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At least 19 recordsLinked to original sources

PubMed1992

Postischemic binding of [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate in the gerbil brain: an autoradiographic study.

Postischemic alteration of second messenger systems was investigated in the Mongolian gerbil, utilizing [3H]phorbol 12,13-dibutyrate and [3H]inositol 1,4,5-trisphosphate receptor autoradiography. Transient ischemia was induced for 10 min, and animals were allowed to survive for various recirculation periods of up to one month. [3H]Phorbol 12,13-dibutyrate binding in selectively vulnerable areas showed no significant change 1-24 h after ischemia except for a transient decline in a few regions. Thereafter, the binding in most of the selectively vulnerable areas showed significant alteration 48 h or seven days after ischemia. Interestingly, dentate molecular layer which was resistant to ischemia showed a significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites. One month after ischemia, [3H]phorbol 12,13-dibutyrate binding showed significant reduction only in the striatum and the hippocampal CA1 sector where severe neuronal damage was seen morphologically. A significant elevation in the number of [3H]phorbol 12,13-dibutyrate binding sites was still seen in the dentate molecular layer one month after ischemia. In contrast, [3H]inositol 1,4,5-trisphosphate binding showed significant reduction in the selectively vulnerable regions 1-24 h after ischemia. Thereafter, [3H]inositol 1,4,5-trisphosphate binding in most of the selectively vulnerable areas markedly decreased up to one month after ischemia. In the dentate molecular layer, [3H]inositol 1,4,5-trisphosphate binding also showed significant reduction during recirculation except for a slight recovery 48 h and seven days after ischemia. One month after ischemia, the binding in all regions showed significant reduction. These results suggest that postischemic alteration of two second messenger (protein kinase C and inositol 1,4,5-trisphosphate) binding sites was produced with different processes in selectively vulnerable areas.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Comparison of the vasoconstrictor responses induced by endothelin and phorbol 12,13-dibutyrate in bovine cerebral arteries.

The vascular effects of endothelin-1 (ET-1) were compared with those elicited by phorbol 12,13-dibutyrate (PDB), an activator of the protein kinase C (PKC), to analyze the involvement of this enzyme on ET-1 responses. PDB and ET-1 caused slow-developing contractions (sustained and transient, respectively), which were reduced by the PKC inhibitor, staurosporine (1 and 10 nM). Only the contractile effects evoked by ET-1 were reduced in Ca-free medium and by the Ca channel antagonist, nifedipine (1 microM), and increased by the Ca channel agonist, BAY K 8644 (10 nM). PDB (10 and 30 nM) preincubation reduced the vasoconstriction elicited by 5-hydroxytryptamine (5-HT; 0.01, 0.1 and 1 microM) in a way dependent on phorbol concentration and preincubation time, whereas ET-1 (1 nM) increased the contractile response to 5-HT (0.1 microM). Furthermore, PDB (0.1 microM) also reduced the responses elicited by ET-1 (30 microM) and vice versa. ET-1 (0.1 microM) induced transient translocation of PKC activity from the cytosol to the membrane, which was less than that produced by PDB (0.1 microM). Electrical stimulation induced [3H]noradrenaline (NA) release, which was increased by PDB (10 and 100 nM) and not affected by ET-1 (10 nM). These results indicate: (1) the responses induced by PDB and ET-1 were independent and dependent on extracellular Ca, respectively; (2) PKC is involved in NA release and 5-HT responses, but mainly in desensitization of these responses, and (3) PKC is activated by ET-1 and is implicated in vascular actions of ET-1, but other mechanisms, such as the activation of ET-1 receptors and opening of dihydropyridine-sensitive Ca channels also appear to be involved.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1992

Phorbol esters induce nitric oxide synthase activity in rat hepatocytes. Antagonism with the induction elicited by lipopolysaccharide.

The incubation of primary cultures of rat hepatocytes with lipopolysaccharide (LPS) or biologically active phorbol esters promotes the release of nitric oxide to the incubation medium. This process is the result of the induction of the Ca(2+)-and calmodulin-independent form of nitric oxide synthase. Both the release of nitric oxide to the incubation medium and the expression of nitric oxide synthase activity exhibited a lag period of about 45-60 min after cell stimulation. Exposure of hepatocytes to both stimuli produced an antagonistic effect on nitric oxide release, with a half-maximal inhibition obtained with 14 nM phorbol 12,13-dibutyrate at saturating concentration of LPS. Incubation of cells with alpha-phorbol 12,13-didecanoate failed to counteract the effect of LPS or to induce nitric oxide synthase, suggesting that activation of protein kinase C was involved in this process.

