calcimycin and phorbol-12-13-didecanoate

Recently, the immediate early gene h-sgk was cloned as a hypertonicity-induced gene from human hepatoma cells. The aim of this study was to localize h-sgk messenger RNA (mRNA) expression in normal and inflamed intestinal mucosa and to identify potential transcriptional regulators.. h-sgk mRNA in small intestinal mucosa from healthy persons and patients with Crohn's disease was determined by in situ hybridization. Transcriptional regulation was studied by Northern blot analysis of total RNA isolated from cultured human Intestine 407, U937, and HepG2 cells.. In normal ileum, h-sgk mRNA was selectively localized to the apical villus enterocytes, whereas no staining was detected in crypt cells. In Crohn's disease, enterocytes of the crypts expressed h-sgk and abundant h-sgk positive inflammatory cells appeared in the lamina propria. Combined h-sgk in situ hybridization and immunohistochemical analysis of CD68 antigen expression identified a part of these cells as macrophages. In addition to spatial correlation of transforming growth factor (TGF)-beta1 protein and h-sgk mRNA expression, h-sgk transcription in human Intestine 407 and HepG2 cells as well as in U937 monocytes/macrophages was strongly induced by TGF-beta1 in vitro.. h-sgk expression in normal and inflamed intestinal mucosa may be regulated by TGF-beta1 and may contribute to the pleiotropic actions of TGF-beta1 in mucosal cell populations.

Topics: Antigens, CD; Antigens, Differentiation, Myelomonocytic; Blotting, Northern; Calcimycin; Cells, Cultured; Crohn Disease; Cycloheximide; Gene Expression Regulation; Humans; Ileum; Immediate-Early Proteins; Immunohistochemistry; In Situ Hybridization; Inflammation; Interleukin-1; Intestinal Mucosa; Ionophores; Nuclear Proteins; Phorbol Esters; Protein Serine-Threonine Kinases; RNA, Messenger; Transcription, Genetic; Transforming Growth Factor beta; Tumor Necrosis Factor-alpha; U937 Cells

Parathyroid hormone acts on the osteoblast to induce osteoclastic bone resorption. Parathyroid hormone utilises cyclic AMP as a second messenger in osteoblasts, but may also cause an increase in cytoplasmatic free calcium ions ([Ca2+]i) in the same cell. To investigate the role of osteoblastic [Ca2+]i in the induction of bone resorption, we have compared the effects of parathyroid hormone and the Ca2+-ionophore, A23187, as well as the adenylate cyclase stimulating agent, forskolin, and the phorbol ester, phorbole 12,13 dibutyrate (PDB), on bone resorption in neonatal mouse calvarial bones. Parathyroid hormone (0.1 and 1 nM) dose dependently stimulated the release of prelabelled 45Ca2+ in 72 h culture. Parathyroid hormone-induced bone resorption was not affected by the addition of 1 microM indomethacin to the incubation media, and was therefore, not mediated by local prostaglandin formation. A23187 stimulated the release of 45Ca2+ at 1-10 nM. Above 100 nM, A23187 inhibited bone resorption. The A23187 (3 and 10 nM)-induced bone resorption was abolished by the cyclooxygenase inhibitor, indomethacin (1 microM), indicating that the stimulatory effect was mediated via prostaglandin formation. The adenylate cyclase stimulating agent, forskolin, dose dependently stimulated bone resorption at and above 1 microM. There was no additive or synergistic effect of forskolin and A23187 on 45Ca2+ release. Forskolin-induced bone resorption was, as with parathyroid hormone but in contrast to ionophore-induced bone resorption, not abolished by indomethacin (1 microM). The protein kinase C activator, PDB, at 10 and 1000 nM stimulated the release of prelabelled 45Ca2+. The stimulatory effect of the protein kinase C stimulating phorbol ester, PDB, on bone resorption was abolished by the addition of indomethacin. In summary, bone resorption induced by a Ca2+-ionophore is abolished by indomethacin. This indicates that bone resorbing agents known to increase [Ca2+]i subsequently enhance local prostaglandin formation. Bone resorption induced by the protein kinase C activator, PDB, was also abolished by indomethacin, whereas, forskolin and parathyroid hormone-induced bone resorption was unaffected. These data indicate that cyclic AMP, but not [Ca2+]i, is involved as a second messenger in parathyroid-induced bone resorption.

