Tumor promoter 12-O-tetradecanoylphorbol 13-acetate inhibits mas/angiotensin receptor-stimulated inositol phosphate production and intracellular Ca2+ elevation in the 401L-C3 neuronal cell line

Stimulation of mas-oncogene transfected 401L-C3 cells by angiotensins leads to the production of inositol phosphates. This response shows dose dependence, and has an apparent rank order of potency angiotensin III greater than or equal to angiotensin II much greater than angiotensin I. Preincubation with 12-O-tetradecanoylphorbol 13-acetate, for 5 min, significantly diminishes both inositol phosphate and intracellular [Ca2+] responses to angiotensins, without affecting those stimulated by the endogenous bradykinin receptor. Incubation of 401L-C3 cells with either phorbol ester or angiotensins leads to elevation of intracellular pH, implying that mas/angiotensin receptor stimulation itself leads to protein kinase C activation. These results suggest the operation of a negative feedback loop specific for the mas/angiotensin receptor signalling pathway, and which may be essential in defining the final biological output response to this receptor stimulation.     

Platelet-activating factor increases inositol phosphate production and cytosolic free Ca2+ concentrations in cultured rat Kupffer cells

Platelet-activating factor (PAF) stimulates glycogenolysis in perfused livers but not in isolated hepatocytes [(1984) J. Biol. Chem. 259, 8685-8688]. PAF-induced glycogenolysis in liver is associated closely with a pronounced constriction of the hepatic vasculature [(1986) J. Biol. Chem. 261, 644-649]. These and other observations suggest that PAF stimulates glycogenolysis in liver indirectly by interactions with cells other than hepatocytes. We have evaluated effects of PAF on hepatic Kupffer cells, which regulate flow through the hepatic sinusoids. Application of PAF to [3H]inositol-labeled Kupffer cells produced dose-dependent increases in [3H]inositol phosphates with an EC50 value of 4 x 10(-10) M. Increases in inositol phosphate production in response to PAF were inhibited by a specific PAF receptor antagonist, SRI 63-675 (2 x 10(-7) M), and stimulus of protein kinase C, phorbol 12-myristate 13-acetate (1 x 10(-7) M). Measurements of cytosolic free Ca2+ concentrations ([Ca2+]i) in single Kupffer cells loaded with Fura-2 demonstrated that application of PAF (2 x 10(-9) M) resulted in significant increases in [Ca2+]i. These observations lead us to propose that interactions of PAF with Kupffer cells may result in the hemodynamic and metabolic responses to PAF in liver.     

Capsaicin inhibits platelet-activating factor-induced cytosolic Ca2+ rise and superoxide production

Platelet-activating factor (PAF) is an important participant in the inflammatory process. We studied the regulation of PAF activity by capsaicin in human promyelocytic leukemia HL-60 cells. Capsaicin inhibited PAF-induced superoxide production in a concentration-dependent manner. In addition to PAF, the fMLP- and extracellular ATP-induced superoxide productions were inhibited by capsaicin, whereas PMA-induced superoxide production was not affected. In the PAF-stimulated cytosolic Ca2+ increase, capsaicin inhibited in particular the sustained portion of the raised Ca2+ level without attenuation of the peak height. In the absence of extracellular Ca2+, the PAF-induced Ca2+ elevation was not inhibited by capsaicin because capsaicin only inhibited the Ca2+ influx from the extracellular space. In addition, capsaicin did not affect PAF-induced inositol 1,4,5-trisphosphate production, suggesting that phospholipase C activation by PAF is not affected by capsaicin. Store-operated Ca2+ entry (SOCE) induced by thapsigargin was inhibited by capsaicin in a concentration-dependent manner. This capsaicin effect was also observed on thapsigargin-induced Ba2+ and Mn2+ influx. Furthermore, capsaicin's inhibitory effect on the thapsigargin-induced Ca2+ rise overlapped with that of SK&F96365, an inhibitor of SOCE. Both capsaicin and SK&F96365 also inhibited PAF-induced cytosolic superoxide generation in HL-60 cells differentiated by all-trans-retinoic acid. Our data suggest that capsaicin exerts its anti-inflammatory effect by inhibiting SOCE elicited via PLC activation, which occurs upon PAF activation and results in the subsequent superoxide production.     

