Inhibition of hCG-stimulated adenylate cyclase in purified mouse Leydig cells by the phorbol ester PMA

The tumour-promoting phorbol ester, PMA (phorbol 12-myristate 13-acetate), markedly reduced the steroidogenic response of mouse Leydig cells to stimulation by hCG and cholera toxin. However, 8Br-cAMP-and forskolin-stimulated steroidogenesis was not inhibited by PMA. PMA did not inhibit hCG-induced steroidogenesis in the simultaneous presence of 1 microM forskolin. The analysis of intracellular cAM P indicated that the PMA-induced inhibition of steroidogenesis was the result of an impaired cAMP accumulation. Adenylate cyclase in membranes prepared from PMA-treated cells showed a diminished response to hCG, GTP, guanosine 5'-[beta, gamma-imido]triphosphate [Gpp(NH)p] or to a combination of the stimulants. PMA, however, was unable to inhibit adenylate cyclase when added directly to the membrane preparation from untreated cells. As previous observations have indicated that 125I-hCG binding and phosphodiesterase activity in mouse Leydig cells are not influenced by PMA, it is concluded from the present study that the site of inhibition has to be localised to the regulatory guanine nucleotide binding protein of the adenylate cyclase system.     

Effect of luteinizing hormone deprivation in situ on steroidogenesis of rat Leydig cells purified by a multistep procedure

Depriving rats of luteinizing hormone (LH) causes Leydig cells to lose smooth endoplasmic reticulum and diminishes their P450 C17-hydroxylase/C17,20-lyase activity (Wing et al., 1984). LH administration to hypophysectomized rats prevents these changes in Leydig cell structure and function (Ewing and Zirkin, 1983). We adopted a multistep procedure of rat Leydig cell isolation to study the trophic effects of LH on steroidogenesis in the Leydig cell. Our method employs vascular perfusion, enzymatic dissociation, centrifugal elutriation, and Percoll gradient centrifugation. The purified Leydig cell fraction obtained after Percoll density-gradient centrifugation contains 95% well-preserved 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD)-staining cells with ultrastructural characteristics of Leydig cells. These Leydig cells produced 248 and 29 ng of testosterone/10(6) Leydig cells when incubated for 3 h with and without a maximally stimulating concentration of ovine LH. Purified Leydig cells obtained from control rats and rats treated with testosterone-estradiol (T-E) implants for 4 days to inhibit LH production were incubated with a saturating concentration (2 microns) of pregnenolone. Leydig cells from control and T-E-implanted rats produced 537 and 200 ng of testosterone/10(6) Leydig cells X 3 h, respectively, suggesting a defect in the steroidogenic reactions converting pregnenolone to testosterone in Leydig cells from T-E-implanted rats. By using rabbit antibodies to the P450 C17-hydroxylase/C17,20-lyase pig microsomal enzyme, immunoblots of one-dimensional sodium dodecyl sulfate polyacrylamide gels of Leydig cell microsomal protein from control and 4- and 12-day T-E implanted rats revealed a continued loss of enzyme as the period of LH withdrawal continues. These results show that Leydig cells from animals deprived of LH had diminished capacity to convert pregnenolone to testosterone and reduced P450 C17-hydroxylase/C17,20-lyase content.     

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.     

Microsomal ethanol-oxidizing system in purified rat Leydig cells

These studies provide evidence for the presence of a microsomal ethanol oxidizing system in rat Leydig cells. Activity of the microsomal ethanol oxidizing system in Leydig cells was 47.4 +/- 4.1 nmol acetaldehyde per 20 min per mg protein, while activity in crude interstitial cells was 26.0 +/- 5.4 nmol. This suggests that among cells comprising interstitial cells, activity is concentrated in Leydig cells. Activity was linear with respect to protein concentration and incubation time. The highest specific activity was observed in the microsomal fraction. The most effective cofactor was NADPH. The apparent Km for ethanol was 4 mM, suggesting that this system could effectively metabolize ethanol at concentrations found in the blood of males who drink. The apparent Km for NADPH was 11 microM. The activity in Leydig cells was unaffected by 4-methylpyrazole or potassium cyanide, which inhibit alcohol dehydrogenase and catalase activities, respectively. These data provide strong evidence for an enzyme system in Leydig cell microsomes which is capable of metabolizing ethanol.     

