Activation of protein kinase C inhibits ATP-induced [Ca2+]i elevation in rat osteoblastic cells: Selective effects on P2Y and P2U signaling pathways

Extracellular ATP elicits transient elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in osteoblasts through interaction with more than one subtype of cell surface P₂-purinoceptor. Elevation of [Ca²⁺]_i arises, at least in part, by release of Ca²⁺ from intracellular stores. In the present study, we investigated the possible roles of protein kinase C (PKC) in regulating these signaling pathways. [Ca²⁺]_i of indo-1-loaded UMR-106 osteoblastic cells was monitored by spectrofluorimetry. In the absence of extracellular Ca²⁺, ATP (100 μM) induced transient elevation of [Ca²⁺]_i to a peak 57 ± 7 nM above basal levels (31 ± 2 nM, means ± S. E. M., n = 25). Exposure of cells to the PKC activator 12-O-tetradecanoyl-β-phorbol 13-acetate (TPA, 100 nM) for 2 min significantly reduced the amplitude of the ATP response to 13 ± 4 nM (n = 11), without altering basal [Ca²⁺]_i. Inhibition was half-maximal at approximately 1 nM TPA. The Ca²⁺ response to ATP was also inhibited by the PKC activators 1,2-dioctanoyl-sn-glycerol or 4β-phorbol 12, 13-dibutyrate, but not by the control compounds 4α-phorbol or 4α-phorbol 12, 13-didecanoate. Furthermore, exposure of cells to the protein kinase inhibitors H-7 or staurosporine for 10 min significantly attenuated the inhibitory effect of TPA. However, these protein kinase inhibitors did not prolong the [Ca²⁺]_i response to ATP alone, indicating that activation of PKC does not account for the transient nature of this response. When the effects of other nucleotides were examined, TPA was found to cause significantly greater inhibition of the response to the P_2Y-receptor agonists, ADP and 2-methylthioATP, than the response to the P_2U-receptor agonist, UTP. These data indicate that activation of PKC selectively inhibits the P_2Y signaling pathway in osteoblastic cells. In vivo, endocrine or paracrine factors, acting through PKC, may regulate the responsiveness of osteoblasts to extracellular nucleotides. © 1995 Wiley-Liss, Inc.     

Cellular uptake and localization of fluorescent derivatives of phorbol ester tumor promoters

The synthetic fluorescent derivatives of 12-O-tetradecanoylphorbol-13-acetate (TPA), dansyl-TPA, dansyl-TPA-20-acetate and dansyl-TPA-13-desacetate, have ID50 values in the [3H]PDBu binding assay of 2nM, 30nM and 1000nM respectively; the ID50 value of TPA is 4nM. Dansyl-TPA is also equipotent with TPA as an activator of protein kinase C(PKC) producing half maximum stimulation at 2nM. Dansyl-TPA-13-desacetate is almost as potent as dansyl-TPA, while dansyl-TPA-20-acetate is completely inactive as an activator of PKC. The cellular uptake of these fluorescent TPA derivatives tends to parallel their activity in the [3H]PDBu binding assay. Treatment of C3H 10T1/2 cells with 100nM dansyl-TPA results in intense fluorescence of the entire cytoplasm, while the nucleus is virtually devoid of fluorescence. The uptake of fluorescence is quenched by an excess of TPA. Thus, dansyl-TPA rapidly enters cells and binds to specific sites distributed throughout the cytoplasm. Presumably these sites reflect the cellular localization of phorbol ester receptors and protein kinase C.     