Atorvastatin inhibits angiotensin II-induced T-type Ca2+ channel expression in endothelial cells

Ca2+ channels are involved in the regulation of vascular functions. Angiotensin II is implicated in the development of atherosclerosis and vascular remodeling. In this study, we demonstrated that angiotensin II preferentially increased the expression of alpha1G, a T-type Ca2+ channel subunit, via AT1 receptors in endothelial cells. Angiotensin II-induced expression of alpha1G was inhibited by pretreatment with atorvastatin and the MEK1/2 inhibitor, PD98059. The effect of atorvastatin was reversed by mevalonate and farnesyl pyrophosphate which implicates the activation of the small GTP-binding protein, Ras. Our data indicate that angiotensin II induces alpha1G expression in endothelial cells via AT1 receptors, Ras and MEK. Angiotensin II-induced migration of endothelial cells in a wound healing model was inhibited by incubation with mibefradil, a T-type Ca2+ channel blocker. Our data indicate that angiotensin II induces T-type Ca2+ channels in endothelial cells, which may play a role in the development of vascular disorders.     

Epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate stimulate lactate production and the pentose phosphate pathway in freshly isolated rat hepatocytes

Epidermal growth factor (EGF) and tetradecanoylphorbol acetate (TPA) rapidly stimulated the production of lactate by hepatocytes isolated from fed rats. Our results indicate that enzymes of both glycolysis and the pentose phosphate pathway are involved in these actions. EGF stimulated CO2 release from the 1-position of glucose, and caused a small but significant increase in pyruvate kinase activity. In addition, EGF caused a rise in fructose 1,6-bisphosphate and fructose 2,6-bisphosphate concentrations, indicating activation of phosphofructokinase. TPA did not alter the concentrations of these sugar phosphates, but did cause an increased lactate production and CO2 production from the 1-position of glucose similar to EGF. Furthermore, the EGF stimulation of lactate formation was independent of the presence of medium Ca2+. Phenylephrine stimulation of this process, in parallel incubations, was entirely dependent upon the presence of Ca2+ in the medium. We conclude that EGF stimulates glycolysis and the pentose phosphate pathway in isolated hepatocytes from fed rats. The duplication of these actions by TPA suggests that protein kinase C is a mediator of EGF action in hepatocytes.     

Ca2+ channel ligand sensitive responses to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in vascular smooth muscle

The action of a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), on isolated rat aortic and tail artery strips has been characterized. TPA (10(-9)-10(-7) M) produced a graded contraction developing maximum tension over 30-40 min. The contraction was irreversible and was not relaxed by prolonged washing with physiologic saline. Relaxation occurred upon washing with Ca2+-free saline but readdition of Ca2+ restored response. TPA was without significant effect in rat tail arteries in physiologic saline but produced responses in saline containing elevated K+ (15 mM). The protein kinase C inhibitor, CP-46,665-1 (4-aminomethyl-1-[2,3-(di-n-decyloxy)n-propyl]-4-phenylpiperidine dihydrochloride) (5 X 10(-5) M), blocked the response to TPA but was without effect on responses to Bay K 8644 (2,6-dimethyl-3-carbomethoxy-5-nitro-4-(2-trifluoromethylphenyl) 1,4-dihydropyridine), KCl, phenylephrine, and B-HT 920 (6-allyl-2-amino-5,6,7,8-tetrahydro-4H-thiazolo[4,5-d]azepin dihydrochloride). The calcium channel antagonist nifedipine and its analogue, 2,6-dimethyl-3,5-dicarbomethoxy-4-(3-cyanophenyl)-1,4-dihydr opyridine, inhibited TPA responses with IC50 values of 9.28 X 10(-9) and 1.96 X 10(-7) M, respectively. Responses to Bay K 8644 in rat aorta were maximum in the presence of elevated KCl (10 mM), but TPA at concentrations of 10(-9) and 3 X 10(-9) M potentiated responses to Bay K 8644 in physiologic saline to levels approximating those in elevated K+ saline. TPA similarly potentiated responses to Ca2+ in Ca2+-free solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Caffeine inhibits the agonist-evoked cytosolic Ca2+ signal in mouse pancreatic acinar cells by blocking inositol trisphosphate production.