Tyrosine protein kinase activity in purified rat Leydig cells

Tyrosine protein kinase activity has been estimated in purified testicular cells with the synthetic peptide substrate NH₂-GLU-ASP-ALA-GLU-TYR-ALA-ALA-ARG-ARG-ARG-GLY-COOH. High levels of enzyme specific activity (56–165 pmol/mg/min) were found in the two populations of Leydig cells isolated by Metrizamide gradient centrifugation. Some activity was also detected in germinal cells, red cells and seminiferous tubules from testis but at levels 6–20 times lower than those found in the Leydig cell fractions. Higher levels of tyrosine protein kinase specific activity were found in population I than in population II Leydig cells.     

Control of steroidogenesis in Leydig cells.

Luteinizing hormone (LH) interacts with its plasma membrane receptor to stimulate steroidogenesis not only via cyclic AMP but also other pathways which include arachidonic acid and leukotrienes and regulation of chloride and calcium channels. The same stimulatory pathways may lead to desensitization and down-regulation of the LH receptor and steroidogenesis. The LH receptor exists in a dynamic state, being truncated, or internalized, degraded or recycled. Desensitization is controlled by protein kinase C (PKC) in the rat and by cyclic AMP dependent protein kinase and PKC in the mouse Leydig cells. Using an adapted anti-sense oligonucleotide strategy we have shown that the cytoplasmic C-terminal sequence of the LH receptor is essential for desensitization to occur. In contrast, these sequences of the LH receptor are not required for the stimulation of cyclic AMP and steroid production. We have also shown that the extracellular domain of the LH receptor is secreted from the Leydig cell and may act as a LH-binding protein.     

Divergent effects of 1-oleoyl-2-acetylglycerol and tumor-promoting phorbol ester on rat granulosa cell steroidogenesis in vitro

The present study was undertaken to compare the effects of diacylglycerol (synthetic 1-oleoyl-2-acetylglycerol; DG) and TPA (12-O-tetradecanoylphorbol 13-acetate) on FSH- and dibutyryl cyclic AMP ((Bu)2cAMP)-stimulated granulosa cell pregnenolone (P5), progesterone (P), and 20 alpha-hydroxypregn-4-en-3-one (20 alpha-OH-P) secretion. Granulosa cells from immature rats pretreated with pregnant mare's serum gonadotropin (PMSG) were incubated for up to 24 h with DG (0-80 micrograms/ml) or TPA (0-80 ng/ml) in the presence or absence of FSH (150 ng/ml) or (Bu)2cAMP (1.5 mM). DG, when continually present in the culture medium (MEM), significantly stimulated basal P5 (in the presence of 25 microM cyanoketone to block further metabolism), P, and 20 alpha-OH-P secretion during 6 h and 24 h of incubation. Pretreatment with TPA for 1 h caused a substantial increase in the subsequent progestin (P + 20 alpha-OH-P) secretion. However, the phorbol ester had little or no effect on steroid secretion during 6 h of incubation, significantly inhibited the secretion of P5 and P, but stimulated 20 alpha-OH-P production in 24 h. DG and TPA exerted divergent effects on FSH- and (Bu)2cAMP-stimulated progestin secretion. Accumulation of P5 throughout the culture periods (1-24 h) was markedly increased by DG (20 micrograms/ml) but significantly inhibited in the presence of TPA (40 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Leydig cells, thyroid hormones and steroidogenesis

