Chelerythrine Chloride Research Articles

17beta-oestradiol acutely regulates Cl- secretion in rat distal colonic epithelium.

In this study we used the short circuit current (ISC) technique to measure the non-genomic effects of the female sex steroid 17beta-oestradiol (E2) on electrogenic transepithelial ion transport in rat distal colonic epithelium. Basal ISC was largely composed of a transepithelial Cl- secretory component with minimal electrogenic Na+ movement. E2 (1-100 nM) caused a significant decrease in basal ISC after 15 min. In addition, pre-treating colonic epithelial tissues with E2 (0.1-100 nM) for 10 min significantly reduced forskolin (20 microM)-induced Cl- secretion. E2 also down-regulated Cl- secretion which was pre-stimulated by forskolin. Cl- secretory responses to the Ca2+-dependent secretagogue carbachol (10 microM) were also significantly reduced in the presence of E2 (10- 100 nM). However, E2 had no effect on amiloride-sensitive Na+ absorption. The rapid anti-secretory effect of E2 was abolished in the presence of the intracellular Ca2+ chelator BAPTA (50 microM) or the protein kinase C (PKC) inhibitor chelerythrine chloride (1 microM). However, in the presence of the nuclear oestrogen receptor antagonist tamoxifen (10 microM), E2 still produced an inhibition of Cl- secretion. Testosterone, progesterone and 17alpha-oestradiol had no significant effect on colonic Cl- secretion. Also, E2 (100 nM) did not alter Cl- secretion in colonic epithelia isolated from male rats. We conclude that E2 inhibits colonic Cl- secretion via a non-genomic pathway that involves intracellular Ca2+ and PKC. It is possible that this gender-specific mechanism contributes to the salt and water retention associated with high E2 states.

Mitogenic effects of oestrogen mediated by a non-genomic receptor in human colon.

Oestrogens are important mitogens in epithelial cancers, particularly where tumours express complementary receptors. While the traditional model of oestrogen action involves gene-directed (genomic) protein synthesis, it has been established that more rapid, non-genomic steroid hormone actions exist. This study investigated the hypothesis that oestrogen rapidly alters cell membrane activity, intracellular pH and nuclear kinetics in a mitogenic fashion. Crypts isolated from human distal colon and colorectal cancer cell lines were used as robust models. DNA replication and intracellular pH were measured by radiolabelled thymidine incorporation (12 h) and spectrofluorescence imaging respectively. Genomic protein synthesis, sodium-hydrogen exchanger (NHE) and protein kinase C (PKC) activity were inhibited with cycloheximide, ethylisopropylamiloride and chelerythrine chloride respectively. Oestrogen induced a rapid (less than 5 min) cellular alkalinization of crypts and cancer cells that was sensitive to NHE blockade (P < 0.01) or PKC inhibition (P < 0.01). Oestrogen increased thymidine incorporation by 44 per cent in crypts and by up to 38 per cent in cancer cells (P < 0.01), and this was similarly reduced by inhibiting the NHE (P < 0.01) or PKC (P < 0.05). Oestrogen rapidly activates cell membrane and nuclear kinetics by a non-genomic mechanism mediated by PKC but not gene-directed protein synthesis.

Role of activator protein 1 transcriptional activity in the regulation of gene expression by transforming growth factor beta1 and bone morphogenetic protein 2 in ROS 17/2.8 osteoblast-like cells.

In osteoblastic cells, transforming growth factor beta1 (TGF-beta1) has been found to regulate the expression of a variety of proto-oncogenes including c-fos, c-jun, and junB. The c-fos in particular has been implicated in the mitogenic effect of TGF-beta1. Here, we examined the role of these early response genes in the regulation of osteoblast (OB) gene expression by two members of the TGF-beta superfamily, TGF-beta1 and bone morphogenetic protein 2 (BMP-2). In ROS 17/2.8 cells, TGF-beta1 as well as BMP-2 up-regulated the expression of junB and c-fos messenger RNAs (mRNAs), and this increase was correlated in both cases with an increase in activator protein 1 (AP-1) DNA-binding activity involving JunB and c-Fos proteins. Protein kinase C (PKC)- and protein tyrosine kinase (PTK)-dependent pathways have been implicated in both TGF-beta1 signaling and AP-1 gene regulation. Therefore, using the kinase inhibitors chelerythrine chloride and genistein, we showed that PKC and PTK activities, respectively, participated in TGF-beta1- and BMP-2-induced increases in junB mRNA levels. Similarly, these kinase activities were involved in the stimulatory effect of BMP-2 on c-fos mRNA expression. Using a natural dominant negative for AP-1 transcriptional activity in ROS 17/2.8 cells, we then showed that AP-1 transcription factors mediated TGF-beta1- and BMP-2-regulated expression of the (alpha1) collagen I gene as well as TGF-beta1-regulated expression of the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor. Our data emphasize the role of the AP-1 transcription factor in TGF-beta1 and BMP-2 signaling and highlight the importance of this transcription factor family in the expression of OB genes.

