Regulated expression of a dominant negative protein kinase C epsilon mutant inhibits the proliferation of U-373MG human astrocytoma cells.

The tight regulation of protein kinase C (PKC) activity is crucial for maintaining normal cell proliferation. Excessive PKC activity leads to uncontrolled growth and malignant transformation. It has been reported that the activity of PKC is higher in astroglial cell lines than in normal astrocytes. Previously, we demonstrated that PKC epsilon is overexpressed in astroglial cell lines and in samples from primary high-grade astroglial brain tumors. Because there are no PKC epsilon isozyme-specific inhibitors, we chose a genetic approach to confirm that PKC epsilon is a potential target for inhibiting astroglial cell proliferation. We regulated the expression of a dominant negative PKC epsilon mutant (PKC epsilon 1-401 encoding amino acid 1-401) in U-373MG human astrocytoma cells using a tetracycline-regulated expression vector and established stable clones. Induction of expression of the dominant negative PKC epsilon mutant by the addition of doxycycline, a tetracycline derivative, completely blocked proliferation of U-373MG cells in proliferation and clonogenic assays. Although the induction of the dominant negative PKC epsilon mutant did not markedly affect mitogen-induced tyrosine phosphorylation of the mitogen-activated protein (MAP) kinases, it inhibited the induction of c-Fos protein expression by substance P (SP) and fetal bovine serum (FBS). These results clearly show that the expression of dominant negative PKC epsilon leads to the inhibition of U-373MG cellular proliferation demonstrating that this isozyme may be a potential therapeutic target for astroglial brain tumors.

Bioactive 6-nitronorepinephrine identified in mammalian brain.

Norepinephrine (NE) (von Euler, U. S. (1972) in Catecholamines (Blaschko, H., and Muscholl, E., eds.) pp. 186-230, Springer-Verlag, Berlin) and nitric oxide (NO.) function as neurotransmitters in the nervous system. We have shown that NE levels in the rat hypothalamic paraventricular nucleus (Shintani, F., Kato, R., Kinoshita, N., Kanba, S., Asai, M., and Nakaki, T.(1995) Proceedings of the Satellite Symposium, 4th IBRO World Congress on Neuroscience, Otsu, 1995) diminish in the presence of NO.. This observation prompted us to explore the possibility of an in vivo interaction between NE and NO. or NO.-related molecules. In fact, nitration of NE has been shown to occur in vitro (d'Ischia, M., and Costantini, C. (1995) Bioorg. Med. Chem. 3, 923-927). We now report the identification of 6-nitronorepinephrine in the mammalian brain. Amounts of 6-nitronorepinephrine in the rat brain were attenuated by intraperitoneal administration of an inhibitor of nitric oxide synthase, NG-nitro-L-arginine methyl ester (L-NAME). This was reversed by coadministration of L-arginine, suggesting that nitric oxide synthase participated in the formation of 6-nitronorepinephrine. Moreover, we found that 6-nitronorepinephrine inhibits the activity of catechol O-methyltransferase, as well as NE transport into rat synaptosomes. A rat brain microdialysis experiment showed that perfusion of 6-nitronorepinephrine into the rat paraventricular nucleus significantly elevated NE while decreasing 3-methoxy-4-hydroxyphenylglycol and that L-NAME administered intraperitoneally decreased NE and increased 3-methoxy-4-hydroxyphenylglycol. These observations suggest that 6-nitronorepinephrine generated in nuclei containing both adrenergic and nitrergic neurons inhibits NE inactivation. We propose that 6-nitronorepinephrine is a potential signal molecule linking the actions of NE and NO..

Attenuation of agonist-induced desensitization of the rat substance P receptor by microinjection of inositol pentakis-and hexakisphosphates in Xenopus laevis oocytes.

