Competitive Inhibitor Of Protein Kinase Research Articles

In vivo study of a novel protein kinase C that mediates immunocompetence and catecholamine biosynthesis in hemocytes of Litopenaeus vannamei by using its potential competitive inhibitor, bisindolylmaleimide I

This study applied bisindolylmaleimide I (BSM), a pharmacological competitive inhibitor of protein kinase C (PKC) enzymatic activity, at 1.25 pmol shrimp−1 for 60 min to investigate the potential involvement of PKC in signal transduction pathways in the hemocytes of Litopenaeus vannamei. A novel PKC in L. vannamei (LvnPKC) was identified and characterized and was determined to be involved in mediating the neuroendocrine-immune regulatory network. The hemocytes of L. vannamei that receive BSM exhibit significantly decreased PKC activity and LvnPKC gene and protein expression levels. Furthermore, the total hemocyte count, hyaline cells, and semigranular cells increased significantly along with significant decreases in granular cells, and meanwhile, the significantly increased phenoloxidase activity, respiratory bursts, superoxide dismutase (SOD) activity, phagocytic activity, and neutrophil extracellular trap were observed; however, phagocytic activity decreased significantly. In a molecular model, the gene expressions of lipopolysaccharide- and β-1,3-glucan-binding protein, peroxinectin, cytosolic manganese SOD, mitochondrial manganese SOD, and copper/zinc SOD in the hemocytes of L. vannamei that had received BSM decreased significantly, but prophenoloxidase I increased significantly. In catecholamine biosynthesis, tyrosine, dopamine, and norepinephrine decreased significantly in the hemocytes of L. vannamei that had received BSM, and l-dihydroxyphenylalanine increased. Moreover, tyrosine hydroxylase (TH) activity increased significantly, whereas TH and dihydroxyphenylalanine decarboxylase gene expression decreased significantly. These findings suggest that BSM inhibits PKC activity in hemocytes in which LvnPKC gene and protein expression are also inhibited. Additionally, the hemocytes’ immunocompetence, including their prophenoloxidase and antioxidant systems, phagocytic activity, and catecholamine biosynthesis, was disrupted, confirming the roles of LvnPKC in mediating the neuroendocrine-immune regulatory network in hemocytes.

Abstract 4186: A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors

Abstract Protein kinases are central to cellular signal transduction and regulation of cellular processes, and are one of the most attractive target classes in modern drug discovery. Multiple kinase inhibitors have already been approved for treatment of various diseases including such severe conditions as cancer. The first and up to today largest group of drugs that effectively inhibits their respective kinase targets belong to the class of ATP competitive compounds. They bind into or near the ATP binding site of the enzymes and inhibit kinase activity by blocking access of ATP to the active site. Although there are numerous examples of highly specific ATP competitive compounds this mode of action is limited by several factors: The ATP binding pocket structures of kinases show a high degree of similarity, which makes finding highly selective compounds challenging. Furthermore, competing with ATP for binding to the same target site, compounds have to be of very high affinity due to the high intracellular ATP concentrations. Therefore, the interest to develop non-ATP competitive inhibitors has risen considerably over the last years. Such inhibitors bind to kinases at sites apart from the ATP binding site, inhibiting their activity e.g. by stabilizing an inactive conformation (like DFG-out state binders), displacing essential cofactors (like cyclins for CDKs) or by blocking activating modifications (like phosphorylation by upstream kinases). We present data of an in-vitro biochemical kinase activity assay setup which is suited to discriminate between ATP-competitive and non-ATP competitive inhibitors. The IC50 of an ATP-competitive inhibitor will increase with increasing ATP concentrations and the IC50 value at a given ATP concentration may be calculated using the equation of Cheng and Prussof: IC50=Ki+(Ki*[ATP]/KM[ATP] (Cheng Y., Prusoff W. H. (1973) Biochem. Pharmacol. 22: 3099-3108). By determining IC50 values for an inhibitor of a specific kinase at different ATP concentrations we examined whether the IC50 value changed according to the Cheng-Prusoff equation, indicating an ATP-competitive mode of action, or if the IC50 values remained unchanged in presence of elevated ATP indicating a non-ATP competitive or mixed type mode of action. In our assay setup we determined the IC50 values at ATP concentrations in a range of 0.1 to 10 fold the ATP KM of the kinase of interest. By comparing the results obtained for the non-ATP competitive MEK1 inhibitor selumetinib and the ATP competitive inhibitor staurosporine we could verify that our assay setup is well suited to discriminate between these different types of kinase inhibitors. Citation Format: Daniel Mueller, Frank Totzke, Thomas Weber, Carolin Heidemann-Dinger, Constance Ketterer, Diane Krämer, Marcel Pathe, Michael H. Kubbutat. A biochemical approach to discriminate between ATP-competitive and non-ATP competitive protein kinase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4186. doi:10.1158/1538-7445.AM2017-4186