Second messengers mediating mechanical responses to the FARP GYIRFamide in the fluke Fasciola hepatica.

Spontaneous phasic contractions recorded from isolated body strips of Fasciola hepatica were increased in frequency and amplitude by GYIRFamide, an FMRFamide-related peptide (FaRP). Superfusion with guanosine 5'-O-(2-thiodiphosphate) (100 microM, n = 5) reduced the effects of GYIRFamide on both frequency (by 82%) and amplitude (by 75%). The adenylate cyclase inhibitor MDL-12330A (25 microM) increased spontaneous activity. MDL-12330A completely inhibited the frequency response to GYIRFamide and reduced the amplitude response by 66% as measured relative to this elevated basal activity (n = 6). Inhibition of phospholipase C (PLC) with neomycin sulfate (1 mM) had no direct effect on activity but reduced the frequency response to GYIRFamide by 64% and the amplitude increase by 95% (n = 9). The protein kinase C (PKC) inhibitor chelerythrine chloride (10 microM) also reduced frequency and amplitude responses by 98 and 99%, respectively, without affecting basal contractility (n = 5). Phorbol 12-myristate 13-acetate, an activator of PKC, increased contraction frequency and amplitude (n = 6). It was concluded that GYIRFamide stimulates mechanical activity in F. hepatica through a G protein, via a PLC- and PKC-dependent second messenger pathway.

Basic fibroblast growth factor inhibits apoptosis of spontaneously immortalized granulosa cells by regulating intracellular free calcium levels through a protein kinase Cdelta-dependent pathway.

Previous studies have shown that basic fibroblast growth factor (bFGF) inhibits primary granulosa cells from undergoing apoptosis. The present studies were designed to determine whether spontaneously immortalized granulosa cells (SIGCs) undergo apoptosis when deprived of growth factors and whether bFGF prevents apoptosis. In the absence of serum, the SIGCs lost cell contact and underwent apoptosis as indicated by the presence of annexin V binding, DNA ladders, and nuclear fragmentation. Basic FGF maintained cell contact and reduced the percentage of apoptotic cells. This antiapoptotic action was not observed ifbFGF was added 30 min after serum withdrawal. Further, intracellular free calcium ([Ca2+]i) levels gradually increased 3- to 4-fold within 10 min of serum withdrawal. This increase was inhibited by bFGF. The intracellular calcium chelator, BAPTA, completely prevented the SIGCs from undergoing apoptosis in the absence of serum. These observations suggest that bFGF's ability to regulate [Ca2+]i is an essential component of its antiapoptotic action. The phorbol ester TPA, an activator of protein kinase C (PKC), blocked apoptosis due to serum deprivation. Conversely, bisindolylmaleimide II, an inhibitor of PKC, completely attenuated, whereas bisindolylmaleimide V, an inactive bisindolylmaleimide analog, did not influence bFGF's antiapoptotic action. Also, treatment with the PKC inhibitor, chelerythrine chloride, interfered with bFGF's ability to maintain calcium homeostasis. Western blot analysis revealed that SIGCs expressed PKCdelta, tau, lambda, and zeta. Of these PKC isoforms, only PKCdelta has been shown to be activated by TPA. In apoptotic SIGCs, PKCdelta levels were depleted. When PKCdelta levels were reduced by pretreatment with 500 nM TPA, neither bFGF nor 10 nM TPA suppressed apoptosis. Collectively, these observations suggest that bFGF maintains [Ca2+]i and thereby SIGC viability through a PKCdelta-dependent pathway.