The inhibitory effects of caffeine on receptor-activated cytosolic Ca2+ signal generation in isolated mouse pancreatic acinar cells were investigated. Using the ability of caffeine to quench Indo-1 fluorescence we measured simultaneously the free intracellular Ca2+ concentration ([Ca2+]i) and the intracellular caffeine concentration ([caffeine]i). We also measured inositol 1,4,5-trisphosphate (InsP3) production with a radioreceptor assay. When caffeine was added to the extracellular solution during a sustained receptor-activated increase in [Ca2+]i, [caffeine]i rose to its steady level within a few seconds. This was accompanied by a decrease of [Ca2+]i, which started only after [caffeine]i had reached an apparent threshold concentration (about 2 mM in the case of 0.5 microM acetylcholine (ACh) stimulation). Above this [caffeine]i level there was a linear relationship between [caffeine]i and [Ca2+]i. Throughout the caffeine exposure [Ca2+]i remained at a steady low level. Following removal of caffeine from the bath, [caffeine]i decreased to zero within seconds. There was no significant increase in [Ca2+]i until [caffeine]i had been reduced to the threshold level (about 2 mM at 0.5 microM ACh). Caffeine inhibited Ca2+ signals evoked by ACh, cholecystokinin, and ATP and also inhibited signals generated in the absence of external Ca2+. Caffeine application had the same effect as removal of agonist allowing recovery from apparent desensitization. Caffeine inhibited the agonist-evoked production of InsP3 in a dose-dependent manner. Our results demonstrate the acute and reversible dose-dependent inhibition of agonist-evoked cytosolic Ca2+ signal generation due to rapid intracellular caffeine accumulation and washout. The inhibition can be explained by the reduction of agonist-evoked InsP3 production.     

Nicardipine-sensitive enhancement of high K+-evoked dopamine release in PC12 cells pretreated with 12-O-tetradecanoylphorbol 13-acetate

The effects of the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) on stimulus-evoked dopamine release were studied in PC12 cells. Pretreatment of the cells with TPA resulted in an enhancement of dopamine release which could be further stimulated by high concentrations of K⁺, A23187, but not with carbamylcholine. TPA-dependent, high-K⁺-evoked enhancement of dopamine release was studied in detail: a maximum release was observed (169% of control) in response to 50 mM KCl upon treatment with 10⁻⁷ M TPA for 5 min at 37°C. This enhancement of dopamine release was associated with the concomitant reduction of the concentration rise of intracellular Ca²⁺ ([Ca²⁺]_i) induced by a high concentration of K⁺ monitored by a fluorescent indicator, fura2. Thus, these data provide an example for alteration in the efficiency of stimulus-secretion coupling as pointed out in our previous paper. Moreover, we have shown that nicardipine, CdCl₂, and CoCl₂ inhibit high-K⁺-evoked dopamine release more effectively in TPA treated cells than that of untreated cells, and that the TPA-dependent, high-K⁺-evoked dopamine release observed in TPA treated cells is completely abolished by the presence of nicardipine, Cd²⁺ or Co²⁺, but is only partially inhibited in the presence of verapamil. These relevant findings suggest the possible involvement of protein kinase C in regulating the efficiency of a high-K⁺-evoked dopamine release through the modification of nicardipine-sensitive Ca²⁺ channels.     

