Inhibitory Effect Of PMA Research Articles

Involvement of Protein Kinase Cε in the Negative Regulation of Akt Activation Stimulated by Granulocyte Colony-Stimulating Factor

Stimulation of cells with G-CSF activates multiple signaling cascades, including the serine/threonine kinase Akt pathway. We show in this study that G-CSF-induced activation of Akt in myeloid 32D was specifically inhibited by treatment with PMA, a protein kinase C (PKC) activator. PMA treatment also rapidly attenuated sustained Akt activation mediated by a carboxy truncated G-CSF receptor, expressed in patients with acute myeloid leukemia evolving from severe congenital neutropenia. The inhibitory effect of PMA was abolished by pretreatment of cells with specific PKC inhibitor GF109203X, suggesting that the PKC pathway negatively regulates Akt activation. Ro31-8820, a PKCepsilon inhibitor, also abrogated PMA-mediated inhibition of Akt activation, whereas rottlerin and Go6976, inhibitors of PKCdelta and PKCalphabetaI, respectively, exhibited no significant effects. Furthermore, overexpression of the wild-type and a constitutively active, but not a kinase-dead, forms of PKCepsilon markedly attenuated Akt activation, and inhibited the proliferation and survival of cells in response to G-CSF. The expression of PKCepsilon was down-regulated with G-CSF-induced terminal granulocytic differentiation. Together, these results implicate PKCepsilon as a negative regulator of Akt activation stimulated by G-CSF and indicate that PKCepsilon plays a negative role in cell proliferation and survival in response to G-CSF.

Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells.

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediated l-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCalpha (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCalpha in PAEC. A selective activator of Ca(2+)-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes in l-arginine uptake and PKCalpha translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 micro M). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 micro M), which activates novel PKC isoforms, did not affect l-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 micro M; 1 h) induced the translocation of PKCepsilon (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCalpha. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCalpha to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCalpha after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.

Uncoupling of ATP-induced inositol phosphate formation and Ca 2+ mobilization by phorbol ester in canine cultured tracheal epithelial cells

The 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 canine cultured tracheal epithelial cells (TECs). Pretreatment of TECs with phorbol 12-myristate 13-acetate (PMA, 1 μM) for 30 min attenuated the ATP- and UTP-induced IPs formation and Ca 2+ mobilization. The concentrations of PMA that gave half-maximal (EC 50) inhibition of ATP- and UTP-induced IPs accumulation and an increase in [Ca 2+] i were 5–10 and 4–12 nM, respectively. Prior treatment of TECs with staurosporine (1 μM), a PKC inhibitor, partially inhibited the ability of PMA to attenuate ATP- and UTP-induced responses, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Furthermore, analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TECs expressed PKC-α, -βI, -βII, -γ, -δ, -ε, -θ, and -ζ. With PMA treatment of the cells for various times, translocation of PKC-α, -βI, -βII, -γ, -δ, -ε, and -θ from the cytosol to the membrane was seen after 5- and 30-min and 2- and 4-h treatment. However, 6-h treatment caused a partial down-regulation of these PKC isozymes. PKC-ζ was not significantly translocated and down-regulated at any of the times tested. In conclusion, these results suggest that activation of PKC may inhibit the phosphoinositide (PI) hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses to ATP and UTP. The translocation of PKC-α, -βI, -βII, -δ, -ε, -γ, and -θ induced by PMA caused an attenuation of ATP- and UTP-induced IPs accumulation and Ca 2+ mobilization in TECs.

Regulation of oligodendrocyte differentiation: protein kinase C activation prevents differentiation of O2A progenitor cells toward oligodendrocytes.

Oligodendrocytes differentiate on a specific schedule in vivo in order to myelinate axons at the precise time and at the appropriate position. The current study was undertaken to obtain further insight as to how this timed appearance is regulated intracellularly. We observed that exposure of O2A progenitor cells in culture to phorbol 12-myristate 13-acetate (PMA; an activator of protein kinase C, PKC) inhibited their differentiation to oligodendrocytes by suppressing the expression of specific myelin markers at the O4-stage. To positively identify a role of PKC per se in differentiation, the use of a minimal medium with low serum content turned out to be essential. This was demonstrated by showing that the inhibitory effect of PMA on oligodendrocyte differentiation could be completely abolished by a combined action of insulin, triiodothyronine (T3), hydrocortisone and other components of a chemically defined medium (CDM). Furthermore, the PMA-mediated inhibition of oligodendrocyte differentiation could be partially restored by activation of the cAMP signal transduction pathway. The results indicate that PKC plays a crucial role in the differentiation of O2A progenitor cells toward oligodendrocytes: PKC activation prevents differentiation of O2A progenitor cells, whereas differentiation toward oligodendrocytes is dependent on other signaling compounds which may counteract the PKC signal transduction route.

