Effects Of Tetradecanoyl Phorbol Acetate Research Articles

Interaction of protein kinases A and C in their effects on the proenkephalin gene in astroglial cells.

In several cell types, the expression of the proenkephalin (PEnk) gene is enhanced after activation of protein kinase A. In the present study, astroglial cells cultured from rat cortex were used to investigate whether protein kinases A and C can act in a synergistic manner on the endogenous proenkephalin gene. The activator of protein kinase C tetradecanoylphorbolacetate (0.001-1 microM) increased the level of proenkephalin-mRNA in a concentration dependent manner. When used together with the phosphodiesterase inhibitor Rolipram (1 microM), the effect of tetradecanoylphorbolacetate (0.01 microM) was potentiated. 8-Bromoadenosine 3',5'-cyclic monophosphate (0.01-1 mM) also enhanced the expression of the proenkephalin gene. When used together with tetradecanoylphorbolacetate (0.01 and 0.1 microM), respectively, both agents had additive effects. Inhibition of protein synthesis with cycloheximide (35 microM) significantly changed the effects of both agents. While the effect of 8Br.cAMP (1 mM) on PEnk-mRNA was enhanced, that of tetradecanoylphorbolacetate (0.1 microM) was abolished. The results provide evidence for a synergistic effect of protein kinase A and C on the expression of the proenkephalin gene in astroglial cells. However, the protein kinases seem to act via different transcription factors on the expression of the proenkephalin gene.

Different responses to mitogenic agents by Adult rat and human chromaffin cells in vitro.

Adrenal medullary hyperplasia and pheochromocytomas occur frequently in laboratory rats, both in the courseof aging and in response to prolonged administration of a variety of drugs and other substances. In contrast, these lesions are rare in humans. Rat chromaffin cells proliferate throughout life, but the proliferative capacities of human chromaffin cells are unknown. To determine whether the difference in prevalence of adrenal lesions might be correlated with differences in cell proliferation, adrenal medullary cells from 3 patients undergoing radical nephrectomy were maintained in vitro for up to 2 weeks in control medium or in the presence of nerve growth factor (NGF) and/or tetradecanoyl phorbol acetate (TPA), an activator of protein kinase C. Both NGF and TPA are known mitogens for neonatal and adult rat chromaffin cells. At intervals, the cultures were pulsed for up to 36 hours with bromodeoxyuridine (BrdU) to label S-phase nuclei. They were then fixed and consecutively stained for BrdU and for tyrosine hydroxylase, to confirm that labeled cells were chromaffin cells. Cells from adult female F344 rats were similarly maintained. Human chromaffin cells labeled with BrdU were extremely rare (less than 0.1 %) under all culture conditions, and effects of NGF or TPA could not be demonstrated. Rat chromaffin cells showed little or no labeling with BrdU in control medium but, in contrast to their human counterparts studied, showed marked increases in the percentages of labeled cells in the presence of NGF (37% ± 3%), TPA (7% ± 1%), or both (31% ± 3%). The apparently lower responsiveness of human chromaffin cells to mitogenic signals, or responses to different types of signals, may contribute to the lower frequency of adrenal medullary hyperplasia and pheochromocytomas in humans compared to rats.

Stimulation of insulin release by vasopressin in the clonal beta-cell line, HIT-T15: the role of protein kinase C.

We have studied the effects of vasopressin and tetradecanoyl phorbol acetate (TPA) on cytosolic free Ca2+ ([Ca2+]i) and insulin release in HIT-T15 beta-cells. Saturable binding of [3H] [Arg8]-vasopressin to HIT cell microsomes indicated a single class of receptors with a dissociation constant (Kd) of 2.5 nM and a total number of binding sites (Bmax) equal to 120 fmol/mg protein. [Arg8]-vasopressin (0.1-100 nM) elicited dose-dependent insulin release from HIT cells by up to 25-fold. This increase was dependent on the presence of extracellular glucose and was blocked by omission of extracellular Ca2+ or addition of verapamil. The stimulation was biphasic; a rapid but short-lived large increase in release was followed by a smaller sustained rise. Vasopressin also evoked a marked, concentration-dependent increase in [Ca2+]i which was also biphasic; an initial spike was followed by a sustained elevation. This increase also required glucose and was blocked by the absence of extracellular Ca2+ or the addition of verapamil. Pretreatment of the cells with TPA overnight to deplete protein kinase C activity did not affect the [Ca2+]i or insulin responses to vasopressin. However, short-term exposure to TPA markedly reduced glucose-induced steady-state [Ca2+]i, despite potentiating glucose-stimulated insulin release sevenfold, and blocked the [Ca2+]i increase induced by vasopressin. These inhibitory effects of TPA were absent in protein kinase C-depleted cells and were prevented by staurosporine. TPA had no significant effect on vasopressin-induced insulin release. Vasopressin did not modify the activity of ATP-sensitive K+ channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin Inhibits while Insulin, Epidermal Growth Factor and Tetradecanoyl Phorbol Acetate Stimulate Insulinlike Growth Factor Binding Protein Secretion from Sheep Thyroid Cells

Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function.

Effect of 12‐O‐tetradecanoyl phorbol 13‐acetate on solute transport and production of cAMP in isolated frog skin

In the present study we have examined the action of the phorbol diester tetradecanoyl phorbol acetate, an activator of protein kinase C, on the transepithelial transport of sodium, chloride and water and the production of cAMP in the isolated frog skin epithelium (Rana esculenta). Addition of tetradecanoyl phorbol acetate to the mucosal solution resulted initially in an increase in the short-circuit current, which was followed by a progressive decrease. If the short-circuit current was first activated by addition of the antidiuretic hormone, arginine vasotocin, then the addition of tetradecanoyl phorbol acetate resulted only in a pronounced inhibition. The changes in the short-circuit current were the result of changes in the active influx of Na+. The effect of tetradecanoyl phorbol acetate on the intracellular potential measured under short-circuited conditions (Vscc) was time-dependent. Just after addition of tetradecanoyl phorbol acetate to the mucosal solution, Vscc depolarized; this was followed by a slight hyperpolarization, after which Vscc continued to decline. The inhibition of the Na+ transport by tetradecanoyl phorbol acetate was associated with a decline in the response to the antidiuretic hormone (arginine vasotocin), but the ability of arginine vasotocin to increase the cellular level of cAMP and to stimulate the osmotic water flow was not affected by the presence of tetradecanoyl phorbol acetate. In skin halves in which the short-circuit current was stimulated with arginine vasotocin, addition of tetradecanoyl phorbol acetate resulted in a dose-dependent inhibition of the short-circuit current, but only minor changes in Vscc were observed. The results presented suggest that the addition of tetradecanoyl phorbol acetate to the isolated frog skin first increases and then decreases the arginine vasotocin-sensitive sodium permeability of the apical membrane. This might be due to a stimulating effect of tetradecanoyl phorbol acetate on both the activation and deactivation (turnover) of the sodium channels.

Effects of tetradecanoyl phorbol acetate, pyrethroids and DDT in the V79.

The effects of the pyrethroids flucythrinate, cyfluthrin, bioallethrin and resmethrin on metabolic cooperation between V79 cells were investigated. Addition of flucythrinate to cocultures of 6-thioguanine-resistant and 6-thioguanine-sensitive V79 cells significantly increased the mutant cell recovery, indicating inhibition of intercellular communication. No such effect was observed by the other pyrethroids tested. To compare the modes of action of TPA-, DDT-, and pyrethroid-induced inhibition of intercellular communication, co-exposure experiments were undertaken. Addition of TPA, together with increasing doses of fenvalerate or flucythrinate, produced a synergistic response. Various combinations of fenvalerate-, flucythrinate- and DDT-exposure gave results in accordance with an additive response. The result suggest different pathways of action for TPA and the insecticides investigated in this study.

Independent and interactive effects of tetradecanoyl phorbol acetate on growth and differentiated functions of FRTL5 cells.