Recently, inositol hexakisphosphate (phytic acid) was shown to bind to photoreceptor arrestin and block its interaction with rhodopsin. Such an interaction might predict that inositol polyphosphates could alter G protein-coupled receptor desensitization. To investigate the possible roles of higher inositol polyphosphates on receptor desensitization, we have expressed the rat substance P receptor in Xenopus laevis oocytes. The functional expression of substance P receptor was monitored by voltage-clamp recording of substance P-induced Ca(2+)-dependent Cl- currents. When control oocytes were stimulated with substance P (30 nM), after 10 min of washing the second responses to substance P were approximately 15% of the first responses. Cytosolic injection of inositol pentakisphosphate (100 microM) or inositol hexakisphosphate (100 microM) inhibited the reduction of the second substance P-induced current responses, maintaining the second responses to 57-58% of the initial responses. The protective effects of inositol pentakisphosphate and inositol hexakisphosphate against agonist-induced desensitization were concentration and time dependent and structurally specific, in that inositol hexasulfate and inositol tris- and tetrakisphosphate isomers were inactive. Microinjection of inositol hexakisphosphate did not (a) change the potency of substance P or the sensitivity of the expressed substance P receptor to substance P, (b) inhibit 12-O-tetradecanoylphorbol-13-acetate-induced loss of substance P-induced current responses, or (c) alter the currents elicited by microinjection of inositol-1,4,5-trisphosphate. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate have specific inhibitory effects on the agonist-induced loss of responsiveness of the rat substance P receptor. Moreover, these protective effects of inositol hexakisphosphate against desensitization were also observed with the endogenous lysophosphatidic acid/phosphatidic acid receptor, indicating that this mechanism is not specific to ectopic receptors. These results suggest that inositol pentakisphosphate and inositol hexakisphosphate may be novel pharmacological tools for the study of agonist-induced desensitization.

Attenuation of agonist-induced desensitization of the rat substance P receptor by progressive truncation of the C-terminus.

We have investigated the C-terminal tail of the rat substance P receptor (SPR) as a domain essential for agonist-induced desensitization. Four progressively shorter mutants, using premature termination in the C-terminus, were constructed and compared with the unaltered SPR using ectopic expression of wild-type and mutant receptors in Xenopus oocytes. These mutants were designated D16, D47, D70 and D96 with 16, 47, 70 and 96 amino acids residues deleted from the tail, respectively. Wild type SPR, D16 and D47 exhibited normal current responses when challenged with substance P, but D70 and D96 had reduced maximal current responses (70% and 5% of wild type SPR, respectively). D70, however, exhibited substantial resistance to substance P-induced desensitization in that 55%, versus 8% for wild type SPR, of the peak current of the first response was preserved on second challenge with substance P. Therefore, a domain from residues 338 to 360 of the rat SPR, though not necessary for the functional activity of the receptor, plays an essential role in agonist-induced desensitization.

Increase in inositol tris-, pentakis- and hexakisphosphates by high K+ stimulation in cultured rat cerebellar granule cells.

Effects of high K+ stimulation on inositol polyphosphate accumulations and intracellular free calcium concentration ([Ca2+]i) were investigated in cultured rat cerebellar granule cells. When the [3H]inositol-labelled cells were stimulated with KCl, concentration-dependent accumulations of [3H]Ins(1,4,5)P3, [3H]InsP5 and [3H]InsP6 were observed. Nifedipine (3 microM), a calcium channel antagonist, inhibited the high (KCl, 90 mM) K(+)-induced accumulations of these inositol polyphosphates. In Ca(2+)-depleted and EGTA-containing (0.1 mM) medium, the high K(+)-induced inositol polyphosphate accumulation were completely inhibited. Similar results were also observed in the case of [Ca2+]i. These results suggest that the rise in [Ca2+]i caused by activation of voltage-dependent calcium channels plays an important roles in the high K(+)-induced accumulation of [3H]Ins(1,4,5)P3, [3H]InsP5 and [3H]InsP6 in cultured rat cerebellar granule cells.

Characteristics of inositol polyphosphate metabolism in cultured adrenal chromaffin cells.

1. Nicotine, high K+ and maitotoxin caused the inositol polyphosphate accumulation concomitant with 45Ca2+ uptake. 2. Angiotensin II (Ang II) and ATP induced the inositol polyphosphate accumulation without 45Ca2+ uptake. 3. Nifedipine-treatment and Ca(2+)-deprivation inhibited the high K(+)-induced inositol polyphosphate accumulation but failed to inhibit the Ang II-induced inositol polyphosphate accumulation. 4. 12-O-tetradecanoylphorbol-13-acetate inhibited the Ang II-induced inositol polyphosphate accumulation but failed to inhibit the high K(+)-induced one. 5. These results suggest that the formation of inositol polyphosphates may be regulated by two mechanisms, i.e. Ca2+ uptake-dependent mechanisms represented by high K+, and Ca2+ uptake-independent mechanisms represented by Ang II.