Stimulation of Na+/phosphate cotransport in LLC-PK1 cells by 12-O-tetradecanoylphorbol 13-acetate (TPA)

We have tested for the effect of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) on Na+/phosphate cotransport in an established epithelial cell line of renal origin (LLC-PK1). Incubation of LLC-PK1 cells with TPA produced an increase in Na+/phosphate (Pi) cotransport. The maximal response was reached at a TPA concentration of 10 ng/ml. Other phorbol esters which have no potency or a smaller one to activate protein kinase C had no effect on Na+/Pi cotransport. Incubation of LLC-PK1 cells with 10 ng/ml TPA for 8 h led to a 300% increase in Na+/Pi cotransport; in the presence of cycloheximide the increase amounted only to a 100% and was reached within 2 h. Kinetic analysis of Na+/Pi cotransport indicated an increase in the apparent Vmax without an effect on the apparent Km. The increased Pi transport was retained in isolated apical vesicles. Na+-dependent alanine transport into LLC-PK1 monolayers was affected by TPA administration in a similar manner. TPA had under the chosen experimental conditions no effect on [3H]thymidine incorporation into DNA excluding a general proliferative effect. We conclude that TPA via activation of protein kinase C regulates the number of operating transport systems. As also other Na+-coupled transport systems are influenced, the TPA effect appears to be related to the expression of a general 'adaptive' alteration of membrane transport in LLC-PK1 cells.     

Pertussis toxin abolishes angiotensin II-induced phosphoinositide hydrolysis and prostaglandin synthesis in rat renal mesangial cells.

Incubation of rat renal mesangial cells with angiotensin II (0.1 microM) resulted in transient breakdown of phosphatidylinositol 4,5-bisphosphate, rapid generation of diacylglycerol and phosphatidic acid, increased 45Ca2+ influx, increased intracellular [Ca2+] as measured by quin 2, and increased prostaglandin E2 synthesis. All of these processes were markedly inhibited time- and dose-dependently by prior exposure of cells to pertussis toxin. In contrast, the effects of the ionophore A23187 on 45Ca2+ influx and prostaglandin E2 synthesis were not altered by the exposure of the cells to pertussis toxin. The action of the toxin was not associated with alterations in cellular concentrations of cyclic AMP. Incubation of membrane fraction of mesangial cells with pertussis toxin resulted in ADP-ribosylation of Mr-42,000 protein. From all these results, it is likely that a G protein is involved in receptor-mediated signal transduction in renal mesangial cells.     

Early rise of cytosolic Ca2+ induced by NGF in PC12 and chromaffin cells

A rise of cytosolic Ca2+ is induced by NGF in rat pheochromocytoma PC12 and bovine chromaffin cells investigated (both in suspension and while attached to polyornithine-coated glass slides) by fluorescence techniques (with quin-2 and fura-2). The effect of NGF on [Ca2+]i is delayed (30-40 s of lag phase), slow (t1/2 = 40 s), relatively small (+50-75%) and persistent (over 10 min). It is due to Ca2+ influx (requires extracellular Ca2+ greater than 10 microM) through a pathway different from the voltage-gated Ca2+ channel, possibly accompanied by intracellular Ca2+ redistribution, and might play a messenger role in NGF action.     

Angiotensin II-induced inositol phosphate production in isolated rat zona glomerulosa and fasciculata/reticularis cells

Angiotensin II (2.5 to 250nM) induced, within 60 sec, a significant increase in [3H]inositol-labeled inositol phosphate, inositol bisphosphate, and inositol trisphosphate in rat zona glomerulosa cells. Neither ACTH (3nM) nor K+ (8.4mM) had any effect, although aldosterone and corticosterone were significantly stimulated by all three agonists (after 30 min incubation). A similar significant dose-dependent increase in the inositol phosphates was observed with angiotensin II in zona fasciculata/reticularis cells after 30 min, but without any effect on corticosterone. In contrast ACTH significantly increased corticosterone with only a small although highly significant increase in inositol trisphosphate and inositol bisphosphate at 0.03nM ACTH. However at the higher dose (3.0nM) only inositol bisphosphate was significantly increased. These results indicate the presence on both zona glomerulosa and zona fasciculata/reticularis cells of AII receptors, which were linked to the formation of the secondary messenger, but only in the zona glomerulosa cells are associated with steroidogenesis.     