Regulation by protein kinase C of organic anion transport driven by rat organic anion transporter 3 (rOAT3)

The organic anion transporter 3 (rOAT3) is a multispecific OAT localized at the basolateral membrane of the proximal tubule. The purpose of this study was to elucidate the role of protein kinase C (PKC) in the regulation of organic anion transport driven by rOAT3 and its mechanism of action. For this purpose, we established and utilized cells derived from the second segment of proximal tubule from mice stably expressing rOAT3 (S2 rOAT3). Phorbol 12-myristate 13-aceate (PMA), a PKC stimulator, attenuated the cellular uptake of estrone sulfate (ES), a prototype organic anion for rOAT3, in a dose- and time-dependent manner. PMA treatment resulted in a decrease in the Vmax, but not the Km of uptake of ES in S2 rOAT3. Treatment of S2 rOAT3 with other PKC stimulators or diacylglycerols also inhibited the uptake of ES, whereas that with an inactive phorbol ester did not. Chelerythrine chloride, a PKC inhibitor, reversed the PMA-induced decrease in uptake of ES in S2 rOAT3. These results suggest that PKC activation downregulates rOAT3-mediated organic anion transport. This downregulation may be due to the inhibition of translocation or internalization of the rOAT3 protein, resulting in the decrease in the Vmax of rOAT3-medaited organic anion transport.

Diacylglycerol and fatty acids synergistically increase cardiomyocyte contraction via activation of PKC

Lipid signaling pathways are thought to play a prominent role in transducing extracellular signals into contractile responses in cardiac muscle. Two putative lipid messengers, diacyglycerol and arachidonic acid, can be generated via distinct phospholipases in separate signaling pathways, but certain stimuli cause them to be elevated in parallel. We tested the hypothesis that these lipids function as comessengers in ventricular myocytes by activating protein kinase C (PKC). In previous work, we demonstrated that the diacylglycerol analog dioctanoylglycerol (diC(8)) can be stimulatory or inhibitory toward myocyte twitches depending on how it is applied. Here we report that arachidonic acid and other cis-unsaturated fatty acids (UFA), at concentrations too low for direct effects, synergistically enhance the stimulatory effects of diC(8) and convert inhibitory effects of diC(8) into stimulation of myocyte twitches. Intracellular Ca(2+) transients changed in parallel with twitch amplitude, suggesting regulation of Ca(2+) homeostasis by these lipids. cis-UFA also interacted synergistically with the PKC activator phorbol 12-myristate 13-acetate to promote positive inotropic responses. Responses were blocked by the PKC antagonists chelerythrine chloride, bisindolylmaleimide, and Gö-6976. DiC(8) and arachidonic acid also synergistically translocated PKC-epsilon and PKC-alpha in intact myocytes. We propose that PKC integrates diacylglycerol and cis-UFA signals in the heart, resulting in preferential activation of positive inotropic mechanisms.

Chelerythrine chloride induces rapid polymorphonuclear leukocyte apoptosis through activation of caspase-3.

Polymorphonuclear leukocytes (PMN) play a primary role in the initiation and propagation of inflammatory responses. PMN apoptosis is a major mechanism associated with the resolution of inflammatory reactions. Understanding mechanisms associated with PMN apoptosis will be of critical value in the development of novel pharmacological treatment strategies for local and/or systemic inflammatory disorders. The present study demonstrates that chelerythrine chloride induces human PMN to undergo rapid and synchronous progression into the apoptotic process via a PKC-independent mechanism. The appearance of the morphological features of apoptosis in chelerythrine-treated PMN is preceded by a significant upregulation in caspase-3 activity. GM-CSF (a cytokine that protects PMN in several models of PMN apoptosis) does not protect PMN from chelerythrine chloride-induced apoptosis.

