293 cells heterologously expressing human D1R (HEK-hD1), fenoldopam, a D1R agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D1R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two PKA inhibitors, H89 (10μmol/L; 88±8.1%) and Rp-cAMP (10μmol/L; 97.7±6.7%), and two PKC inhibitors, bisindolylmaleimide I (1μmol/L; 94±6%) and staurosporine (10nmol/L; 93±8%), which by themselves had no effect (n=4–8/group). The inhibitory effect of PMA (1μmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%; n=5 or 6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθS676, an effect that was also blocked by H89. Fenoldopam (1μmol/L) also increased PKCθS676 in HEK-hD1 and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross talk between PKA and PKCθ plays an important role in the D1R-mediated negative regulation of NADPH oxidase activity in human kidney cells.

Modulation of ileal apical Na+-dependent bile acid transporter ASBT by protein kinase C

Ileal apical Na(+)-dependent bile acid transporter (ASBT) is responsible for reabsorbing the majority of bile acids from the intestinal lumen. Rapid adaptation of ASBT function in response to physiological and pathophysiological stimuli is essential for the maintenance of bile acid homeostasis. However, not much is known about molecular mechanisms responsible for acute posttranscriptional regulation of ileal ASBT. The protein kinase C (PKC)-dependent pathway represents a major cell signaling mechanism influencing intestinal epithelial functions. The present studies were, therefore, undertaken to investigate ASBT regulation in intestinal Caco-2 monolayers by the well-known PKC activator phorbol 12-myristate 13-acetate (PMA). Our results showed that Na(+)-dependent [(3)H]taurocholic acid uptake in Caco-2 cells was significantly inhibited in response to 2 h incubation with 100 nM PMA compared with incubation with 4alpha-PMA (inactive form). The inhibitory effect of PMA was blocked in the presence of 5 microM bisindolylmaleimide I (PKC inhibitor) but not 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM (Ca(2+) chelator) or LY-294002 (phosphatidylinositol 3-kinase inhibitor). PMA inhibition of ASBT function was also abrogated in the presence of myristoylated PKCzeta pseudosubstrate peptide, indicating involvement of the atypical PKCzeta isoform. The inhibition by PMA was associated with a significant decrease in the maximal velocity of the transporter and a reduction in ASBT plasma membrane content, suggesting a modulation by vesicular recycling. Our novel findings demonstrate a posttranscriptional modulation of ileal ASBT function and membrane expression by phorbol ester via a PKCzeta-dependent pathway.

The regulation of glycine transporter GLYT1 is mainly mediated by protein kinase Cα in C6 glioma cells

Glycine has been shown to possess important functions as a bidirectional neurotransmitter. At synaptic clefts, the concentration of glycine is tightly regulated by the uptake of glycine released from nerve terminals into glial cells by the transporter GLYT1. It has been recently demonstrated that protein kinase C (PKC) mediates the downregulation of GLYT1 activity in several cell systems. However, it remains to be elucidated which subtypes of PKC might be important in the regulation of GLYT1 activity. In this study, we attempted to make clear the mechanism of the phorbol 12-myristate 13-acetate (PMA)-suppressed uptake of glycine in C6 glioma cells which have the native expression of GLYT1. In C6 cells, the expression of PKCα, PKCδ, and PKCɛ of the PMA-activated subtypes was detected. The PMA-suppressed action was fully reversed by the removal of both extracellular and intracellular Ca 2+. Furthermore, the inhibitory effects of PMA or thymeleatoxin (THX), which is a selective activator of conventional PKC (cPKC), were blocked by the downregulation of all PKCs expressed in C6 cells by long-term incubation with THX, or pretreatment with GF109203X or Gö6983, which are broad inhibitors of PKC, or Gö6976, a selective inhibitor of cPKC. On the other hand, treatment of C6 cells with ingenol, a selective activator of novel PKCs, especially PKCδ and PKCɛ, did not affect the transport of glycine. Silencing of PKCδ expression by using RNA interference or pretreatment with the inhibitor peptide for PKCɛ had no effect on the PMA-suppressed uptake of glycine. Together, these results suggest PKCα to be a crucial factor in the regulation of glycine transport in C6 cells.