Topics: Animals; Animals, Newborn; Bone Resorption; Calcimycin; Calcium; Calcium Radioisotopes; Colforsin; Cyclooxygenase Inhibitors; Humans; Indomethacin; Ionophores; Mice; Parathyroid Hormone; Phorbol 12,13-Dibutyrate; Phorbol Esters; Recombinant Proteins; Second Messenger Systems

Treatment of metaphase II-arrested hamster eggs with activators of protein kinase C has been reported to promote resumption of the cell cycle, second polar body emission, and pronucleus formation (G.I. Gallicano, S.M. Schwarz, R.W. McGaughey, and D.G. Capco, 1993, Dev. Biol. 156, 94-106). In contrast, we have not observed these responses in mouse eggs obtained from CF-1 mice treated with these activators. In this report, we evaluated if this difference was due to differences in the technique used for PKC stimulation in the two different laboratories or due to species differences. Metaphase II-arrested hamster or mouse eggs were treated with phorbol diesters for 5 min or with a membrane-permeable diacylglycerol for 1 hr. Treatment of hamster eggs resulted in (1) the formation of "second polar body-like structures" commencing 5 min after treatment and reaching a maximum by 20-40 min; (2) a remarkable increase in the staining of filamentous actin in the region of these polar body-like structures; and (3) the disassembly of spindle microtubules. A reduction in cdc2/cyclin B1 kinase activity, as assessed by a decrease in H1 kinase activity, as well as progression from metaphase to anaphase were not observed. Treatment of mouse eggs from either CF-1 or CD-1 mice with these activators of PKC did not result in the formation of these polar body-like structures, did not cause an increase in filamentous actin, and did not result in a reduction in histone H1 kinase activity. This treatment, however, did induce disassembly of the spindle microtubules and the formation of multiple "pronucleus-like structures" that were more discernible in eggs from CD-1 mice. We conclude that the "apparent" activation of hamster eggs by activators of PKC is due to the effect of these agents on the cytoskeleton, which gives rise to structures that appear similar to polar bodies, but without any evidence of cell cycle resumption. The different responses seen in mouse and hamster eggs are mainly due to differences in the sensitivity of the cytoskeleton to rearrangements induced by these agents.

Topics: Amino Acid Sequence; Animals; Calcimycin; Cricetinae; Cytoskeleton; Diglycerides; Enzyme Activation; Female; Mesocricetus; Mice; Molecular Sequence Data; Ovum; Peptides; Phorbol Esters; Protamine Kinase; Protein Kinase C; Species Specificity; Substrate Specificity; Tetradecanoylphorbol Acetate

The regulation of beta-amyloid precursor protein (beta-APP) secretion was compared in differentiated and nondifferentiated PC12 and B104 cells. Phorbol esters stimulated the release of beta-APP in all cells examined. However, differentiated PC12 cells were much more sensitive to phorbol ester treatment than nondifferentiated PC12 cells and their beta-APP release was also induced by the protein phosphatase inhibitor okadaic acid and the Ca(++)-ionophore A23187. In contrast, beta-APP release from B104 cells was strongly stimulated by A23187 and to lesser degree by phorbol esters. This effect was most pronounced in nondifferentiated B104 cells, which might be due to a higher basic release of beta-APP from differentiated B104 cells. Thus, the regulation of beta-APP cleavage and release varies depending on the cell type and differentiation state of the cell.

Topics: Amyloid beta-Protein Precursor; Animals; Bucladesine; Calcimycin; Carcinogens; Cell Differentiation; Cell Line; Culture Media; PC12 Cells; Phorbol 12,13-Dibutyrate; Phorbol Esters; Tetradecanoylphorbol Acetate

We investigated the signal transduction pathways that mediate activation of Syrian hamster eggs. Under conditions in which the concentration of intracellular free calcium ([Ca2+]i) is clamped low, activation of protein kinase C (PKC) can induce second polar body formation, reformation of the nuclear envelope, and decondensation of chromatin, as well as golgi reformation. However, calcium is necessary for normal transition from meiotic metaphase II to anaphase II. Conversely, under conditions in which the level of PKC activity is clamped low, induction of a rise in [Ca2+]i, using the calcium ionophore A23187, does not induce egg activation. These results strongly suggest that PKC acts after the calcium signal as a proximal inducer of egg activation. This suggestion is supported by the kinetics of egg activation; PKC stimulators activate the eggs at a significantly enhanced rate (P < 0.01) compared with activation by calcium ionophore. We show here that PKC stimulators induce emission of the second polar body, but that subsequently, with longer culture, the emitted polar body is absorbed. Our results suggest that the rise in [Ca2+]i serves two functions, to activate PKC and to induce the transition from metaphase II to anaphase II. PKC, once activated, mediates several other events of egg activation.