Adenosine A 1 Receptor Induced Delayed Preconditioning in Rabbits

Transient adenosine A(1) receptor (A(1)R) activation in rabbits induces delayed preconditioning against myocardial infarction 24 to 72 hours later. The cellular mechanisms downstream of A(1)R mediating this delayed cardioprotection have not been elucidated. This study examined the role of protein kinase C (PKC) and tyrosine kinases (TKs) in the signaling cascade mediating A(1)R-induced late preconditioning in rabbits. The small heat shock protein Hsp27 has been shown to confer cytoskeletal protection when in the phosphorylated state. We therefore also evaluated the potential role of the p38 mitogen-activated protein kinase (p38 MAPK) and Hsp27 as distal mediators of A(1)R-induced delayed preconditioning. Pharmacological preconditioning of rabbits with the selective A(1) agonist 2-chloro-N(6)-cyclopentyladenosine (CCPA; 100 microgram/kg) significantly reduced myocardial infarct size compared with control animals, after 30-minute regional ischemia/2-hour reperfusion in vivo 24 hours later (23.7+/-3.1 versus 43.0+/-4.1%; P<0.05). This delayed protection was abrogated by prior inhibition of either PKC with chelerythrine chloride (5 mg/kg) or of TKs with lavendustin A (1.3 mg/kg), suggesting that both PKC and TK are crucial for the development of delayed preconditioning after A(1) receptor activation in the rabbit. Myocardial tissue extracts obtained 24 hours after CCPA treatment were analyzed for p38 MAPK catalytic activity using an in vitro kinase assay. This showed an almost 7-fold increase in p38 MAPK activity in myocardial samples pretreated with CCPA compared with control hearts. Two-dimensional gel electrophoresis revealed an increase in the phosphorylated isoforms of Hsp27 in hearts pretreated with CCPA compared with control hearts. Prior inhibition of either PKC or TK prevented the CCPA-induced increase in p38 MAPK activity and phosphorylation of Hsp27. This study identifies new components of the signaling mechanism of A(1)R-induced delayed preconditioning. Our results suggest an important role for both PKC and TK as mediators of late preconditioning against infarction after A(1)R activation and, although correlative, point to the p38 MAPK/Hsp27 pathway as a potential distal effector of this protection.

Soluble vascular cell adhesion molecule 1 mediation of monocyte chemotaxis in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by infiltration of leukocytes, including monocyte/ macrophages, into synovial tissue (ST), but factors mediating the ingress of these cells are poorly understood. Vascular cell adhesion molecule 1 (VCAM-1) plays an important role in adhesion of leukocytes to the vasculature. This study was undertaken to test the hypothesis that soluble VCAM-1 (sVCAM-1) might mediate chemotaxis of monocytes in RA. Chemotaxis assays were performed using a modified Boyden chamber to determine the effects of sVCAM-1 on and the role of very late activation antigen 4 (VLA-4) in peripheral blood (PB) monocyte migration. Synovial fluids (SF) were immunodepleted of sVCAM-1 to identify a role for sVCAM-1 in RA. Immunohistochemistry and flow cytometry analyses were performed to show the expression of VLA-4 in ST, SF, and PB. Tyrosine phosphorylation was studied by Western blot analysis on PB monocyte lysates in the presence of signaling inhibitors. Soluble VCAM-1 induced monocyte migration in the nM range, in a concentration-dependent manner. Anti-VLA-4 significantly inhibited sVCAM-1-induced monocyte migration, suggesting that sVCAM-1 acts in part via a VLA-4-dependent mechanism. In RA SF, incubation with anti-VCAM-1 resulted in a reduction in the ability to induce monocyte migration (mean 28%). VLA-4 immunolocalized to RA ST, SF, or PB, monocytes, macrophages, and lymphocytes. Soluble VCAM-1 stimulated tyrosine phosphorylation in monocytes, and pertussis toxin, chelerythrine chloride, and staurosporine significantly reduced sVCAM-1-mediated monocyte chemotaxis, suggesting that signaling pathways via G proteins and protein kinase C are required for sVCAM-1-mediated monocyte migration. These results demonstrate a novel function for sVCAM-1 as a monocyte chemotactic agent in RA and suggest a new potential target for modulating monocyte ingress into inflamed RA ST.

Involvement of spinal protein kinase C in induction and maintenance of both persistent spontaneous flinching reflex and contralateral heat hyperalgesia induced by subcutaneous bee venom in the conscious rat

