Membrane-associated Protein Kinase C Activity Research Articles

Sex-specific effects of 17β-estradiol and dihydrotestosterone (DHT) on growth plate chondrocytes are dependent on both ERα and ERβ and require palmitoylation to translocate the receptors to the plasma membrane

Rat costochondral cartilage growth plate chondrocytes exhibit cell sex-specific responses to 17β-estradiol (E2), testosterone, and dihydrotestosterone (DHT). Mechanistically, E2 and DHT stimulate proliferation and extracellular matrix synthesis in chondrocytes from female and male rats, respectively, by signaling through protein kinase C (PKC) and phospholipase C (PLC). Estrogen receptors (ERα; ERβ) and androgen receptors (ARs) are present in both male and female cells, but it is not known whether they interact to elicit sex-specific signaling. We used specific agonists and antagonists of these receptors to examine the relative contributions of ERs and ARs in membrane-mediated E2 signaling in female chondrocytes and DHT signaling in male chondrocytes. PKC activity in female chondrocytes was stimulated by agonists of ERα and ERβ and required intact caveolae; PKC activity was inhibited by the E2 enantiomer and by an inhibitor of ERβ. Western blots of cell lysates co-immunoprecipitated for ERα suggested the formation of a complex containing both ERα and ERß with E2 treatment. DHT and DHT agonists activated PKC in male cells, while AR inhibition blocked the stimulatory effect of DHT on PKC. Inhibition of ERα and ERβ also blocked PKC activation by DHT. Western blots of whole-cell lysates, plasma membranes, and caveolae indicated the translocation of AR to the plasma membrane and specifically to caveolae with DHT treatment. These results suggest that E2 and DHT promote chondrocyte differentiation via the ability of ARs and ERs to form a complex. The results also indicate that intact caveolae and palmitoylation of the membrane receptor(s) or membrane receptor complex containing ERα and ERβ is required for E2 and DHT membrane-associated PKC activity in costochondral cartilage cells.

Resveratrol Attenuates Adenosine Diphosphate-Induced Platelet Activation by Reducing Protein Kinase C Activity

Inappropriate platelet activation is the key point of thrombogenesis. The aim of the present study was to investigate the effects of resveratrol (RESV), a compound extracted from the Chinese medicinal herb Polygonum cuspidatum sieb et Zucc, on the platelet activation induced by adenosine diphosphate (ADP) and its possible mechanism. The percentage of platelet aggregation and surface P-selectin-positive platelets, and the activity of protein kinase C (PKC) of platelet were observed with platelet aggregometer, flow cytometry and phosphorimaging system, respectively. RESV at 25, 50 and 100 microM showed anti-platelet aggregation and inhibition of surface P-selectin-positive platelets in a concentration-dependent manner. RESV (50 microM) inhibited the activity of PKC in the membrane fraction of platelets and decreased the percentage of membrane associated PKC activity in total PKC activity. Moreover, DL-erythro-1,3-Dihydroxy-2-aminooctadecane, an elective protein kinase C inhibitor (PKCI), and RESV had additive effects of inhibiting the percentage of platelet aggregation and surface P-selectin-positive platelets. It is suggested that RESV may inhibit platelet aggregation, the percentage of surface P-selectin-positive platelets and subsequent thrombus formation. The mechanisms may be partly relative to the decrease of the activity of PKC of platelets.

Requirement of Aldose Reductase for the Hyperglycemic Activation of Protein Kinase C and Formation of Diacylglycerol in Vascular Smooth Muscle Cells