Heterologous,PKC-Mediated Desensitization of Human Histamine H3 Receptors Expressed in CHO-K1 Cells

Desensitization is a major mechanism to regulate the functional response of G protein-coupled receptors. In this work we studied whether the human histamine H₃ receptor of 445 amino acids (hH₃R₄₄₅) experiences heterologous desensitization mediated by PKC activation. Bioinformatic analysis indicated the presence of Serine and Threonine residues susceptible of PKC-mediated phosphorylation on the third intracellular loop and the carboxyl terminus of the hH₃R₄₄₅. In CHO-K1 cells stably transfected with the hH₃R₄₄₅ direct PKC activation by phorbol 12-myristate 13-acetate (TPA, 200 nM) abolished H₃R-mediated inhibition of forskolin-stimulated cAMP accumulation. Activation of endogenous purinergic receptors by ATP (adenosine 5′-triphosphate, 10 μM) increased the free calcium intracellular concentration ([Ca²⁺]i) confirming their coupling to phospholipase C stimulation. Incubation with ATP also abolished H₃R-mediated inhibition of forskolin-induced cAMP accumulation, and this effect was prevented by the PKC inhibitors Ro-31-8220 and Gö-6976. Pre-incubation with TPA or ATP reduced H₃R-mediated stimulation of [³⁵S]-GTPγS binding to membranes from CHO-K1-hH₃R₄₄₅ cells by 39.7 and 54.2 %, respectively, with no change in the agonist potency, and the effect was prevented by either Ro-31-8220 or Gö-6976. Exposure to ATP or TPA also resulted in the loss of cell surface H₃Rs (−30.4 and −45.1 %) as evaluated by [³H]-NMHA binding to intact cells. These results indicate that the hH₃R₄₄₅ undergoes heterologous desensitization upon activation of receptors coupled to PKC stimulation.

     

Effects of 12-0-tetradecanoylphorbol-13-acetate (TPA) on rat pheochromocytoma (PC12) cells: Interactions with epidermal growth factor and nerve growth factor

The phorbol ester tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA) specifically inhibited the binding of radioiodinated epidermal growth factor (¹²⁵I-EGF) to rat pheochromocytoma (PC12) cells in a noncompetitive fashion with an apparent K_i of 11–26 nM. Both TPA and EGF elicited similar biological responses in PC12 cells including enhanced incorporation of ³H-choline and ³²P-orthophosphate into macromolecules, induction of ornithine decarboxylase, and stimulation of the phosphorylation of a 30,000 MW nonhistone, chromosome-associated protein. These effects were also elicited by nerve growth factor (NGF) which, in contrast to the former agents, is a differentiating stimulus for the PC12 cells. The effects of TPA were additive or more than additive to the effects of NGF and EGF. When PC12 cells were induced to differentiate by treatment with NGF for 72 hours, the binding of ¹²⁵I-EGF and responses to EGF were reduced by approximately 70%. The response of PC12 cells to the tumor promoter TPA was unaffected by treatment with NGF. Thus, the qualitatively similar effects of TPA and EGF seemed to be mediated through separate receptor systems with only the EGF receptor system reduced by NGF treatment.     

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin, 12-O-tetradecanoylphorbol-13-acetate and 17β-estradiol on estrogen receptor regulation in MCF-7 human breast cancer cells

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF-7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER-ICA), and on ER function by competitive binding assays under conditions of saturating 17β-estradiol (E₂). Comparative studies were conducted with E₂ and 12-O-tetradecanoylphorbol-13-acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E₂ and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER-ICA after 72 h exposure to 1 nM E₂ and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [³H]E₂ under saturating conditions (10 nM E₂) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [³H]E₂ was depressed by 49% in MCF-7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1-h treatment with 5 μM α-naphthoflavone, which inhibits TCDD-induced, P450-mediated, E₂ metabolism, and subsequent E₂ depletion. In conclusion, while TPA and E₂ effectively down-regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF-7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF-7 cells by TCDD. © 1996 Wiley-Liss, Inc.     