To further study the roles of spinal protein kinase C (PKC) in induction and maintenance of both the persistent spontaneous nociception and the contralateral heat hyperalgesia induced by subcutaneous (s.c.) bee venom injection, the effects of intrathecal (i.t.) treatment with a PKC inhibitor, chelerythrine chloride (CH), were evaluated in conscious rats. Pre-treatment i.t. with CH at three doses of 0.01, 0.1 and 1 nmol produced a dose-dependent suppressive effect on the flinching reflex with the inhibitory rates of 39, 48 and 59%, respectively, when compared with the pre-saline control group. Post-treatment i.t. with the drug at the highest dose used (1 nmol) also resulted in a 42% suppression of the flinching reflex compared with the control. Moreover, pre-treatment i.t. with CH at three doses of 0.01, 0.1 and 1 nmol also produced 12, 22 and 48% inhibition of the contralateral heat hyperalgesia in the pre-saline control group. Post-treatment i.t. with the drug at the highest dose used (1 nmol) also resulted in a 35% reversal effect on the established contralateral heat hyperalgesia. The present result suggests that activation of PKC in the spinal cord contributes to the induction and maintenance of both peripherally-dependent persistent spontaneous pain and contralateral heat hyperalgesia which is dependent upon central sensitization.

Nongenomic effects of aldosterone on Ca2+ in M-1 cortical collecting duct cells

Aldosterone at physiological levels induces rapid (<5 min) increases in intracellular protein kinase C (PKC) activity and a rise in calcium and pH in mineralocorticoid hormone target epithelia, such as distal colon and sweat gland. The end targets of these rapid responses in epithelia are Na+/H+ exchange and K+ channels. The mouse cortical collecting duct (CCD) M-1 cell line was grown to confluency and loaded with Fura-2 for spectrofluorescence measurements of intracellular free calcium at 37 degrees C bathed in Krebs solution. Aldosterone (1 nmol/L) produced a rapid, transient peak increase in [Ca2+]i in M-1 cells. This effect was abolished upon removal of extracellular Ca2+, but was unaffected by pretreatment with spironolactone (10 micromol/L) or actinomycin D (10 micromol/L). However, pretreatment with the specific PKC inhibitor chelerythrine chloride (1 micromol/L) prevented the aldosterone-induced rise in [Ca2+]i. Dexamethasone, at a concentration 10,000-fold higher than aldosterone (10 micromol/L), also produced a transient increase in [Ca2+]i, but this response was significantly smaller than that of aldosterone. In contrast, hydrocortisone had no effect on [Ca2+]i at either nmol/L or micromol/L concentrations. Both of the sex steroids, 17beta-estradiol (10 nmol/L) and progesterone (10 nmol/L), induced protein kinase C-dependent increases in [Ca2+]i. Aldosterone and sex steroid hormones activate intracellular calcium signaling in CCD cells via a nongenomic PKC-dependent pathway, which may have important implications for renal transport.

ICAM-1 expression by vascular smooth muscle cells is phenotype-dependent

Atherosclerosis is an inflammatory disease characterised by increased expression of adhesion molecules for leukocytes on both the surface of dysfunctional endothelium and on smooth muscle cells (SMC) within the lesion. It is also characterised by altered SMC phenotypic expression, indicated by a decreased volume fraction of myofilaments (V vmyo) [1,2] and changes in gene expression [3]. The present study used an in vitro model to investigate, by immunofluorescence staining and flow cytometry, the influence of phenotype on vascular SMC expression of the adhesion molecule for leukocytes, intracellular adhesion molecule-1 (ICAM-1), and the regulatory mechanisms involved in this process. Smooth muscle cells with a high V vmyo, freshly isolated from rat aortic media, expressed little or no ICAM-1 and this could not be induced by interleukin-1β (IL-1β). As SMC modulated phenotype, indicated by decreasing V vmyo over the first 5 days of culture, there was a concomitant increase in ICAM-1 expression. At day 9 of primary culture, when SMC cultures had returned to the high V vmyo phenotype, ICAM-1 expression was markedly lower. However, these cells retained the capacity to express ICAM-1 in response to IL-1β. After several passages in culture, cells (with a low V vmyo) constitutively expressed ICAM-1, with levels further up-regulated in response to IL-1β. These changes in ICAM-1 expression were not related to proliferative state, since similar results were obtained with growth arrested SMC. Investigation of signalling pathways involved in regulating ICAM-1 expression by primary vascular SMC suggested a complex regulatory mechanism. Activation of adenyl cyclase (with forskolin) caused a significant increase in cells expressing ICAM-1. Treatment with inhibitors of protein kinase C (chelerythrine chloride), protein tyrosine kinase (genistein), or the transcription factor NF-κB (PDTC) had no significant effect on IL-1-induced ICAM-1 expression. However, in the presence of serum, both genistein and PDTC caused a significant increase in basal expression. The results indicate that ICAM-1 expression by SMC is phenotype-dependent, with expression evident only after cells have modulated to a low V vmyo phenotype. They also indicate the existence of complex regulatory mechanisms, possibly involving the SMC cytoskeleton.