Inhibition of 5-hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilization in canine cultured tracheal smooth muscle cells by phorbol ester.

1. Regulation of the increase in inositol-1,4,5-trisphosphate (IP3) production and intracellular Ca2+ concentration ([Ca2+]i by protein kinase C (PKC) was investigated in canine cultured tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by 5-hydroxytryptamine (5-HT) caused an initial transient [Ca2+]i peak followed by a sustained elevation of [Ca2+]i in a concentration-dependent manner. 2. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 microM) for 30 min blocked the 5-HT-induced IP3 formation and Ca2+ mobilization. This inhibition was reduced after the cells had been incubated with PMA for 8 h, and within 48 h the 5-HT-induced Ca2+ mobilization reached the same extent as control cells. 3. The concentration of PMA that gave half-maximal inhibition of 5-HT-induced increase in [Ca2+]i was 4 nM. Pretreatment of TSMCs with staurosporine (1 microM) of GF109203X (0.1 microM), PKC inhibitors, inhibited the ability of PMA to attenuate 5-HT-induced responses, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. In parallel with the effect of PMA on 5-HT-induced IP3 formation and Ca2+ mobilization, the translocation and down-regulation of PKC isozymes were determined by Western blot analysis in TSMCs. Analysis of cell extracts by Western blotting with antibodies against different PKC isozymes revealed that TSMCs expressed PKC-alpha, beta I, beta II, delta, epsilon, theta and zeta. With PMA treatment of the cells for various times, translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta 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 PKC-zeta was not significantly translocated and down-regulated at any of the times tested. 5. In conclusion, these results suggest that activation of PKC may inhibit the receptor-mediated phosphoinositide hydrolysis and consequently attenuate the [Ca2+]i increase or inhibit both responses independently. The translocation of PKC-alpha, beta I, beta II, delta, epsilon, and theta induced by PMA caused an attenuation of 5-HT-stimulated IP3 accumulation and Ca2+ mobilization in TSMCs.

Bradykinin regulates the histamine-induced Ca 2+ mobilization via protein kinase C activation in human gingival fibroblasts

We previously demonstrated that histamine and bradykinin evoke an increase in intracellular Ca 2+ ([Ca 2+] i) in human gingival fibroblasts by using a fluorescent Ca 2+ indicator Fura-2. In this paper, we further demonstrate the regulation of the histamine-induced Ca 2+ mobilization by bradykinin. In fibroblasts stimulated with bradykinin (1 μM), subsequent stimulation with histamine (100 μM) failed to mobilize Ca 2+, whereas bradykinin induced an increase in [Ca 2+] i in the cells pre-stimulated with histamine. The attenuation of the histamine response was dependent on the concentration of bradykinin for the first stimulation. Histamine also failed to induce the formation of inositol 1,4,5-trisphosphate in fibroblasts pretreated with bradykinin. In fibroblasts pretreated with bradykinin (1 μM) for 3 min and then washed with fresh medium, the effect of histamine on [Ca 2+] i quickly returned to the control level. The activation of protein kinase C by phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (PMA) elicited a marked decrease in histamine-induced Ca 2+ mobilization. When the protein kinase C activity was inhibited with H7, a protein kinase C inhibitor, or was down-regulated by pretreatment with PMA for 20 h, the inhibitory effect of PMA on the histamine response was relieved. In the fibroblasts pretreated with H7 or PMA for 20 h, histamine evoked Ca 2+ mobilization even after bradykinin stimulation. These results suggest that the histamine response is regulated by bradykinin receptor activation via the activation of protein kinase C in human gingival fibroblasts.