Leydig cells are the primary source of androgens in the mammalian testis. It is established that the luteinizing hormone (LH) produced by the anterior pituitary is required to maintain the structure and function of the Leydig cells in the postnatal testis. Until recent years, a role by the thyroid hormones on Leydig cells was not documented. It is evident now that thyroid hormones perform many functions in Leydig cells. For the process of postnatal Leydig cell differentiation, thyroid hormones are crucial. Thyroid hormones acutely stimulate Leydig cell steroidogenesis. Thyroid hormones cause proliferation of the cytoplasmic organelle peroxisome and stimulate the production of steroidogenic acute regulatory protein (StAR) and StAR mRNA in Leydig cells; both peroxisomes and StAR are linked with the transport of cholesterol, the obligatory intermediate in steroid hormone biosynthesis, into mitochondria. The presence of thyroid hormone receptors in Leydig cells and other cell types of the Leydig lineage is an issue that needs to be fully addressed in future studies. As thyroid hormones regulate many functions of Sertoli cells and the Sertoli cells regulate certain functions of Leydig cells, effects of thyroid hormones on Leydig cells mediated via the Sertoli cells are also reviewed in this paper. Additionally, out of all cell types in the testis, the thyrotropin releasing hormone (TRH), TRH mRNA and TRH receptor are present exclusively in Leydig cells. However, whether Leydig cells have a regulatory role on the hypothalamo-pituitary-thyroid axis is currently unknown.     

Different regulation of phospholipase D activity in glioma C6 cells by sphingosine, propranolol, imipramine and phorbol ester

In has been found that sphingosine, propranolol, imipramine and phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) have a stimulatory effect on phospholipase D activity in glioma C6 cells. The cells were prelabelled with [1-(14)C]palmitic acid and phospholipase D-mediated synthesis of [(14)C]phosphatidylethanol was measured. The enhancing effect of TPA was almost completely blocked by a specific protein kinase C inhibitor, GF 109203X. In contrast, GF 109203X failed to inhibit the sphingosine, imipramine and propranolol stimulatory effects, indicating that their stimulation was independent of protein kinase C. The effect of TPA on phospholipase D was also blocked by imipramine and propranolol, whereas sphingosine additively potentiated TPA-mediated phospholipase D activity, both at shorter and longer (2-60 min) times of incubation. These results suggest that in glioma C6 cells, sphingosine is not only involved in a different phospholipase D activation than the TPA regulatory system, but also that it operates in a different compartment of the cell.     

Acquisition of estradiol-mediated regulatory mechanism of steroidogenesis in cultured fetal rat Leydig cells

The inability of the fetal and immature Leydig cell to be desensitized by gonadotropin treatment, a characteristic of the adult cell, is attributed to the absence of an estrogen-mediated regulation of the androgen pathway. Cultures of fetal rat Leydig cells were employed to analyze this differential response. The fetal rat Leydig cells revealed low aromatization capacity, undetectable estradiol production, a low level of estrogen receptors, and a minimally detectable level of an estradiol-regulated protein. However, exogenous estradiol caused up-regulation of its own receptor, increase of an estradiol-regulated protein, and induction of a steroidogenic lesion at the microsomal level, resulting in decreased androgen production. This estrogen-mediated enzymatic inhibition resembles that observed in gonadotropin-desensitized adult Leydig cells. The absence of this regulation in fetal life is likely due to insufficient aromatase activity, with lack of consequent receptor-mediated estrogen action. The cultured fetal Leydig cell provides a useful model to elucidate the molecular mechanism involved in the development of estradiol-mediated desensitization.     

Chronic administration of corticosterone impairs LH signal transduction and steroidogenesis in rat Leydig cells