Mechanisms of ischemic preconditioning effects on Ca(2+) paradox-induced changes in heart.

The effects of ischemic preconditioning (IP) on changes in cardiac performance and sarcoplasmic reticulum (SR) function due to Ca(2+) paradox were investigated. Isolated perfused hearts were subjected to IP (three cycles of 3-min ischemia and 3-min reperfusion) followed by Ca(2+)-free perfusion and reperfusion (Ca(2+) paradox). Perfusion of hearts with Ca(2+)-free medium for 5 min followed by reperfusion with Ca(2+)-containing medium for 30 min resulted in a dramatic decrease in the left ventricular (LV) developed pressure and a marked increase in LV end-diastolic pressure. Alterations in cardiac contractile activity due to Ca(2+) paradox were associated with depressed SR Ca(2+)-uptake, Ca(2+)-pump ATPase, and Ca(2+)-release activities as well as decreased SR protein contents for Ca(2+)-pump and Ca(2+) channels. All these changes due to Ca(2+) paradox were significantly prevented in hearts subjected to IP. The protective effects of IP on Ca(2+) paradox changes in cardiac contractile activity as well as SR Ca(2+)-pump and Ca(2+)-release activities were lost when the hearts were treated with 8-(p-sulfophenyl)-theophylline, an adenosine receptor antagonist; KN-93, a specific Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) inhibitor; or chelerythrine chloride, a protein kinase C (PKC) inhibitor. These results indicate that IP rendered cardioprotection by preventing a depression in SR function in Ca(2+) paradox hearts. Furthermore, these beneficial effects of IP may partly be mediated by adenosine receptors, PKC, and CaMK II.

Stimulation of nucleoside efflux and inhibition of adenosine kinase by A 1 adenosine receptor activation

Adenosine is produced intracellularly during conditions of metabolic stress and is an endogenous agonist for four subtypes of G-protein linked receptors. Nucleoside transporters are membrane-bound carrier proteins that transfer adenosine, and other nucleosides, across biological membranes. We investigated whether adenosine receptor activation could modulate transporter-mediated adenosine efflux from metabolically stressed cells. DDT 1 MF-2 smooth muscle cells were incubated with 10 μM [ 3H]adenine to label adenine nucleotide pools. Metabolic stress with the glycolytic inhibitor iodoacetic acid (IAA, 5 mM) increased tritium release by 63% ( P < 0.01), relative to cells treated with buffer alone. The IAA-induced increase was blocked by the nucleoside transport inhibitor nitrobenzylthioinosine (1 μM), indicating that the increased tritium release was primarily a purine nucleoside. HPLC verified this to be [ 3H]adenosine. The adenosine A 1 receptor selective agonist N 6-cyclohexyladenosine (CHA, 300 nM) increased the release of [ 3H]purine nucleoside induced by IAA treatment by 39% ( P < 0.05). This increase was blocked by the A 1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (10 μM). Treatment of cells with UTP (100 μM), histamine (100 μM), or phorbol-12-myristate-13-acetate (PMA, 10 μM) also increased [ 3H]purine nucleoside release. The protein kinase C inhibitor chelerythrine chloride (500 nM) inhibited the increase in [ 3H]purine nucleoside efflux induced by CHA or PMA treatment. The adenosine kinase activity of cells treated with CHA or PMA was found to be decreased significantly compared with buffer-treated cells. These data indicated that adenosine A 1 receptor activation increased nucleoside efflux from metabolically stressed DDT 1 MF-2 cells by a PKC-dependent inhibition of adenosine kinase activity.

Involvement of protein kinase C in superoxide anion-induced activation of nuclear factor-kappa B in human endothelial cells.