Marine Derived Hamacanthins as Lead for the Development of Novel PDGFRβ Protein Kinase Inhibitors

In this study, we report on pyrazin-2(1H)-ones as lead for the development of potent adenosine triphosphate (ATP) competitive protein kinase inhibitors with implications as anti-cancer drugs. Initially, we identified the pyrazin-2(1H)-one scaffold from hamacanthins (deep sea marine sponge alkaloids) by Molecular Modeling studies as core binding motif in the ATP pocket of receptor tyrosine kinases (RTK), which are validated drug targets for the treatment of various neoplastic diseases. Structure-based design studies on a human RTK member PDGFR (platelet-derived growth factor receptor) suggested a straight forward lead optimization strategy. Accordingly, we focused on a Medicinal Chemistry project to develop pyrazin-2(1H)-ones as optimized PDGFR binders. In order to reveal Structure-Activity-Relationships (SAR), we established a flexible synthetic route via microwave mediated ring closure to asymmetric 3,5-substituted pyrazin-2(1H)-ones and produced a set of novel compounds. Herein, we identified highly potent PDGFR binders with IC50 values in an enzymatic assay below µM range, and possessing significant activity against PDGFR dependent cancer cells. Thus, marine hamacanthin-derived pyrazin-2(1H)-ones showing interesting properties as lead for their further development towards potent PDGFR-inhibitors.

Non-ATP Competitive Protein Kinase Inhibitors

Protein kinases represent an attractive target in oncology drug discovery. Most of kinase inhibitors are ATP-competitive and are called type I inhibitors. The ATP-binding pocket is highly conserved among members of the kinase family and it is difficult to find selective agents. Moreover, the ATP-competitive inhibitors must compete with high intracellular ATP levels leading to a discrepancy between IC50s measured by biochemical versus cellular assays. The non-ATP competitive inhibitors, called type II and type III inhibitors, offer the possibility to overcome these problems. These inhibitors act by inducing a conformational shift in the target enzyme such that the kinase is no longer able to function. In the DFG-out form, the phenylalanine side chain moves to a new position. This movement creates a hydrophobic pocket available for occupation by the inhibitor. Some common features are present in these inhibitors. They contain a heterocyclic system that forms one or two hydrogen bonds with the kinase hinge residue. They also contain a hydrophobic moiety that occupies the pocket formed by the shift of phenylalanine from the DFG motif. Moreover, all the inhibitors bear a hydrogen bond donor-acceptor pair, usually urea or amide, that links the hinge-binding portion to the hydrophobic moiety and interacts with the allosteric site. Examples of non ATP-competitive inhibitors are available for various kinases. In this review small molecules capable of inducing the DFG-out conformation are reported, especially focusing on structural feature, SAR and biological properties.

The cAMP-Dependent Protein Kinase Inhibitor H-89 Attenuates the Bioluminescence Signal Produced by Renilla Luciferase