Stimulus-induced accumulation of inositol tetrakis-, pentakis-, and hexakisphosphates in N1E-115 neuroblastoma cells.

When [3H]inositol-prelabelled N1E-115 cells were stimulated with carbamylcholine (CCh) (100 microM), high K+ (60 mM), and prostaglandin E1 (PGE1) (10 microM), a transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached its maximum level at 15 s and had declined to the basal level at 2 min. CCh, high K+, and PGE1 also caused accumulations of [3H]inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], [3H]inositol 1,3,4,6-tetrakisphosphate [Ins(1,3,4,6)P4], and [3H]inositol hexakisphosphate (InsP6). Muscarine and CCh induced accumulations of [3H]Ins(1,4,5)P3, [3H]-Ins(1,3,4,6)P4, [3H]InsP5, and [3H]InsP6 with a similar potency and exerted these maximal effects at 100 microM, whereas nicotine failed to do so at 1 mM. With a slower time course, CCh, high K+, and PGE1 caused accumulations of [3H]-inositol 1,3,4-trisphosphate [Ins(1,3,4)P3] and [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4]. In an N1E-115 cell homogenate, [3H]Ins(1,4,5)P3, [3H]Ins(1,3,4,5)P4, and [3H]Ins(1,3,4)P3 were converted to [3H]InsP5 through [3H]-Ins(1,3,4,6)P4. The above results indicate that Ins(1,3,4,6)P4, InsP5, and InsP6 are rapidly formed by several kinds of stimulants in N1E-115 cells.

Two possibly distinct prostaglandin E1 receptors in N1E-115 clone: one mediating inositol trisphosphate formation, cyclic GMP formation, and intracellular calcium mobilization and the other mediating cyclic AMP formation.

Prostaglandin E1 (PGE1)-mediated transmembrane signal control systems were investigated in intact murine neuroblastoma cells (clone N1E-115). PGE1 increased intracellular levels of total inositol phosphates (IP), cyclic GMP, cyclic AMP, and calcium ([Ca2+]i). PGE1 transiently increased inositol 1,4,5-trisphosphate formation, peaking at 20 s. There was more than a 10-fold difference between the ED50 for PGE1 at cyclic AMP formation (70 nM) and its ED50 values at IP accumulation (1 microM), cyclic GMP formation (2 microM), and [Ca2+]i increase (5 microM). PGE1-mediated IP accumulation, cyclic GMP formation, and [Ca2+]i increase depended on both the concentration of PGE1 and extracellular calcium ions. PGE1 had more potent intrinsic activity in cyclic AMP formation, IP accumulation, and cyclic GMP formation than did PGE2, PGF2 alpha, or PGD2. A protein kinase C activator, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, had opposite effects on PGE1-mediated IP release and cyclic GMP formation (inhibitory) and cyclic AMP formation (stimulatory). These data suggest that there may be subtypes of the PGE1 receptor in this clone: a high-affinity receptor mediating cyclic AMP formation, and a low-affinity receptor mediating IP accumulation, cyclic GMP formation, and intracellular calcium mobilization.

Vascular smooth muscle contraction induced by Na+ channel activators, veratridine and batrachotoxin.

The effects of the sodium channel activators veratridine and batrachotoxin on isolated rat aorta were investigated. Veratridine caused gradual contraction, independent of the presence of endothelium, with an EC50 of 35 microM. Batrachotoxin (1 microM) also induced contraction. Both effects were completely inhibited by the sodium channel blocker tetrodotoxin (1 microM). The veratridine (60 microM)-induced contraction was inhibited by nifedipine (0.1 microM). In the absence of extracellular Ca2+, veratridine (60 microM) did not cause contraction. Sodium nitroprusside (80 nM), acetylcholine (10 microM) and isoproterenol (1 microM) caused relaxation of rings precontracted with veratridine (60 microM). An inhibitor of endothelium-derived relaxing factor (EDRF) synthase, N omega-nitro-L-arginine methyl ester (L-NAME) (0.65 mM), enhanced the veratridine-induced contraction in rings with an intact endothelium, which suggests that EDRF was being released during the veratridine-induced contraction. These results show that the activation of sodium channels on smooth muscle cells induces a contraction that is probably mediated by Ca2+ influx through voltage-dependent Ca2+ channels.