Role of Protein Kinase C and Second Messengers in Regulation of the Norepinephrine Transporter

Publisher Summary Transport of NE by the neuronal NE transporter (NET) is absolutely dependent on extracellular Na+ and CI- and is selectively inhibited by, for example, desipramine and nisoxetine, and Na+ and Cl- dependent binding of these inhibitors to the NET has been used to localize the tissue distribution of NET. In spite of its physiological and therapeutic importance, little is known about the involvement of second messengers in the regulation of NET expression and function. Cloning of the human and bovine NET cDNAs has revealed one common consensus sequence for phosphorylation by protein kinase C (PKC) within the second intracellular loop and, for the bovine NET (bNET), two additional PKC sites at the C-terminal end of bNET. In this chapter, evidence that stimulation of PKC by a phorbol ester causes downregulation of NE transport was provided and that the second-messenger cyclic adenosine monophosphate (cAMP) may be involved in tissue-specific regulation of NETs. Uptake of [3]NE by all three transporters was not different from controls if cells were exposed to both PMA and staurosporin; that is, the inhibitory effect of PMA was antagonized by the PKC inhibitor staurosporin. The results of this study indicate that human NET (and NET in COS-7 cells) is downregulated in a staurosporin-sensitive way by the activation of PKC. Because NE transport in a variant hNET, lacking a potential phosphorylation site for PKC, was also downregulated by the PKC-activating phorbol ester PMA, a direct phosphorylation of the transporter at the PKC site(s) of NETS can be excluded.

Role of conserved peptide in taurine transporter inactivation modulated by protein kinase C.

Activation of protein kinase C (PKC) by the active phorbol ester 12-myristate 13-acetate (PMA, 100 nM) or phorbol-12, 13-dibutyrate (100 nM) reduced taurine uptake by 80% in oocytes given injections with cRNA from and expressing the Madin-Darby canine kidney cell taurine transporter pNCT. Inhibition of PKC by calphostin C or staurosporine increased taurine uptake by 20% and 400%, respectively. The inhibitory effect of PMA on taurine uptake was blocked by calphostin C, a specific inhibitor of PKC. Modulation by PMA mainly affected the apparent affinity K(m) (from 5.6 microM to 18.1 microM) with minimal effect on the maximal velocity (25% decrease) of the transporter. A polyclonal antibody (AbS4) directed against a conserved intracellular segment (S4) of the Madin-Darby canine kidney cell taurine transporter enhanced taurine uptake by pNCT cRNA-treated oocytes. The effect of AbS4 was blocked by incubation with the corresponding peptide antigen. Preimmune IgG and peptide antigen had no effect on taurine transporter activity expressed in oocytes. Modulation seemed to occur through phosphorylation of a consensus PKC site located on segment S4 of the transporter, because downregulation of the transporter by PMA (100 nM) was abolished by preinjection of AbS4 (12 ng/ oocyte). In contrast, downregulation of the transporter by PMA could not be restored by AbS4 when pNCT-expressing oocytes were pretreated with PMA (50 nM). In conclusion, the peptide segment recognized by this antibody appears to participate directly in taurine transporter inactivation that is modulated by PKC phosphorylation.

Effect of phorbol ester on phosphoinositide hydrolysis and calcium mobilization induced by endothelin-1 in cultured canine tracheal smooth muscle cells

Regulation of the increase in inositol 1,4,5-trisphosphate (IP 3) production and intracellular Ca 2+ concentration ([Ca 2+] i) by protein kinase C (PKC) was investigated in cultured canine tracheal smooth muscle cells (TSMCs). Stimulation of TSMCs by endothelin-1 (ET-1) led to IP 3 formation and caused an initial transient peak followed by a sustained elevation of [Ca 2+] i in a concentration-dependent manner. Pretreatment of TSMCs with phorbol 12-myristate 13-acetate (PMA, 1 μM ) for 30 min blocked the ET-1-induced IP 3 formation and Ca 2+ mobilization. However, this inhibition was reduced after incubating the cells for 8 h with PMA. Following preincubation, ET-1-induced Ca 2+ mobilization recovered with time and reached the same extent of control cells within 48 h. The concentrations of PMA that gave half-maximal inhibition (-IogEC 50) of ET-1-induced IP 3 formation and increase in [Ca 2+] i were 8.6 and 8.4 M, respectively. Prior treatment of TSMCs with staurosporine (1 μM), a PKC inhibitor, inhibited the ability of PMA to attenuate ET-1-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 ET-1-induced IN formation and Ca 2+ mobilization, a change of PKC activity was observed in TSMCs. PMA rapidly decreased PKC activity in the cytosol of TSMCs, while increasing it transiently in the membranes within 30 min. Thereafter the membrane-associated PKC activity decreased and persisted for at least 24 h of PMA treatment. Taken together, these results suggest that activation of PKC may inhibit the phosphoinositide hydrolysis and consequently attenuate the [Ca 2+] i increase or inhibit independently both responses. The PMA-induced inhibition of responses to ET-1 was associated with an increase in membranous PKC activity.