Activation of protein kinase C (PKC) has been linked to the development of secondary diabetes complications. However, the underlying molecular mechanisms remain unclear. We examined the contribution of aldose reductase, which catalyzes the first, and the rate-limiting, step of the polyol pathway of glucose metabolism, to PKC activation in vascular smooth muscle cells (VSMCs) isolated from rat aorta and exposed to high glucose in culture. Exposure of VSMCs to high glucose (25 mmol/l), but not iso-osmotic mannitol, led to an increase in total membrane-associated PKC activity, which was prevented by the aldose reductase inhibitors tolrestat or sorbinil or by the ablation of aldose reductase by small interfering RNA (siRNA). The VSMCs were found to express low levels of sorbitol dehydrogenase, and treatment with the sorbitol dehydrogenase inhibitor CP-166572 did not prevent high-glucose-induced PKC activation. Stimulation with high glucose caused membrane translocation of conventional (alpha, beta1, beta2, and gamma) and novel (delta and epsilon) isoforms of PKC. Inhibition of aldose reductase prevented membrane translocation of PKC-beta2 and -delta and delayed the activation of PKC-beta1 and -epsilon, whereas membrane translocation of PKC-alpha and -gamma was not affected. Treatment with tolrestat prevented phosphorylation of PKC-beta2 and -delta. High glucose increased the formation of diacylglycerol (DAG) and enhanced phosphorylation of phospholipase C-gamma1 (PLC-gamma1). Inhibition of aldose reductase prevented high glucose-induced DAG formation and phosphorylation of PLC-gamma1 and PLC-beta2 and -delta. Inhibition of phospholipid hydrolysis by D609, but not by the synthetic alkyl-1-lysophospholipid 1-O-octadecyl-2-O-methyl-rac-glycerophosphocholine, or edelfosine, prevented DAG formation. Treatment with sorbinil decreased the levels of reactive oxygen species in high-glucose-stimulated VSMCs. Hence, inhibition of aldose reductase, independent of sorbitol dehydrogenase, appears to be effective in diminishing oxidative stress and hyperglycemic changes in signaling events upstream to the activation of multiple PKC isoforms and PLC-gamma1 and may represent a useful approach for preventing the development of secondary vascular complications of diabetes.

Lithium and valproic acid treatments reduce PKC activation and receptor-G protein coupling in platelets of bipolar manic patients

Dysregulated protein kinase C (PKC) distribution and activation, and abnormal receptor-G protein coupling, have been implicated in the pathophysiology of bipolar affective disorder (BD). The therapeutic effectiveness of lithium has also been correlated with its ability to reduce PKC activation and G protein-mediated signaling. We examine the cellular distribution and activation of PKC and receptor-G protein coupling in blood platelets from normal controls, patients with BD mania or schizophrenia during treatment-free state, and after lithium or valproic acid administration. PKC activity was measured under basal and 50 nM phorbol 12-myristate, 13-acetate (PMA), 1 μM serotonin or 0.5 U/ml thrombin-stimulated conditions. The coupling of G proteins to serotonin or thrombin receptors were assessed by serotonin or thrombin-mediated [ 35S]GTPγS binding to membrane Gα proteins. The results demonstrate that membrane-associated PKC activity and stimulus-induced PKC translocation are increased in BD manic, whereas stimulus-elicited PKC translocation is attenuated in schizophrenic patients. Lithium and valproic acid treatments attenuated the stimulus-induced PKC translocations to a similar degree and decreased PKC activity in both cytosolic and membranous fractions after two weeks of drug administration. An increase in 5-HT or thrombin stimulated [ 35S]GTPγS binding to Gα proteins was detected in BD manic but not in schizophrenic patients although basal [ 35S]GTPγS binding was not different across the diagnostic groups. Lithium and valproic acid treatments similarly reduced receptor-G protein coupling with comparable time courses. Thus, increased membrane-associated PKC, cytosol to membrane PKC translocation and receptor-G protein coupling in platelets of BD manic patients were alleviated by lithium or valproic acid treatments.