In studies of regulation of the growth and differentiated function of the thyroid follicular cell, we have employed the FRTL5 cell line to evaluate both the effects of agents that activate protein kinase-C (PKC) and their interaction with other agents that influence the growth and/or function of the FRTL5 cell. The PKC activator tetradecanoyl-phorbol acetate (TPA) alone induced a time- and concentration-dependent stimulation of the incorporation of [3H]thymidine into the DNA of quiescent FRTL5 cells, an effect anteceded by an increase in the levels of the mRNAs of the proto-oncogene c-myc and associated with a stimulation of cell replication. TPA also produced a dose-dependent inhibition of the low levels of radioiodine uptake in quiescent FRTL5 cells. These effects of TPA were unaccompanied by any change in the cellular cAMP concentration. TPA also modified a variety of responses to TSH, attenuating the TSH-induced stimulation of [3H]thymidine incorporation into DNA, cell replication, cAMP generation, and iodine uptake. Inhibition of TSH-stimulated growth and iodine uptake by TPA could not be ascribed solely to a decrease in cAMP generation, since TPA also inhibited the increase in [3H]thymidine incorporation and iodide uptake induced by the cAMP analog (Bu)2cAMP. In contrast, the independent stimulatory effects of TPA and insulin-like growth factor I (IGF-I) on [3H]thymidine incorporation and cell replication were at least additive when the two stimulators were added together. We have previously reported that both TSH and (Bu)2cAMP amplify the enhancement of DNA synthesis and cell replication in FRTL5 cells induced by IGF-I, and that the response of DNA synthesis to IGF-I is also enhanced if cells are preincubated with either TSH or (Bu)2cAMP. Both the former amplification of mitogenesis and the latter priming effect were decreased by exposing cells to TPA concomitant with their exposure to TSH or (Bu)2cAMP. The effects of TPA were mimicked by other activators of PKC, but not by a phorbol ester that fails to activate this enzyme. In general, we conclude that in the FRTL5 cell, regulation of cell growth is extremely complex; there are at least three mitogenic pathways that are separate from but interact with one another. The first is the well known cAMP-dependent pathway, which is activated by TSH. The second is activated by IGF-I and is cAMP independent. These two pathways interact to produce a marked amplification of their individual mitogenic effects. The third pathway is that stimulated by TPA and involves activation of PKC.(ABSTRACT TRUNCATED AT 400 WORDS)

The influence of tetradecanoyl-phorbol-acetate (TPA) on histamine release from isolated rat mast cells

A detailed investigation of the influence of tetradecanoyl-phorbol-acetate (TPA) on isolated rat mast cells was undertaken in order to explore the possible involvement of protein kinase C in histamine release. TPA alone could induce histamine release in a medium without calcium, whereas 1 mM CaCl2 suppressed the release. TPA in combination with a low concentration of the ionophore A23187 induced a considerable histamine release. Preincubation with TPA followed by incubation with the ionophore induced a similar release at low concentrations of TPA (less than or equal to 2.5 nM) whereas the response was reduced at higher concentrations of TPA. The inhibition after preincubation with TPA was almost at a maximum within 2 min and was due to a decreased rate of release. TPA could also increase antigen-induced histamine release. After preincubation the potency of low concentrations of TPA increased, whereas higher concentrations (50 nM) became inhibitory. The effects of preincubation were almost fully expressed after 2 min and were not due to altered kinetics of the release. The interaction of oleoylacetylglycerol (OAG) with the ionophore A23187 and with antigen resembled that of TPA, but OAG was considerably less potent. Preincubation with TPA was inhibitory to the histamine release induced by compound 48/80, particularly in the absence of calcium. The release induced by TPA and the ionophore or antigen was calcium-dependent and energy-requiring, and the effects of TPA persisted after washing the cells before exposure to antigen or the ionophore. Preincubation with the protein kinase C inhibitor isoquinolinesulfonyl-methylpiperazine (H7) slightly enhanced the histamine release induced by the combination of TPA and the ionophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Effects of Thyrotropin, Phorbol Esters, Norepinephrine, and Carbachol on Iodide Organification in Dog Thyroid Slices, Follicles, and Cultured Cells*