Binding of curcumin and its long chain derivatives to the activator binding domain of novel protein kinase C

Protein kinase C (PKC) is a family of serine/threonine kinases that play a central role in cellular signal transduction. The second messenger diacylglycerol having two long carbon chains acts as the endogenous ligand for the PKCs. Polyphenol curcumin, the active constituent of Curcuma longa is an anti-cancer agent and modulates PKC activity. To develop curcumin derivatives as effective PKC activators, we synthesized several long chain derivatives of curcumin, characterized their absorption and fluorescence properties and studied their interaction with the activator binding second cysteine-rich C1B subdomain of PKCδ, PKCε and PKCθ. Curcumin (1) and its C16 long chain analog (4) quenched the intrinsic fluorescence of PKCδC1B, PKCεC1B and PKCθC1B in a manner similar to that of PKC activator 12-O-tetradecanoylphorbol 13-acetate (TPA). The EC₅₀s of the curcumin derivatives for fluorescence quenching varied in the range of 4–11 μM, whereas, EC₅₀s for TPA varied in the range of 3–6 μM. Fluorescence emission maxima of 1 and 4 were blue shifted and the fluorescence anisotropy values were increased in the presence of the C1B domains in a manner similar to that shown by the fluorescent analog of TPA, sapintoxin-D, confirming that they were bound to the proteins. Molecular docking of 1 and 4 with novel PKC C1B revealed that both the molecules form hydrogen bonds with the protein residues. The present result shows that curcumin and its long chain derivatives bind to the C1B subdomain of novel PKCs and can be further modified structurally to improve its binding and activity.     

Tumour promoter-mediated reversible inhibition of cell-cell communication (electrical coupling) : Relationship with phorbol ester binding and de novo macromolecule synthesis

A tumour promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), reversibly inhibits the onset and maintenance of cell-cell communication measured by electrophysiological method. We have now studied the mechanism by which TPA inhibits communication of human cells (FL) in culture. Using [³H]phorbol-12,13-dibutyrate ([³H]PDBu), we found a class of specific, high-affinity, saturable binding sites in intact FL cells; they have a dissociation constant of 15.4 nM, and at saturation about 3 × 10⁵ PDBu molecules were bound to each cell. The binding of [³H]PDBu to FL cells was inhibited by TPA, phorbol-12-13-didecanoate and mezerein, whereas phorbol and 4α-phorbol-12-13-didecanoate had no effect. There is a close correlation between the ability of the former compounds to inhibit [³H]PDBu binding and their capacity to inhibit cell-cell communication. When FL cells are dispersed with EDTA and plated onto a culture dish, they start to couple electrically within 2 h; such cell coupling was not affected by the presence of cycloheximide or actinomycin D. TPA inhibits the formation of electrical cell coupling as well as its maintenance, even in the presence of cycloheximide; the recovery of cell-cell communication after the removal of TPA was not significantly affected by the addition of cycloheximide or actinomycin D. Taken together, these results suggest that TPA-mediated reversible inhibition of intercellular communication is mediated by specific binding of TPA to cellular receptors and that macromolecular synthesis is not necessary.     

GM-CSF and phorbol esters modulate GM-CSF receptor expression by independent mechanisms.

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 nM) down-modulates its receptor in IL-3/GM-CSF dependent M-07e cells, in KG-1 cells and normal granulocytes, whereas phorbol esters 12-O-tetradecanoylphorbol-13-acetate (TPA) (2 nM) down-modulates the GM-CSF receptor in M-07e cells and granulocytes but not in KG-1 cells. As data analysis shows by nonlinear regression, the decreased binding ability depends on a reduction of the binding sites with no significant change of their dissociation constant. To gain insight into the mechanisms involved in the GM-CSF receptor regulation, we investigated the role of protein kinase C (PKC). GM-CSF, unlike TPA, was unable to activate PKC in all the cells studied. Moreover, unlike TPA, GM-CSF was still able to down-modulate its receptor in cells where PKC was inhibited by 1-(5-isoquinolonesulphonyl)-2-methylpiperazine (H7) and staurosporine or in cells where PKC was exhausted by prolonged incubation with 1 microM TPA. Finally, the receptor re-expression rate was accelerated by protein kinases inhibitors. These results, taken together, indicate the presence of a PKC-dependent and -independent down-modulation mechanism and a negative role of the endogeneous protein kinases in GM-CSF receptor re-expression.     