Advanced glycosylation end products stimulate collagen mRNA synthesis in mesangial cells mediated by protein kinase C and transforming growth factor–beta

Advanced glycosylation end products (AGE) seem to be implicated in the pathogenesis of diabetic nephropathy. The present study has examined the effects of AGE on protein kinase C (PKC) activity and transforming growth factor–β1 (TGF-β1) in relation to collagen gene regulation in cultured human mesangial cells (HMCs). Quiescent HMCs were exposed to serum-free media containing bovine serum albumin (BSA), AGE-modified BSA (AGE-BSA), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM). AGE-BSA (200 μg/mL) induced a peak membrane-associated PKC activity, particularly PKC-a, at 4 hours. AGE-BSA stimulated α1(I) and α1(IV) collagen mRNA expression after 24-hour incubation with HMCs, which remained elevated until hour 60. HMCs incubated with AGE-BSA induced a significant inhibition of cell proliferation compared with cells incubated with BSA. AGE-BSA stimulated TGF-β mRNA and protein expression in HMCs. The TGF-β secreted by HMCs was shown by CCL-64 mink lung cell assay to be bioactive. In contrast, BSA-AM did not affect either collagen or TGF-β mRNA or protein expression in HMCs. The stimulatory effects of AGE-BSA on collagen gene regulation in HMCs could be negated by the pretreament of HMCs with GF 109203X for 30 minutes or with phorbol myristate acetate for 24 hours before AGE-BSA administration. Neutralizing antibody to TGF-β inhibited increased collagen mRNA expression by HMCs exposed to AGE-BSA. These results suggest that AGE-BSA stimulates collagen mRNA expression by activating PKC and the transcriptional upregulation of TGF-β1 in HMCs. Thus, PKC and TGF-β may function as key signaling intermediaries in the AGE–up-regulated collagen gene expression pathway in HMCs. (J Lab Clin Med 2001;138:59-68)

ATP-Sensitive potassium channels modulate glucose transport in cultured human skeletal muscle cells.

Several lines of evidence suggest that ATP-sensitive potassium (KATP) channels are involved in glucose uptake by insulin target tissues. The aim of the present study was to prove directly the effect of KATP channel activity on glucose transport into cultured human skeletal muscle cells. We used potassium channel openers PCO-400 and nicorandil alone or in combination with channel blockers glibenclamide and gliclazide to examine their effects on insulin- or high glucose concentration-induced glucose uptake using 2-deoxy-D-3H-glucose or 3-O-methyl-D-3H-glucose as tracer, respectively. PCO-400 inhibited the basal (non-stimulated) uptake of 2-DG or 3-OMG at the glucose concentration of 5 mM. PCO-400 and nicorandil dose-dependently inhibited insulin-stimulated glucose uptake, and their inhibitory effects were reversed by glibenclamide or gliclazide. In addition, PCO-400 inhibited high glucose concentration-facilitated glucose transport in the absence of insulin, and this effect was also antagonized by both sulfonylurea drugs. Regarding the mechanism by which KATP channels modulate glucose transport, we focused on protein kinase C (PKC), because PKC has been supposed to participate in both insulin- and high glucose concentration-stimulated glucose transport. PMA (phorbol 12-myristate 13-acetate) dose-dependently reversed the PCO-400-induced suppression of insulin-stimulated glucose uptake. On the other hand, PCO-400 at the concentration that inhibited glucose uptake caused no alteration of membrane-associated PKC activity in the presence of insulin or PMA. From these results we conclude that KATP channels modulate the basal and insulin-or high glucose level-stimulated glucose transport in skeletal muscle through a mechanism independent of PKC.

1,25(OH)(2)-vitamin D(3) affects the subcellular distribution of protein kinase C isoenzymes in rat duodenum: influence of aging.