Amino Acid Oxidoreductases↗

PubMed1992

Prolonged activation of alpha 1 adrenoceptors induces down-regulation of protein kinase C in vascular smooth muscle.

Sustained exposure of vascular smooth muscle to catecholamines results in desensitization of alpha 1-adrenoreceptor-mediated vascular smooth muscle contraction. The present study was designed to determine the effects of prolonged exposure of blood vessels to catecholamines on protein kinase C (PKC) activity. Incubation of rat aortic smooth muscle with 10 microM norepinephrine (NE) for 4 h resulted in a threefold decrease in sensitivity of the contractile response of rat aortic smooth muscle to the phorbol ester 4 beta-phorbol 12,13-dibutyrate (PDBu); this loss in sensitivity was dependent on the presence of endothelium. NE induced a 45% decrease in enzymatic activity of the soluble and particulate forms of PKC. With [3H]PDBu used to label phorbol ester receptor binding sites in the aorta, there was a 34% decrease in [3H]PDBu binding sites in NE-treated blood vessels without change in binding affinity for the ligand. To determine whether this loss in enzymatic activity and [3H]PDBu binding resulted from a decrease in the quantity of the enzyme, Western blot analyses were performed using a monoclonal antibody (MoAb) against PKC. This approach confirmed the presence of an 80-Kd immunoreactive PKC in the soluble fraction of rat aortic smooth muscle and demonstrated a 44% decrease in the amount of PKC in blood vessels after sustained exposure to catecholamines. Our results demonstrate that prolonged activation of alpha-adrenoceptors in blood vessels leads to down-regulation of PKC which may contribute to desensitization of contraction mediated by vasoconstrictors.

Animals↗

PubMed1992

Inhibition of human neutrophil protein kinase C activity by the antimalarial drug mefloquine.

Mefloquine (alpha-(2-piperidyl)-2,8-bis(trifluoromethyl)-4-quinolinemethanol) , an antimalarial drug, has been shown to inhibit human neutrophil functions, particularly oxygen-dependent bactericidal activity. Since calcium- and phospholipid-dependent protein kinase C (PKC) has a central role in the regulation of this function, we hypothesized that its activity might be altered by mefloquine. We found that mefloquine directly inhibited PKC in a dose-dependent manner, with an IC50 of 45 microM. This inhibition appeared to be non-competitive with respect to ATP, histone and phosphatidylserine. In addition, mefloquine inhibited the binding of [3H]phorbol 12,13 dibutyrate to PKC, indicating that it interacts with the regulatory domain of PKC. By contrast, mefloquine had little or no effect on neutrophil cAMP-dependent protein kinase or its catalytic subunit. Phorbol myristate acetate-induced protein phosphorylation in intact neutrophils was also inhibited by preincubation with mefloquine at concentrations similar to those inhibiting superoxide anion production. These data suggest that inhibition of neutrophil functions by mefloquine may be due to the inhibition of cellular PKC and that mefloquine could have further biological effects in situations in which PKC is involved.

Dose-Response Relationship, Drug↗

PubMed1992

Purification and characterization of protein kinase C from the nematode Caenorhabditis elegans.

Protein kinase C (PKC) of Caenorhabditis elegans was identified by enzymatic activity and [3H]phorbol 12,13-dibutyrate binding after DEAE-Sephacel column chromatography of a crude cytosolic extract. Ca(2+)-dependent activation of nematode PKC was observed in the presence of phosphatidylserine. The enzyme was maximally activated by 1,2-dioleoylglycerol or phorbol 12-myristate 13-acetate in the presence of phosphatidylserine and Ca2+. Hydroxyapatite column chromatography showed only one peak of PKC activity with histone H1 and myelin basic protein as substrates. The enzyme was purified to near homogeneity by sequential chromatography on polylysine-agarose and phosphatidylserine affinity columns. The purified protein showed a molecular mass of 79 kDa on SDS/PAGE. The substrate specificity of the C. elegans enzyme was shown to be different from that of mammalian PKCs. Here we describe some of the properties of the nematode enzyme.