Phorbol Ester-induced G1 Phase Arrest Selectively Mediated by Protein Kinase Cδ-dependent Induction of p21

Although protein kinase C (PKC) has been widely implicated in the positive and negative control of proliferation, the underlying cell cycle mechanisms regulated by individual PKC isozymes are only partially understood. In this report, we show that PKCdelta mediates phorbol ester-induced G1 arrest in lung adenocarcinoma cells and establish an essential role for this novel PKC in controlling the expression of the cell cycle inhibitor p21. Activation of PKC with phorbol 12-myristate 13-acetate (PMA) in early G1 phase impaired progression of lung adenocarcinoma cells into S phase, an effect that was completely abolished by specific depletion of PKCdelta, but not PKCalpha. Although the PKC effect was unrelated to the inhibition of cyclin D1 expression, PKC activation significantly up-regulated p21 and down-regulated Rb hyperphosphorylation and cyclin A expression. Elevations in p21 mRNA and protein by PMA were mediated by PKCdelta but not PKCalpha. Studies using luciferase reporters also revealed an essential role for PKCdelta in the PMA-induced inhibition of Rb-dependent cyclin A promoter activity. Finally, we showed that the cell cycle inhibitory effect of PKCdelta is greatly attenuated by RNA interference-mediated knock-down of p21. Our results identify a novel link between PKCdelta and G1 arrest via p21 up-regulation and highlight the complexities in the downstream effectors of PKC isozymes in the context of cell cycle progression and proliferation.

Phosphorylation of Coronin 1B by Protein Kinase C Regulates Interaction with Arp2/3 and Cell Motility

Coronins are a conserved family of WD repeat-containing, actin-binding proteins that regulate cell motility in a variety of model organisms. Our results show that Coronin 1B is a ubiquitously expressed member of the mammalian Coronin gene family that co-localizes with the Arp2/3 complex at the leading edge of fibroblasts, and co-immunoprecipitates with this complex. Pharmacological experiments show that the interaction between Coronin 1B and the Arp2/3 complex is regulated by protein kinase C (PKC) phosphorylation. Coronin 1B is phosphorylated by PKC both in vitro and in vivo. Using tryptic peptide mapping and mutagenesis, we have identified serine 2 (Ser-2) on Coronin 1B as the major residue phosphorylated by PKC in vivo. Rat2 fibroblasts expressing the Coronin 1B S2A mutant show enhanced ruffling in response to phorbol 12-myristate 13-acetate (PMA) and increased speed in single cell tracking assays. Cells expressing the Coronin 1B S2D mutant have attenuated PMA-induced ruffling and slower cell speed. Expression of the S2A mutant partially protects cells from the inhibitory effects of PMA on cell speed, whereas expression of the S2D mutant renders cells hypersensitive to its effects. These data demonstrate that Coronin 1B regulates leading edge dynamics and cell motility in fibroblasts, and that its ability to control motility and interactions with the Arp2/3 complex are regulated by PKC phosphorylation at Ser-2. Furthermore, Coronin 1B phosphorylation is responsible for a significant fraction of the effects of PMA on fibroblast motility.

Involvement of PKCe in the Negative Regulation of Akt Activation Stimulated by G-CSF.