We have previously shown that the steroid hormone 1, 25-dihydroxy-vitamin D(3) [1,25(OH)(2)D(3)] stimulates total cell protein kinase C (PKC) activity in rat duodenum, an effect that is severely impaired in old animals. We further examined the role of 1, 25(OH)(2)D(3) on PKC as it relates to aging by measuring hormone-induced changes in subcellular localization of PKC activity and isoenzymes in duodenal mucosae from young (three-month-old) and aged (24-month-old) rats. Short treatment of duodenum with 1, 25(OH)(2)D(3) (0.1 nM, 1 min) increased membrane-associated PKC activity, whereas it decreased the activity in the cytosol of young rats but was without significant effect in aged animals. Furthermore, the ability to translocate was present in young animals after a short treatment with the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA; 100 nM) or dioctanoyl-glycerol (50 microM), whereas the ability was absent in aged rats, suggesting that PKC function was impaired with aging independent of agonist stimulation. The expression of specific PKC isoenzymes and changes in their subcellular distribution after short exposure of the duodenum to the hormone were determined. Western blot analysis of total homogenates using antibodies to various PKC isoforms allowed detection of PKC alpha, beta, and delta. The expression of the straight theta and the zeta isoforms was in addition demonstrated by reverse transcription-polymerase chain reaction. The pattern of isoenzymes present in the duodenum was unaffected by aging. In young rats, 1, 25(OH)(2)D(3) translocates PKC alpha, beta, and delta to the membrane and nucleus; however, no translocation of PKC isoforms was observed in 24-month-old animals in response to the hormone. In summary, in rat duodenum, 1,25(OH)(2)D(3) modulation of PKC activity and isoenzyme subcellular distribution are impaired with aging and may explain age-induced alterations in the intestinal processes under the control of the hormone.

Inhibition of TPA-induced protein kinase C and transcription activator protein-1 binding activities by theaflavin-3,3'-digallate from black tea in NIH3T3 cells.

Tea is one of the most popular beverages in the world. Several reports have shown that both green tea and black tea were able to inhibit tumor cell proliferation in animal models. In this study, we investigated the inhibitory effects of black tea polyphenols including theaflavin (TF-1), the mixture (TF-2) of theaflavin-3-gallate (TF-2a), and theaflavin-3'-gallate (TF-2b), theaflavin-3,3'-digallate (TF-3), thearubigin (TR), and a major green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) on 12-O-tetradecanoylphorbol 13-acetate (TPA)-induced protein kinase C (PKC) and transcription activator protein-1 (AP-1) binding activities in NIH3T3 cells. On analysis of PKC activity with partial purified preparation, TPA (100 ng/mL) treatment was able to elevate membrane-associated PKC activity approximately 3-fold, and treatment with TF-3 (20 microM) and EGCG (20 microM) showed 94.5% and 9.4% suppression on TPA-induced PKC activity, respectively. Translocation of PKCalpha protein from cytosol to membrane was detected in TPA-treated NIH3T3 cells, and TF-3 was able to block its translocation. By in vitro kinase assay using myelin basic protein (MBP) as a PKC-specific substrate, we found that TPA treatment was able to increase PKC kinase activity by detection of phosphorylated MBP protein and TF-3 showed strongest inhibitory effect on its phosphorylation while EGCG was shown to be less effective. We also analyzed the AP-1 binding activity by electrophoretic mobility shift assay and c-Jun gene expression by northern blot and western blot, the results showed that TF-3 is the most potent inhibitor on TPA-induced AP-1 binding activity and c-Jun gene expression among these five tea polyphenols. Our results might provide new molecular basis for understanding the inhibitory effects of tea polyphenols on TPA-mediated tumor promotion.

Stimulation of Membrane-Associated Protein Kinase-C Activity in Spleen Lymphocytes by hPTH-(1–31)NH 2, its Lactam Derivative, [Leu 27]-cyclo(Glu 22-Lys 26)-hPTH-(1–31)NH 2, and hPTH-(1–30)NH 2

Human parathyroid hormone, hPTH-(1–34), stimulates adenylyl cyclase and phosphatidylinositol- bisphosphate-specific phospholipase-C (PIP 2-PLC), as indicated by increased membrane-associated protein kinase C (PKC) activity in ROS 17/2 rat osteosarcoma cells. The C-terminally truncated hPTH-(1–31)NH 2 stimulates adenylyl cyclase as strongly as hPTH-(1–34) in these cells, but it does not stimulate PKC activity. Even [Leu 27]-cyclo(Glu 22-Lys 26)-hPTH-(1–31)NH 2, a 6-fold stronger adenylyl cyclase stimulator than hPTH-(1–34), cannot stimulate PKC activity in ROS cells. Therefore PTH required its 32–34 region to stimulate PIP 2-PLC/PKCs in this osteosarcoma line. In contrast, hPTH-(1–31)NH 2 [Leu 27]-cyclo(Glu 22-Lys 26)-hPTH-(1–31)NH 2 and even hPTH-(1–30)NH 2 can stimulate PKC activity in freshly isolated rat spleen lymphocytes as strongly as hPTH-(1–34)NH 2. The difference in the ability of membrane-associated PKC activity in spleen lymphocytes, but not in ROS cells, to be stimulated by C-terminally truncated PTH fragments might be due to different receptor densities or to the lymphocyte's atypical PTH/PTHrP receptor.

