Enzymatic oxidation and reduction of retinal by mouse epidermis

Evidence is presented for the ability of mouse epidermis to oxidize retinal by the action of a cytosolic enzyme with a pH optimum of about 8.5. The enzyme activity was slightly enhanced by the addition of either NAD or FAD. The reaction exhibited Michaelis-Menton kinetics with a km of 1.627 × 10^?4M for retinal. Mouse epidermal cytosol was also capable of reducing retinal. This enzyme had an optimum pH of 6.0 and the activity of the undialyzed enzyme was enhanced 5- to 7-fold by the addition of reduced NAD or NADP. Possible roles of these enzymic activities in epidermis are discussed.     

Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester

The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.     

Visualization of protein kinases in lymphocytes stimulated to proliferate with concanavalin A or inhibited with a phorbol ester

Stimulation of lymphocytes with a mitogenic lectin such as concanavalin A (ConA) results in differentiation and cell division. Among the changes which occur after stimulation are increases in phosphorylation of proteins and in protein kinase activity. We used a high-resolution, nondenaturing gel system to separate and visualize protein kinases in situ. We have clearly identified both autophosphorylating and substrate-dependent kinases. One band of cyclic AMP-dependent kinase activity was significantly enhanced in lectin-stimulated cells. In contrast, treatment of the cells with phorbol ester under conditions which depress stimulation caused a decrease in the activity of one kinase.     

Insulin secretion: Combined effects of phorbol ester and A23187

The effect of the ionophore, A23187, and/or the phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA), on insulin secretion were compared with those of glucose. Glucose induces a biphasic pattern of insulin secretion; A23187 a comparable initial spike but no second phase; and TPA a slowly progressive increase. Combined A23187 and TPA evoke a pattern similar to that induced by glucose. Forskolin enhances both phases of glucose-induced and of TPA-A23187-induced insulin secretion. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system, the calmodulin branch and the C-kinase branch, control, respectively, the initial and sustained phases of insulin secretion.     

Aldosterone secretion: effect of phorbol ester and A23187

The effects of the divalent ionophore, A23187, the phorbol ester, and/or 12-0-tetradecanoyl-phorbol-13-acetate on aldosterone secretion from adrenal glomerulosa cells were compared to those of angiotensin II (AII). AII causes a prompt and sustained increase in secretion. A23187 causes an initial increase followed by a gradual decline to values less than 25 percent of those seen with AII. TPA causes no initial increase but a slowly progressive rise in secretion rate to a less than maximal value. When TPA and A23187 act together, there is a prompt and sustained increase in aldosterone production rate similar to that seen after AII addition. The effect of TPA is dependent on the free Ca2+ concentration of the cell cytosol. These results are interpreted in terms of a model of cell activation in which two branches of the calcium messenger system operate to control respectively the initial and sustained phases of the secretory response. The first phase occurs as a consequence of amplitude modulation of the calmodulin branch of the system by a rise in [Ca2+]c, and the second phase as a consequence of the sensitivity modulation of the C-kinase branch by diacylglycerol.     

Synergistic effect of TPA and T-cell mitogens in nonmammalian vertebrates

Summary The phorbol ester, 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was used as a comitogen with the plant lectins phytohemagglutinin (PHA) and concanavalin A (ConA) in short-term cultures of whole blood from nonmammalian vertebrates. Stimulation with TPA in addition to standard mitogens resulted in a synergistic effect, consistently yielding more metaphases than cultures stimulated with either PHA, ConA, or TPA alone and is successful with blood samples as small as 0.1 ml. The increased mitotic index makes it possible to use different banding procedures for systematic studies. Also, because the amount of blood needed is so small, this procedure, unlike other published techniques, does not require the destruction of smaller animals to do chromosome studies. This work was supported in part by National Institute of Environmental Health Science Grant 5-T32-ES07015-08 to the Environmental Toxicology Center at the University of Wisconsin-Madison.     

Inhibition of respiration by a phorbol ester tumor promoter in murine cultured cells

The tumor promoter 12-O-tetradecanoyl phorbol-13-acetate is a potent inhibitor of mitochondrial respiration in both normal and methylcholanthrene-transformed C3H $\text{10T1}\text{2}$ mouse fibroblasts. This inhibition is seen at concentrations of tumor promoter in the range of 10^?8M, occurs within a few minutes after exposure of the intact cells, and is not seen with a biologically inactive analog. The effect appears to be exerted through inhibition of the function of an oligomycin-sensitive ATPase. It is possible, therefore, that alterations in mitochondrial function are associated with the process of tumor promotion.     