Granulocyte colony-stimulating factor (G-CSF) supports the proliferation, differentiation and survival of myeloid cells by stimulating the activation of several signaling cascades including the serine/threonine kinase Akt pathway. Akt activation has been shown to be important for G-CSF-induced survival and granulocytic differentiation. Although significant progresses have been made in our understanding of the molecular mechanisms by which Akt is activated, much less is known about the signaling events that negatively regulate Akt activation. Interestingly, G-CSF-induced activation of Akt was completely inhibited when myeloid 32D cells transfected with the wild type G-CSF receptor were incubated with phorbol-12-myristate 13-acetate (PMA), a PKC activator. PMA-mediated inhibition of Akt activation occurred with 5 min and lasted at least 1 hour. Previously, it has been shown that a carboxyl terminally truncated G-CSF receptor (D715), whose expression is associated with the development of acute myeloid leukemia in patients with severe congenital neutropenia (SCN), mediates significantly prolonged Akt activation. Notably, Akt activation by G-CSF in 32D cells expressing the D715 receptor mutant was rapidly downregulated by PMA treatment. The inhibitory effect of PMA on Akt activation was abolished by pretreatment of cells with the specific PKC inhibitor GF109203X, suggesting that PKC-dependent pathway negatively regulates Akt activation. Ro-31-7549, a specific inhibitor of PKCe, also abrogated PMA-mediated inhibition of Akt activation whereas rottlerin and Go6976, inhibitors of PKCd and PKC a/bI, respectively, displayed no effect. Together, these results identified PKCe as being critically involved in PMA-mediated inhibition of Akt activation. Experiments are currently under way to determine the mechanism by which PKCe downregulates Akt activation and the role of PKCe in the regulation of cell proliferation, differentiation and survival in response to G-CSF.

THE IMPACT OF PHORBOL ESTER ON THE REGULATION OF AMILORIDE-SENSITIVE EPITHELIAL SODIUM CHANNEL IN ALVEOLAR TYPE II EPITHELIAL CELLS

Amiloride-sensitive sodium channel (ENaC) plays an important role in recovery from pulmonary edema. Recently, it has been shown that an activation of protein kinase C (PKC) could affect the mRNA expression of ENaC in rat parotid gland cells and A6 distal nephron epithelial cells. To determine whether an activation of PKC would regulate the mRNA expression or the function of ENaC, we stimulated rat alveolar type II epithelial cells with phorbol 12-myristate 13-acetate (PMA), a potent PKC activator, at a concentration of 100 nM. The mRNA expression of α -, β -, and γ -ENaC subunits and amiloride-sensitive current were measured. PMA inhibited the mRNA expression of all 3 ENaC subunits (α -ENaC: 56.0% ± 12.1%; β -ENaC: 62.6% ± 15.9%; γ -ENaC: 68.5% ± 10.6%, respectively) and amiloride-sensitive current (control = 7.0 ± 1.5 μ A/cm 2 ; PMA = 1.7 ± 0.9 μ A/cm 2) significantly at 24 hours. On the other hand, 4 α -phorbol didecanoate 4 α -PDD, inactive form of PMA, had no inhibitory effect on α - and γ -ENaC expression or amiloride-sensitive current. However, no significant difference was seen in β -ENaC expression between PMA and 4 α -PDD. GF 109203X, a wide-range PKC inhibitor, blocked the inhibitory effect of PMA on all ENaC subunits mRNA expression. These results suggest that an activation of PKC may play an important role in the regulation of ENaC mRNA expression and function.

Protein kinase C activation reduces microglial cyclic AMP response to prostaglandin E2 by interfering with EP2 receptors.

We studied the modulation by protein kinase C (PKC) of the cyclic AMP (cAMP) accumulation induced by prostaglandin (PG) E2 in rat neonatal microglial cultures. Short pretreatment of microglia with phorbol 12-myristate 13-acetate (PMA) or 4beta-phorbol 12,13-didecanoate, which activate PKC, but not with the inactive 4alpha-phorbol 12,13-didecanoate, substantially reduced cAMP accumulation induced by 1 microM PGE2. The action of PMA was dose and time dependent, and the maximal inhibition (approximately 85%) was obtained after 10-min preincubation with 100 nM PMA. The inhibitory effect of PMA was mimicked by diacylglycerol and was prevented by the PKC inhibitor calphostin C. As PMA did not affect isoproterenol- or forskolin-stimulated cAMP accumulation, we investigated whether activation of PKC decreased cAMP production by acting directly at PGE2 EP receptors. Neither sulprostone (10(-9)-10(-5) M), a potent agonist at EP3 receptors (coupled to adenylyl cyclase inhibition), nor 17-phenyl-PGE2 (10(-6)-10(-5) M), an agonist of EP1 receptors, modified cAMP accumulation induced by forskolin. On the contrary, 11-deoxy-16,16-dimethyl PGE2, which does not discriminate between EP2 and EP4 receptors, both coupled to the activation of adenylyl cyclase, and butaprost, a selective EP2 agonist, induced a dose-dependent elevation of cAMP that was largely reduced by PMA pretreatment, as in the case of PGE2. These results indicated EP2 receptors as a possible target of PKC and suggest that PKC-activating agents present in the pathological brain may prevent the cAMP-mediated microglia-deactivating function of PGE2.