The mechanism involved in the inhibitory actions of chronic corticosterone treatment on Leydig cell steroidogenesis was studied in adult Wistar rats. Rats were treated with corticosterone-21-acetate (2 mg/100 g body weight, i.m., twice daily) for 15 days and another set of rats was treated with corticosterone plus ovine luteinizing hormone (oLH) (100 microg/kg body weight, s.c., daily) for 15 days. Chronic treatment with corticosterone increased serum corticosterone but decreased serum LH, testosterone, estradiol and testicular interstitial fluid (TIF) testosterone and estradiol concentrations. Administration of LH with corticosterone partially prevented the decrease in serum and TIF testosterone and estradiol. Leydig cell LH receptor number, basal and LH-stimulated cAMP production were diminished by corticosterone treatment which remained at control level in the corticosterone plus LH treated rats. Activities of steroidogenic enzymes, 3beta- and 17beta-hydroxysteroid dehydrogenase (3beta-HSD and 17beta-HSD) were significantly decreased in corticosterone treated rats. LH plus corticosterone treatment did not affect 3beta-HSD activity but decreased 17beta-HSD activity, indicating a direct inhibitory effect of excess corticosterone on Leydig cell testosterone synthesis. The indirect effect of corticosterone, thus, assume to be mediated through lower LH which regulates the activity of 3beta-HSD. Basal, LH and cAMP-stimulated testosterone production by Leydig cells of corticosterone and corticosterone plus LH treated rats were decreased compared to control suggesting the deleterious effect of excess corticosterone on LH signal transduction and thus steroidogenesis.     

Optimizing testosterone production by purified adult rat Leydig cells in vitro

Summary We sought to establish conditions that increased the duration of testosterone production by fully differentiated adult rat Leydig cells in primary culture. A freshly isolated suspension of highly purified adult rat Leydig cells produced 83 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated in Medium 199 in a 1.5 ml microfuge tube with shaking for 3 h with a maximally stimulating concentration of ovine luteinizing hormone (LH). Unfortunately, adult rat Leydig cells that were allowed to attach only to a plastic culture dish flattened out, and testosterone production diminished rapidly. Leydig cells in Dulbecco's modified Eagles' medium-Ham's F12 (1∶1; vol/vol) containing Cytodex 3 beads pre-equilibrated in culture medium containing fetal bovine serum attached to the beads and remained viable, but produced only 30 ng testosterone/10⁶ Leydig cells·h⁻¹ when incubated for 24 h with similar stimulation. Leydig cells similarly cultured and maximally stimulated with LH, responded to bovine lipoproteins (<1.222 g/ml) producing 105 ng of testosterone/10⁶ Leydig cells·h⁻¹ when incubated with 1 mg/ml bovine lipoprotein. Therefore, lipoproteins maintain the steroidogenic capacity of purified adult rat Leydig cells in primary culture for 24 h. Paper presented at the 38th Annual Meeting of the Tissue Culture Association in Arlington, Virginia, in May 1987. The session was chaired by Dr. Carlton H. Nadolney, member of the TCA Committee on Toxicity, Carcinogenesis and Mutagenesis Evaluation. This research was supported in part by the National Institutes of Health (grant HD-07204), The Population Center (grant HD-06268), and EPA cooperative agreement (CR81-2765), an NSF equipment grant, and a Mellon Foundation Postdoctoral Fellowship for Gary Klinefelter. Although the research described herein has been funded in part by the U.S. Environmental Protection Agency through cooperative agreement (CR81-2765) to the Division of Reproductive Biology at Johns Hopkins University, it has not been subjected to the agency's peer and policy review, and therefore, does not necessarily reflect the views of the agency and no official endorsement should be inferred.     

Protein kinase C activity and phorbol ester binding to rat myogenic cells during growth and differentiation

Phorbol esters have been reported to induce opposite responses in fetal myoblasts and in satellite cells isolated from adult skeletal muscles. We examined the possibility that different levels of protein kinase C (PKC) activity and different phorbol ester binding characteristics account for these responses. For this purpose, the subcellular distributions of PKC were compared in primary cultures of myogenic cells from fetal and adult rat muscles and in the L6 cell line. Cells were used at the proliferative stage or after differentiation into myotubes. Binding of phorbol dibutyrate (PDBu) was assayed. In all three cell types, the levels of PKC specific activity were comparable at the proliferating and the differentiated stages, and partial translocation of PKC activity from the membrane to the cytosolic compartment was observed after differentiation into myotubes. PDBu binding, which had a Kd of 6 to 13 nM in proliferative cells, rose to between 30 and 52 nM in myotubes. Simultaneously, a small increase was observed in the total number of PDBu binding sites. These results suggest that the role of PKC might change with the stage of differentiation. They also imply that the difference described by others between the sensitivity to phorbol esters of fetal myoblasts and satellite cells is not connected with the phorbol ester receptor (i.e., PKC), but might be caused by events subsequent to PKC activation.     

