Particulate Protein Kinase C Activity Research Articles

Benzo[a]pyrene inhibits protein kinase C activity in subcultured rat aortic smooth muscle cells

Recent studies in this laboratory have shown that benzo[ a]pyrene (BaP) interferes with protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins in vivo. To evaluate the biochemical basis of this response, the present studies have been conducted to examine the time- and concentration-dependent effects of BaP on PKC activity in vitro. Growth-arrested subcultures of rat aortic SMCs were exposed to 0.3, 3, or 30 μM BaP in the presence of fetal bovine serum for various times and then processed for measurements of exogenous histone Type III-S phosphorylation under PKC-activating conditions. Challenge of SMCs with BaP for 8 h was associated with a concentration-dependent inhibition of PKC activity in both cytosolic and particulate fractions. While no changes of enzymatic activity were observed in either fraction following exposure of SMCs to 0.3 μM BaP, higher concentrations of BaP inhibited PKC in both cytosolic and particulate fractions. A 49% and 68% reduction of cytosolic PKC activity was observed in SMCs treated with 3 and 30 μM BaP, respectively. The inhibitory response elicited by BaP was more pronounced in the particulate fraction where 61% and 89% decreases in PKC activity were observed in cultures treated with 3 and 30 μM BaP, respectively. Time course studies revealed that inhibition of PKC activity by 30 μM BaP occurred as early as 30 min and was sustained for up to 24 h in both fractions. Benzo[ a]pyrene (30 μM) did not interfere with the ability of phorbol-12-myristate-13-acetate to induce PKC translocation from the cytosolic to particulate compartment since maximal translocation occurred by 5 min and lasted for up to 60 min in both control and BaP-treated cultures. The inhibitory effects of BaP were independent of new protein or RNA synthesis, but appear to involve oxidative metabolism of the parent compound since 3-hydroxy-BaP, the major P450-derived BaP metabolite in SMCs, also inhibited cytosolic and particulate PKC activity. Collectively, these data demonstrate that BaP and its 3-hydroxy metabolite inhibit PKC activity in rat aortic SMCs and raise the possibility that interference with PKC-mediated protein phosphorylation participates in the deregulation of SMC growth and differentiation induced by BaP.

Activation of D1 and D2 dopamine receptors inhibits protein kinase C activity in striatal synaptoneurosomes.

The effects of D1 and D2 dopamine ligands on protein kinase C (PKC) activity were examined in synaptoneurosomes. Incubation with D1 agonists (SKF 38393, fenodopam), in the presence of calcium, decreased the soluble and increased the particulate PKC activity. These effects were reversed by SCH 23390, which by itself had the opposite effect of increasing the soluble and decreasing the particulate PKC activity. In contrast, incubation with the D2 agonists [LY 171555, (+)-3-(3-hydroxyphenyl)-N-n-propylpiperidine, RU 24213] increased the soluble and decreased the particulate PKC activity. These effects were reversed by sulpiride. (-)-3-(3-Hydroxyphenyl)-N-n-propylpiperidine had a D2 antagonist profile. Apomorphine showed a biphasic dose-response change; i.e., it decreased particulate PKC activity at the D2 receptor at low concentrations (0.1 microM) and increased it at the D1 receptor at higher concentrations (10 microM). Pretreatment with tetrodotoxin or omission of calcium in the incubation medium did not alter the responses of the D2 agonists, but it reversed the changes in PKC activity induced by the D1 agonists and converted the biphasic response of apomorphine to a monophasic inhibition. These results indicate that (1)D1 and D2 dopamine receptors are negatively coupled to PKC and (2) the increase in particulate PKC activity seen with the D1 drugs in the presence of calcium is mediated indirectly via a transneuronal effect.