Protein kinase C-dependent and -independent inhibition of Ca 2+ influx by phorbol ester in rat pancreatic β-cells

Phorbol esters were used to investigate the action of protein kinase C (PKC) on insulin secretion from pancreatic β-cells. Application of 80 nM phorbol 12-myristate 13-acetate (PMA), a PKC-activating phorbol ester, had little effect on glucose (15 mM)-induced insulin secretion from intact rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, PMA significantly reduced the glucose-induced insulin secretion. PMA decreased the level of intracellular Ca 2+ concentration ([Ca 2+] i) elevated by the glucose stimulation when tested in isolated rat β-cells. This inhibitory effect of PMA was not prevented by BIM. PMA inhibited glucose-induced action potentials, and this effect was not prevented by BIM. Further, 4α-phorbol 12,13-didecanoate (4α-PDD), a non-PKC-activating phorbol ester, produced an effect similar to PMA. In the presence of nifedipine, the glucose stimulation produced only depolarization, and PMA applied on top of glucose repolarized the cell. When applied at the resting state, PMA hyperpolarized β-cells with an increase in the membrane conductance. Recorded under the voltage-clamp condition, PMA reduced the magnitude of Ca 2+ currents through L-type Ca 2+ channels. BIM prevented the PMA inhibition of the Ca 2+ currents. These results suggest that activation of PKC maintains glucose-stimulated insulin secretion in pancreatic β-cells, defeating its own inhibition of the Ca 2+ influx through L-type Ca 2+ channels. PKC-independent inhibition of electrical excitability by phorbol esters was also demonstrated.

Modulation of lysophosphatidic acid-induced Cl − currents by protein kinases A and C in the Xenopus oocyte

The roles of protein kinase C (PKC) and protein kinase A (PKA) in the regulation of lysophosphatidic acid (LPA)-induced Cl − currents in Xenopus oocytes were examined. PKC activation by phorbol 12-myristate 13-acetate (PMA) treatment completely blocked LPA-induced Cl − currents by inhibiting inositol 1,4,5-trisphosphate (IP 3) elevation. This inhibitory effect of PMA on the LPA response was blocked by pretreatment of oocytes with staurosporine and 3-[ N-(dimethylamino)propyl-3-indolyl]-4-[3-indolyl]maleimide (GF109203X), PKC inhibitors. In addition, treatment of oocytes with GF109203X enhanced the LPA response by increasing IP 3 production. Elevation of the intracellular adenosine 3′,5′-cyclic monophosphate (cAMP) concentration by treating oocytes with either forskolin (FK) plus isobutylmethylxanthine (IBMX) or 2′- O-dibutyryl-cAMP (dB-cAMP) reduced LPA-induced Cl − currents. The effect of activation of the cAMP pathway appears to be mediated by PKA, since treatment of oocytes with FK plus IBMX or dB-cAMP enhanced PKA activity. Furthermore, the inhibitory effect of dB-cAMP on the LPA response was blocked by treatment of oocytes with N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinolinesulframide-2HCl (H-89), a selective inhibitor of PKA. Both FK plus IBMX and dB-cAMP treatment reduced IP 3 generation in response to LPA stimulation. Inhibition of PKA activity with H-89 or Rp-cyclic 3′,5′-hydrogen phosphorothioate adenosine triethylammonium had no effect on LPA-induced Cl − currents. Finally, inhibition of the LPA response by activation of PKA was independent of extracellular Ca 2+. These results demonstrate that both PKC and PKA play active roles in modulating the LPA-induced signaling pathway.