The effect of TSH, phorbol ester, norepinephrine (NE), and carbachol, agents known to influence thyroid metabolism, was compared on iodide organification in dog thyroid slices, freshly isolated follicles, and cultured cells. TSH stimulated iodide organification in all three types of preparations, and this effect was mimicked by (Bu)2cAMP. In contrast, the phorbol ester, tetradecanoyl phorbol acetate (TPA) stimulated iodide organification in slices and follicles but inhibited it in cells. The dose and time required for these divergent effects were similar. Other stimulators of protein kinase C such as aplysiatoxin and teleocidin mimicked the effects of TPA, and these effects were partially reversed by H-7, an inhibitor of protein kinase C. Pretreatment of cells with TPA for 4 h did not affect TSH-stimulated cAMP production, but TPA inhibited iodide organification in cells even in the presence of TSH or (Bu)2cAMP. Similarly NE and carbachol stimulated iodide organification in follicles but inhibited it in cells under basal as well as TSH-stimulated conditions. These effects of NE and carbachol were via alpha 2-adrenergic and muscarinic cholinergic receptors, respectively. However, NE and carbachol inhibited TSH-stimulated cAMP production in both follicles and cells. In thyroid cells, carbachol inhibited uptake and increased efflux of iodide within minutes. TPA also produced similar effects after longer periods of incubation, where an inhibition of uptake was seen by 1 h and an increase in efflux by 2 h. NE had a marginal inhibitory effect on uptake and had no effect on efflux of iodide. In contrast to these agents, TSH increased uptake but not efflux of iodide. The present results suggest that the response of the freshly isolated tissue to phorbol esters, NE and carbachol differs from that of cells in culture with respect to an important metabolic function of the thyroid gland. These agents seem to have direct effects on iodide transport and organification unrelated to their effects on cAMP production.

Synergism between vasopressin and phorbol esters in stimulation of insulin secretion and phosphatidylcholine metabolism in RIN insulinoma cells

The tumor promoter, tetradecanoylphorbolacetate (TPA), causes a significant increase in both insulin secretion and the incorporation of 32Pi into phosphatidylcholine (PC) in RIN insulinoma cells. The peptide hormone, arginine vasopressin (AVP), also stimulates these functions, although to a lesser degree. When added together, the effects on secretion and PC metabolism are synergistic. At the same time, TPA inhibits the AVP-stimulated rise in phosphoinositide (PI) metabolism. Neither phloretin nor tamoxifen, reported to be inhibitors of protein kinase C activity, are able to block the effects of TPA on secretion, although both influence PC metabolism.

Prostaglandin E1 and F2 alpha stimulate differentiation and proliferation, respectively, of clonal osteoblastic MC3T3-E1 cells by different second messengers in vitro.

The effect of several prostaglandins (PGs) on osteoblastic cells was investigated using clone MC3T3-E1 under serum-free conditions. PGA1, A2, B1, and B2 had little effect on intracellular cAMP, alkaline phosphatase (ALP) activity, and DNA synthesis in the cells. At 4-2000 ng/ml, PGE1 among PG analogs tested had a dose-dependent stimulatory effect on ALP activity in the cells, and this effect was amplified by isobutyl methylxanthine. Also, PGE1 strongly augmented the amount of intracellular cAMP over the same concentration range. However, PGE1 had little effect on ornithine decarboxylase activity and DNA synthesis, and at high doses it rather depressed DNA synthesis. Furthermore, PGE1 did not affect the intracellular cGMP level. The effect of PGE1 on the cells closely mimics that of forskolin, suggesting that the PG stimulates the differentiation of the osteoblastic cells predominantly via the stimulation of adenylate cyclase. In contrast with PGE1, PGF2 alpha strongly increased ornithine decarboxylase activity and DNA synthesis in the cells in a dose-related fashion at low concentrations (4-100 ng/ml), at which concentrations it had little effect on the intracellular cAMP or cGMP level and depressed ALP activity. Moreover, PGF2 alpha depressed the stimulatory effect of PGE1 on ALP activity but did not affect the elevation of cAMP level by PGE1. The accumulation of inositol phosphates was greatly increased by PGF2 alpha in the concentration range effective in stimulating DNA synthesis, but was increased little by PGE1, suggesting that PGF2 alpha is a potent stimulator of phosphatidyl inositol turnover in the cells. In addition, A23187, a Ca ionophore, alone did not influence the DNA synthesis, but the effects of tetradecanoyl phorbol acetate, a direct activator of protein kinase C, were very similar to those of PGF2 alpha. Moreover, the stimulation of DNA synthesis or the inhibition of ALP activity by PGF2 alpha was partially counteracted by H-7, a strong inhibitor of protein kinase C. These results suggest that PGF2 alpha stimulates the proliferation of osteoblastic cells predominantly through the phosphatidyl inositol turnover system following in part the activation of protein kinase C. Our data presented here indicate that PGE1 and PGF2 alpha are closely involved in the differentiation and proliferation, respectively, of osteoblasts in vitro and that their action may be mediated by second messengers which differ from each other.

Tetradecanoyl phorbol acetate suppresses follicle-stimulating hormone-induced synthesis of the cholesterol side-chain cleavage enzyme complex in rat ovarian granulosa cells.