A review of the evidence from in vitro and in vivo studies for a role for phorbol ester tumour promoters from the Euphorbiales in the selection and clonal expansion of specific cell populations

Review of the evidence from in vitro and in vivo studies for a role for phorbol ester tumour promoters from the Euphorbiales in the selection and clonal expansion of specific cell populations. The identification of the active principle of croton oil as 12-0-tetradecanoylphorbol-13-acetate (TPA) and the advent of in vitro studies, facilitated the identification of a plethora of biochemical effects, certain of which could be correlated with biological and differentiation end points. Evidence is presented for the ability of TPA to select and expand specific cell populations in two different cell culture systems. The roles of differentiation state and second messenger systems will be discussed in relation to models of multi-step carcinogenesis.     

Activation of calcium-activated, phospholipid-dependent protein kinase (protein kinase C) by new classes of tumor promoters: teleocidin and debromoaplysiatoxin

The new potent tumor promoters teleocidin and debromoaplysiatoxin , which are structurally unrelated to phorbol esters, activate Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C). The concentrations of 12-O-tetradecanoylphorbol-13-acetate, teleocidin and debromoaplysiatoxin for half-maximum activation of protein kinase C were found to be approximately 3 ng/ml, 40 ng/ml and 400 ng/ml, respectively. These three types of tumor promoters bind to protein kinase C, and appear to exhibit their pleiotropic actions through activation of this enzyme.     

The tumor promoting phorbol diester, 12-0-tetradecanoylphorbol-13-acetate (TPA) increases glyoxalase I and decreases glyoxalase II activity in human polymorphonuclear leukocytes

Glyoxalase I and II catalyze the formation and breakdown of S-lactoylglutathione respectively. Recent studies have implicated this com-pound as a possible mediator of immune and inflammatory responses. Incubation of human polymorphonuclear leukocytes with the tumor promoter, 12-0-tetradecanoylphorbol-13-acetate has been found to affect the activities of both glyoxalase enzymes in an interrelated manner. The diester either increases the activity of glyoxalase I or decreases the activity of glyoxalase II or has both effects. It is suggested that a subsequent increase in S-lactoylglutathione might mediate some or all of the effects of the phorbol diesters.     

Ca2+-dependent stimulation of hexose transport by A23187, 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor in mouse fibroblasts

Ca2+ ionophore A23187 stimulated 2-deoxy-D-glucose (2DG) uptake in Swiss 3T3 mouse fibroblasts. Chelation of extracellular Ca2+ with ethylene-glycol-bis-(beta-aminoethylether) N,N'-tetraacetic acid (EGTA) inhibited the effect of A23187. Similarly, the stimulation of 2DG uptake by a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was prevented by EGTA, whereas the epidermal growth factor (EGF)-stimulated 2DG uptake was not affected by EGTA alone, but in the presence of both EGTA and A23187 which effectively depleted cellular Ca2+ content, EGF could no longer stimulate 2DG uptake. These results suggest that Ca2+ regulates hexose transport system in Swiss 3T3 mouse fibroblasts, the activation of which by TPA and EGF differently depends on Ca2+.     

A simultaneous protein-binding assay for adenosine 3':5'-monophosphate in biological materials.

Adenosine 3′:5′-monophosphate (cyclic AMP) and guanosine 3′:5′-monophosphate (cyclic GMP) have been determined simultaneously by combining individual protein binding assays using different isotopically labeled cyclic nucleotides. Preparations of cyclic AMP-binding protein from beef adrenal cortex and cyclic GMP-binding protein from the fat body of silkworm pupae ($\text{Bombyx mori}$) have been used for the assay. The method allows the analysis of cyclic AMP and cyclic GMP levels in crude extracts without any purification. The assay has been applied to hormone-stimulated Mouse liver and phorbol ester-treated Rat embryo cells.     