Topics: Animals; Calcimycin; Calcium; Cells, Cultured; Cricetinae; Diglycerides; Egtazic Acid; Enzyme Activation; Female; Kinetics; Mesocricetus; Ovum; Phorbol Esters; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Topics: Amino Acid Oxidoreductases; Animals; Arginine; Calcimycin; Carcinogens; Cells, Cultured; Cyclic GMP; Diglycerides; Enzyme Induction; Kinetics; Lipopolysaccharides; Liver; Nitric Oxide Synthase; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rats; Tetradecanoylphorbol Acetate

Calcyclin is a member of the S-100 family of calcium-binding proteins, whose expression is enhanced when quiescent cells are exposed to mitogenic signals. The function of calcyclin is unknown, but it is thought to be involved in modulating the intracellular calcium concentration following mitogenic stimuli. Since activation of protein kinase C (PKC) also occurs following stimulation of quiescent cells by a variety of mitogens, we have investigated the relationship between calcyclin expression and PKC activation in three human endometrial adenocarcinoma cell lines. The addition of 10(-7) M 4 beta-phorbol 12-myristate 13-acetate (PMA) to HEC-50 and HEC-1B cell cultures resulted in a change in cell morphology, an inhibition of proliferation, an increase in calcyclin transcription rate, and an increase in calcyclin mRNA and calcyclin protein levels. In contrast, PMA had no effect on cell morphology or cell proliferation in the Ishikawa adenocarcinoma cell line but enhanced calcyclin expression. Another bioactive phorbol ester had the same effect, whereas the calcium ionophore A23187 and the non-phorbol-ester-type tumor promoter thapsigargin had no effect on calcyclin expression. The effect of PMA on calcyclin expression was blocked by the simultaneous addition of the PKC inhibitor staurosporine and by protein synthesis inhibition with cycloheximide. RNase protection assays and primer extension analysis demonstrated that PMA enhanced transcription from all three of the previously identified transcription start sites in the calcyclin gene. These data clearly demonstrate a dissociation between calcyclin expression and cellular proliferation and suggest that the enhanced calcyclin expression which is seen in quiescent cells following mitogenic stimuli may result from activation of the PKC system.

Topics: Adenocarcinoma; Alkaloids; Calcimycin; Calcium-Binding Proteins; Cell Cycle Proteins; Cell Division; Cycloheximide; Enzyme Activation; Female; Gene Expression Regulation, Neoplastic; Humans; Neoplasm Proteins; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; RNA, Messenger; RNA, Neoplasm; S100 Calcium Binding Protein A6; S100 Proteins; Signal Transduction; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate; Thapsigargin; Uterine Neoplasms

The abundance of 1,25-dihydroxyvitamin D3 receptors (VDR) in cultured cells has been shown to vary in direct relation to the rate of cell proliferation. This study examines the question of whether the growth-factor mediated up-regulation of VDR is due to direct modulation of VDR gene expression or is secondary to the stimulation of cell cycle events. Mitogenic agents, such as basic fibroblast growth factor and phorbol esters, were found to cause significant decreases in VDR abundance, while substantially stimulating proliferation of NIH-3T3 cells. Potent phorbol esters, such as phorbol myristate acetate (PMA) and phorbol-12,13-dibutyrate, whose biological actions have been shown to be mediated through the activation of protein kinase-C, down-regulated VDR in a time- and dose-dependent manner. An inactive phorbol ester, 4 alpha-phorbol-12,13-didecanoate, which does not activate protein kinase-C, did not alter VDR levels. Desensitization of protein kinase-C by prolonged exposure of cells to phorbol esters eliminated the PMA-mediated down-regulation of VDR. Staurosporine, an inhibitor of protein kinase-C, blocked the actions of PMA. Oleoyl acetyl glycerol, a synthetic diacyl glycerol, and A23187, a calcium ionophore, were both able to suppress VDR abundance alone and were additive in combination. The results suggest that activation of the protein kinase-C pathway and elevation of intracellular Ca2+ lead to significant down-regulation of VDR. The inhibitory effect of PMA appears to be exerted at the level of VDR mRNA expression. Northern blot analysis revealed significant decreases in steady state levels of VDR mRNA species that qualitatively corresponded to the decrease in VDR protein concentration seen on a Western blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Alkaloids; Animals; Blotting, Northern; Blotting, Southern; Calcimycin; Cell Division; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Down-Regulation; Fibroblast Growth Factor 2; Gene Expression Regulation, Enzymologic; Mice; Phorbol 12,13-Dibutyrate; Phorbol Esters; Protein Kinase C; Receptors, Calcitriol; Receptors, Steroid; RNA, Messenger; Staurosporine; Tetradecanoylphorbol Acetate