Biphasic modulation of protein kinase C (PKC) activity by polychlorinated dibenzo‐p‐dioxins (PCDDs) in serum‐deprived rat aortic smooth muscle cells

Previous studies in this laboratory have shown that benzo(a)pyrene (BaP) modulates protein kinase C (PKC)-mediated phosphorylation of aortic smooth muscle cell (SMC) proteins. This observation is consistent with the ability of other aromatic hydrocarbons (AHs), such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to modulate kinase activities in cells of hepatic, testicular, and thymic origin. Because all these chemicals share the ability to bind the aryl hydrocarbon receptor (AhR), the present studies were conducted to determine if changes in PKC activity by AHs conform with established structure-activity relationships. Experiments were conducted to examine the effects of TCDD, 2,3,7,8-tetrachlorodibenzofuran (TCDF), and 2,8-dichlorodibenzodioxin (DCDD) on the phosphorylation of exogenous histone type-III under basal and PKC-activating conditions. These congeners exhibit both high (TCDD and TCDF) and low (DCDD) AhR agonist activities. Measurements of kinase activity were conducted in the cytosolic and particulate fractions of growth-arrested (i.e., serum-deprived) cultured rat aortic SMCs incubated with 10 nM TCDD, TCDF, and DCDD for 0.5, 12, or 24 hours. No changes in basal kinase activity were induced by these chemicals at any of the times tested. Significant decreases in cytosolic and particulate PKC activity relative to controls were observed upon exposure of SMCs for 0.5 hours to 10 nM TCDD, TCDF, and DCDD. In contrast, SMCs exposed to TCDD and TCDF for 12 hours exhibited a significant increase in PKC activity in both cytosolic and particulate fractions. The PKC activity in cells exposed to DCDD for 12 hours was not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein Kinase C Activity Is Reduced in Epidermal Cells from Energy-Restricted SENCAR Mice ,

Female SENCAR mice were pre-fed a control or 40% energy-restricted (ER) diet with energy removed from fat and carbohydrate, or a control, balanced high fat (BHF, with similar energy from fat and carbohydrate), 35% energy restricted from fat (HCR) or 35% energy restricted from carbohydrate (HFR) diet. Epidermal cells were isolated by trypsin digestion for measurement of protein kinase C (PKC) activity, lipid composition or lipid metabolism. Dietary restriction of fat or carbohydrate energy (HFR or HCR group) reduced particulate PKC activity in epidermal cells compared with cells from control mice. The ratio of soluble particulate PKC activity was higher in epidermal cells from mice fed the HCR diet compared with those fed the HFR diet. Diet did not affect soluble PKC activity. Inositol accumulation was measured in the water- or lipid-soluble fractions of prelabeled ([3H]inositol) epidermal cells following a 1-h incubation in media with LiCl. Phosphatidylinositol, inositol biphosphate and inositol triphosphate fractions were more heavily labeled in cells from mice fed the ER diet. Energy restriction did not modify epidermal total lipid or phospholipid composition, but 1,2-diacylglycerol levels were elevated in relation to cell number in epidermal cells from mice fed the ER diet. These data suggest that dietary energy restriction modified PKC activity through a pathway other than alteration in membrane lipid composition or inositol lipid metabolism.

Effect of fatty acids and their acyl-CoA esters on protein kinase C activity in fibroblasts: Possible implications in fatty acid oxidation defects

We studied the effect of fatty acids and their acyl-CoA esters on protein kinase C (PK-C) activity in human skin fibroblasts. Butyrate, octanoate, palmitate and oleate did not alter PK-C activity in either cytosolic or particulate fraction. In the presence of calcium, phosphatidylserine and diacylglycerol, both palmitoyl-CoA (Pal-CoA) and oleoyl-CoA (Ole-CoA) enhanced particulate PK-C activity by approx. 70% and octanoyl-CoA (Oct-CoA) by approx. 35%. Partially purified cytosolic PK-C activity was enhanced by 60–70% by 13.5 μM of either Pal-CoA or Ole-CoA. Basal histone phosphorylation (i.e., PK-C-independent phosphorylation) was decreased in the particulate fraction in the presence of these esters in a concentration-dependent manner. Both Pal-CoA and Ole-CoA fully substituted diacylglycerol in activating the kinase in both the cytosolic and particulate fractions, whereas Oct-CoA had a moderate effect. The pattern of endogenous cytosolic and particulate protein phosphorylation was altered in the presence of either Pal-CoA or Ole-CoA. We conclude that long-chain fatty acyl-CoA esters may activate PK-C in non-stimulated fibroblasts, i.e., in the absence of physiological diacylglycerol formation. Activation of PK-C in stimulated fibroblasts, i.e., in the presence of an elevated diacylglycerol concentration, is less pronounced. These results support the hypothesis that activation of PK-C and alteration of endogenous protein phosphorylation may play a role in the pathogenesis of diseases in which there is intracellular accumulation of fatty acyl-CoA esters, such as in inborn fatty-acid oxidation defects.