Formation of inositol polyphosphates in cultured adrenal chromaffin cells.

Formation of inositol polyphosphates has been characterized in cultured bovine adrenal chromaffin cells in terms of calcium dependency and isomers of inositol polyphosphates. There are two distinct pathways of generation of InsP3. Stimulants such as high K+ induce InsP3 accumulation by a calcium uptake-dependent mechanism. Stimulants such as Ang II induce InsP3 accumulation by a calcium uptake-independent mechanism. Both mechanisms are involved in nicotinic stimulation. These results suggest that calcium entry as well as receptor-mediated mechanisms play a significant role in phosphoinositides hydrolysis through phospholipase C in adrenal chromaffin cells. Nicotinic receptor stimulation induces a rapid and transient increase in Ins(1,4,5)P3 accumulation followed by a slower accumulation of Ins(1,3,4)P3. Moreover, nicotine induces a large and rapid increase in Ins(1,3,4,5,6)P5 accumulation with an extent and time course similar to Ins(1,4,5)P3, which peaks at 15 sec after stimulation. Nicotine also induced Ins(1,3,4,5)P4 and InsP6 accumulation with a slower time course and a lesser magnitude than Ins(1,3,4,5,6)P5. These results indicate that adrenal chromaffin cells possess fine regulation of inositol polyphosphates metabolism and that inositol polyphosphates are involved with the control of cellular function in these cells.

Stimulus-responsive and rapid formation of inositol pentakisphosphate in cultured adrenal chromaffin cells.

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with high K+ (56 mM) and nicotine (10 microM), a large and transient increase in [3H]inositol 1,3,4,5,6-pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s and then declined to the basal level at 2 min. The time course of accumulation of InsP5 was parallel to that of [3H]inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). Angiotensin II (Ang II) (10 microM) rapidly accumulated InsP5, but the level was sustained for 2 min. With a slower time course and a lesser amount than InsP5, high K+, nicotine, and Ang II caused an accumulation of [3H]inositol 1,3,4,5-tetrakisphosphate and [3H]inositol hexakisphosphate. Veratridine (100 microM), maitotoxin (10 ng/ml), ATP (30 microM), platelet-derived growth factor (10 ng/ml), and endothelin (10 ng/ml) also induced the InsP5 accumulation. High K+, nicotine, veratridine, and maitotoxin induced an increase in 45Ca2+ uptake, whereas Ang II, ATP, platelet-derived growth factor, and endothelin did not cause 45Ca2+ uptake. Nifedipine, a calcium channel antagonist, inhibited the high K(+)-induced InsP5 accumulation but failed to affect the Ang II-induced InsP5 accumulation. In an EGTA-containing and Ca2(+)-depleted medium, the high K(+)-induced InsP5 accumulation was completely inhibited, whereas the InsP5 accumulation induced by Ang II was not significantly inhibited. 12-O-tetradecanoylphorbol-13-acetate inhibited partially the Ang II-induced InsP5 accumulation but failed to inhibit the high K(+)-induced accumulation. In those experiments, the changes of InsP5 accumulation were closely correlated to those of Ins(1,4,5)P3. In the chromaffin cell homogenate, [3H] Ins(1,4,5)P3 was converted eventually to [3H]InsP5 through [3H]inositol 1,3,4,6-tetrakisphosphate. Taken together, the above results suggest that InsP5 is rapidly formed by a variety of stimulants and that the formation of InsP5 may occur through two mechanisms, i.e. Ca2+ uptake-dependent and Ca2+ uptake-independent ones in cultured adrenal chromaffin cells.

Rapid increase in inositol pentakisphosphate accumulation by nicotine in cultured adrenal chromaffin cells.

When [3H]inositol-prelabeled cultured bovine adrenal chromaffin cells were stimulated with nicotine (10 microM), a large and transient increase in [3H]inositol pentakisphosphate (InsP5) accumulation was observed. The accumulation reached the maximum level at 15 s, then declined to the basal level at 2 min. Nicotine also induced [3H]inositol tetrakisphosphate (InsP4) and [3H]inositol hexakisphosphate (InsP6) accumulation with a slower time course and a lesser magnitude than [3H]InsP5. The peaks of [3H]InsP4, [3H]InsP5 and [3H]InsP6 coincided with those of 32P radioactivity, when cells were doubly labeled with [3H]inositol and inorganic 32P. These results suggest that inositol pentakisphosphate is rapidly increased by nicotine, a cholinergic agonist, in cultured adrenal chromaffin cells.