The activation of protein kinase C (PKC) by phorbol myristate acetate (PMA) rapidly inhibits the phagocytosis of rod outer segments (ROS) by cultured rat retinal pigment epithelial (RPE) cells. PMA, at a concentration between 3.3 and 10 nM, blocks ROS ingestion by 50%, but does not inhibit the binding of ROS. The Ca2+ ionophore, A23187, also inhibits ROS phagocytosis, with an IC50 of about 0.5-1.0 microM and interferes with the ability of RPE cells to bind ROS. The effects of both of these drugs are reversible after drug washout. When PMA and A23187 are applied to cells consecutively, the effects are additive. These results suggest either that PMA and A23187, act upon the same proteins in the pathway which controls ROS ingestion, or that A23187 affects phagocytosis at the ROS binding level, while PKC affects steps further along the ingestion path. The effect of this process is to shut down the ingestion of ROS, as is seen during the prolonged feeding of ROS to RPE cells in culture.

Topics: Animals; Calcimycin; Calcium; Culture Techniques; Phagocytosis; Phorbol Esters; Pigment Epithelium of Eye; Protein Kinase C; Rats; Rats, Inbred Strains; Rod Cell Outer Segment; Tetradecanoylphorbol Acetate

In the present study mechanisms of signal transduction were investigated by treatment of NCC with phorbol esters and calcium ionophore. NCC were stimulated to produce 3-fold increased cytotoxicity by 10(-4) to 10(-5) M A23187. PMA, PDD, and 4-beta phorbol either suppressed NCC lysis (PMA) or had no enhancing effects (PDD and 4-beta). At a concentration of 10(-4) M increased cytotoxicity occurred after 0.5 to 3 hours treatment. Four-hour preincubation enhanced cytotoxicity at suboptimal A23187 concentrations. A23187 produced increased NCC activity at each E:T ratio tested, however, greatest differences in A23187 concentrations were observed at 20:1 and 40:1. The effects of simultaneous treatment of NCC and/or sequential with PMA and A23187 indicated that different transduction pathways might operate to augment cytotoxicity. PMA, 10(-8) M, combined with 10(-4) M concentrations of A23187 produced a 164.7% increase in cytotoxicity compared to media controls, and a 37.7% increase compared to A23187 controls. These data indicate that teleost cytotoxic cells, unlike mammalian NK, are augmentable by increased levels of extracellular calcium without the presence of any other comodulating signals.

Topics: Animals; Calcimycin; Catfishes; Cell Line; Cytotoxicity, Immunologic; Humans; Phorbol Esters; Tetradecanoylphorbol Acetate; Time Factors

Protein kinase C modulates the activity of a highly specific uptake mechanism for queuine in cultured human fibroblasts. Activators of protein kinase C induce an increased uptake rate for the radiolabeled analog of queuine, rQT3. The protein kinase C inhibitors, H-7, staurosporine and sphingosine all induced a dramatic decrease in the uptake rate of rQT3. This suggests that protein kinase C is tied to efficient cellular uptake of queuine. Uptake is prerequisite to the modification of transfer RNA with queuine. Perturbation of queuine-modified transfer RNA levels has been associated with neoplastic transformation, differentiation and growth control.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Alkaloids; Calcimycin; Cells, Cultured; Diglycerides; Enzyme Activation; Fibroblasts; Growth Substances; Guanine; Humans; In Vitro Techniques; Isoquinolines; Male; Phorbol Esters; Phosphatidylserines; Piperazines; Protein Kinase C; Sphingosine; Staurosporine

Prostaglandin F2 alpha (PGF2 alpha) release from the uterus causes luteolysis in ruminants, and oxytocin is thought to be a regulator of this release. In the present study, we have examined the mechanisms involved in oxytocin stimulation of PGF2 alpha secretion by bovine endometrium in vitro. Endometrial tissue explants, obtained from heifers at Day 19 or 20 (n = 3) and Day 0 (estrus, n = 5) of the estrous cycle, were incubated for 2 h and 6 h, and PGF2 alpha concentration in the medium was determined by radioimmunoassay (RIA). Basal PGF2 alpha release increased for up to 6 h and was significantly stimulated after 2 h of incubation with 100 microU and 1000 microU of oxytocin at Day 0 but not at Day 19 or 20. Secretion of PGF2 alpha was not affected by cholera toxin (10 ng/ml) or the cyclic nucleotide analogs dibutyryl cyclic adenosine 3',5'-monophosphate and dibutyryl cyclic guanosine 3',5'-monophosphate at a concentration of 1 mM. A protein kinase A inhibitor (500 microM) had no effect on the oxytocin-induced release of PGF2 alpha. Both the phorbol ester, 12-myristate-13-acetate (100 mM), and the non-phorbol stimulator of protein kinase C, 1-octanoyl-2-acetylglycerol (500 microM), significantly stimulated PGF2 alpha secretion to the same extent as oxytocin. Neither basal nor stimulated PGF2 alpha release was affected by the calcium ionophore A23187 (0.1-5.0 microM). However, PGF2 alpha secretion was sensitive to cycloheximide (1 microgram/ml) suggesting that protein synthesis may be involved. In conclusion, these data suggest that the stimulation of PGF2 alpha by oxytocin is via the protein kinase C effector pathway.