Role of protein kinase C activation in oocyte maturation and steroidogenesis in ovarian follicles of Rana pipiens: Studies with phorbol 12-myristate 13-acetate

In ovarian follicles of Rana pipiens, frog pituitary homogenates (FPH) elevate intrafollicular progesterone levels which in turn is thought to induce meiotic resumption in the prophase I arrested oocytes. Calcium plays a role in FPH and steroid-provoked responses in the somatic and gametic components of the follicle, presumably via effects exerted at the plasma membrane of their respective target cells. Many membrane active hormones which utilize Ca²⁺ in their intracellular transduction also provoke membrane phosphoinositide hydrolysis yielding inositol triphosphate (IP₃) and diacyl glycerol (DAG), an activator of the CA²⁺-dependent protein kinase C (PKC). The actions of phorbol 12-myristate 13-acetate (TPA), a potent synthetic activator of PKC, on progesterone production and oocyte maturation was examined in in vitro cultured ovarian follicles. TPA induced germinal vesicle breakdown (GVBD) in intact follicles and in oocytes denuded of somatic components, while the inactive compound phorbol 13-monoacetate was ineffective. Further, TPA induction of GVBD exhibited similarities to progesterone-induced GVBD, being inhibited by treatments which elevate cAMP or inhibit protein synthesis. TPA alone did not elevate intrafollicular or medium progesterone levels, as occurred in FPH-treated follicles. TPA partially inhibited intrafollicular progesterone accumulation induced by FPH or treatments which elevate cAMP levels. These data suggest that activation of PKC plays a role in oocyte maturation independent of follicular progesterone production as occurs in response to FPH. Further, it appears that the somatic cells of the amphibian follicle also possess PKC which when activated, antagonizes cAMP generating pathway in these cells. Results indicate that protein kinase can influence oocyte maturation in Rana follicular oocytes by several mechanisms.     

Protein kinase C activator phorbol 12, 13-dibutyrate inhibits platelet activating factor-stimulated Ca2+ mobilization and phosphoinositide turnover in neurohybrid NG108-15 cells

The protein kinase C (PKC) activator, phorbol 12, 13-dibutyrate (PDBa) dose-dependently inhibited platelet-activating factor (PAF)-induced [Ca²⁺]_i elevation and inositol monophosphate (IP₁) accumulation in neurohybrid NG108-15 cells with IC₅₀ values of 162 nM and 35 nM, respectively. Pretreatment of NG108-15 cells with PKC inhibitor H-7 partially prevented the inhibitory effect of PDBu on PAF-induced [Ca²⁺]_i elevation as well as PI metabolism in NG108-15 cells. Pretreatment of the cells with pertussis toxin (PTX) resulted in a dose-dependent inhibition of PAF-induced IP₁ and IP₃ accumulation but only slightly affected PAF-induced [Ca²⁺]_i elevation in NG108-15 cells. The results reveal that PAF receptor-mediated Ca²⁺ mobilization and PI metabolism in NG108-15 cells are regulated by PKC while a PTX-sensitive G protein is coupled to PAF receptor for inducing activation of phospholipase C.     

Modulation of endothelial autacoid release by protein kinase C: Feedback inhition or non-specific attenuation of receptor-dependent cell activation?