Nuclear factor-kappa B (NF-kappa B) plays an important role in the regulation of redox-sensitive genes which are related to the pathogenesis of various vascular diseases. Although oxygen free-radicals are known to activate NF-kappa B, the signaling pathway of oxygen free radical-induced NF-kappa B activation remains largely unclear. Thus, this study was performed to examine the possible involvement of protein kinase C (PKC) in the oxygen free radical-induced NF-kappa B activation in human umbilical vein endothelial cells (HUVECs). Superoxide anion was generated by xanthine and xanthine oxidase. An electrophoretic mobility shift assay (EMSA) was performed using a kappa B-motif oligonucleotide and nuclear extracts from HUVECs. Immunoblot analysis using an antibody against I kappa B alpha, phosphorylated by I kappa B alpha kinase, or myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylated by protein kinase C was carried out. An NF-kappa B luciferase reporter gene assay was also performed. The treatment of the cells with superoxide anion for 60 min increased the NF-kappa B/DNA binding activity. Immunoblot analysis showed that superoxide anion induced phosphorylation of I kappa B alpha within 10 min. Furthermore, phosphorylation of MARCKS occurred more rapidly than phosphorylation of I kappa B alpha. Pretreatment of the cells with calphostin C (100-400 nmol/l) and chelerythrine chloride (5-10 mumol/l), inhibitors of PKC, abolished the superoxide anion-induced NF-kappa B activation. Down-regulation of endogenous PKC by long-term exposure to phorbol 12-myristate 13-acetate decreased the superoxide anion-induced NF-kappa B activation to a basal level. Superoxide anion induced the luciferase reporter gene and this induction was completely inhibited by calphostin C (200 nmol/l) and 4,5-dihydroxy-1,3-benzene disulfonic acid (tiron). These results suggest that PKC is involved in the activation of NF-kappa B by superoxide anion in human endothelial cells.

The effect of PKC activation on the survival of rat retinal ganglion cells in culture

Natural cell death is a degenerative phenomenon occurring during the development of the nervous system. Approximately half the neurons initially generated during this period die. The role of trophic molecules produced by target and afferent neurons as well as by glial cells controlling this regressive event has been extensively demonstrated. The aim of this work was to study the role of activated protein kinase C (PKC), an enzyme involved in apoptosis regulation, on the survival of retinal ganglion cells kept “in vitro” for 48 h. For this purpose, we used the phorbol 12-myristate 13-acetate (PMA), a tumor promoter agent that activates PKC. Our results showed that PMA increases the survival of ganglion cells. The effect was dose-dependent and PMA concentrations of 10 or 100 ng/ml produced the maximal effect (a two-fold increase on ganglion cells survival compared with 48 h control). This effect was totally abolished by 1.25 μM chelerythrine chloride (an inhibitor of PKC) and 30 μM genistein (an inhibitor of tyrosine kinase enzymes). Otherwise, PMA was effective only when it was chronically present in the cultures. On the other hand, treatment with 20 μM 5-fluoro-2′-deoxyuridine, an inhibitor of cell proliferation, or 25 μM BAPTA-AM, an intracellular calcium chelator, did not block PMA effect. Our results suggest that the survival of retinal ganglion cells “in vitro” may be mediated by a mechanism that involves PKC activation.

Different roles of protein kinase C α and δ isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells

The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and interferon γ (IFN-γ) in HL-60 cells were investigated. IFN-γ was found to increase selectively the activity of cytosolic, Mg2+-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)2D3 and IFN-γ had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg2+-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)2D3 and IFN-γ. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca2+-dependent PKC isoforms, inhibited the effect of 1,25(OH)2D3 but had no effect on the IFN-γ-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)2D3, arachidonic acid (AA) and PMA. The depletion of PKC isoforms β1, β2, ϵ, η, μ, ζ and λ had no effect on the activation of sphingomyelinase induced by 1,25(OH)2D3 or by AA. The depletion of PKC α from the cytosol completely abolished the effect of 1,25(OH)2D3 on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or α-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC ∆. Moreover, PMA inhibited the effect of 1,25(OH)2D3 on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC ∆ from the cytosol. These studies demonstrate that 1,25(OH)2D3-induced activation of sphingomyelinase is mediated by PKC α. Furthermore, PKC ∆ had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)2D3- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC α and PKC ∆ isoforms.