Modulation of protein kinase C by taurolithocholic acid in isolated rat hepatocytes.

The protein kinase C (PKC) family of isoenzymes plays a key role in the regulation of hepatocellular secretion. The hydrophobic and cholestatic bile acid, taurolithocholic acid (TLCA), acts as a potent Ca++ agonist in isolated hepatocytes. However, its effect on PKC isoforms has not been elucidated. Here we investigate the effects of TLCA at low micromolar concentrations on the distribution of PKC isoforms and on membrane-associated PKC activity. The distribution of PKC isoforms was determined in isolated rat hepatocytes in short-term culture using Western blotting and immunofluorescence techniques. PKC activity was measured radiochemically. TLCA (10 micromol/L) induced selective translocation of epsilon-PKC by 47.9% +/- 20.5% (P <.02 vs. controls; n = 7), but not of alpha-, delta-, and zeta-PKC to the hepatocellular membranes, whereas the phorbol ester, phorbol 12-myristate 13-acetate (PMA) (1 micromol/L) caused translocation of all mobile isoforms, alpha-, delta-, and epsilon-PKC, as shown by immunoblotting. Immunofluorescence studies demonstrated selective translocation of epsilon-PKC to the canalicular membranes of isolated rat hepatocyte couplets by TLCA (10 micromol/L), but predominant translocation to intracellular and basolateral membranes by PMA (1 micromol/L). Both TLCA (10 micromol/L) and PMA (1 micromol/L) stimulated membrane-bound PKC activity by 60.5% +/- 45. 8% (P <.05 vs. controls; n = 5) and 72.4% +/- 37.2% (P <.05; n = 5), respectively. TLCA at lower concentrations (5 micromol/L) was less effective. Because activation of epsilon-PKC has been associated with impairment of vesicle-mediated targeting and insertion of membrane proteins in secretory cells, it is attractive to speculate that TLCA reduces bile secretory capacity of the liver cell by activation of epsilon-PKC at the canalicular membrane.

Comparison of superoxide production, protein kinase C and tyrosine kinase activities in neutrophils from neonatal calves and cows

Superoxide (O 2 −) production and intracellular signal transduction of neutrophils were evaluated in five Holstein dairy calves and five lactating cows. Opsonised zymosan (OPZ)-induced O 2 − production by neutrophils from neonatal calves was significantly higher (P<0·01) than that of neutrophils of cows, whereas heat-aggregated IgG (H-agg.IgG)- and phorbol myristate acetate (PMA)-induced O 2 − production of neutrophils were significantly lower (P≤0·01) than those of cows. To clarify the functional differences of intracellular signal transduction in neutrophils between neonatal calves and cows, the activities of protein kinase C (PKC) and tyrosine phosphorylation were evaluated in OPZ-, H-agg.IgG- and PMA-stimulated neutrophils. Membrane-associated PKC activity of OPZ-stimulated neutrophils from neonatal calves was significantly higher (P<0·05) than that of cows, whereas PKC activity in membrane-associated fractions of H-agg.IgG-stimulated neutrophils from neonatal calves was significantly lower (P<0·05) than that of cows. A significant difference was not found in membrane-associated PKC activity of neutrophils stimulated with PMA between neonatal calves and cows. The amount of tyrosine phosphorylated 100 kDa protein in neutrophils from neonatal calves stimulated with OPZ, H-agg.IgG and PMA were 1926, 67·8 and 97·2 per cent of those of cows, respectively. These results indicate that complement receptor type 3 (CR3)- and Fc receptor (FcR)-mediated O 2 − producing activities of neutrophils are clearly different between neonatal calves and cows. This phenomenon may be associated with the age-related changes in intracellular signal transduction of neutrophils including PKC activity and tyrosine phosphorylation of cellular protein.