Spatial changes of [Ca2+]i and contraction caused by phorbol esters in vascular smooth muscle cells.

In A7r5 smooth muscle cell suspensions, 12-deoxyphorbol 13-isobutyrate (DPB) and phorbol 12,13-dibutyrate (PDB), active phorbol esters for protein kinase C (PKC), increased the intracellular Ca2+ concentration ([Ca2+]i), but 4 alpha-PDB, an inactive phorbol ester, did not. Digital images of the fura 2 fluorescence from single cells revealed that DPB caused elevation of Ca2+ in localized peripheral regions, followed by expansion of this elevated Ca2+ level throughout the cytoplasm. High K+ also induced a dynamic change of [Ca2+]i. DPB and high K+ increased the wrinkles and distortions in the flexible growth surface beneath the cells. In Ca(2+)-depleted media, DPB did not affect [Ca2+]i but substantially increased wrinkles. The increase could be eliminated by staurosporine, a specific inhibitor of PKC. DPB increased the particulate PKC activity in a concentration-dependent manner. These results suggest that PKC activation induces cellular contractions through two distinct mechanisms, one dependent on and another independent of the [Ca2+]i increase.

PKC translocation in rat brain cortex is promoted in vivo and in vitro by alpha-glycerylphosphorylcholine, a cognition-enhancing drug.

Protein kinase C (PKC) activity was measured in soluble and particulate fractions of rat individual brain areas after treatment with alpha-glycerylphosphorylcholine (GPC), a cognition-enhancing drug which promotes acetylcholine synthesis and release. The drug induced both in vivo and in vitro PKC translocation. In vivo, an increase of particulate PKC activity was observed 1 hour following the acute oral administration of a behaviorally active dose (600 mg/kg); the effect was transient. In vitro, GPC promoted PKC translocation in cortical slices at concentrations as low as 50 nM; the concentration-response curve was bell shaped. The increased PKC activity may be related to the cortical effects of GPC.

Protein Kinase C Inhibitors Suppress Cell Growth in Established and Low-Passage Glioma Cell Lines. A Comparison between Staurosporine and Tamoxifen

We have previously demonstrated that the proliferation of established human glioma cell lines correlated with protein kinase C (PKC) activity and that a relatively selective PKC inhibitor, staurosporine, inhibits glioma cell proliferation. The purpose of this study was to determine whether low-passage glioma cell lines were also sensitive to staurosporine and to compare the antimitotic effects of staurosporine with tamoxifen, an antiestrogen with a known PKC inhibitory effect presently being investigated in the treatment of recurrent glioma. We measured the effects of treatment with staurosporine or tamoxifen on the proliferation rate of five established glioma cell lines (A172, U251, U87, U373, U563) and four low-passage glioma cell lines. The proliferation of all cell lines was inhibited by staurosporine, at an IC50 value (concentration at which activity is 50% inhibited) of approximately 2 nmol/L. All established lines, but only one low-passage line, were susceptible to tamoxifen, with an IC50 value of 10 mumol/L. Three of the four low-passage lines were poorly inhibited by tamoxifen. The IC50 values for the inhibition of cellular proliferation by staurosporine and tamoxifen closely corresponds to the IC50 data for the inhibition of particulate PKC activity in gliomas. We conclude that staurosporine is more effective in the inhibition of glioma proliferation than tamoxifen and that staurosporine is potentially useful in the adjuvant treatment of gliomas. The correspondence in IC50 results for proliferation and PKC activity further strengthens the hypothesis that an aberrant PKC system in gliomas drives their hyperproliferative state.

Mechanism and regulation of neutrophil priming by platelet-activating factor.