Receptor-mediated elevations of intracellular Ca²⁺ in endothelial cells may be controlled by a negative feedback mechanism through activation of protein kinase C (PKC). To test this hypothesis, we studied the effects of an activation or inhibition of PKC on the release of nitric oxide (NO) and prostacyclin (PGI₂) from cultured bovine and porcine aortic endothelial cells (EC). Preincubation with the PKC activators phorbol-12-myristate-13-acetate (PMA) (3-300 nM) or 1-oleyl-2-acetyl-glycerol (OAG) (30 μM) significantly attenuated the release of NO and PGI₂ from EC stimulated with bradykinin (0.3–30 nM), whereas phorbol-12, 13-didecanoate (PDD) (30–300 nM), which does not activate PKC, had no effect. UCN-01 (10 nM), a specific PKC inhibitor, significantly augmented the bradykinin-stimulated release of NO from EC. These effects were correlated with a reduced (PMA) or enhanced (UCN-01) elevation of intracellular Ca²⁺ in response to bradykinin in both types of EC. Neither the PKC activators nor the inhibitor had any effect on resting intracellular Ca²⁺ or basal endothelial autacoid release. Several isoforms of PKC (namely PKC_α, PKC_δ, PKC_?, and PKC_ζ) were detected in bovine, human, and porcine EC by immunoblotting analysis with isotype-specific anti-PKC antibodies, which, except PKC_?, were predominantly located in the cytosol. Incubation of bovine EC with PMA elicited a significant increase in membrane-bound PKC_α immunoreactivity, whereas there was no translocation of PKC_α from the cytosolic to the membrane fraction with bradykinin. As determined by histone phosphorylation, PKC activity was similarly reduced in the cytosol, but increased in the membrane fraction of bovine EC exposed to PMA, whereas bradykinin had no significant effect. These findings indicate that endothelial autacoid release can be modulated by activators and inhibitors of PKC. However, stimulation of EC with bradykinin does not lead to a detectable activation of PKC, suggesting that PKC does not exert a negative feedback in the signal transduction pathway of this receptor-dependent agonist. © 1993 Wiley-Liss, Inc.     

Regulation of the Cell Cycle at the G2/M Boundary in Metastatic Melanoma Cells by 12-O-Tetradecanoyl Phorbol-13-acetate (TPA) by Blocking p34Kinase Activity

12-O-Tetradecanoyl phorbol-13-acetate (TPA) inhibits the growth of most malignant melanoma cells but stimulates the growth of normal human melanocytes. We previously showed that addition of TPA inhibits the growth of the human metastatic melanoma cell line, Demel, by blocking cells at both the G1/S and G2/M cell cycle transitions (D. L. Coppocket al.,1992,Cell Growth Differ.3, 485–494). To examine the G2/M transition, we developed a method to synchronize the cells in early S phase using Lovastatin and mevalonate, followed by treatment with hydroxyurea (HU). TPA (30 nM) was effective in blocking cells from entering mitosis and reentering G1 when added up to the end of G2. These cells arrested in G2. Examination of the levels of cyclins A and B1 demonstrated that the levels of these cyclins were not limiting for entrance into M. However, the addition of TPA blocked the increase in p34^cdc2/cyclin B1 kinase activity. In cells treated with TPA, most p34^cdc2was found in the slowly migrating forms on Western blots, which contained increased levels of phosphotyrosine. In addition, the level of the cyclin-dependent kinase inhibitor p21^Cip1/Waf1, but not of p27^Kip1, was increased. We examined the expression of protein kinase C (PKC) isoforms in Demel cells using Western blots to understand which types were involved in the G2 arrest. Demel cells expressed the PKC α, βI, βII, δ, ε, ι/λ, ζ, and μ isozymes. PKC η and PKC θ were not detected. Addition of TPA did not completely down regulate any PKC isozymes over a 12-h period in these synchronized cells. PKC α, βI, βII, δ, and ε isozymes were translocated to the membrane fraction from the cytosolic fraction when treated with TPA. PKC δ appeared as a doublet and the addition of TPA shifted a majority to the slower migrating form. The level of PKC μ was constant; however, a slow mobility form was observed in TPA-treated cells. This reduced mobility was at least partially due to phosphorylation. Thus, the arrest of growth in G2 appears to be due to the inhibition of the p34^cdc2kinase activity which is associated with the increased expression of p21^Cip1/Waf1and increased phosphorylation on tyrosine of p34^cdc2. This arrest, in turn, is associated with a shift of PKC isozymes PKC α, PKC βI, PKC βII, PKC δ, PKC ε, and PKC μ to the membrane fraction which is induced by addition of TPA.     