BackgroundInvestigations into the regulation and functional roles of kinases such as cAMP-dependent protein kinase (PKA) increasingly rely on cellular assays. Currently, there are a number of bioluminescence-based assays, for example reporter gene assays, that allow the study of the regulation, activity, and functional effects of PKA in the cellular context. Additionally there are continuing efforts to engineer improved biosensors that are capable of detecting real-time PKA signaling dynamics in cells. These cell-based assays are often utilized to test the involvement of PKA-dependent processes by using H-89, a reversible competitive inhibitor of PKA.Principal FindingsWe present here data to show that H-89, in addition to being a competitive PKA inhibitor, attenuates the bioluminescence signal produced by Renilla luciferase (RLuc) variants in a population of cells and also in single cells. Using 10 µM of luciferase substrate and 10 µM H-89, we observed that the signal from RLuc and RLuc8, an eight-point mutation variant of RLuc, in cells was reduced to 50% (±15%) and 54% (±14%) of controls exposed to the vehicle alone, respectively. In vitro, we showed that H-89 decreased the RLuc8 bioluminescence signal but did not compete with coelenterazine-h for the RLuc8 active site, and also did not affect the activity of Firefly luciferase. By contrast, another competitive inhibitor of PKA, KT5720, did not affect the activity of RLuc8.SignificanceThe identification and characterization of the adverse effect of H-89 on RLuc signal will help deconvolute data previously generated from RLuc-based assays looking at the functional effects of PKA signaling. In addition, for the current application and future development of bioluminscence assays, KT5720 is identified as a more suitable PKA inhibitor to be used in conjunction with RLuc-based assays. These principal findings also provide an important lesson to fully consider all of the potential effects of experimental conditions on a cell-based assay readout before drawing conclusions from the data.

Non-ATP competitive protein kinase inhibitors as anti-tumor therapeutics

Alternative approaches for inhibitor development in targeting sites other than the ATP cleft are increasingly being pursued in the search for new therapeutics based on inhibition of protein kinases. While recently approved kinase inhibitor drugs offer benefit in cancer treatment, further advances are required to affect tumor selective cell killing, avoid off-target related toxicities and improve survival rates. Protein–protein interactions involved in kinase regulation and substrate recognition as well as exploiting allosteric pockets, offer the potential for selectivity and avoid decreased efficacy as a result of competition with high intracellular ATP concentrations. We discuss several preliminary examples where regulatory and substrate binding sites present potential druggable interfaces. These include the cell cycle targets which are the cyclin-dependent and polo-like kinases among several others.

Effects of Enzastaurin, Alone or in Combination, on Signalling Pathway Controlling Growth and Survival of B-Cell Lymphoma Cell Lines

Background. Although, the recent introduction of Rituximab in combination with chemotherapy has improved outcome of patients with indolent lymphomas, in particular FL, these diseases are still considered incurable and the majority of patients still have a fatal course. Enzastaurin, an acyclic bisindolylmaleimide, is a potent and selective competitive inhibitor of protein kinase C beta, which has been shown to inhibit cell proliferation and angiogenesis in human cancer cell lines. The purpose of the present study was to test enzastaurin for its effects on proliferation and survival of B-cell lines established from NHL patients.Methods. The WSU-NHL and Karpas 422 were kindly provided Dr M Introna ( Division of Haematology, Ospedali Riuniti, Bergamo, Italy); the RL was purchased by DSMZ. All three lines are carrying the t(14; 18) and Karpas 422 is EBV+. We decided to conduct all the experiments under the optimal culture conditions with 10% FCS to avoid subjecting the cells to further stress stimuli. IC50 values were calculated from curves based on enzastaurin concentration ranging from 1 to 10μM using MTT assay and cell viability assessment by Trypan Blue exclusion. Cell apoptosis was assessed by flow cytometer after staining with Annexin V-FITC and propidium iodide. The effects of enzastaurin on caspases activation as well as on AKT phosphorilation were evaluated by Western blotting. We also investigated the interaction of enzastaurin with chlorambucil and fludarabine. Results using MTT assay were expressed as fraction of cells killed by the individual drug or the combination in the drug-treated versus untreated cells. The interaction between drugs was analyzed by isobologram analysis using the StaCorp8.2 software program based upon the Chou-Talalay method to determine if the combinations were additive or synergistic.Results. We found that enzastaurin alone inhibits the proliferation of B-cell lymphomas cell lines at IC50 values ranging from 5 to 7.5 μM after 48 h and from 2.5 to 3.2 μM after 72 h. Induction of apoptosis by enzastaurin evaluated on WSU-NHL and RL, by flow cytometry analysis of membrane permeability, showed that enzastaurin induces an increase in the percentage of apoptotic cells compared with untreated controls in a time-dependent fashion. Furthermore, enzastaurin induces the appearance of the cleaved caspase-3 fragment in the same cell lines in a time dependent fashion. Activation of the apoptotic pathway was confirmed by cleavage of the PARP enzyme. The apoptosis is partially prevented by the ZVAD–fmk broad caspase inhibitor. Phosphorilation status analysis of AKT up to 72h of treatment showed a decrease of AKT phosphorilation starting from 48h after treatment. We tested the effect of enzastaurin combined with chlorambucil and fludarabine,drugs that are active against B-cell lymphoma and we demonstrated that these agents enhanced the cytotoxicity triggered by enzastaurin in a dose-dependent fashion. A clear synergistic interaction (CI=0.87) appeared using low concentrations of the drugs and increased (CI=0.1) at high concentrations.Conclusions. Our data suggest that in B lymphoma cell lines carrying t(14; 18), enzastaurin elicits its antitumor effect suppressing AKT phosphorilation, inducing apoptosis and inhibiting proliferation. Furthermore, enzastaurin synergizes with chlorambucil and fludarabine. These results support the potential use of enzastaurin in patients with NHL, and in particular provide a rationale for combination with chlorambucil and fludarabine.