Animals↗

PubMed1992

Regulation of M-phase progression in Chaetopterus oocytes by protein kinase C.

We have examined the presence of protein kinase C in oocytes of Chaetopterus pergamentaceus and its role in the initiation of germinal vesicle breakdown (GVBD). First, we demonstrated that the oocytes contain a phospholipid- and calcium-dependent protein kinase, protein kinase C (PKC). Since PKC is the primary intracellular receptor for phorbol esters, we tested the ability of phorbol 12,13-dibutyrate (PDBu) to induce GVBD and compared several critical events and processes involved in GVBD induced by PDBu to those induced normally (by seawater). Seawater and 100-200 nM PDBu induced chromosome condensation, spindle formation, and spindle migration over a similar time course. Both treatments induced similar alterations in the SDS-PAGE pattern of newly synthesized proteins. The synthesis of polypeptides of approximately 46 and 54 kDa increased specifically. Both treatments increased oocyte protein phosphorylation, especially of proteins of 22, 32, 46, 55, 64, and 84 kDa. Both treatments resulted in the activation of an M-phase-specific histone H1 kinase activity, which demonstrates the appearance of maturation-promoting factor. Staurosporine, a potent protein kinase C inhibitor, blocked GVBD and the activation of M-phase-specific H1 kinase, whereas HA1004, which preferentially antagonizes protein kinase A, had no effect. The results of this study demonstrate that protein kinase C can activate a wide spectrum of essential biochemical and morphological processes involved in GVBD. Further, these studies suggest that protein kinase C elicits GVBD by activating maturation-promoting factor and support the hypothesis that protein kinase C plays an essential role in oocyte maturation in this species.

Alkaloids↗

PubMed1992

Platelet-activating factor stimulates phosphoinositide turnover in neurohybrid NCB-20 cells: involvement of pertussis toxin-sensitive guanine nucleotide-binding proteins and inhibition by protein kinase C.

Platelet-activating factor (PAF) is an unusually potent phospholipid known to be produced by neuronal cells and to modulate cerebral blood flow and metabolism. In previous studies with NCB-20 cells, we reported that PAF induced a significant mobilization of intracellular free Ca2+ ([Ca2+]i), which was inhibited by PAF antagonists. The increase was the result of release from intracellular stores and influx from extracellular sources. The present study was designed to characterize further PAF receptor-mediated cellular signal-transduction mechanisms in myo-[3H]inositol-labeled cells. PAF induced a concentration-dependent increase in phosphatidylinositol (Pl) metabolism, with EC50 values of 1.96 +/- 0.62 nM and 1.12 +/- 0.50 nM for inositol trisphosphate (IP3) and inositol monophosphate (IP1) formation, respectively (four experiments). The maximal production of IP3 and IP1 induced by 50 nM PAF was 254 +/- 34% and 178 +/- 25% over the basal, respectively (four experiments). PAF-induced Pl metabolism was concentration-dependently inhibited by the PAF antagonist BN50739, with an IC50 value of 6.48 +/- 0.52 nM (four experiments). The protein kinase C (PKC) activator phorbol 12,13-dibutyrate concentration-dependently inhibited PAF-induced Pl metabolism and [Ca2+]i mobilization in NCB-20 cells, of NCB-20 cells with pertussis toxin (PTX) resulted in a concentration-dependent inhibition of PAF-induced IP3 production and intracellular Ca2+ release, with a maximal reduction of 66.9 +/- 3.5% and 63 +/- 6.1%, respectively, at 300 ng/ml PTX. PTX in the presence of [32P]NAD specifically [32P]ADP-ribosylated a 38-kDa protein in membranes prepared from NCB-20 cells. Pretreatment of the cells with PTX resulted in a concentration-dependent inhibition of subsequent 32P-labeling of the toxin substrate in the membranes and correlated with the uncoupling of PAF-induced IP3 formation. PAF (0.01-10 nM) elicited a concentration-related stimulation in guanosine 5'-O-(3-[35S]) triphosphate ([35S]GTP gamma S) binding to G alpha i(1,2) proteins, which was inhibited by the PAF antagonist BN50739. PAF at 10 nM also increased [35S]GTP gamma S binding to G alpha s and G alpha o. PAF-evoked activation of G alpha i(1,2) and G alpha o was reduced by preincubation with PTX. Our results reveal that neuronal cells possess PAF receptors linked through guanine nucleotide-binding proteins to phospholipase C and receptor-operated Ca2+ channels that are regulated by PKC. Both PTX-sensitive and -insensitive guanine nucleotide-binding proteins appear to couple the PAF receptor to activation of phospholipase C and the increase in [Ca2+]i. These results contribute to the further understanding of the mechanisms behind PAF actions on neuronal cells.