Effects of sarpogrelate on serotonin-induced increases in cytosolic Ca2+ in cultured rat mesangial cells

The effects of the new 5-HT_2A receptor antagonist sarpogrelate on the cellular action of serotonin were examined in cultured rat mesangial cells by measuring cytosolic free calcium concentration ([Ca²⁺]i). Sarpogrelate inhibited serotonin-induced increases in [Ca²⁺]i in a concentration-dependent manner. M1, a major metabolite of sarpogrelate, also exhibited an inhibitory effect exceeding that of sarpogrelate. The inhibitory effects of sarpogrelate and M1 were abolished by washing out these compounds. In contrast, the increase in [Ca²⁺]i induced by angiotensin II or arginine vasopressin was not affected by pretreatment of the cells with sarpogrelate or M1. These results suggest that sarpogrelate and its major metabolite (M1) act as reversible and specific 5-HT_2A receptor antagonists against the contractile action of platelet-derived serotonin in mesangial cells.     

A mutagenesis-enhancing activity of 12-O-tetradecanoylphorbol 13-acetate detected in Chinese hamster ovary cells

Tumour-promoting agents may bring about the completion of multi-step carcinogenesis by acting as enhancers of mutagenesis, recombinogens or clastogens. We report here that the classical mouse skin tumour promoter TPA, although non-mutagenic per se, can enhance the induction of OuaR CHO-K1 cell mutants by MNNG approximately 2-fold. This observation was made at a concentration approaching the compounds aqueous solubility limit which was non-cytotoxic. Mutagenesis enhancement was dependent on TPA being present throughout mutation expression and mutant selection. It was not accompanied by any modification of cell sensitivity to mutagen killing. In the same treatment protocol TPA did not enhance either EMS- or UV-induced mutagenesis. TPA exposure over 2 rounds of cell replication failed to produce an increase in the frequency of SCE in control or mutagen-treated CHO-K1 cultures. Likewise TPA exposure over 1 round of cell replication failed to produce an increase in the frequency of chromosomal aberrations. Apparently TPA is not a recombinogen or clastogen but in the right exposure regime is capable of acting to enhance mutagenesis by certain genotoxic agents, an action which may contribute to tumour promotion.     

Regulation of protein phosphorylation in pancreatic acini. Distinct effects of Ca2+ ionophore A23187 and 12-O-tetradecanoylphorbol 13-acetate.

Regulation of protein phosphorylation in isolated pancreatic acini by the intracellular messengers Ca2+ and diacylglycerol was studied by using the Ca2+ ionophore A23187 and the tumour-promoting phorbol ester 12-O-tetradecanoylphorbol 13-acetate. As assessed by two-dimensional polyacrylamide-gel electrophoresis, the phorbol ester (1 microM) and Ca2+ ionophore (2 microM) altered the phosphorylation of distinct sets of proteins between Mr 83,000 and 23,000 in mouse and guinea-pig acini. The phorbol ester increased the phosphorylation of four proteins, whereas the ionophore increased the phosphorylation of two proteins and, in mouse acini, decreased the phosphorylation of one other protein. In addition, the phorbol ester and ionophore each caused the dephosphorylation of two proteins, of Mr 20,000 and 20,500. Administered together, these agents reproduced the changes in phosphorylation induced by the cholinergic agonist carbamoylcholine. The effects of the phorbol ester and ionophore on acinar amylase release were also studied. In mouse pancreatic acini, a maximally effective concentration of phorbol ester (1 microM) produced a secretory response that was only 28% of that produced by a maximally effective concentration of carbamoylcholine, whereas the ionophore (0.3 microM) stimulated amylase release to two-thirds of the maximal response to carbamoylcholine. In contrast, in guinea-pig acini, the phorbol ester and carbamoylcholine evoked similar maximal secretory responses, whereas the maximal secretory response to the ionophore was only 35% of that to carbamoylcholine. Combination of phorbol ester and ionophore resulted in a modest synergistic effect on amylase release in both species. It is concluded that cholinergic agonists act via both diacylglycerol and Ca2+ to regulate pancreatic protein phosphorylation, but that synergism between these intracellular messengers is of limited importance in stimulating enzyme secretion.     