Although a weak direct stimulus of superoxide anion (O2-) production, platelet-activating factor (PAF) markedly enhances responses to chemotactic peptides (such as n-formyl-met-leu-phe, FMLP) and phorbol esters (such as phorbol myristate acetate, PMA) in human neutrophils. The mechanism of priming was explored first through inhibition of steps in the signal transduction pathway at and following PAF receptor occupation. Priming was not altered by pertussis toxin or intracellular calcium chelation, but the PAF receptor antagonist WEB 2086 and the protein kinase C (PKC) inhibitors sphinganine and staurosporine significantly inhibited the primed response. In order to study the regulation of PAF priming, the effect of PAF alone was desensitized by exposure to escalating doses of PAF prior to exposure to the secondary stimuli. The priming effect of PAF was not desensitized under these conditions. The role of PKC in desensitization was also studied. Prior exposure to PAF also desensitized the increase in membrane PKC activity evoked by a single concentration of PAF. However, when the PAF response was desensitized, PKC priming of the response to FMLP or PMA still occurred, suggesting that PKC activity may play a role in the maintenance of the primed state despite PAF desensitization. These data suggest that: (1) PAF priming is receptor- and PKC-mediated but is independent of pertussis toxin-inhibitable G-proteins or intracellular calcium, (2) during migration in vivo, neutrophils may be desensitized to the direct effects of PAF but maintain the capacity for enhanced responses to other stimuli, (3) desensitization of PAF-induced particulate PKC activity also occurs, but PAF primes PKC activity despite PAF desensitization, and (4) distinct mechanisms govern the direct and priming effects of PAF on oxidative metabolism.

Hydroperoxide-Induced Diacylglycerol Formation and Protein Kinase C Activation in Vascular Endothelial Cells

We examined the effects of hydrogen peroxide (H2O2) and other hydroperoxides on endothelial cell protein kinase C (PKC) activity. Bovine pulmonary artery endothelial cells (BPAEC) were grown to confluent monolayers and PKC activity was determined in an in vitro assay by measuring phosphorylation of H1 histone. In control unstimulated BPAEC, PKC was primarily localized in the cytosol and treatment of BPAEC with H2O2 resulted in a concentration (10−5 M-10−3 M) and time (15 min-60 min.) dependent translocation of the enzyme from the cytosol to the membrane fraction. In addition to H2O2, linoleic acid hydroperoxide treatment of BPAEC also resulted in PKC activation while tert-butyl hydroperoxide and cumene hydroperoxide were not effective. In addition to causing an increase in membrane-associated PKC activity, H2O2 treatment also resulted in the partial loss of cytosolic PKC activity. As diacylglycerol (DAG) is a critical endogenous activator of PKC, we evaluated whether H2O2 exposure resulted in the increased production of DAG. Exposure to 1.0 mM H2O2 resulted in biphasic accumulation of DAG (two- to threefold). The first phase of DAG formation occurred within 5 min of H2O2 exposure while the second phase of accumulation began at 15 min of treatment and plateaued at about 45 min. The metal ions Cu2+ and Fe3+ augmented the H2O2-induced loss of total (cytosolic and particulate) PKC activity in BPAEC. These studies suggest that oxidants modulate PKC activity and increase DAG levels in vascular endothelial cells.

Effect of P2Y Agonists on Adenosine Transport in Cultured Chromaffin Cells

Adenosine transport in cultured chromaffin cells was inhibited by purinergic P2y-receptor agonists without significant changes in the affinity constant, the values being between 1 +/- 0.4 and 1.6 +/- 0.6 microM. The Vmax parameter was modified significantly, being 40 +/- 1.0, 26 +/- 5.0, 32 +/- 3.0, and 22 +/- 4.7 pmol/10(6) cells/min for control, adenosine-5'-O-(2-thiodiphosphate), 5'-adenylylimidodiphosphate, and P1,P4-di(adenosine-5'-) tetraphosphate (Ap4A) (100 microM for every effector), respectively. Ap4A, a physiological ligand for P2y receptors in chromaffin cells, showed the highest inhibitory effect (45%). This transport inhibition is explained by an increase in the cytosolic Ca2+ concentration ([Ca2+]i) and the activation of protein kinase C (PKC). Experiments of [Ca2+]i measurement with the fura-2 technique showed that P2y agonists, as well as bradykinin, were able to increase [Ca2+]i, this effect being independent of the presence of extracellular Ca2+. The peptide bradykinin, determined to be coupled to phosphatidylinositol hydrolysis and internal Ca2+ mobilization in chromaffin cells, exhibited a behavior similar to that of P2y agonists in adenosine transport inhibition (39%). P2y agonists and bradykinin increased PKC activity associated with the membrane fraction (about 50% increase in particulate PKC activity with respect to controls). The present studies suggest that adenosine transport is regulated by P2y-purinergic receptors mediated via Ca2+ mobilization and PKC activation.