Topics: Animals; Arachidonic Acids; Bucladesine; Calcimycin; Carcinogens; Cattle; Cholera Toxin; Cycloheximide; Dibutyryl Cyclic GMP; Diglycerides; Dinoprost; Endometrium; Female; Nucleotides, Cyclic; Oxytocin; Phorbol Esters; Protein Kinase Inhibitors; Tetradecanoylphorbol Acetate; Uterus

Prophase I oocytes, free of follicle cells, and metaphase II eggs of the amphibian Xenopus laevis were subjected to transient treatments with the protein kinase C activators, phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-didecanoate, and 1-olyeoyl-2-acetyl-sn-glycerol. In both oocytes and eggs, these treatments triggered early events of amphibian development: cortical granule exocytosis, cortical contraction, and cleavage furrow formation. Surprisingly, activation of oocytes occurred in the absence of meiotic resumption, resulting in cells with an oocytelike nucleus and interior cytoplasm, but with a zygotelike cortex. PMA-induced activation of oocytes and eggs did not require external calcium, a prerequisite for normal activation of eggs. PMA-induced activation of eggs was inhibited by retinoic acid, a known inhibitor of protein kinase C. In addition, pretreatment of eggs with retinoic acid prevented activation by mechanical stimulation and inhibited activation by calcium ionophore A23187. The results suggest that protein kinase C activation is an integral component of the Xenopus fertilization pathway.

Topics: Animals; Calcimycin; Calcium; Diglycerides; Enzyme Activation; Exocytosis; Oocytes; Ovum; Phorbol Esters; Protein Kinase C; Tetradecanoylphorbol Acetate; Tretinoin; Xenopus laevis

To clarify the possible role of protein kinase C in the control of parathyroid hormone (PTH)-degrading activity (PTHDA) in a PTH-responsive opossum kidney (OK) cell line, we investigated the effects of protein kinase C activators, 12-O-tetradecanoyl phorbol 13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 4 beta-phorbol 12, 13-didecanoate (4 beta-PDD). TPA, OAG, and 4 beta-PDD enhanced PTHDA in a dose-dependent fashion (10-50 ng/ml, 10-100 microgram/ml, and 10-50 nM, respectively), whereas 4 alpha-PDD, a non-activator of protein kinase C, did not affect it. HPLC analysis of TPA-treated samples revealed increase of all immunoreactive PTH fragments produced by OK cells. These findings suggested that activation of protein kinase C in OK cells would augment PTHDA in the cells.

Topics: Animals; Bucladesine; Calcimycin; Cell Line; Chromatography, High Pressure Liquid; Colforsin; Diglycerides; Enzyme Activation; Humans; Kidney; Opossums; Parathyroid Hormone; Peptide Fragments; Phorbol Esters; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate

To explore the possible role of protein kinase C and calcium in the luteolytic process, we treated luteal cells with a protein kinase C activator, the phorbol ester, phorbol 12-myristate 13-acetate (PMA), and with the calcium ionophore, A23187. Lower concentrations of PMA could clearly mimic the inhibitory, luteolytic effects of prostaglandin F2 alpha (PGF2 alpha) on LH-induced cAMP and progesterone production. A nontumor promoting phorbol ester, 4 alpha-phorbol 12,13-didecanoate, had no inhibitory effect, indicating a specific PMA effect. The calcium ionophore, A23187, also gave a marked inhibition of LH-induced cAMP and progesterone production. Hormone-stimulated adenylate cyclase activity was markedly impaired after preincubation of the cells with PGF2 alpha, PMA, or A23187, but no effect was seen when the substances were added to isolated membranes. In addition, the stimulation of progesterone production in the luteal cells with the cAMP analogs, 8-bromo-cAMP and (Bu)2cAMP, was almost totally abolished when PMA or A23187 was present. We conclude that PMA and A23187 in many ways mimic the effect of PGF2 alpha in luteal cells. The inhibition of steroidogenesis is partly dependent on depressed activity of the hormone-sensitive adenylate cyclase, but also obtained by inhibiting steps distal to cAMP formation. Both points of action seem to be calcium and/or protein kinase C dependent. In contrast, higher concentrations of PMA markedly stimulated steroidogenesis without affecting the cAMP level, a stimulation not seen after incubation with 4 alpha-phorbol 12,13-didecanoate, suggesting again a specific PMA effect. The stimulation of steroidogenesis by higher concentrations of PMA seems to be specific, but the interpretation of this finding is unclear at present. In conclusion, PMA and A23187 mimic some of the luteolytic properties of PGF2 alpha, not only inhibiting the luteal cAMP system, but also by inducing lesions in the steroidogenic steps beyond the cAMP system.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Adenylyl Cyclases; Animals; Calcimycin; Corpus Luteum; Cyclic AMP; Dinoprost; Female; In Vitro Techniques; Kinetics; Luteinizing Hormone; Phorbol Esters; Progesterone; Prostaglandins F; Rats; Rats, Inbred Strains; Tetradecanoylphorbol Acetate