Adenosine Diphosphate↗

PubMed1992

Ba2+ current oscillations modulated by cyclic AMP and phorbol esters in ras-transformed fibroblasts.

An oscillatory influx of divalent cations was measured as Ba2+ inward currents (Ba2+ current oscillations) by voltage-clamp recording in v-Ki-ras-transformed NIH/3T3 (DT) fibroblasts after activation with bradykinin or serum. Application of forskolin or dibutyryl cyclic AMP onto DT cells initiated Ba2+ current oscillations. Increasing intracellular cyclic AMP reduced the amplitude but increased the frequency of the Ba2+ current oscillations. Activation of protein kinase C by phorbol esters terminated Ba2+ current oscillations. No inhibition of Ba2+ current oscillations by phorbol esters was observed in down-regulated cells that had been pretreated with phorbol esters for 24 hrs. The results suggest that Ba2+ current oscillations are regulated by intracellular second messengers.

3T3 Cells↗

PubMed1992

Enhanced expression of complement regulatory proteins on thyroid epithelial cells of Graves' disease.

Cytotoxic anti-thyroid microsomal autoantibodies are highly prevalent in sera of patients with Graves' disease, but in Graves' disease thyroid tissues rarely show destructive changes. We postulated that this might be due to membrane-associated complement regulatory proteins which protect target cells from injury by complement activation. We, therefore, investigated the expression of membrane attack complex inhibitory factor (MACIF) and decay accelerating factor (DAF) in the thyroid tissues from patients with Graves' disease, Hashimoto's thyroiditis, thyroid adenocarcinoma and normal human thyroid tissues. We found a high level of expression of MACIF and DAF in Graves' thyroid tissues. Using the membrane immunofluorescence and cell-ELISA techniques, we also investigated the factors which enhanced the MACIF and DAF expression in cultured thyroid cells. Thyroid stimulating hormone, phorbol 12, 13-dibutyrate and thyroid stimulating autoantibody enhanced the MACIF and DAF expression. These findings suggest that the membrane complement regulatory proteins increase in response to the thyroid stimulating factors such as thyroid stimulating autoantibody in Graves' disease and that this increase then protects the cells from damage due to complement activation by thyroid autoantibodies.

Antigens, CD↗

PubMed1992

Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinase in the rat hippocampal slice.

Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylated of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Extracellular ATP stimulates increases in Na+/K+ pump activity, intracellular pH and uridine uptake in cultures of mammalian cells.

Using 3T3 and 3T6 mouse fibroblasts and A431 epidermoid carcinoma cells, we previously observed that extracellular ATP and ADP were mitogens and they synergized with other growth factors (Huang, N., Wang, D. and Heppel, L. A. (1989) Proc. Natl. Acad. Sci. USA 86, 7904-7908). We now report that ATP and ADP stimulated Na+ entry, intracellular alkalinization and Na+/K+ pump activity, which are early events that had been proposed to play a central role in DNA synthesis. In addition, ATP, ADP and AMPPNP stimulated uridine uptake by a pathway involving arachidonic acid metabolism. In A431 cells, activation of protein kinase C also contributed to ATP-dependent stimulation of uridine uptake. Concentrations of indomethacin and pertussis toxin which inhibited uridine uptake also blocked arachidonic acid metabolism and DNA synthesis. ATP acted as a competence factor. Interestingly, ATP did not have to be continuously present to stimulate uridine uptake. It was equally effective even when it was washed away after brief treatment of cells.