Cognition stimulating drugs modulate protein kinase C activity in cerebral cortex and hippocampus of adult rats

The in vivo and in vitro effect of oxiracetam, aniracetam and α-glicerylphosphorylcholine (αGPC) on protein kinase C (PKC) activity was studied in rat brain cortex and hippocampus. Administration of oxiracetam and αGPC in vivo elicited an early increase of particulate histone-directed PKC activity accompanied by a decrease of soluble activity and followed a few hours later by a down regulation of the enzyme. The effect was also observed in vitro when either oxiracetam or αGPC were administered at nanomolar concentrations to rat brain cortex slices. Aniracetam had no effect in the cortex but promoted PKC translocation both in vivo and in vitro in the hippocampus. In cortex slices the effect of oxiracetam was antagonized by the addition of AP-5, an NMDA receptor blocker, but not by CNQX and L-AP3, antagonists of AMPA and metabotropic glutamate receptors, respectively. Scopolamine also prevented the increase of particulate PKC elicited by oxiracetam in vitro. In the hippocampus the increase of particulate PKC activity was antagonized by AP-5, CNQX and L-AP3, indicating participation by both ionotropic and metabotropic glutamate receptors in the action of aniracetam. The data support the hypothesis that PKC activation may be a common mechanism amongst cognition stimulating drugs from different chemical classes.

Activation of protein kinase C isozymes by contractile stimuli in arterial smooth muscle

Protein kinase C (PKC) has been proposed to be involved in the regulation of vascular smooth muscle (VSM) contractile activity. However, little is known in detail about the activation of this kinase or specific isozymes of this kinase by contractile stimuli in VSM. As an index of PKC activation, Ca 2+- and phospholipid-dependent histone IIIS kinase activity was measured in the particulate fraction from individual strips of isometrically contracting carotid arterial smooth muscle. Phorbol 12,13-dibutyrate (PDB) increased PKC activity in the particulate fraction (155% over resting value by 15 min) with a time course which paralleled or preceded force development. Stimulation with the agonist histamine (10 −5 m) resulted in rapid increases in both force and particulate fraction PKC activity which was maximal by 2 min (increase of 139%) and partially sustained over 45 min (increase of 41%). KCl (109 m m), which evokes a sustained contractile response, caused a slow increase (124% by 45 min) in particulate fraction PKC activity. No significant increases in activator-independent histone kinase activity were observed in response to any stimulus tested. PKCα and PKCβ were identified as the principal Ca 2+/phospholipid-dependent PKC isozymes expressed in this tissue. In unstimulated arterial tissue, the ratio of immunodetectable isozyme content (α:β) was estimated to be 1:1 in the particulate and 1.5:1 in the cytosolic fractions. Upon stimulation with each of the three contractile stimuli, particulate fraction PKC content assessed by immunoblotting increased with a time course and to an extent comparable to the observed changes in PKC activity. There was no evidence of differential regulation of the PKCα or -β isozymes by PDB compared to the other contractile stimuli. These results indicate that diverse contractile stimuli are capable of tonically activating PKC in preparations of functional smooth muscle, and are consistent with a functional role for PKCα and/ or -β in the regulation of normal smooth muscle contractile activity.

Increase in protein kinase C activity is associated with human fibroblast growth inhibition

Protein kinase C(PKC) activity and DNA synthesis were measured in human fetal bone marrow fibroblasts following treatment with tumor necrosis factor alpha (TNFα)(500 U/ml) or conditioned media containing natural cell proliferation inhibitor (CM-NCPI). Treatment with TNFα led to growth stimulation (120 ± 7% of control in 24 h, 141 ± 6% in 72 h). At the same time particulate PKC activity diminished, reaching 55 ± 8% of control in 24 h and remaining at this level at 72 h. CM-NCPI treatment of the cells resulted in a decrease in DNA synthesis (by 39 ± 6% in 2 h, by 58 ± 5% in 24 h, and by 78 ± 8% in 72 h). This was accompanied by a significant rise in particulate PKC activity which increased over 3-fold in 2 h, over 5-fold in 24 h, and up to 11-fold in 72 h. This 11-fold elevation was maintained after 2 week exposure of the fibroblasts to CM-NCPI. The PKC inhibitor neomycin abolished CM-NCPI induced growth inhibition, whereas PKC activator 12- O-tetradecanoylphorbol 13-acetate intensified it. These results suggest that CM-NCPI acts as PKC activator and that negative growth regulation by extracellular agents may involve stimulation of PKC activity.