Hydrogen Peroxide Formation by Cells Treated with a Tumor Promoter

To determine whether oxidants capable of DNA modification are produced by cells treated with tumor promoters, we adapted a fluorometric method to our needs. HeLa cells were preincubated with 2‘,7‘-dichlorofluorescin diacetate (DCFdAc), treated with various agents, sonicated. centrifuged and fluorescence of the oxidized product (DCF) was determined in supernatants. When cells were exposed to H₂O₂ in the presence of azide (catalase inhibitor) or o-phenanthroline (a lipophilic Fe chelator), an increase in fluorescence was observed. These results show that some Fe ions were interacting with the H₂O₂ which entered the cells, thus decreasing its levels available for oxidation of the substrate and potentially increasing formation of OH, known DNA-damaging species. Glutathione (GSH). which is present in cells in substantial amounts, was found to reduce DCF whereas azide counteracted GSH-mediated reduction.

Treatment of HeLa cells with 12–0-tetradecanoyl-phorbol-13-acetate (TPA) in the presence of DCFdAc and azide resulted in dose-and time-dependent formation of DCF. Even when cells were sonicated prior to incubation with TPA, DCF was formed at levels proportional to the number of cells as well as dose of TPA. Flow cytometry of TPA-treated cells confirmed these findings.

These results demonstrate that tumor promoters can cause oxidative activation of HeLa cells, which produce active oxygen species, most likely H₂O₂, that ultimately contribute to the formation of oxidized bases such as 5-hydroxymethyl uracil in cellular DNA. They also show that this fluorometric method can be utilized for determination of cellular H₂O₂ formation at nM concentrations.     

Prolactin-Stimulated Mitogenesis of Cultured Astrocytes Is Mediated by a Protein Kinase C-Dependent Mechanism

Abstract: Prolactin (PRL) has been reported to activate cellular proliferation in nonreproductive tissue, such as liver, spleen, and thymus. Recently, we have extended the possible role of PRL as a mammalian mitogen by demonstrating a mitogenic effect of PRL in cultured astrocytes. Although the cellular mechanisms by which PRL regulates cell growth are not fully understood, protein kinase C (PKC) has been implicated as one of the transmembrane signaling systems involved in the regulation of PRL-induced cell proliferation in Nb2 lymphoma cells and liver. In the present studies, we examined the possible role of PKC in PRL-induced proliferation of cultured astrocytes. Incubation of cultured astrocytes with 1 nM PRL resulted in a rapid translocation of PKC from the cytosol to the membrane, with maximal PKC activity in the membrane occurring 30 min after exposure to PRL. Translocation of PKC activity occurred over a physiological range of PRL, with maximal PKC activation occurring at 1 nM. At concentrations greater than 10 nM PRL, there was a decrease in the amount of PKC activity associated with the membrane fraction compared with that of cells stimulated with 1 nM PRL. Incubation of astrocytes with PRL in the presence of the PKC inhibitors staurosporine, 1-(-5-isoquinolinesulfonyl)-2-methylpiperazine, or polymyxin B blocked the PRL-induced increase in cell number with IC₅₀ values of approximately 2 nM, 10 μM, and 6 μM, respectively. PKC is the only known cellular receptor for 12-O-tetradecanoylphorbol 13-acetate (TPA), which stimulates the translocation of PKC from the cytosol to the membrane. Incubation of astrocytes with 20 nM TPA resulted in an increase in the expression of proliferating cell nuclear antigen and cell number, whereas 4α-phorbol 12,13-didecanoate, an inactive phorbol ester, was ineffective. To examine further the effect of TPA and PRL on cellular proliferation, cultured astrocytes were incubated with increasing concentrations of TPA in the presence or absence of a minimal effective dose of PRL (100 pM). In the absence of PRL, incubation with TPA resulted in an inverted U-shaped dose-response curve, with 100 nM TPA resulting in a maximal increase in cell number. In the presence of 100 pM PRL, the TPA dose-response curve was shifted to the left, with maximal activity occurring with 10 nM TPA. Chronic stimulation of astrocytes with 500 nM TPA depleted the cells of PKC and blocked the PRL-induced increase in cell number. Finally, TPA treatment decreased cell-surface binding of ¹²⁵I-PRL. These data indicate that the PKC is involved in the mitogenic effect of PRL in cultured astrocytes.     