Different roles of protein kinase C α and δ isoforms in the regulation of neutral sphingomyelinase activity in HL-60 cells

The signalling mechanisms responsible for the hydrolysis of sphingomyelin mediated by 1,25-dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] and interferon gamma (IFN-gamma) in HL-60 cells were investigated. IFN-gamma was found to increase selectively the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The treatment of HL-60 cells with the combination of 1,25(OH)(2)D(3) and IFN-gamma had an additive effect on sphingomyelin hydrolysis, ceramide release and the activity of cytosolic, Mg(2+)-independent, neutral sphingomyelinase. The pretreatment of HL-60 cells with staurosporine, chelerythrine chloride and bisindolylmaleimide abolished the activity of sphingomyelinase in response to 1,25(OH)(2)D(3) and IFN-gamma. Calphostin C, which acts on the regulatory site of protein kinase C (PKC), and Gö 6976, a selective inhibitor of Ca(2+)-dependent PKC isoforms, inhibited the effect of 1,25(OH)(2)D(3) but had no effect on the IFN-gamma-mediated increase in activity of sphingomyelinase. Isoform-specific antibodies were used to deplete different PKC isoforms from cytosol before the treatment of the cytosolic fraction with 1,25(OH)(2)D(3), arachidonic acid (AA) and PMA. The depletion of PKC isoforms beta(1), beta(2), epsilon, eta, mu, zeta and lambda had no effect on the activation of sphingomyelinase induced by 1,25(OH)(2)D(3) or by AA. The depletion of PKC alpha from the cytosol completely abolished the effect of 1,25(OH)(2)D(3) on sphingomyelinase activity but had no effect on the AA-induced activity of sphingomyelinase. PMA had no effect on the activity of sphingomyelinase in either untreated or alpha-depleted cytosol but significantly increased the activity of sphingomyelinase when added to cytosol depleted of PKC delta. Moreover, PMA inhibited the effect of 1,25(OH)(2)D(3) on sphingomyelinase activation but the inhibitory effect was abolished by prior depletion of PKC delta from the cytosol. These studies demonstrate that 1,25(OH)(2)D(3)-induced activation of sphingomyelinase is mediated by PKC alpha. Furthermore, PKC delta had an inhibitory effect on sphingomyelinase, suggesting that the difference between the 1,25(OH)(2)D(3)- and PMA-mediated effects on sphingomyelin turnover depends on the specific regulation of the PKC alpha and PKC delta isoforms.

Neurotrophin‐3 (NT‐3) modulates early differentiation of oligodendrocytes in rat brain cortical cultures

Multiple extracellular signals are required for oligodendroglia survival, proliferation and differentiation, and increasing evidence has accumulated that also neurotrophins regulate glial cell development in the central nervous system (CNS). In the present study we have investigated the influence of neurotrophin-3 (NT-3) on the in vitro differentiation and proliferation of oligodendrocytes prepared from the brains of newborn rats. Cells were grown in chemically defined growth medium, in the absence of fetal calf serum (FCS). RT-PCR analysis confirmed the expression of mRNA encoding the NT-3 receptor trkC in oligodendrocytes throughout in vitro development. Cell morphology was observed by phase contrast microscopy and indirect immunofluorescence staining using anti-galactocerebroside (GalC) antibodies. An increase in process formation and arborization was observed 8–24 h after the treatment with NT-3 (5–50 ng/ml). Concomitantly, NT-3 caused an increase in the appearance of GalC-positive cells. Long-term treatment with NT-3 (up to seven days) did not yield any further improvement of process formation. To elucidate the molecular mechanisms and signal transduction pathways underlying the effect of NT-3 in oligodendrocytes, the time- and concentration-dependent effect of NT-3 on c-Fos protein expression was studied by Western blot analysis. The data show that NT-3 stimulated the appearance of two c-Fos immunoreactive polypeptides with apparent molecular weights of 62 and 55 kDa, respectively. This effect was maximal at a concentration of 50 ng/ml of NT-3 after 8–24 h . NT-3-modulated morphological differentiation and c-Fos protein expression was regulated by protein kinases. Whereas the protein kinase C (PKC) inhibitors staurosporine and chelerythrine chloride had a stimulatory effect on NT-3-promoted process formation, the tyrosine kinase inhibitor genistein had an inhibitory effect and mainly cells with a bipolar and immature morphology were observable. The inhibition of tyrosine kinase activity prevented NT-3-promoted induction of c-Fos protein. Thus, in addition to its mitogenic effects, NT-3 during early time points influences the in vitro differentiation of oligodendrocytes. This process involves the induction of c-Fos protein and is mediated by PKC and trosine kinase activities. GLIA 28:244–255, 1999. © 1999 Wiley-Liss, Inc.