[Signal transduction and calcium homeostasis in stimulus-secretion coupling].

Cultured adrenal chromaffin cells are regarded as a suitable system for studying the regulatory mechanism of "stimulus-secretion coupling". Indeed, the term "stimulus-secretion coupling" was originally coined by Douglas and Rubin for the chromaffin cells. Although it has been suggested that calcium plays a central role in this coupling process, there still remain many important and unresolved issues on the molecular mechanisms of "stimulus-secretion coupling" such as (1) the regulatory mechanisms of the calcium uptake, (2) the mechanism by which calcium entry into the cell induces membrane fusion and exocytosis, and (3) the roles of phospholipase C and C-kinase in mediating intracellular calcium homeostasis and catecholamine secretion. In this review, roles of intracellular calcium and inositol phosphate formation in "stimulus-secretion coupling" in cultured bovine adrenal chromaffin cells are discussed, mainly on the basis of the biochemical and pharmacological differences between agonist- and potassium depolarization-induced cellular responses.

Stimulation by ATP of inositol trisphosphate accumulation and calcium mobilization in cultured adrenal chromaffin cells.

Effects of ATP on accumulation of inositol phosphates and Ca2+ mobilization were investigated in cultured bovine adrenal chromaffin cells. When the cells were stimulated with 30 microM ATP, a rapid and transient rise in intracellular Ca2+ concentration was observed. At the same time, ATP rapidly increased accumulation of inositol phosphates. The concentration-response curve for the ATP-induced Ca2+ mobilization was similar to that for inositol trisphosphate (IP3) accumulation. ATP exerted its maximal effects at 30 microM for either IP3 accumulation or Ca2+ mobilization. The order of the efficacy of the agonists for IP3 accumulation and Ca2+ mobilization at 100 microM was ATP greater than ADP greater than AMP approximately adenosine, AMP (100 microM) and adenosine (300 microM) failed to induce IP3 accumulation and Ca2+ mobilization. Although 100 microM GTP and 100 microM UTP also induced IP3 accumulation and Ca2+ mobilization, their efficacy was less than that of ATP. CTP (100 microM) induced a slight IP3 accumulation, but it did not induce Ca2+ mobilization. Nifedipine (10 microM), a Ca2+ channel antagonist, and theophylline (100 microM), a P1-purinergic receptor antagonist, failed to inhibit the ATP-induced IP3 accumulation and Ca2+ mobilization. The above two cellular responses induced by ATP were also observed in the Ca2+-depleted medium. ATP induced a rapid and transient accumulation of 1,4,5-IP3 (5s), followed by a slower accumulation of 1,3,4-IP3. These results suggest that ATP induces the formation of 1,4,5-IP3 through the P2-purinergic receptor and consequently promotes Ca2+ mobilization from intracellular storage sites in cultured adrenal chromaffin cells.

Calcium uptake-dependent and -independent mechanisms of inositol trisphosphate formation in adrenal chromaffin cells: comparative studies with high K+, carbamylcholine and angiotensin II.