The effect of tetradecanoylphorbol acetate (TPA) on follicle-stimulating hormone (FSH)-induced synthesis of the cholesterol side-chain cleavage (SCC) enzyme complex was studied in rat ovarian granulosa cells cultured for 48 h in serum-free medium. Cell proteins were radiolabeled with [35S]methionine, followed by immunoprecipitation of cholesterol side-chain cleavage cytochrome P-450 (P-450SCC) as well as the iron-sulfur protein adrenodoxin. Polyacrylamide gel electrophoresis and fluorography of the immunoprecipitates showed that TPA, when added in combination with FSH (50 ng/ml) or dibutyryl cAMP (Bt2cAMP; 1 mM), suppressed the stimulatory effects of these compounds on the synthesis of the SCC components in a concentration-dependent fashion. The effect of TPA was accompanied by decreased progesterone formation and decreased cAMP accumulation. The structural analog of TPA, phorbol-4 alpha-didecanoate, which does not activate protein kinase C (Ca2+/phospholipid-dependent enzyme), had no effect on the FSH- or Bt2cAMP-stimulated synthesis of SCC and progesterone or on cAMP formation. In addition to inhibiting the synthesis of these proteins, TPA greatly reduced the FSH- and Bt2cAMP-induced increase in levels of mRNA encoding the precursor form of P-450SCC. It is concluded that the effect of the phorbol ester TPA to inhibit FSH-stimulated progesterone formation in cultured ovarian granulosa cells of the rat involves decreased synthesis of the components of the SCC enzyme complex due to reduced levels of mRNA encoding the precursor forms of these proteins. The results are indicative that TPA not only inhibits FSH-mediated stimulation of cAMP formation but also may block cAMP-mediated induction of SCC synthesis. It is postulated that the effects of TPA may reflect the physiological role of protein kinase C in the regulation of ovarian steroidogenesis.

Protein kinases induce isoproterenol desensitization of β-adrenoceptor-coupled adenylate cyclase system: significance of receptor occupancy

Treatment of rat reticulocytes with isopreterenol resulted in dose- and time-dependent desensitization of adenylate cyclase to β-adrenoceptor agonist stimulation. Cyclic AMP-dependent protein kinase was possibly involved in this desensitization. Treatment of rat reticulocytes with a phorbol ester, tetradecanoyl phorbol acetate (TPA), also induced desensitization to β-adrenoceptor agonists, indicating the possible involvement of protein kinase C. The addition of a β-adrenoceptor agonist enhanced the desensitizing effect of dibutyryl cyclic AMP or TPA. T 1 2 (time for induction of half maximal desensitization) was decreased from 30 to 2.5 min in accordance with an increase in the concentration of the agonist. The extent of the desensitization was also increased by addition of the agonist. The potency with which the increase was induced was compatible with the potency for binding of the agonist to β-adrenoceptors (K D values). These results suggest that cyclic AMP- or phorbol ester-induced desensitization is enhanced by receptor occupation by β-adrenoceptor agonists.

Role of non-TSH factors in thyroid cell growth.

The effects of insulin, the tumour promotor tetradecanoyl phorbol acetate (TPA), TSH and combinations of these factors on growth and DNA synthesis have been examined in the FRTL-5 cell strain and in sheep thyroid cells. In addition the regulation of the production by sheep thyroid cells of the insulin-like growth factors (IGF) by TSH and their possible autocrine roles have been investigated. We found that insulin and the IGF's stimulated DNA synthesis in both rat FRTL-5 cells and sheep cells. TPA also stimulated growth in both cell types, and its effects were additive to those of insulin. In the FRTL-5 cells, TPA was a less potent stimulator of growth than TSH, but the effects of TPA and TSH were not additive which may imply growth stimulation through a common pathway. In sheep cells TSH was not mitogenic and did not appear to activate protein kinase C, the receptor for TPA. Sheep cells, unlike FRTL-5 cells, were found to produce IGF-I and IGF-II, and their syntheses were regulated by TSH. Sheep cells were also found to produce IGF-binding proteins which may modulate the biologic effects of the IGF's. Sheep thyroid IGF binding proteins were found to copurify with urokinase-like plasminogen activator on immunoaffinity chromatography. The production of this serine protease has also been shown to be regulated by TSH.