3T3 Cells↗

PubMed1992

Preferential activation of [3H]phorbol-12,13-dibutyrate binding by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) in neonatal striatal cell cultures.

Activation of excitatory amino acid receptors increased [3H]phorbol-12,13-dibutyrate ([3H]PdBu) binding in four week cultures of striatal cells from postnatal day 7 rat pups (PN7), and in PN7 cells co-cultured the fourth week with striatal cells from postnatal day 1 rat pups. Kainate (KA), trans-1-amino-cyclopentyl-1,3-dicarboxylate (ACPD), and N-methyl-D-aspartate (NMDA) increased [3H]PdBu binding equally in both types of cultures, but alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) increased binding by 3-fold in the co-cultures. Thus, [3H]PdBu binding in these two types of striatal cultures offers a simple model system for studying the regulation of AMPA/KA receptor responses.

Animals↗

PubMed1992

Cholera toxin promotes the proliferation of anti-mu antibody-prestimulated human B cells.

The predominant effect of cholera toxin (CT) on cell growth has been postulated to be inhibitory as a result of its induction of intracellular cAMP. We have recently reported that CT selectively enhances surface DR expression while it inhibits anti-mu antibody-induced B lymphocyte proliferation. In the present series of experiments we studied the effect of CT on in vitro preactivated highly purified (greater than 95% CD20+) human B cells. Cholera toxin enhanced thymidine incorporation of anti-mu antibody-preactivated but not of Staphylococcus aureus Cowan I or PMA + ionomycin-preactivated B cells. Concentrations of 100 pg/ml CT stimulated an enhancement of thymidine incorporation equivalent to that of optimal doses of BCGF. The growth factor-like effect of CT required the complete molecule, since binding of purified B subunit (B-CT) to GM1 ganglioside by itself did not reproduce the holotoxin effect. Moreover, B-CT pretreatment of anti-mu antibody-primed cells completely neutralized the holotoxin-enhancing effect. Both PGE2, a physiological agent that stimulates intracellular cAMP elevation, and the cAMP analogue, 8-bromo-cAMP, mimicked the growth-promoting effect of CT. However, the ED50 of CT required to augment proliferation in anti-mu antibody-preactivated human B cells was approximately 100 times less than the ED50 for cAMP formation. These results demonstrate a specific growth factor-like promoting effect of CT on sIg-preactivated highly purified human B cells that may be mediated at least in part through elevation in intracellular cAMP levels. Increased DR expression and stimulation of growth of sIg preactivated B cells may explain some of the adjuvant properties of CT following orally or parenterally administered antigens.

Antibodies, Monoclonal↗

PubMed1992

Phorbol ester-induced redistribution of the ASGP receptor is independent of receptor phosphorylation.

Like virtually all endocytic receptors, the human asialoglycoprotein (ASGP) receptor is phosphorylated by protein kinase C at serine residues within the cytoplasmic domains of its two subunits H1 and H2. Activation of protein kinase C by phorbol esters results in hyperphosphorylation and in a concomitant net redistribution of receptors to intracellular compartments (down-regulation) in HepG2 cells. To test whether there is a causal relationship between receptor hyperphosphorylation and redistribution, we examined the effect of phorbol ester treatment on the ASGP receptor composed of either wild-type subunits or of mutant subunits lacking any cytoplasmic serine residues in transfected NIH3T3 fibroblast and COS-7 cells. Although the wild-type subunits were hyperphosphorylated in fibroblast cells, the distribution of neither the wild-type nor the mutant receptors was affected. In contrast, phorbol ester treatment of transfected COS-7 cells induced down-regulation of both wild-type and mutant receptors. These findings indicate that redistribution of the receptor is independent of its cytoplasmic serines and is not caused by receptor phosphorylation.

3T3 Cells↗

PubMed1992

Simultaneous redistribution of mannose 6-phosphate and transferrin receptors by insulin-like growth factors and phorbol ester.