Nicotine-like effects of cotinine on protein kinase C activity and noradrenaline release in bovine adrenal chromaffin cells.

1. We studied the effect of cotinine, a slowly eliminated metabolite of nicotine, on protein kinase C (PKC) distribution and noradrenaline release in primary cultured bovine adrenal chromaffin cells. Changes in PKC activity were detected by [3H]-phorbol-12,13-dibutyrate binding, histone phosphorylation assay and by Western blot. 2. Cotinine (10-32 mM) increased phorbol binding to chromaffin cells in response to 10 min but not to 24 h exposure. The increased binding was reversed by a nicotinic antagonist hexamethonium (10 microM). 3. Cotinine (10 mM, 30 min) also increased membrane-associated PKC activity and membrane-associated PKC alpha and epsilon immunoreactivity. 4. Cotinine (0.1-32 mM for 10 s to 20 min) dose- and time-dependently increased the release of preloaded [3H]-noradrenaline from the cultured cells. The release increased with increasing duration of the contact period. In treatments lasting 1 min or longer, a peak effect was followed by a reduced response at higher concentrations. 5. We confirm the earlier findings that cotinine is biologically active, and conclude that its effects are at least partly mediated via nicotinic cholinergic receptors and through PKC.

Enhancement of cytolytic activity of human peripheral blood lymphocytes by sodium periodate (IO4) possible involvement of protein kinase C

Sodium periodate (IO 4) exerts a number of biological effects including the enhancement of lymphocyte activation. In this study, we investigated its effects on cytotoxicity of human peripheral blood lymphocytes (PBL) and explored the mechanism whereby it exerted these effects. In vitro treatment of human PBL with IO 4 augmented their cytotoxicity against K562 myelogenous leukemia cells. IO 4 oxidative treatment increased the frequency of effector-to-target cell binding. It also increased cellular ATP levels in effector cells, suggesting that the post-binding cytolytic functions of these cells were also enhanced after treatment with IO 4. Moreover, IO 4 treatment significantly increased the protein kinase C (PKC) activity of effector cells and induced the translocation of activity in the membrane fraction from the cytosol. H-7, a potent PKC inhibitor, significantly reduced this enhancement of membrane-associated PKC activity at 10 μM and significantly reduced the enhanced cytotoxicity of PBL at the same concentration. These results indicated that IO 4 enhanced the binding capacity and post-binding cytolytic functions of PBL and that PKC activation was one mechanism to explain the IO 4-induced cellular activation.

Protein kinase C activity is increased in rat heart during the early hyperdynamic phase of sepsis.

Changes in protein kinase C (PKC) (calcium- and phospholipid-dependent protein kinase) activity in rat heart during different cardiodynamic phases of sepsis were studied in an attempt to understand the pathophysiology of altered myocardial function during sepsis. Sepsis was induced by cecal ligation and puncture. Experiments were divided into three groups: control, early sepsis, and late sepsis. Early and late sepsis refers to those animals sacrificed at 9 and 18 h, respectively, after cecal ligation and puncture. Cardiac PKC was extracted and partially purified by ammonium sulfate fractionation and diethylaminoethyl-cellulose chromatography. PKC activity was assayed on the basis of the rate of incorporation of 32P from [gamma-32P]adenosine triphosphate into histone. The results show that during early sepsis, cytosolic PKC activity was increased by 42-73%, whereas membrane associated PKC activity was unchanged. During late sepsis, both cytosolic and membrane associated PKC activities remained unchanged. Kinetic analysis of the data on cytosolic PKC during the early phase of sepsis reveals that the Vmax (maximal velocity) values for Ca2+, phosphatidylserine, and diacylglycerol were increased by 58, 42, and 50%, respectively, with no changes in their Km (substrate concentration required for half-maximal enzyme activity) values. These data indicate that cytosolic PKC activity was activated in rat heart during the early hyperdynamic phase of sepsis. Because PKC mediated phosphorylation plays an important role in regulating myocardial contractility, an activation in cytosolic PKC may contribute to the development of a hypercardiodynamic state during the early phase of sepsis.