Colony size, cell density and nature of the tumor promoter are critical variables in expression of a transformed phenotype (focus formation) in co-cultures of UV-TDTx and C3H10T1/2 cells.

UV-TDTx cells are cloned from foci arising after C3H10T1/2 cells are sequentially exposed to u.v. irradiation followed by tetradecanoylphorbol acetate (TPA). When grown in pure culture, UV-TDTx cells appear transformed. Co-culture with C3H10T1/2 cells suppresses focus formation by the UV-TDTx cells. In the presence of TPA, however, focus formation by UV-TDTx cells occurs in C3H10T1/2 co-cultures. We now demonstrate that only tumor promoters that activate protein kinase C (TPA, teleocidin) can reverse C3H10T1/2 suppression of UV-TDTx focus formation in co-culture; other promoters (diethyl stilbestrol, dioxin, saccharin, cadmium) are inactive. Retinoic acid, a potent inhibitor of many biological effects of TPA, blocks the action of TPA in UV-TDTx:C3H10T1/2 co-cultures. Focus formation by UV-TDTx cells in co-culture is dependent on the size of the UV-TDTx colony at confluence; if the UV-TDTx colony is below a minimal size when the co-cultures reach density-dependent growth arrest, suppression of focus formation by C3H10T1/2 cells occurs even in the presence of TPA. Finally, TPA must be present prior to confluence to relieve suppression of focus formation. If TPA is added to co-cultures after density arrest, UV-TDTx cells will not subsequently form foci.

The effect of tetradecanoylphorbol acetate on calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly induced by thrombin or arachidonate

Tetradecanoylphorbol acetate (TPA) activates primarily only the protein kinase C pathway not the calcium ion-dependent pathway in platelets. The net effect of this split activation is that only the pseudopodal cytoskeleton assembles, not the contractile cytoskeleton needed for rapid secretion. In this study, platelets were first activated with TPA, then activated secondarily with either thrombin or arachidonate and the subsequent dense body secretion, calcium-ion mobilization, protein phosphorylation and cytoskeletal assembly compared to these same processes in control platelets activated solely with either thrombin or arachidonate. Secretion was reduced as the length of time between the primary and secondary activation was increased; but at a 2–3 min interval, where the activation by TPA was essentially complete, the reduction in the total radiolabeled serotonin secreted was small. Furthermore, nearly normal cytosolic calcium-ion increases, phosphorylation of myosin light chain and contractile cytoskeletal development were induced by thrombin or arachidonate after this interval. Prior treatment of the platelets with 100 μM acetylsalicylate to block the cyclooxygenase-dependent pathway caused minor reduction in dense-body secretion induced by TPA or thrombin or the combination of both, but otherwise the relative results were comparable to the untreated platelets. Therefore, short-term prior activation of gel-filtered platelets with TPA, even at concentrations in excess of 100-times that required to saturate protein kinase C, does not prevent normal activation of the calcium ion dependent processes through either the cyclooxygenase-dependent or -independent pathway. Longer-term preincubations with TPA differentially inhibit the secretion response induced by thrombin and arachidonate.

Peroxide formation and glucose oxidation in calf thyroid slices: regulation by protein kinase-C and cytosolic free calcium.

We have determined the effects of tetradecanoyl phorbol acetate (TPA) and of the calcium ionophore A23187 on two thyroid responses to TSH previously reported to be cAMP-independent. We observed that TPA and A23187, at doses of 1.0 microM, stimulated both hydrogen peroxide generation and glucose oxidation in calf thyroid slices. A subthreshold dose of A23187 (0.1 microM) added to a submaximal dose of TPA (0.5 microM) acted synergistically, stimulating H2O2 production to the same degree as a maximally effective dose of TSH (50 mU/ml). Forskolin (25 microM), a direct stimulator of adenylate cyclase, actually inhibited both glucose oxidation and hydrogen peroxide generation. Lithium chloride (25 mM) had no effect on either response, either in the basal state or with TSH stimulation. The calcium channel antagonist verapamil (50 microM) decreased the basal activity of glucose oxidation and peroxide generation but did not substantially inhibit the effect of TSH on H2O2 generation under the conditions studied. These data support the concept that TSH induces changes in the thyroid phosphatidylinositol metabolism which activates protein kinase-C (c-kinase) and raises cytosolic free calcium. These events appear to act in concert to mediate certain metabolic responses in differentiated thyroid tissue.