Both 4 beta-phorbol 12,13-didecanoate (PDD) and calcium ionophore A23187 induced c-fos mRNA accumulation with similar kinetics in human monocyte-like cells (U937). Their effects were additive. Cells pretreated with PDD were not induced to accumulate c-fos mRNA by PDD but remained responsive to the induction by A23187. Similarly cells pretreated with A23187 responded to PDD but not to A23187. Nuclear run-off transcription assay indicated that increase in the c-fos mRNA level after the second inducer treatment corresponded to transcriptional activation of the c-fos gene. The accumulation of c-fos mRNA and the transcriptional activation of c-fos induced by PDD or A23187 were inhibited by the protein kinase inhibitor H-7 and by quinidine and amiloride. The induction by A23187, but not that by PDD, was also inhibited by 4-aminopyridine, or tetraethylammonium. From these results, it is proposed that activation of protein kinase C and Na+ or K+ transport are required for c-fos induction caused by PDD and A23187.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; 4-Aminopyridine; Amiloride; Aminopyridines; Biological Transport, Active; Calcimycin; Humans; Isoquinolines; Phorbol Esters; Piperazines; Potassium; Protein Kinase C; Proto-Oncogene Mas; Proto-Oncogenes; Quinidine; RNA, Messenger; Sodium; Tetraethylammonium Compounds; Transcription, Genetic

The effects of phorbol esters and diacylglycerol on phosphate uptake in opossum kidney (OK) cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on renal phosphate handling. OK cells are widely used as a model of proximal renal tubular cells and are reported to possess a Na+-dependent phosphate transport system. Phorbol-12,13-dibutyrate (PDBu) inhibited phosphate uptake. This inhibitory effect was synergistically enhanced with A23187. 4 beta-phorbol 12,13-didecanoate inhibited phosphate uptake, while 4 alpha-phorbol 12,13-didecanoate did not. 1-oleoyl-2-acetyl-glycerol (OAG), a synthetic diacylglycerol, also exhibited an inhibitory effect on phosphate uptake. These data suggest the possible involvement of protein kinase C in proximal renal tubular phosphate transport.

Topics: Animals; Calcimycin; Cells, Cultured; Diglycerides; Kidney; Kidney Tubules, Proximal; Kinetics; Opossums; Phorbol 12,13-Dibutyrate; Phorbol Esters; Phosphates

The effect of the phorbol ester phorbol-12,13-dibutyrate (PDB), an activator of protein kinase C, on endothelium-dependent relaxation was studied in noradrenaline-constricted isolated aortic ring preparations of the rabbit. Endothelium-dependent relaxation induced by acetylcholine or substance P was inhibited by PDB (greater than or equal to 10(-7) M). Endothelium-dependent relaxation induced by the calcium ionophore A23187 (7.5 X 10(-8) and 10(-7) M) was unaffected by PDB (to 10(-6) M). The mechanical responses to acetylcholine or sodium nitroprusside in endothelium-denuded rings were not altered by PDB (to 10(-6) M). The results suggest a role for protein kinase C in receptor-mediated EDRF release mechanisms.