Long-term activation of protein kinase C by nicotine in bovine adrenal chromaffin cells.

Previous results from our laboratory suggest that long-term treatment of primary cultured bovine adrenal medullary (BAM) chromaffin cells with nicotine or phorbol 12-myristate 13-acetate, either of which directly activates protein kinase C (PKC), increases the mRNA levels encoding catecholamine-synthesizing enzymes and proenkephalin. In the present study, we have examined the effects of nicotine on BAM cell PKC activity with special emphasis on long-term effects. Nicotine increased particulate PKC activity in a concentration-dependent manner when measured using in vitro enzyme assay with histone as the substrate. This effect is mediated through nicotinic cholinergic receptors, because 1,1-dimethylphenylpiperazinium, a nicotinic agonist, had a similar effect. In addition, chlorisondamine, a specific nicotine-receptor blocking drug, antagonized the effect of nicotine. Nicotine also increased specific [3H]phorbol 12,13-dibutyrate ([3H]PdBu) binding within 1 min, the effect of which was maximal between 3 and 12 min. This effect was reversed by chlorisondamine similarly after 12 min and after 18 h of nicotine treatment, indicating that continual nicotinic-receptor occupancy is required for persistent PKC activation. Compared to PKC activation, the onset of nicotine-stimulated diacylglycerol production was slow, and it was observed after 12 min of incubation with nicotine. The diacylglycerol levels, specific [3H]PdBu binding, and PKC activity remained significantly elevated for at least 18 h with continuous nicotine incubation. Furthermore, nicotine increased the PKC immunoreactivity of a particulate protein with a molecular mass of 82 kDa in the western blot. These results suggest that nicotinic-receptor activation increases PKC activity and immunoreactivity in BAM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activity in permanent focal cerebral ischemia.

Protein kinase C (PKC) activity was investigated in a model of focal stroke in the rat. Following 6 h of left middle cerebral artery occlusion, rat brains were frozen in situ. In the peripheral ischemic zone, total PKC activity declined by close to two-thirds (1.07 +/- 0.35 vs 2.77 +/- 0.12 nmol/min/mg protein; p less than 0.05, n = 4), and the proportion of total activity associated with the particulate fraction decreased from 33.3 +/- 1.5% to 16.2 +/- 1.4% (p less than 0.01, n = 4). Thus, overall particulate PKC activity in the ischemic zone was less than 20% of control. The cerebral energy metabolite profile of tissue from the ipsilateral hemisphere, corresponding to the region where samples were obtained for PKC activity assay, suggests that this tissue may have been part of the ischemic penumbra before further deterioration.

Protein kinase C activity in human colonic adenoma and colorectal carcinoma

To additionally understand the molecular mechanisms and biologic indicators of colonic tumorigenesis through the adenoma-carcinoma sequence, protein kinase C (PKC) activity was examined in the cytosol and particulate fraction of specimen homogenates from 18 human colonic carcinomas and seven coexisting colonic adenomas and was compared with the adjacent normal mucosal tissues. This study showed that PKC activity could be detected precisely using mini DEAE-Sephacel column purification and histone III-S as a substrate. The PKC activity in both colonic adenoma and carcinoma progressively was reduced in the particulate fraction compared with that of the adjacent normal mucosa from each patient (74.9 +/- 11.3 and 42.4 +/- 9.37 versus 112 +/- 16.8 pmol/min/mg, P less than 0.001), although PKC activity in the cytosolic fraction was not significantly different (62.6 +/- 17.7 and 63.1 +/- 8.08 versus 56.4 +/- 7.32 pmol/min/mg) with respect to protein concentration. Both colonic adenomas and carcinomas showed a significant progressive decrease in total particulate PKC activity compared with the adjacent normal mucosa of each patient (13.5 +/- 2.18 and 7.64 +/- 1.35 versus 19.8 +/- 2.74 pmol/min/g tissue, P less than 0.001) and no difference in total cytosolic PKC activity (15.2 +/- 3.80 and 16.5 +/- 2.02 versus 14.6 +/- 1.81 pmol/min/g tissue). Among PKC activities in carcinomas, there was no difference related to histologic type, Dukes' staging, or carcinoembryonic antigen values. Among PKC activities in colonic adenomas, a significant decrease in particulate PKC correlated with size. The specific PKC activity in the particulate fraction decreased with advancing adenoma size (P less than 0.05). This study showed that colonic carcinogenesis might be associated with alterations in cellular levels of PKC activity and that the decrease in particulate PKC activity in the adenoma had a possible correlation with adenoma size.