Protein kinase C activation inhibits cytokine-induced nitric oxide synthesis in vascular smooth muscle cells

Vascular smooth muscle cells (SMC) respond by relaxation to nitric oxide (NO) released from the endothelium which expresses a constitutive, Ca 2+-dependent NO synthase (cNOS). SMC can, however, produce NO themselves upon stimulation by proinflammatory cytokines which induce expression of an inducible, Ca 2+-independent NO synthase (iNOS). Protein kinase C represents another important second messenger system involved in the regulation of SMC contraction. We have investigated iNOS expression and NO synthesis in rat vascular SMC treated with the cytokines, IFNγ and TNFα, in the presence or absence of the activator of protein kinase C, β-phorbol-12-myristate 13-acetate (PMA). Treatment with PMA did not induce any significant accumulation of nitrite, a major stable metabolite of NO, in SMC. When added simultaneously with the cytokines, PMA significantly reduced nitrite accumulation induced by cytokine stimulation in a dose-dependent fashion. This inhibitory effect was mediated by activation of PKC since calphostin C, a specific PKC inhibitor, abolished the PMA effect. Further analysis of iNOS mRNA with a rat iNOS cDNA probe demonstrated that addition of PMA reduced expression of SMC iNOS mRNA, indicating that the antagonism in induction of NO synthesis between PMA and the proinflammatory cytokines acts on the transcriptional level. The inhibitory effect of PMA may be mediated via induction of a suppressor of iNOS expression, since pretreatment with PMA reduced NO production after subsequent treatment with cytokines. These observations suggest that activation of the PKC pathway is involved in a negative regulation of iNOS gene expression and this is compatible with the observation that vascular SMC contraction can be induced by PKC activation.

Phorbol esters alter functions of the expressed dopamine transporter

Recent elucidation of the amino acid sequences of the neurotransmitter transporters reveals several consensus sequences for phosphorylation by kinases including protein kinase C. Protein kinase C activation did modulate the function of the rat dopamine transporter expressed in COS cells. Cell treatment with the protein kinase C activator 12-myristate 13-acetate (PMA) reduced the affinity of binding of the radiolabeled cocaine analog [ 3H](-)-2β-carbomethoxy-3β-(4-fluorophenyl)tropane (WIN 35,428) without affecting its B max. The uptake of [ 3H]dopamine was reduced by treatment with PMA in a staurosporine-sensitive manner. Kinetic analysis revealed that the inhibitory effect of PMA on [ 3H]dopamine uptake was due to reduced uptake velocity and a small reduction of affinity for Na +, without changed affinity for dopamine. 1-Oleoyl-2-acetyl- sn-glycerol (OAG) mimicked these actions of PMA. These results demonstrate that activation of protein kinase C alters dopamine transporter functions in both ligand recognition and substrate translocation. These phosphorylation phenomena in vitro suggest the possibility that phosphorylation could modulate the activity of this important dopaminergic synaptic regulator under physiological conditions.

Endothelin‐ and Sarafotoxin‐Induced Phosphoinositide Hydrolysis in Cultured Canine Tracheal Smooth Muscle Cells

The endothelins (ETs) and sarafotoxin are two structurally related classes of potently contractile peptides. To understand the mechanism of action of ETs, we have examined the effect of ETs and sarafotoxin on phosphoinositide (PI) hydrolysis in cultured canine tracheal smooth muscle cells (TSMCs). ET-1, ET-2, ET-3, and sarafotoxin caused dose-dependent accumulation of inositol phosphatase (IPs) and tracheal smooth muscle contraction. BQ-123, an ETA receptor antagonist, had a high affinity to block the ET-1-induced IP accumulation and tracheal smooth muscle contraction with pKB values of 7.3 and 7.4, respectively. Pretreatment of TSMCs with cholera toxin impaired the ability of ET-1 and ET-2 to stimulate IP formation, whereas there was no effect by treatment with pertussis toxin. Stimulation of PI turnover by these peptides required the presence of extracellular Ca2+ and was blocked by treatment with EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IP accumulation. A further Ca(2+)-dependent increase in IP formation was obtained by inclusion of either GTPrS or ET-1. The combined presence of GTPrS and ET-1 elicited an additive effect on IP formation. Short-term exposure to phorbol 12-myristate 13-acetate (PMA, 1 microM) abolished the stimulation of PI hydrolysis induced by these peptides. The inhibitory effect of PMA on ET-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. Prolonged incubation of TSMCs with PMA resulted in a recovery of receptor responsiveness that may be due to downregulation of PKC.(ABSTRACT TRUNCATED AT 250 WORDS)