The kinetics of fibronectin synthesis and release in normal and tumor promoter-treated human lung fibroblasts

Summary The present study is a detailed kinetic analysis of the synthesis, release and multimerization of fibronectin (FN) in normal and tumor promoter-treated human lung fibroblasts. Pulse/chase and surface labeling experiments were performed to follow the fate of both newly synthesized and pre-existing cell-surface FN over time. The majority of FN (80%) left the intracellular compartment within one hour of synthesis. However, the rate of direct secretion was very low and after one hour, 70% of newly synthesized FN was still at the cell surface. This material was primarily dimeric. Dimeric and multimeric (very high molecular weight) FN was detectable at the cell surface and in the medium 4 hours after synthesis. Pulse-labeled FN multimer levels peaked at 12 hours and declined thereafter. After 24 hours, 85% of pulse-labeled FN had been shed into the medium and the labeled FN remaining at the cell surface was primarily multimeric. Surface labeling experiments confirmed that the majority of FN resides at the cell surface prior to release into the medium.One hour treatment with the phorbol ester tumor promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA), stimulated a nine-fold increase in release of preexisting, dimeric cell-surface FN (¹²⁵I-labeled). The major effect of longer term TPA treatment up to nine hours was continued depletion of dimeric cell-surface FN. Increased release of cell-surface multimeric FN was also stimulated by TPA, but to a much lesser extent. Release of newly synthesized (pulse-labeled) dimeric FN was also stimulated by TPA though much less than pre-existing FN, and TPA treatment produced a small decrease in the steady-state level of multimeric FN. Thus, preexisting cell-surface IN and newly synthesized FN differ dramatically in their susceptibility to TPA treatment.Abbreviations FN fibronectin - HLF human lung fibroblasts - PMSF phenylmethylsulfonylfluoride - TPA 12-O-tetradecanoyl phorbol-13-acetate - DMSO dimethylsulfoxide - MEM minimal essential medium - ELISA enzyme linked immunosorbent assay - PBS phosphate-buffered saline - BSA bovine serum albumin - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - DOC deoxycholate - DTT dithiotreitol     

Inhibition of histidine decarboxylase and tumour promoter-induced arachidonic acid release by lecanoric acid analogues

Lecanoric acid analogues containing benzanilide structure inhibited histidine decarboxylase and arachidonic acid release from the cell membrane phospholipids induced by a tumour promoter, 12-O-tetradecanoylphorbol-13-acetate. But they did not inhibit cellular binding of phorbol-12,13-dibutylate. Lecanoric acid analogues also inhibited prostaglandin synthetase and delayed-type hypersensitivity responses against sheep red blood cells in mice. Thus, lecanoric acid analogues antagonized several enzymic and cellular effects of the tumour promoter.     

Effects of L-carnitine on urea synthesis following acute ammonia intoxication in mice

L-Carnitine protects mice against acute ammonia intoxication. The effect of L-carnitine on in vivo incorporation of [14C] bicarbonate into urea has been investigated in mice given large doses of ammonium acetate. The hepatic content of N-acetylglutamate has been measured. Following ammonia administration the animals given L-carnitine have much higher production of urea than the unprotected mice. The marked protective effect of L-carnitine on ammonium acetate-induced hyperammonemia and on the increase in urea synthesis is not due primarily to activation of N-acetylglutamate synthetase.     

A novel protamine kinase activity in human promyelocytic leukemia cells

A novel protamine kinase activity is present in human promyelocytic leukemia cells (HL60). The enzyme is clearly distinct from cyclic AMP-dependent protein kinase and is not calcium activated. The enzyme requires Mg²⁺, is sulfhydryl dependent and is strongly inhibited by fluoride. Activity is 80% cytosolic and 20% particulate but appears to be the same enzyme in the two fractions. Enzyme activity is markedly stimulated during differentiation by retinoic acid and dimethylformanide but not by a phorbol diester. However the latter treatment does lead to a redistribution of the kinase so that it becomes predominantly particulate.     

Involvement of calcium, calmodulin and phospholipase A in the alteration of membrane dynamics and superoxide production of human neutrophils stimulated by phorbol myristate acetate

We have reported an increased fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene in the phorbol myristate acetate-stimulated plasma membrane of human neutrophils [FEBS Lett. (1982) 144, 199–203]. We now present evidence that both the increased fluorescence polarization and the production of super-oxide radicals by human neutrophils require calcium, calmodulin and phospholipase activity.