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells

The role of chloride ions in the regulation of steroidogenesis in rat Leydig cells and adrenal cells has been investigated. It was found that the chloride channel blocker 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (DIDS) inhibited LH but not dibutyryl cAMP (dbcAMP)-stimulated steroidogenesis in the Leydig cells. This was found to be via an inhibition of cAMP production, because both LH- and forskolin-stimulated cAMP productions were inhibited by DIDS. The exclusion of chloride ions enhanced steroidogenesis during incubation of Leydig cells and adrenal cells with dbcAMP. The adrenal cells were found to be more sensitive to dbcAMP than Leydig cells and the enhancing effects of chloride removal were higher. In the presence of chloride ions, near maximum steroidogenesis was achieved with approximately 60 μM and 1 mM dbcAMP in the adrenal and Leydig cells, respectively. In the absence of chloride ions the concentrations required decreased approximately 50-fold and 10-fold, respectively. It is concluded that although LH may regulate DIDS sensitive chloride channels, the enhanced stimulation of cAMP-mediated steroidogenesis by chloride exclusion is not mediated via these channels. We propose a model based on the present and previous studies [1] with Leydig tumour (MA10) cells i.e. that intracellular chloride ion depletion enhances the action of cAMP on protein synthesis which results in increased synthesis of the Steroidogenic Acute Regulator (StAR) protein and consequently increased steroidogenesis.     

Nicotine and cotinine inhibit steroidogenesis in mouse Leydig cells

Cigarette smoking alters plasma testosterone concentrations in men. The objectives of this study were to determine if nicotine and cotinine, two alkaloid products of cigarettes, affect luteinizing hormone(LH)-stimulated steroidogenesis in isolated adult mouse Leydig cells. Leydig cells from adult Swiss-Webster mice were isolated by linear density gradient and incubated (95% O2, 5% CO2) in minimum essential medium at 37 C for 3 hours with LH (10 ng) and with or without nicotine or cotinine (10(-5)-10(-7) M). Both nicotine and cotinine produced dose response inhibition (P less than 0.05) of LH-stimulated testosterone production (50-70%). The addition of 8-bromo-3',5'-cyclic monophosphate (cAMP, 500 uM) stimulated steroidogenesis comparable to LH in the absence of the alkaloids, but both nicotine and cotinine significantly (P less than 0.05) reduced testosterone production in response to cAMP, suggesting that the alkaloids inhibit testosterone production in response to LH distal to the formation of cAMP. In MEM without calcium, LH-stimulated testosterone synthesis was decreased, and neither nicotine nor cotinine significantly affected steroidogenesis. The addition of a calcium ionophore in MEM with normal calcium content enhanced (P less than 0.05) the inhibitory effects of nicotine and cotinine on LH-responsive steroidogenesis. A calcium channel blocking agent, verapamil, at 10uM significantly (P less than 0.05) reversed the inhibition of LH-stimulated testosterone production produced by both alkaloids when incubated in the medium with a normal calcium concentration. These results suggest that nicotine and cotinine either affect intracellular calcium content or block the effects of calcium on steroidogenesis in mouse Leydig cells.     

Synergism between lysophosphatidylserine and the phorbol ester tetradecanoylphorbolacetate in rat mast cells

In rat peritoneal mast cells tetradecanoylphorbolacetate (TPA) induced a non cytotoxic histamine release in the absence of extracellular calcium. The addition of calcium prevented the TPA effect but micromolar concentrations of lysophosphatidylserine (lysoPS) converted the calcium-induced inhibition into a stimulation. Other lysophospholipids were inactive. In agreement with a mutual influence between lysoPS and TPA, minimal TPA concentrations enhanced the calcium-dependent histamine release induced by lysoPS in the presence of nerve-growth factor. It is proposed that the calcium-dependent pathway promoted by lysoPS and the activation of protein kinase C by TPA act synergically to induce histamine release from mast cells.     