The effects of acute and repeated cocaine injections on protein kinase C activity and isoform levels in dopaminergic brain regions

The present study was designed to assess the effects of acute and repeated cocaine exposure on protein kinase C (PKC) activity and the levels of calcium-dependent isoforms of PKC in mesocorticolimbic and nigrostriatal dopamine brain regions. Animals received repeated injections of saline or cocaine and were challenged with saline or cocaine 24 h or 7 days after the last of their daily injections. Animals were sacrificed 2, 6 or 24 h after the challenge injection and their brains were dissected and used in PKC studies. The data demonstrated that previously reported cocaine-induced increases in PKC activity in the ventral tegmental area are transient and not associated with changes in the levels of calcium-dependent isoforms of PKC. In addition, there was a decrease in membrane-associated PKC activity, with a concomitant increase in the levels of PKC β I in the medial prefrontal cortex 24 h after the last injection of cocaine. These data suggest that changes in PKC activity in the ventral tegmental area may be involved in the initiation of sensitization whereas changes in PKC activity in the medial prefrontal cortex may be related to the expression of the sensitized response to cocaine.

Antiproliferative effect in human prostatic smooth muscle cells by nitric oxide donor.

We obtained a primary culture of prostatic cells through explantation from patients with benign prostatic hyperplasia. Structural morphology, immunohistochemical staining, and growth characteristics of these cells demonstrate that they are consistent with the population of smooth muscle cells (SMCs). We examined the influence of a nitric oxide donor, sodium nitroprusside (SNP), on the regulation of human prostatic SMC proliferation. SNP exhibited a concentration-dependent (0.1-10 microM) inhibition of fetal calf serum-induced proliferation in human prostatic SMCs. In addition, growth-inhibitory responses to 8-bromo-cGMP (1-30 muM) were observed. However, the responses to SNP were significantly diminished by the presence of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (3 microM; a selective guanylate cyclase inhibitor). Furthermore, SNP induced an increased concentration-dependent accumulation of intracellular cGMP in human prostatic SMCs. After 48-hr period of deprivation of serum, cells were restimulated with serum to permit cell cycle progression. The addition of SNP (10 microM) at various times after the addition of serum to serum-deprived cells showed maximal inhibition of cell proliferation even when added 6 hr after the serum. This blocking effect of cell cycle progression was lost gradually as the delay from serum to SNP application increased from 6 to 18 hr. The membrane-associated protein kinase C (PKC) activity was studied in human prostatic SMCs; results showed that fetal calf serum (10%, v/v) significantly increased membrane-associated PKC activity. SNP (10 muM), which had little effect on basal kinase activity, completely abolished serum-induced augmentation of PKC activity. Therefore, we suggest that SNP mediates its antiproliferative effect by the inhibition of PKC activity on human prostatic SMCs; furthermore, its antiproliferative effect occurs at the early G1 phase of the cell cycle.

Ceramide Promotes Calpain-Mediated Proteolysis of Protein Kinase C β in Murine Polymorphonuclear Leukocytes

Ceramide has been recognized as an important second messenger in intracellular signaling. We demonstrate here that ceramide promotes the down-regulation of protein kinase C (PKC) activity in phorbol ester-stimulated murine polymorphonuclear leukocytes (PMNs). As reported previously, treatment of PMNs with phorbol ester caused a translocation of PKC from the cytosolic to the membrane fractions. When PMNs were pretreated with cell-permeable ceramide analogue, C2-ceramide, the membrane-associated PKC activity was rapidly down-regulated by phorbol ester stimulation. E64-d, a potent inhibitor of calpain which proteolyzes PKC, eliminated the rapid down-regulation of PKC activity. By hydroxyapatite column chromatography and Western blotting, the predominant PKC isoform was PKC β with a small amount of PKC α in murine PMNs. We found that ceramide strikingly promoted calpain-mediated proteolysis of PKC βin vitro.Ceramide was also shown to inhibit [3H]phorbol 12,13-dibutyrate(PDBu) binding to PKC β. Moreover, we show that ceramide stimulates PKC β autophosphorylation. These results suggest that ceramide directly activates PKC β and promotes calpain-mediated proteolysis in murine PMNs.