Neuropeptide Y mobilizes intracellular Ca2+ and increases inositol phosphate production in human erythroleukemia cells

The intracellular concentration of free Ca2+ was monitored by measuring the fluorescence of fura-2 loaded Human Erythroleukemia Cells. Neuropeptide Y (NPY) increased intracellular Ca2+ in a dose-dependent manner and the 50% effective concentration was 2 nM. Chelation of extracellular Ca2+ by EGTA did not reduce the NPY-mediated increase in cytoplasmic Ca2+, indicating that the increase in fluorescence was due to the release of intracellular Ca2+. A second dose of NPY, after intracellular Ca2+ had returned to basal levels, failed to elicit a response, indicating that the NPY receptor had undergone desensitization. In similar experiments, NPY increased the formation of inositol phosphates, suggesting that the mobilization of Ca2+ from intracellular stores in HEL cells was secondary to the generation of inositol phosphates and stimulation of phospholipase C.     

Effect of phorbol 12-myristate 13-acetate on Ca2+-ATPase activity in rat liver nuclei

The effect of phorbol 12-myristate 13-acetate (PMA) on Ca²⁺-ATPase activity in rat liver nuclei was investigated. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺-Mg²⁺)-ATPase activity. The nuclear Ca²⁺-ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)-increased Ca²⁺-ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca²⁺-ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca²⁺-ATPase independent of protein kinase. © 1994 Wiley-Liss, Inc.     

Proteome analysis of antiproliferative mechanism of 12-O-tetradecanoylphorbol 13-acetate on cultured nasopharyngeal carcinoma CNE2 cells

12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is a plant derivative with multiple function as tumor promoter, differentiation revulsant or leukemia therapy drug. The molecular mechanism of its function is perplexing. Many studies have focused on the mechanism of TPA stimulation in tumor promotion of mouse models or terminal differentiation of leukemia cells, but the effect of TPA on nasopharyngeal carcinoma (NPC) remains unclear, while TPA was considered to be associated with NPC development. In the present study, we employed proteomics techniques to study protein changes of a poorly differentiated squamous carcinoma cell line-CNE2 of human NPCs cells induced by TPA. Six significantly and reproducibly changed proteins were identified and their functional implications were discussed in some details.     

Na+/H+ exchange in Ehrlich ascites tumor cells. Regulation by extracellular ATP and 12-O-tetradecanoylphorbol 13-acetate

The effects of extracellular ATP and/or the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on the intracellular pH of Ehrlich ascites tumor cells were measured using both distribution of [14C]5,5-dimethyloxazolidine-2,4-dione, and the fluorescent indicator 5(6)-carboxyfluorescein. Micromolar concentrations of extracellular ATP induce a biphasic change in the intracellular pH characterized by a rapid acidification of 0.04 pH units followed by an alkalinization of 0.11 pH units. Concurrently with the alkalinization, an increase in the total cellular [Na+] from 37.5 to 45.0 mM is observed. The pH change is half-maximally activated by 0.5-2.5 microM extracellular ATP. The intracellular alkalinization, but not the initial acidification, phase requires extracellular Na+, with half-maximal alkalinization in the presence of 24-32 mM Na+, and is inhibited by amiloride. Exposure of Ehrlich ascites tumor cells to TPA alone produces a slight alkalinization of approximately 0.04 pH units. Conversely, preincubation of the cells with TPA partially inhibits the ATP-induced changes in intracellular pH. Under identical conditions TPA also inhibits the ATP-induced increase in the cytosolic [Ca2+]. The half-maximal dose for both effects is produced by 3-10 nM TPA. These data indicate that extracellular ATP triggers the activation of Na+/H+ exchange. Furthermore, activation of protein kinase C mediates at least part of the Na+/H+ exchange, although a second mechanism may also exist.