Insulin-like growth factors I and II (IGF-I and IGF-II) and phorbol ester are known to induce in fibroblasts a rapid redistribution of mannose 6-phosphate (M6P)/IGF II-receptors to the cell surface. We compared the redistribution of the M6P/IGF-II receptor with that of the 46 kDa M6P receptor (MPR46) and of receptors for transferrin, low-density lipoprotein (LDL) and epidermal growth factor (EGF) in human fibroblasts under the influence of these effectors. None of the effectors altered the surface expression of receptors for LDL or EGF, which are predominantly located at the cell surface. IGF-I, IGF-II and phorbol ester increased the surface expression of the M6P/IGF-II receptor and of MPR46. The concentration of the transferrin receptor at the cell surface was increased only by IGF-I and IGF-II, with similar kinetics as for the M6P/IGF-II receptor, suggesting that the same mechanism causes redistribution. The increased surface expression of M6P receptors was accompanied by an increased uptake of receptor ligands. The number of transferrin receptors did not correlate with iron uptake, although neither the rate nor the extent of transferrin internalization was changed. These results indicate that the redistribution of several endocytic receptors induced by IGF-I, IGF-II and phorbol ester shows selectivity, and that the uptake of receptor ligand may become uncoupled from the surface expression of the receptors via distinct mechanisms.

Cells, Cultured↗

PubMed1992

Vascular smooth muscle proliferation in hypertensive transgenic rats.

In vascular smooth muscle cell (VSMC) cultures from Sprague-Dawley (SD) and hypertensive transgenic rats for the mouse renin gene Ren-2 (TGR), the DNA synthesis, which was analyzed by the uptake of [3H]thymidine, was higher in TGR than SD VSMCs (2.5- to 8-fold, mean of 5.6-fold) under basal conditions. DNA synthesis was increased by fetal calf serum (10%) in SD cells more than in TGR VSMCs, and was decreased by heparin (400 micrograms/ml) and by phorbol-12,13-dibutyrate (10(-7) M) in TGR VSMCs to a higher degree than in SD cells. Neither endothelin (10(-7) M), angiotensinogen (10(-8) M), the renin inhibitor CGP 29,287 (10(-4) M), angiotensin I (10(-7) M), captopril (10(-5) M), angiotensin II (10(-7) M), nor saralasin (10(-6) M) modified DNA synthesis in either type of VSMCs. Sodium nitroprusside (10(-4) and 10(-3) M) increased DNA synthesis in both kinds of VSMCs but in TGR cultures it became toxic at 10(-3) M. 8-Bromocyclic GMP (10(-7) to 10(-5) M) reduced DNA synthesis in SD cells more than in TGR VSMCs. These results suggest that (a) cellular mechanisms of proliferation appear to be more activated in TGR VSMCs, likely involving a protein kinase C-dependent pathway but not the renin-angiotensin system, and (b) in both type of cells, sodium nitroprusside possesses proliferative properties whereas 8-bromocyclic GMP has antiproliferative properties.

Animals↗

PubMedDate not supplied

Ubenimex (Bestatin), an aminopeptidase inhibitor, modulates protein kinase C in K562 cells.

Ubenimex (Bestatin) is a potent inhibitor of aminopeptidases (APase) including APase N (EC 3.4.11.2), a widely distributed membrane-bound metalloprotease. Binding of Ubenimex (UBX) to cells has been implicated in a variety of its biological activities, while little evidence has yet been provided as to any subsequent mechanisms of intracellular signal transduction. We now examined the possible involvement of protein kinase C (PKC), a key regulator in transmembrane signaling. Human leukemia K562 cells were cultured in the presence or absence of UBX (1 to 50 micrograms.ml-1, 1 to 72 h), and the subcellular distribution as well as phorbol-12, 13-dibutyrate (PDBu)-induced redistribution of PKC activities were assessed. The membrane-bound enzymatic activity tended to increase in the presence of UBX, while a significant loss of the activity was demonstrable upon subsequent exposure to PDBu (100 nM, 10 min) in both the cytosolic and membrane fractions. Specific binding of [3H]PDBu to intact K562 cells was also down-modulated with UBX concentration- and time-dependently, suggesting loss of PKC enzyme protein on the cell surface. Western blot analysis of the total cell extracts disclosed no appreciable alteration in the amount of PKC protein. APase inhibition with UBX was observable independently of PKC modulation. The present findings were discussed with reference to the possible differential mechanisms of PKC-mediated regulation of cellular responses depending on cell types.

Aminopeptidases↗