Urinary trypsin inhibitor, a Kunitz-type protease inhibitor, modulates tumor necrosis factor-stimulated activation and translocation of protein kinase C in U937 cells.

Urinary trypsin inhibitor (UTI) is a Kunitz-type protease inhibitor. We have reported that UTI inhibited TNF-induced urokinase (uPA) production via a protein kinase C (PKC)-dependent mechanism. It is likely that UTI suppresses tumor cell invasion and metastasis by a mechanism, possibly by inhibiting uPA production. In the present study, we attempted to determine how UTI is associated with PKC, and how UTI is involved in uPA-dependent tumor cell invasion and metastasis. The increments of membrane-associated PKC activity by TNF were subsequently accompanied by a rapid loss of cytosol-associated PKC activity in U937 leukemia cells. Semi-quantitative immunoblotting of membrane and cytosol fractions showed that the translocation of PKC-alpha, -beta, and -epsilon were blocked by the addition of UTI in cells stimulated with TNF but not in cells stimulated with PMA, demonstrating that PKC itself is not sensitive to UTI. This effect was dependent on the carboxyl-terminus of UTI. In addition, UTI neither inhibited TNF binding to cellular receptors nor inactivate PKC and uPA activities directly. Taken together, the experiments suggest that the carboxyl-terminus of UTI may inhibit the PKC-signalling pathways upstream of diacylglycerol by a mechanism, possibly by interrupting the coupling of receptor and effector systems. UTI was shown to have an interesting new function besides being a protease inhibitor. This is the first report that UTI has a selective inhibition of TNF-activated PKC. We conclude that UTI suppresses tumor cell invasion and metastasis by a mechanism that UTI inhibits TNF-stimulated uPA production via a PKC-dependent mechanism.

Transforming growth factor-beta1 inhibits membrane association of protein kinase C alpha in a human prostate cancer cell line, PC3.

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-beta1 (TGF-beta1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-beta1 in PC3, a human prostate cancer cell line. PKC alpha, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKC alpha were decreased by a growth-inhibitory dose of TGF-beta1. The response to TGF-beta1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKC alpha protein. TGF-beta1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKC alpha's membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKC alpha, is an early event associated with TGF-beta1 postreceptor signaling that might mediate suppression of cell proliferation.

Transforming Growth Factor- 1 Inhibits Membrane Association of Protein Kinase C in a Human Prostate Cancer Cell Line, PC3

The postreceptor signaling pathway(s) that mediates the effects of transforming growth factor-β1 (TGF-β1) is incompletely understood. The present study investigated the involvement of protein kinase C (PKC) in the growth-inhibitory action of TGF-β1 in PC3, a human prostate cancer cell line. PKCα, the only conventional PKC isoform detected in PC3 cells, appeared to be constitutively active based on its presence in both Triton-soluble membrane fraction and cytosol. However, levels of membrane-associated PKCα were decreased by a growth-inhibitory dose of TGF-β1. The response to TGF-β1 was rapid (within 5 min), time dependent, isoform specific, and occurred without apparent changes in levels of total PKCα protein. TGF-β1 also decreased the levels of membrane-associated PKC activity coincident with its inhibitory effect on PKCα’s membrane association. Inhibition of PKC activity appeared to be associated with growth inhibition in PC3 cells, because chelerythrine (a specific PKC inhibitor) likewise decreased cell proliferation. Taken together, our data suggest that inhibition of PKC activity, at least in part due to inactivation of PKCα, is an early event associated with TGF-β1 postreceptor signaling that might mediate suppression of cell proliferation.