Topics: Animals; Calcimycin; Carcinogens; Female; In Vitro Techniques; Male; Muscle Relaxation; Nitric Oxide; Nitroprusside; Norepinephrine; Phorbol 12,13-Dibutyrate; Phorbol Esters; Rabbits; Vasodilator Agents

Astrocytes metabolize arachidonic acid via the cyclooxygenase pathway to prostanoids. We examined whether primary culture astrocytes from neonatal rat brain can be induced to generate and release the lipoxygenase derivative leukotriene C4 (LTC4). While there was only minute constitutive production of immunoreactive LTC4 this metabolite was liberated by astroglial cells in response to calcium ionophore A23187. The phorbol diester 12-O-tetradecanoylphorbol 13-acetate (TPA) failed to precipitate leukotriene release. However, when threshold doses of A23187 were added to astrocyte cultures challenged with TPA, LTC4 was recovered from their supernatants. It is suggested that leukotriene generation by astrocytes bears relevance to immunoinflammatory responses in the central nervous system and may be involved in brain edema formation.

Topics: 4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyrazol-3-amine; Animals; Animals, Newborn; Anti-Inflammatory Agents, Non-Steroidal; Astrocytes; Calcimycin; Carcinogens; Cells, Cultured; Kinetics; Phorbol Esters; Pyrazoles; Rats; SRS-A; Tetradecanoylphorbol Acetate

The regulation of T4 5'-deiodinase activity was studied in cultured GH4C1 cells. Enzyme activity was measured in cell sonicates as the release of radioiodide from [125I]T4. Enzyme activity was stimulated 2- to 3-fold by hypothyroid serum and activators of protein kinase C, such as TRH and phorbol esters. The hypothyroid serum effect was maximal by 3 h, whereas TRH and phorbol esters required 6 h to achieve a maximal effect. The hypothyroid serum effect was gone within 4 h of returning the cells to control medium. In contrast, the TRH and phorbol ester effects persisted 24-48 h after removal of those agents. Both T4 and rT3 were at least as potent as T3 in blocking the effect of hypothyroid serum. The stimulation of 5'-deiodinase induced by hypothyroid serum was additive with that induced by kinase C activators. Trifluoperazine blocked the effect of TRH and phorbol esters, but not that of hypothyroid serum. It is concluded that stimulation of 5'-deiodinase activity can occur by at least two independent mechanisms: one involving hypothyroidism and another involving activation of protein kinase C. The relative potencies of various iodothyronines for abolishing the hypothyroid effect differ markedly from the relative binding affinities of these agents for the nuclear T3 receptor, suggesting that this thyroid hormone effect may not be mediated by the classical nuclear thyroid hormone receptor.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Calcimycin; Cell Line; Colforsin; Cyclic GMP; Dose-Response Relationship, Drug; Enzyme Activation; Hypothyroidism; Iodide Peroxidase; Phorbol Esters; Pituitary Neoplasms; Protein Kinase C; Rats; Tetradecanoylphorbol Acetate; Thyroid Hormones; Thyrotropin-Releasing Hormone; Thyroxine; Trifluoperazine; Triiodothyronine; Triiodothyronine, Reverse

The effects of phorbol esters and diacylglycerol on phosphate accumulation in the cultured mouse kidney cells were investigated to assess the possible role of Ca2+-activated, phospholipid dependent protein kinase (protein kinase C) on the renal phosphate handling. 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated phosphate accumulation dose-dependently. TPA-induced phosphate accumulation was synergistically enhanced with A23187. 4 alpha-phorbol 12,13-didecanoate did not stimulate the phosphate accumulation, while 4 beta-phorbol 12,13-didecanoate stimulated it. Additionally, 1-oleoyl-2-acetyl-glycerol exhibited a stimulatory effect on phosphate accumulation. These data indicated that protein kinase C is one of possible regulators of phosphate transport at the renal tubules.

Topics: Animals; Calcimycin; Cells, Cultured; Diglycerides; Dose-Response Relationship, Drug; Drug Synergism; Isomerism; Kidney Tubules; Mice; Phorbol Esters; Phosphates; Protein Kinase C; Tetradecanoylphorbol Acetate

The tumor-promoting phorbol diester 4 beta-phorbol 12-myristate 13-acetate (PMA) inhibited mobilization of intracellular Ca2+ in platelets by thrombin (also trypsin and 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine). PMA was effective over the same concentration range that activates protein kinase C in intact platelets; IC50 vs. thrombin = 2 ng/ml, 3.4 nM: greater than 90% inhibition at 10-20 ng/ml. Suppression of thrombin-induced Ca2+ mobilization was evident within 30 sec of pretreatment with PMA and was essentially complete by 6-10 min at 10-20 ng of PMA per ml. Thrombin-induced secretion was initially accelerated in the presence of PMA, but after 1 min it was progressively inhibited when Ca2+ mobilization was depressed by greater than 60%. PMA did not inhibit Ca2+ mobilization or secretion caused by A23187. Thrombin-induced phosphatidylinositol 4,5-[32P]bisphosphate breakdown and [32P]phosphatidic acid production were also initially increased by PMA and then progressively depressed. Inhibition of thrombin-induced lipid metabolism required higher concentrations of PMA (IC50 = 10 ng/ml), and it was not overcome by A23187. 4 alpha-Phorbol 12,13-didecanoate, which lacks the ability to activate protein kinase C, did not inhibit any responses to thrombin. These results suggest that activation of protein kinase C, which initially fosters secretion and aggregation, may subsequently exert negative feedback on the receptor-mediated mobilization of intracellular Ca2+ and the hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Topics: Aminoquinolines; Blood Platelets; Calcimycin; Calcium; Humans; Lipid Metabolism; Phorbol Esters; Phorbols; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Tetradecanoylphorbol Acetate; Thrombin