5‐Hydroxytryptamine receptor‐mediated phosphoinositide hydrolysis in canine cultured tracheal smooth muscle cells

1. 5-Hydroxytryptamine (5-HT) has been shown to induce contraction of tracheal smooth muscle. However, the mechanisms of action of 5-HT are not known. We therefore investigated the effects of 5-HT on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and its regulation in canine cultured tracheal smooth muscle cells (TSMCs) labelled with [3H]-inositol. 5-HT-induced inositol phosphates (IPs) accumulation was time- and dose-dependent with a half-maximal response (EC50) and a maximal response at 0.38 +/- 0.05 and 10 microM, respectively. 2. Ketanserin and mianserin (10 and 100 nM), 5-HT2 receptor antagonists, were equipotent in blocking the 5-HT-induced IPs accumulation with pKB values of 8.46 and 8.21, respectively. In contrast, the dose-response curves of 5-HT-induced IPs accumulation were not shifted until the concentrations of NAN-190 and metoclopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased up to 10 microM. 3. Pretreatment of TSMCs with pertussis toxin or cholera toxin did not inhibit the 5-HT-induced IPs accumulation, but partially inhibited the AlF(4-)-induced IPs response. 4. Stimulation of IPs accumulation by 5-HT required the presence of external Ca2+ and was blocked by EGTA. The addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs directly stimulated IPs accumulation. A further Ca(2+)-dependent increase in IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphoshate) (GTP gamma S) or 5-HT. The combination of GTP gamma S and 5-HT elicited an additive effect on IPs accumulation. 5. Treatment with phorbol 12-myristate 13-acetate (PMA, 1 microM, 30 min) abolished the 5-HT-induced IPs accumulation. The concentrations of PMA that gave a half-maximal and maximal inhibition of 5-HT-induced IPs accumulation were 2.2 +/- 0.4 nM and 1 microM, n = 3, respectively. The protein kinase C (PKC) activator, 4 alpha-phorbol 12,13-didecanoate, at 1 microM, did not influence this response. The inhibitory effect of PMA was reversed by staurosporine, a PKC inhibitor, suggesting that the inhibitory effect of PMA is mediated through the activation of PKC. 6. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the effect of PMA is distal to the 5-HT receptor. 7. Acetylcholine-induced IPs accumulation was completely inhibited by atropine, but not affected by ketanserin or mianserin, suggesting that 5-HT-induced IPs accumulation is not due to release of acetylcholine.8. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis via a pertussis toxin- and cholera toxin-insensitive GTP binding protein in canine TSMCs and that this coupling process is negatively regulated by PKC. 5-HT2 receptors may be predominantly mediating IPs accumulation and presumably IP-induced Ca2+ release may function as the transducing mechanism for 5-HT stimulated contraction of tracheal smooth muscle.

Bradykinin-stimulated phosphoinositide metabolism in cultured canine tracheal smooth muscle cells.