Enhanced protein kinase C activity correlates with the growth rate of malignant gliomas in vitro

Direct measurement of protein kinase C (PKC) activity in vitro revealed a significant increase in the activity of the enzyme in all human malignant glioma lines examined and the rat C6 tumor in comparison with control nonneoplastic astrocyte and mixed glial cultures. The total and particulate PKC activity in these cell types correlated strongly [r = 0.98 (P less than 0.001) and 0.94 (P = 0.002), respectively] with the maximal growth rates as measured by 3H-thymidine incorporation in each of the samples. An alteration in the growth rate of an individual glioma line (A172) by varying the serum concentration in the growth medium produced comparative changes in the measured PKC activity. The addition of the phorbol ester phorbol-12-myristate-13-acetate to this tumor line under high serum conditions produced down-regulation of the enzyme, which was accompanied by a corresponding reduction in thymidine incorporation. The administration of the PKC inhibitor staurosporine produced a dose-related decrease in the basal proliferation rate of glioma lines A172 and C6, as measured by 3H-thymidine uptake and confirmed by flow cytometry, indicating that the high intrinsic PKC activity is amenable to pharmacological manipulation. Cytofluorometric deoxyribonucleic acid cell cycle analysis of the tumors treated with PKC modulators demonstrated that reduced proliferation rates were caused by an inhibition of entrance into the deoxyribonucleic acid synthesis (S) phase (decrease in proliferative index), supporting the evidence that these modulators are not slowing the tumor growth in a nonspecific cytotoxic manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Endothelium‐derived relaxing factor inhibits the endothelin‐1‐induced increase in protein kinase C activity in rat aorta

1. Particulate and cytosolic protein kinase C (PKC) activity was measured in rat aortae with and without endothelium, following exposure to endothelin-1 (10(-8) M) for various time intervals. 2. Endothelin-1 induced two peaks of particulate PKC activity, occurring at 30 s and 10 min exposure times in both endothelium-intact and endothelium-denuded preparations. Cytosolic PKC activity fell below baseline at all incubation times studied. 3. In endothelium-denuded preparations, elevation of guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels with sodium nitroprusside (10(-6) M) or atrial natriuretic peptide (10(-6) M) and, in endothelium-intact preparations with the calcium ionophore A23187 (10(-6) M), inhibited the activation of particulate PKC activity seen after incubation with endothelin-1 for 30 s. The inhibitory effect of A23187 was prevented by prior incubation of the endothelium-intact vessels with the nitric oxide synthetase inhibitor, L-NG-nitro arginine (5 x 10(-5) M). 4. These results indicate that EDRF acting via cyclic GMP can inhibit the activation of PKC induced by endothelin-1 in rat aorta.

Prolactin activates protein kinase C and stimulates growth-related gene expression in rat liver

We have examined the effect of prolactin (PRL) on growth-related gene expression, protein kinase C (PKC) activity and diacylglycerol (DAG) mass in rat liver. Hepatic levels of messenger (m)RNA for c- myc, ornithine decarboxylase (ODC) and β-actin increased in a dose-dependent manner within 1 h after PRL administration. Prolactin also caused a transient elevation of liver DAG levels and particulate-associated PKC activity. The PRL-provoked increases in DAG mass and particulate PKC activity were coincident and maximal at 20 min and began declining toward control levels by 30 min. These results suggest a temporal relationship between PRL-stimulated DAG accumulation and PKC activation. Furthermore, the subsequent rapid induction of growth-related gene expression provides new information on the role of PRL as a hepatic mitogen.