Stimulation and inhibition of Leydig cell steroidogenesis by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate: similarity to the effects of gonadotropin-releasing hormone

To explore the mechanism of gonadotropin-releasing hormone (GnRH) action on Leydig cell steroidogenesis the effects of a GnRH analog (GnRHa) were compared to those of 12-O-tetradecanoylphorbol 13-acetate (TPA). Both compounds acutely stimulated androgen production 2-4 fold with EC50's of 9 nM (TPA) and 0.2 nM (GnRHa). The effects of TPA and GnRHa were not additive and neither compound acutely altered the luteinizing hormone (LH) concentration-response relationship. After 24 h of exposure to TPA or GnRHa the ability of LH to stimulate androgen production was impaired. The parallel effects of TPA and GnRHa on Leydig cell steroidogenesis suggest that they are acting via similar mechanisms; presumably the activation protein kinase C.     

Release of arachidonic acid and the effects of corticosteroids on steroidogenesis in rat testis Leydig cells.

The release of arachidonic acid by luteinizing hormone (LH) and the effects of inhibiting phospholipase A2 (PLA2) in vivo and in vitro on LH stimulated steroidogenesis in rat testis Leydig cells has been investigated. It was found that arachidonic acid is rapidly incorporated into phospholipids and is released within 1 min after addition of LH. The effects of treating adult rats with dexamethasone and human chorionic gonadotropin (hCG) in vivo on steroidogenesis and prostaglandin synthesis in Leydig cells isolated 6 h later were determined. It was found that hCG caused a marked increase in prostaglandin F2 alpha formation which was inhibited by treatment with dexamethasone. LH-stimulated testosterone production was inhibited in the hCG treated rats and dexamethasone caused a further decrease. Treatment with dexamethasone alone also caused a decrease in the response to LH. HCG, but not dexamethasone, had similar inhibitory effects on LH-stimulated cyclic AMP production. Similarly, the PLA2 inhibitors quinacrine, dexamethasone and corticosterone, added to the Leydig cells in vitro, inhibited LH-stimulated testosterone production but not cyclic AMP production. 11-Dehydrocorticosterone also inhibited LH-stimulated testosterone production, but higher concentrations were required to give 50% inhibition compared to corticosterone (50 and 25 microM, respectively). Ring A-reduced metabolites of corticosterone and progesterone were also found to inhibit LH-stimulated steroidogenesis. The results obtained in this and previous studies are consistent with the activation of PLA2, (either directly by LH and/or via cyclic AMP), which results in the release of arachidonic acid and the formation of leukotrienes, which stimulate steroidogenesis in the Leydig cell. This study also indicates that corticosteroids and their metabolites may exert inhibitory effects at other sites in the steroidogenic pathways, in addition to PLA2.     

Altered catalytic properties of protein kinase C in phorbol ester treated cells

Exposure of various cell types (rat-1 fibroblasts, bovine adrenocortical cells, human lymphoid cells) to nanomolar concentrations of TPA, resulted in a rapid, apparent loss of cellular protein kinase C content, when the enzyme was assayed by its phospholipid and Ca2+-dependent histone (H1)-kinase activity, following solubilization and DEAE-cellulose chromatography isolation. By contrast, no loss of protein kinase C was detected when the enzyme was probed by its high affinity PDBu binding capacity nor when the kinase activity was assayed with protein substrates other than histones, such as vinculin and a cytochrome P-450. It is concluded that, in addition to the previously reported enzyme subcellular redistribution, following TPA treatment, the phorbol ester induces striking alterations of the cellular protein kinase C catalytic activities. The molecular mechanisms of these changes and their implication in the tumor promotion process remain to be clarified.