The co-carcinogenic compound phorbol 12-myristate 13-acetate but not its inactive analogue 4 alpha-phorbol 12,13-didecanoate causes the phosphorylation of several rabbit neutrophil polypeptides whose molecular weights and isoelectric points (pI) are as follows: Mr = 40,000, pI = 6.4; Mr = 50,000, pI = 4.9; Mr = 55,000, pI = 6.3; Mr = 64,000, pI = 6.0; Mr = 70,000, pI = 5.6; Mr = 90,000, pI = 6.0. Most of these phosphorylated proteins are located exclusively in the cytosol; the 64,000 molecular weight protein is found both in the cytosol and the cytoskeleton, and the 40,000 molecular weight protein is found in the nuclear pellet. The 50,000 molecular weight protein is also phosphorylated in whole cells by the chemotactic peptide fMet-Leu-Phe and in cell-free systems by protein kinase C. Using limited proteolysis, one phosphopeptide fragment was phosphorylated by the three stimuli. In addition, phorbol 12-myristate 13-acetate but not 4 alpha-phorbol 12,13-didecanoate causes cell aggregation and the exocytotic release of the specific granules of rabbit neutrophils. In contrast, both compounds increase the amount of actin associated with the cytoskeleton. The divalent cation ionophore A23187 at low concentration and the compound phorbol 12-myristate 13-acetate act synergistically in causing neutrophil degranulation. Lysosomal enzyme release and the phosphorylation of the 50,000 molecular weight polypeptide produced by phorbl 12-myristate 13-acetate are inhibited by trifluoperazine, and these two responses seem to be causally related. These results are discussed in terms of the role of 1,2-diacylglycerol and activation of protein kinase C in specific granule release from rabbit neutrophils.

Topics: Animals; Blood Proteins; Calcimycin; Carcinogens; Cytochalasin B; Kinetics; Lysosomes; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phorbol Esters; Phorbols; Phosphorylation; Protein Kinases; Rabbits; Tetradecanoylphorbol Acetate; Trifluoperazine

The influence of phorbol esters upon the thermotropic behaviour of multilamellar liposomes formed of dipalmitoylphosphatidylcholine (DPPC) or dimyristoylphosphatidylcholine (DMPC) was investigated, as a model for possible interferences of the phorbol esters with the phospholipid domain of biological membranes. Both biologically active (TPA, 12-O-tetradecanoylphorbol-13-acetate, PDD, phorbol-12,13-didecanoate) and inactive (4 alpha-PDD, 4 alpha-phorbol-12,13-didecanoate) phorbol esters lowered the temperature required to cause a fall in fluorescence polarization of a fluorescent probe inserted in the lipid matrix of the DMPC or DPPC liposomes and facilitated the process of calcium exchange-diffusion in DPPC liposomes containing the ionophore A23187. Both of these effects could be due to a decrease in viscosity of the liposomal matrix. However, differential scanning calorimetry revealed that the thermotropic changes evoked by each of these phorbol esters were not identical. In most cases, the phorbol esters decreased both the main phase transition temperature and enthalpy of melting. However, when TPA was incorporated in DMPC liposomes, i.e. when a myristoyl chain was present in both the phorbol ester and phospholipid, no change in the enthalpy of melting could be detected, whereas the main phase transition temperature decreased in proportion to the TPA content of the liposomes. These findings emphasize the view that phorbol esters indeed interact with phospholipids and that the characteristics of such an interaction may tightly depend on the precise chemical structure of both the phorbol ester and phospholipid under consideration.

Topics: Calcimycin; Calcium; Carcinogens; Diffusion; Dimyristoylphosphatidylcholine; Lipid Bilayers; Phorbol Esters; Phorbols; Phosphatidylcholines; Pulmonary Surfactants; Spectrometry, Fluorescence; Structure-Activity Relationship; Temperature; Tetradecanoylphorbol Acetate