Protein kinase C-mediated regulation of the renal Na +/dicarboxylate cotransporter, NaDC-1

The Na +/dicarboxylate cotransporter of the renal proximal tubule, NaDC-1, reabsorbs Krebs cycle intermediates, such as succinate and citrate, from the tubular filtrate. Although long-term regulation of this transporter by chronic metabolic acidosis and K + deficiency is well documented, there is no information on acute regulation of NaDC-1. In the present study, the transport of succinate in Xenopus oocytes expressing NaDC-1 was inhibited up to 95% by two activators of protein kinase C, phorbol 12-myristate, 13-acetate (PMA) and sn-1,2-dioctanoylglycerol (DOG). Activation of protein kinase A had no effect on NaDC-1 activity. The inhibition of NaDC-1 transport by PMA was dose-dependent, and could be prevented by incubation of the oocytes with staurosporine. Mutations of the two consensus protein kinase C phosphorylation sites in NaDC-1 did not affect inhibition by PMA. The inhibitory effects of PMA were partially prevented by cytochalasin D, which disrupts microfilaments and endocytosis. PMA treatment was also associated with a decrease of approximately 30% in the amount of NaDC-1 protein found on the plasma membrane. We conclude that the inhibition of NaDC-1 transport activity by PMA occurs by a combination of endocytosis and inhibition of transport activity.

Regulation of 5-Hydroxytryptamine-Induced Signal Transduction in Canine Cultured Aorta Smooth Muscle Cells by Phorbol Ester

Regulation of the increase in inositol phosphates (IPs) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine aorta smooth muscle cells (ASMCs). Stimulation of ASMCs by 5-hydroxytryptamine (5-HT) led to IPs formation and caused an initial transient [Ca 2+] i peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of ASMCs with phorbol 12-myristate 13-acetate (PMA) for 30 min almost abolished the 5-HT- induced IPs formation and Ca 2+ mobilization. This inhibition was reduced after long-term incubating the cells with PMA. Prior treatment of ASMCs with staurosporine or GF109203X, PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. In parallel with the effect of PMA on the 5-HT-induced IP formation and Ca 2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blotting with antibodies against different PKC isozymes. The results revealed that treatment of ASMCs with PMA for various times, translocation of PKC-α, βI, βII, δ, ϵ, θ, and ζ isozymes from the cytosol to the membrane was seen after 5-min, 30-min, 2-h, and 4-h treatment. However, 24-h treatment caused a partial down-regulation of these PKC isozymes. In conclusion, these results demonstrate that translocation of PKC-α, βI, βII, δ, ϵ, θ, and ζ induced by PMA caused an attenuation of 5-HT-induced IPs accumulation and Ca 2+ mobilization in ASMCs.

Molecular Characterization of the Human CRT-1 Creatine Transporter Expressed inXenopusOocytes

The protein sequence encoded by a creatine transporter cDNA cloned from a human heart library was identical to that cloned from a human kidney library (Nashet al., Receptors Channels2, 165–174, 1994), except that at position 285 the former contained anAlaresidue and the latter contained aProresidue. Expression of this human heart cDNA clone inXenopus laevisoocytes induced a Na+- and Cl−-dependent creatine uptake activity that saturated with aKmof ∼20 μM for creatine. The induced uptake was inhibited by β-guanidinopropionic acid (IC50∼44.4 μM), 2-amino-1-imidazolidineacetic acid (cyclocreatine; IC50∼369.8 μM), γ-guanidinobutyric acid (IC50∼697.9 μM), γ-aminobutyric acid (IC50∼6.47 mM), and amiloride (IC50∼2.46 mM). The inhibitors β-guanidinopropionic acid, cyclocreatine, and γ-guanidinobutyric acid also inhibited the uptake activity of theAla285toPro285(A285P) mutant as effectively as that of the wild type. In contrast, guanidinoethane sulfonic acid, a potent inhibitor of taurine transport, inhibited the uptake activity of the A285P mutant approx. two times more effectively than that of the wild type. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), but not its inactive analog, 4α-phorbol 12,13-didecanoate, inhibited the creatine uptake, and the inhibitory effect of PMA was both time and concentration dependent. The protein kinase A activator 8-bromo-cyclic AMP, however, had no effect on the creatine uptake. The rate of uptake increased hyperbolically with the increasing concentration of the external Cl−(equilibrium constantKCl−∼5 mM) and sigmoidally with the increasing concentration of the external Na+(equilibrium constantKNa+∼56 mM). Further analyses of the Na+and Cl−concentration dependence data suggested that at least two Na+and one Cl−were required to transport one creatine molecule via the creatine transporter.

Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney

The functional characteristics of rat organic anion transporter OAT1 were investigated using Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) by the oocytes expressing OAT1 was markedly inhibited by glutarate, α-ketoglutarate and probenecid, moderately inhibited by folate and methotrexate, but not inhibited by taurocholate or tetraethylammonium. Methotrexate and folate were transported by OAT1, but probenecid, a typical inhibitor of organic anion transporter, was not transported. Inhibition of PAH uptake by aliphatic dicarboxylates with various alkyl chain lengths was maximal at 5 (glutarate) and 6 (adipate) carbon atoms. OAT1-mediated PAH uptake was markedly inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and mezerein, but not by 4α-phorbol 12,13-didecanoate. The inhibitory effect of PMA was attenuated in the presence of staurosporine, suggesting that OAT1 is regulated by protein kinase C. These results suggest that the substrate recognition of OAT1 is comparable to that of renal basolateral organic anion transporter, and the transport activity is regulated by protein kinase C.

Consensus phosphorylation sites of human GABA C/GABA ρ receptors are not critical for inhibition by protein kinase C activation

The mechanism of inhibition of human GABA C/GABA ρ receptors by protein kinase C (PKC) activation was investigated in Xenopus oocytes. Phorbol 12-myristate 13 acetate (PMA), a potent PKC activator, at 25 nM inhibited the currents through GABA ρ2 receptors, which have one consensus phosphorylation site by PKC in the predicted intracellular loops. The time-courses and amplitudes of inhibition were not significantly different from those occurring through GABA ρ1 receptors, which have six such sites. The inhibitory effect of PMA was also observed after removing each consensus phosphorylation site in both GABA ρ1 and ρ2 receptors by site-directed mutagenesis. These results suggest that phosphorylation of consensus sites in the intracellular loops is not involved in the inhibition of human GABA C/GABA ρ receptors by PKC activation.

Loss of protein kinase C inhibition in the beta-T594M variant of the amiloride-sensitive Na+ channel.

We previously reported the presence of a novel variant (beta-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the beta-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein-Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the beta-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the beta-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

Regulation of p-aminohippurate transport by protein kinase C in OK kidney epithelial cells.

We studied the effect of phorbol 12-myristate 13-acetate (PMA), a phorbol ester which activates protein kinase C, on p-aminohippurate (PAH) transport in OK cells. PMA (10(-7) M) almost completely inhibited the transcellular transport of PAH across OK cell monolayers from the basal to the apical side, as well as the accumulation of PAH in the cells. The uptake of PAH across the basolateral membrane of OK cells was inhibited by PMA in a time-and dose-dependent fashion. Exposing the cells with other protein kinase C activators such as active phorbol esters and diacylglycerols also resulted in a significant inhibition of basolateral PAH uptake, but the inactive phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no effect. The inhibition of basolateral PAH uptake by PMA was blocked by staurosporine, an inhibitor of protein kinase C. Cycloheximide, actinomycin D, colchicine, and cytochalasin D did not affect the inhibitory effect of PMA on basolateral PAH uptake. These results suggested that the PAH transport system in OK cells is under the regulatory control of protein kinase C.