1. Stimulation of bradykinin (BK) receptors coupled to phosphoinositide (PI) hydrolysis was investigated in canine cultured tracheal smooth muscle cells (TSMCs). BK, kallidin, and des-Arg9-BK, stimulated [3H]-inositol phosphates (IPs) accumulation in a dose-dependent manner with half-maximal responses (EC50) at 20 +/- 5, 13 +/- 4, and 2.3 +/- 0.7 nM, (n = 5), respectively. 2. D-Arg[Hyp3, D-Phe7]-BK and D-Arg[Hyp3, Thi5,8, D-Phe7]-BK, B2 receptor antagonists, were equipotent in blocking the BK-induced IPs accumulation with pKB = 7.1 and 7.3, respectively. 3. Short-term exposure of TSMCs to phorbol 12-myristate 13-acetate (PMA, 1 microM) attenuated BK-stimulated IPs accumulation. The concentrations of PMA that gave half-maximal and maximal inhibition of BK-induced IPs accumulation were 15 +/- 4 nM and 1 microM, n = 3, respectively. The inhibitory effect of PMA on BK-induced response was reversed by staurosporine, a protein kinase C (PKC) inhibitor, suggesting that the inhibitory effect of PMA was mediated through the activation of PKC. 4. Prolonged incubation of TSMCs with PMA for 24 h, resulted in a recovery of receptor responsiveness which may be due to down-regulation of PKC. The inactive phorbol ester, 4 alpha-phorbol 12, 13-didecanoate at 1 microM, did not inhibit this response. 5. The site of this inhibition was further investigated by examining the effect of PMA on AlF(4-)-induced IPs accumulation in canine TSMCs. AlF(4-)-stimulated IPs accumulation was inhibited by PMA treatment, suggesting that the G protein(s) can be directly activated by AlF4-, which is uncoupled from phospholipase C by PMA treatment. 6. Incubation of TSMCs in the absence of external Ca2+ or upon removal of Ca2+ by addition of EGTA, caused a decrease in IPs accumulation without changing the basal levels. Addition of Ca2+ (3-620 nM) to digitonin-permeabilized TSMCs stimulated IPs accumulation was obtained by inclusion of either guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or BK. The combination of GTP gamma S and BK caused an additive effect on IPs accumulation.7. Pretreatment of TSMCs with cholera toxin enhanced BK-stimulated IPs accumulation, whereas there was no effect with pertussis toxin.8. These data suggest that BK-stimulated PI metabolism is mediated by the activation of BK B2 receptors coupling to a G protein which is not blocked by cholera toxin or pertussis toxin treatment and dependent on external Ca2+. The transduction mechanism of BK coupled to PI hydrolysis is sensitive to feedback regulation by PKC.

Sphingosine reverses growth inhibition caused by activation of protein kinase c in vascular smooth muscle cells

In certain cell systems, including neonatal vascular smooth muscle (VSM) cells, phorbol esters are growth inhibitory. Here we show that 1,2-dioctanoyl-sn-glycerol (DiC8), when added 2 h after alpha-thrombin, reverses by greater than 95% the induction of DNA synthesis in VSM cells by alpha-thrombin. Sphingosine, a naturally occurring lysosphingolipid inhibitor of protein kinase C, and its synthetic analogues N-acetylsphingosine and C11-sphingosine were used to investigate this phenomenon further. Neither phorbol 12-myristate 13-acetate (PMA;200 ng/ml) nor sphingosine (up to 10 microM) alone had any effect upon basal DNA synthesis in VSM cells. Like DiC8, PMA totally blocked the induction of DNA synthesis by alpha-thrombin. This inhibitory effect of PMA was reversed by sphingosine in a dose-dependent manner with complete reversal at 10 microM. Neither N-acetylsphingosine nor C11-sphingosine exhibited any effect on DNA synthesis in VSM cells. The effect of sphingosine and its analogues on the activity of protein kinase C extracted from VSM cells was measured by histone III-S phosphorylation. Protein kinase C activity was inhibited 50% by 300 microM sphingosine, but less than 15% by similar concentrations of N-acetylsphingosine and C11-sphingosine. To assess the effects of sphingosine and analogues on protein kinase C in intact cells, we examined the effect of the lipids on [3H]phorbol dibutyrate binding. Sphingosine (at greater than 5 microM), but not N-acetylsphingosine or C11-sphingosine, blocked [3H]phorbol dibutyrate binding in a dose- and time-dependent fashion. Thus the mechanism of growth inhibition by DiC8 and PMA in neonatal VSM cells appears to be through activation of protein kinase C by these compounds. Sphingosine reverses this growth inhibition through interference with the binding to protein kinase C of phorbol esters or other activators of this enzyme.

Endocytosis and Degradation of Ovine Prolactin by Nb2 Lymphoma Cells: Characterization and Effects of Agents Known to Alter Prolactin-Induced Mitogenesis*