When [3H]inositol prelabelled cultured bovine adrenal chromaffin cells were stimulated with 56 mM KCl (high K+), 300 microM carbamylcholine (CCh) or 10 microM angiotensin II (Ang II), a rapid accumulation of [3H]IP3 was observed. At the same time, high K+ or CCh induced rapid increases in 45Ca2+ uptake, but Ang II did not induce a significant 45Ca2+ uptake. The concentration-response curve for KCl-induced [3H]IP3 accumulation coincided well with that for KCl-induced 45Ca2+ uptake into the cells. Nifedipine, a Ca2+ channel antagonist, inhibited the high K(+)-induced [3H]IP3 accumulation and 45Ca2+ uptake with a similar potency. Nifedipine at a similar concentration range also inhibited CCh-induced 45Ca2+ uptake. Although nifedipine inhibited CCh-induced [3H]IP3 accumulation, the potency was approximately 300-fold less than that for the inhibition of 45Ca2+ uptake. Nifedipine failed to affect the Ang II-induced [3H]IP3 accumulation. BAY K 8644 (2 microM), a Ca2+ channel activator, plus partially depolarizing concentration of KCl (14 mM), induced 45Ca2+ uptake and [3H]IP3 accumulation. Ionomycin (1 microM and 10 microM), a Ca2+ ionophore, also induced 45Ca2+ uptake and [3H]IP3 accumulation in a concentration-dependent manner. Pretreatment of the cells with protein kinase C activator, 100 nM 12-O-tetradecanoyl phorbol-13-acetate, for 10 min, partially inhibited CCh and Ang II-induced [3H]IP3 accumulation, but failed to inhibit the high K(+)-induced accumulation. Furthermore, the effects of high K+ and Ang II on the IP3 accumulation was additive. Ang II and CCh induced a rapid and transient increase in inositol 1,4,5-trisphosphate (1,4,5-IP3) accumulation (5 s) followed by a slower accumulation of inositol 1,3,4-trisphosphate (1,3,4-IP3). High K+ evoked an increase in 1,3,4-IP3 accumulation but obvious accumulation of 1,4,5-IP3 could not be detected. In Ca2(+)-depleted medium, high K(+)-induced [3H]IP3 accumulation was completely abolished, whereas [3H]IP3 accumulation induced by CCh and Ang II was partially inhibited. These results demonstrate the existence of the Ca2+ uptake-triggered mechanism of IP3 accumulation represented by high K+, and also the Ca2+ uptake-independent mechanism of IP3 accumulation represented by Ang II in cultured bovine adrenal chromaffin cells. Mechanism of CCh-induced IP3 accumulation has an intermediate property between those of high K+ and Ang II.

Differential effects of diacylglycerols and 12-O-tetradecanoylphorbol-13-acetate on protein phosphorylation and cell proliferation of tumor promoter-dependent leukemia cell line A65T.

The growth of A65T cells, a mouse thymic leukemia cell line, was strictly dependent on the presence of tumor promoters, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), teleocidin and mezerein. In the presence of the tumor promoters, A65T cells proliferated rapidly in a concentration-dependent manner. When the cells were cultured with a synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol (OAG) or 1,2-dicaprylin, cell proliferation was not stimulated. In addition, bihourly cumulative addition of OAG or 1,2-dicaprylin again failed to induce A65T cell proliferation. Phospholipase C (PLC) induced a concentration-dependent stimulation of A65T cell proliferation, though the effect was slight compared with that of the tumor promoters. Protein kinase C activity was detected both in cytosol and particulate fractions of A65T cells. When the cells were incubated in the TPA-free medium for 5 h, the protein kinase C activity in the cytosol fraction increased, whereas the activity in the particulate fraction decreased. Treatment of the cells with the tumor promoters mentioned above resulted in the increased phosphorylation of proteins with apparent mol. wts of 27,000 and 68,000. The effects were concentration-dependent and the half maximal phosphorylation was observed either with 3.6 nM TPA, 4.5 ng/ml teleocidin or 0.33 microM mezerein, respectively. On the other hand, a half maximal effect on cell proliferation was observed either with 0.14 nM TPA, 47 pg/ml teleocidin or 6.3 nM mezerein, respectively. At these concentrations, these tumor promoters never induced the detectable stimulations of the protein phosphorylation. A synthetic diacylglycerol 1,2-dicaprylin failed to stimulate the phosphorylation of 27- and 68-kd proteins, but stimulated the phosphorylation of proteins with apparent mol. wts of 100,000 and 54,000. The effect was concentration-dependent and a half maximal stimulation was observed with 35 micrograms/ml 1,2-dicaprylin. Neither TPA, teleocidin nor mezerein stimulated the phosphorylation of these 100- and 54-kd proteins. OAG and PLC failed to induce any detectable alterations in the protein phosphorylation in A65T cells. Both OAG and 1,2-dicaprylin, which we used, actually activated partially purified mouse brain protein kinase C in a concentration-dependent manner. These results clearly demonstrate that in A65T cells TPA and diacylglycerol mutually stimulate the phosphorylation of the completely different set of endogenous proteins, and also suggest that the cell-proliferating effects of the tumor promoters do not appear to be mediated through the phosphorylation of 27- and 68-kd proteins.