Differential effects of phorbol ester on apoptosis in HL-60 promyelocytic leukemia cells

The role of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) in apoptosis of HL-60 cells was investigated. PMA inhibited DNA fragmentation induced by thapsigargin (TG) and 4-bromo-calcium ionophore (Br-A23187). The inhibitory effect of PMA was concentration-related and was abolished by a specific PKC inhibitor, bisindolylmaleimide (GF109203X). In addition, TG-induced apoptosis was decreased in cells in which PKC activity was down-regulated by long-term pretreatment with PMA. These results indicate that PKC activation by PMA inhibits HL-60 cell apoptosis induced by TG and Br-A23187, and that this inhibition is not influenced by the down-regulation of PKC. However, PMA did not inhibit DNA fragmentation induced by 1-β- d-arabinofuranosylcytosine (Ara-C) and cycloheximide. PMA suppressed TG- or Br-A23187-induced DNA fragmentation probably by interfering in the modulation of calcium homeostasis by TG and Br-A23187. Our results indicate that PKC participates in the regulation of apoptosis only by some pathways. Down-regulation of PKC is not responsible for the diverse effects of PKC activators on apoptosis. The effect of a PKC modulator on apoptosis is dependent upon interaction with individual apoptotic stimulus.

Protein kinase C regulation of the adenylyl cyclase system in rat prostatic epithelium.

In the context of the crosstalk between transmembrane signalling pathways, we studied the loci within the stimulatory receptor/Gs protein/adenylyl cyclase system at which protein kinase C (PKC) exerts regulatory effects in rat prostatic epithelial cells. The treatment of cells with the PKC activator phorbol 12-myristate 13-acetate (PMA) resulted in an impairment of the stimulation of adenylyl cyclase activity in terms of both potency, as seen with both vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase-activating peptide (PACAP-27), and efficacy, as seen with the beta-adrenergic agonist isoproterenol. This inhibitory effect of PMA could be prevented by cell incubation with pertussis toxin but not with cholera toxin, pointing to a Gi- but not Gs-dependent mechanism. This hypothesis was reinforced by ADP-ribosylation experiments that showed a low extent of alpha i with pertussis toxin but no change of alpha s with cholera toxin, as well as by the observation of the loss of the ability of low Gpp[NH]p doses to inhibit forskolin-stimulated adenylyl cyclase activity (a measure of Gi function) after cell treatment with PMA. However, the phorbol ester did not modify the adenylyl cyclase catalytic subunit, as shown by experiments on direct stimulation of the enzyme by forskolin. Whatever the exact mechanisms, the results support a crosstalk between the PKC and the adenylyl cyclase systems in rat prostatic epithelial cells in terms of an impairment of adenylyl cyclase stimulation, due presumably to phosphorylation of both membrane receptors (coupled to Gs) and Gi protein, but not of Gs protein or the adenylyl cyclase itself.

Stimulatory and Inhibitory Effects of a Phorbol Ester on Chloride Secretion by Rat Epididymal Epithelium1

The effects of a protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), on Cl- secretion by rat cauda epididymal epithelium were studied through use of the short-circuit current (Isc) technique. PMA alone could stimulate the Isc in a dose-dependent manner. The PMA-induced Isc was blocked by the Cl channel blocker, diphenylamine-2-carboxylate, but not by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid. PMA also exerted an inhibitory effect on the subsequent Ca(2+)-activated Isc. ATP or ionomycin-induced Isc was significantly reduced by treatment with PMA (5-10 min). Both stimulatory and inhibitory effects of PMA could be mimicked by a diacylglycerol analog, 1,2-dioctanoyl-sn-glycerol, but not an inactive analog of PMA, 4 alpha-phorbol 12,13-didecanote (4 alpha D). Down-regulation of protein PKC by prolonged treatment of epididymal cells with PMA (12 h) diminished both stimulatory and inhibitory effects of PMA on Isc. These results suggest that the dual effect of PMA on Isc was mediated by PKC. However, the PKC inhibitor, calphostin C, could block the inhibitory effect of PMA on ATP-induced Isc but not the stimulatory effect of PMA alone on Isc. The stimulatory effect of PMA was apparent only when PMA was applied to the apical aspect; in contrast, the inhibitory effect of PMA on ATP-induced Isc was readily seen with application of PMA to either side of the epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)