A combination of calcium ionophore and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulates the growth of purified resting B cells.

In this study we investigated whether the calcium ionophores A23187 and ionomycin can act synergistically with the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) to stimulate the growth of resting B lymphocytes purified from human tonsil cells. Ionomycin, A23187, and TPA added separately to cultures at doses of 0.4-1.6 micrograms/ml, 0.2-0.8 micrograms/ml, and 0.05-0.25 ng/ml respectively, did not induce DNA synthesis in resting B lymphocytes. In contrast, calcium ionophores at concentrations of 0.4-1.6 micrograms/ml ionomycin and 0.2-0.8 micrograms/ml A23187, in the presence of 0.05-4 ng/ml TPA, induced marked DNA synthesis and B-cell proliferation, as shown by analyses of incorporation of [3H]thymidine, growth kinetics, and the percentage of cells in the S and G2 + M phases of the cell cycle. These results show that the synergistic effects of calcium ionophores and TPA can bypass the requirement for antigen and exogenous growth factors in B-cell activation. These observations are similar to those obtained from studies of T lymphocytes by other workers.

305 REGULATION OF SURFACTANT SECRETION FROM TYPE II CELLS MECHANISM OF TUMOR PROMOTER ACTION

Tumor promoter 12–0–tetradecanoylphorbol 13-acetate (TPA) is a potent secretagogue for surfactant release from lung: its mechanism of action remains unknown. Since TPA activates a Ca2+, phospholipid (PL) C-kinase in other cells we tested whether TPA alters surfactant release from isolated Type II cells in a Ca2+ dependent fashion and whether polymixin B, a known inhibitor of C-kinase, blocks effects of TPA. TPA caused a dose dependent release of 3H-choline labelled phosphatidylcholine (3H-PC) from purified rat Type II cells in primary culture:TPA was not toxic as monitored by lactate dehydrogenase release. Since C-kinase is a Ca2+ PL dependent enzyme, effects of calcium ionophore A23187 and TPA together on surfactant release were studied. TPA-induced (100nM) 3H-PC release was significantly potentiated by A23187 (10nM): Control 1.40±0.23%; A23187 1.61±.24%; TPA 3.30±0.40%; A23187+TPA 5.63±0.39% (p<0.001 for A23187+TPA vs. controU p<0.05 for TPA vs. control). Polymixin B, a known inhibitor of Ca2+ -PL kinase, partially blocked TPA-induced 3H-PC release: Control 1.17±0.17%; polymixin B 0.40±0.08%; TPA 4.07±0.34%; TPA+polymixin B 2.43±0.29% (p<0.05 for TPA vs. control and TPA+polymixin B vs. polymixin B alone). These data provide support for C-kinase involvement in regulation of TPA-induced surfactant secretion. Supported by the American Lung Association, NIH-HL-28623 and HD-11725.

Inhibition of the effects of 12-O-tetradecanoylphorbol-13-acetate on mouse epidermal glutathione peroxidase and ornithine decarboxylase activities by glutathione level-raising agents and selenium-containing compounds.

The present study was undertaken to determine the effect of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter known to inhibit superoxide dismutase (SOD) (superoxide: superoxide oxidoreductase, EC 1.15.1.1) and catalase (CAT) (H2O2: H2O2 oxidoreductase, EC 1.11.1.6) activities, on mouse epidermal glutathione (GSH) peroxidase (glutathione: H2O2 oxidoreductase, EC 1.11.1.9) activity in vivo and in vitro. TPA led to a rapid and transient increase in GSH peroxidase specific activity within 30 min followed by a decrease from 1 to 12 h. Incubation of isolated epidermal cells with GSH level-raising agents and/or selenium-containing compounds increased remarkably basal GSH peroxidase activity, and thus, abolished totally the prolonged inhibitory effects of TPA on this enzyme. The inhibitory effects of 0.2 mM cysteine (Cys) or 0.5 mM GSH and 2.5 microM Na2 SeO3 or 50 microM selenocystamine on TPA-decreased GSH peroxidase activity were additive, in relation with their additive inhibitory effects on TPA-induced ornithine decarboxylase (ODC) (L-ornithine carboxylase, EC 4.1.1.17) activity. These data support the hypothesis that the stimulators of the GSH-dependent antioxidant protective system of the epidermal cells may inhibit the oxidative challenge linked to skin tumor promotion by TPA.