Salviae Miltiorrhizae BGE Radix Increases Rat Striatal K+-Stimulated Dopamine Release and Activates the Dopamine Release with Protection Against Hydrogen Peroxide-Induced Injury in Rat Pheochromocytoma PC12 Cells

The present study investigated the effect of the medicinal plant Salviae miltiorrhizae radix (SMR) on dopaminergic neurotransmission in comparison with amphetamine. The effect of SM (0.1 g/ml) on K⁺ (20 mM)-stimulated dopamine (DA) release from rat striatal slices was compared with amphetamine (10⁻⁴ M). Amphetamine and SMR significantly increased K⁺-stimulated DA release (P<0.001) from rat striatal slices when compared with K⁺-stimulated alone. On the other hand, to examine whether in vitro SMR treatment induces DA release in PC12 cells, the role of protein kinases has been investigated in the induction of the SMR-mediated events by using inhibitors of protein kinase C (PKC), mitogen activated protein kinase (MAP kinase) or protein kinase A (PKA). PKC inhibitors chelerythrine (50 and 100 nM), Ro31-8220 (100 nM) and the MAP kinase inhibitor, PD98059 (20 μM) inhibited the ability of SMR to elicit the SMR-stimulated DA release. The direct-acting PKC activator, 12-O-tetradecanoyl phorbol 13-acetate (TPA, 100 nM) mimicked the ability of SMR to elicit DA release. On the contrary, a selective PKA inhibitor, 50 μM Rp-8-Br-cAMP, blocked the development of SMR-stimulated DA release. The results demonstrated that SMR may stimulate DA release and that SMR-induced increases in MAP kinase and PKC are important for induction of the enhancement in transporter-mediated DA release and PKA was also required for the enhancement in SMR-stimulated DA release. SMR treatment (0.1–10 μg/ml) to the hydrogen peroxide (H₂O₂)-treated PC12 cells activated the enzyme activities such as catalase, superoxide dismutase and glutathione peroxidase, and decreased the malondialdehyde level, indicating that SMR has also protective effects against free radical-induced cell toxicity. Therefore, the mechanism by which SMR induces the enhancement in SMR-stimulated DA release is apparent. It remains to be determined whether the effect of SMR on DA function is important in its therapeutic use in the treatment of drug addiction.     

P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration-dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2-methylthioadenosine 5′-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin-insensitive G_q protein. Short-term (10 min) treatment of cells with 1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of UTP- or ATP-stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF₄^?, but not Ca²⁺ ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of PKCε in the P2Y receptor/G_q protein/phospholipase Cβ pathway might be the G_q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor-mediated phosphoinositide turnover.     

Changes in the Susceptibility of 12-O-Tetradecanoylphorbol 13-Acetate (TPA)-Treated HL-60 Cells to Staphylococcal Leukocidin

Susceptibility of human promyelocytic leukemia HL-60 cells to staphylococcal leukocidin following treatment of cells with 12-O-tetradecanoylphorbol 13-acetate (TPA) was examined. TPA treatment for 6 hr rendered the cells very resistant transiently to leukocidin. There was no change in binding of leukocidin to the cells, but leukocidin-induced ⁴⁵CaCl₂ influx, phospholipase A₂ and C activities were inhibited. Further incubation with TPA rendered the cells sensitive again and then more sensitive than original HL-60 cells following increase of the binding, and leukocidin-induced activities described above appeared again. Those cells treated with TPA for more than 18 hr started to differentiate to macrophages morphologically and functionally. These data suggest that the differentiated cells were more sensitive than original HL-60 cells because of increased binding of leukocidin and that treatment of TPA for 6 hr may transiently impair the signal transduction system of leukocidin after binding of leukocidin to the specific receptor of the cell membrane. Using these TPA-treated cells, it was shown in this report that calcium influx, phospholipase A₂ and C activities were important to induce cytotoxic action of leukocidin after binding of leukocidin to specific receptors on the cells.