Rat Nb2 node lymphoma cells proliferate in response to lactogens, but the signal transduction mechanism involved remains unclear. Specific binding, internalization, and degradation of ovine PRL (oPRL) were examined under a variety of experimental conditions to characterize the metabolism of receptor-bound hormone by these cells. Stationary-phase cells were incubated with [125I]oPRL in Fischer's medium containing horse serum. Cell suspensions were centrifuged, and the cell pellets were assayed to determine specific cell-associated radioactivity. Internalized ligand was measured by exposing the cells to an acidic buffer before centrifugation to dissociate hormone from plasma membrane receptors, and cell-surface ligand was calculated by subtracting internalized hormone from the total [125I]oPRL bound by the cells. Hormone degradation was assessed by measuring the radioactivity in an acid-soluble fraction prepared from the incubation medium. Endocytosis of [125I]oPRL was observed within 30 min at 37 C, and the internalized component accounted for approximately 50% of the bound hormone under steady-state conditions. Hormone degradation was detectable within 1 h at 37 C and continued at a relatively linear rate thereafter; by 4 h, 8% of the added [125I]oPRL was acid soluble. Chloroquine (0.2 mM), methylamine (20 mM) and monensin (20 microM) prevented [125I]oPRL degradation and elevated both cell-surface and intracellular hormone 2-fold during a 4-h incubation. Leupeptin (0.2 mM) decreased degradation by only 15% under the same conditions. Phorbol 12-myristate 13-acetate (PMA; 20 nM), a comitogen for lactogen-stimulated Nb2 cells, increased cell-surface hormone by 20% and decreased intracellular hormone by a corresponding amount 1 h after administration. Calcium ionophore A23187 (1 microM) produced similar changes, and a synergistic effect was noted when cells were exposed to both agents for 4 h. Amiloride (125 microM), an inhibitor of Nb2 cell mitogenesis, decreased [125I]oPRL degradation by 25% during a 4-h incubation. This response was abolished when the cells were exposed simultaneously to PMA. These experiments demonstrate that receptor-bound oPRL is rapidly internalized and extensively degraded via the endosome-lysosome pathway when Nb2 cells are maintained at 37 C. The inhibitory effect of PMA on oPRL internalization may help to explain the comitogenic action of this phorbol on Nb2 cells. Since amiloride also produced major changes in oPRL metabolism, post-binding events in lactogen processing by target cells could play an important role in the mitogenic response elicited by such hormones.

Phorbol Ester-induced Inhibition of the Beta-adrenergic System in Pulmonary Endothelium: Role of a Pertussis Toxin-sensitive Protein

To investigate possible cellular mechanisms for how activation of protein kinase C inhibits the relaxation caused by isoproterenol, we studied the effect of the protein kinase C activator 4 beta-phorbol-12 beta-myristate-13 alpha-acetate (PMA) on the increase in cyclic AMP (cAMP) production and adenylate cyclase activity caused by isoproterenol in bovine pulmonary artery endothelial cells. Treatment of intact cells with PMA prevented in a time- and dose-dependent manner the increase in cAMP production caused by isoproterenol, whereas 4 alpha-phorbol-12 beta-myristate-13 alpha-acetate (4 alpha-PMA), which does not activate protein kinase C, did not affect isoproterenol-induced cAMP production. PMA also reduced the increase in adenylate cyclase activity caused by isoproterenol, forskolin, and Gpp(NH)p. To test the hypothesis that the inhibitory effect of PMA is mediated via a pertussis toxin-sensitive G protein, we determined whether pretreatment of the cells with pertussis toxin would prevent the inhibitory effects of PMA. In pulmonary endothelial cells, pertussis toxin ADP-ribosylated an Mr 40,000 peptide that comigrated with the pertussis toxin substrate of human erythrocytes. Pertussis toxin treatment eliminated the inhibitory effect of PMA on isoproterenol-stimulated cAMP production and adenylate cyclase activity. Thus, the protein kinase C activator PMA inhibits the increase in cAMP production and adenylate cyclase caused by isoproterenol. This inhibitory effect in endothelial cells appears to be mediated via a pertussis toxin-sensitive protein.

The effect of some experimental parameters on the inhibition of metabolic cooperation by phorbol myristate acetate.

The effect of cell density, PMA exposure time, concentration, pre-exposure and binding activity on the recovery of V 79 HGPRT- Chinese hamster cells in the metabolic cooperation assay was determined. A PMA exposure interval of only 1 min resulted in maximum recovery of HGPRT- cells. PMA began to inhibit metabolic cooperation at a dose as low as 0.1 ng/ml final media concentration. The recovery of HGPRT- cells varied according to cell density in the presence or absence of PMA, although the magnitude of this effect was much greater in untreated cells. Pre-exposure of cells to PMA increased the recovery of both post-PMA-treated and non-treated HGPRT- cells in a dose-dependent manner. [3H]PMA was rapidly bound to or taken up by V 79 cells. These results suggest that the inhibitory effect of PMA on metabolic cooperation in V 79 cells involves receptor binding.