Synthesis and Characterization of 5′-p-fluorosulfonylbenzoyl-2′ (or 3′)-(biotinyl)adenosine as an Activity-Based Probe for Protein Kinases

Most of the kinase inhibitors that are approved for therapeutic uses or that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. 5'-Fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an activitybased probe (ABP) that covalently modifies a conserved lysine present in the nucleotide binding site of most kinases. Here the authors describe synthesis of FSBA derivatives, 2'-biotinyl-FSBA and 3'-biotinyl-FSBA as kinase ABPs, and delineate a Western blot method to screen and validate ATP competitive protein kinase inhibitors using biotinyl-FSBA as a nonselective activity-based probe for protein kinases.

A phase I clinical study of safingol followed by cisplatin: promising activity in refractory adrenocortical cancer with novel pharmacology

13044 Background: Safingol (L-threo-dihydrosphingosine), originally identified as a competitive inhibitor of protein kinase C, has been re-identified as an inhibitor of sphingosine kinase (SK). Inhibition of SK induces apotosis and autophagy by increasing intracellular conversion of sphingosine to ceramide. In vivo, safingol enhances the antitumor activity of cisplatin in a sequence dependent manner (safingol→cisplatin). Methods: We designed a phase I trial of escalating doses of safingol (60→120→240→ 360→480→600 mg/m2) followed 1 hr later by fixed-dose cisplatin 75 mg/m2 given q21 days in pts with advanced solid tumors. Standard phase I eligibility critieria apply. Prior cisplatin is allowed. Results: 13 evaluable pts have been enrolled: 5 male/8 female, median age 54 (range 36–68), median KPS 90% (range 80–100%), mean number of priors 2.8 (range 1–6). 2 dose limiting toxicities (DLT) were observed with escalation of safingol to 240 mg/m2 (gr 3 fatigue, gr 3 hyponatremia), both felt to be due primarily to cisplatin. The protocol was amended to reduce cisplatin to 60 mg/m2, given with 240 mg/m2 of safingol. We observed 1 DLT (persistent gr 3 thrombocytopenia) at this dose level. Pharmacokinetic (PK) analysis by mass spectroscopy shows nearly complete clearance of safingol from circulation within 24 hours of treatment with a 10-fold greater amount of safingol in whole blood than in plasma. An increase in sphingosine and dihydroceramide levels were observed with therapy, and N-acyl- and N-methyl- metabolites of safingol were detected. We observed 1 PR (4+ months) in a pt with cisplatin-refractory adrenocortical cancer metastatic to the liver and lung after only 1 cycle of therapy. 1 additional pt with adrenocortical cancer has SD (2+ months) on therapy. Conclusions: Escalating doses of safingol can be safely administered with a fixed dose of cisplatin. PK data reveal acyl- and methyl-safingol for the first time in human plasma, with a significant fraction of safingol bound to human blood cells. We observed unusual activity in pts with adrenocortical cancer. Further dose escalation of safingol with cisplatin 60 mg/m2 is ongoing, and additional PK data and complete sphingolipid profiles will be presented. (Supported by NCI R21-CA112910). No significant financial relationships to disclose.

Calcitonin stimulates the secretion of urokinase‐type plasminogen activator from prostate cancer cells: Its possible implications on tumor cell invasion

Calcitonin (CT) is synthesized and secreted in prostate epithelium, and its secretion from malignant prostates is several folds higher than that in benign prostates. CT receptor (CTR) is expressed in malignant prostate epithelium, and its activation increases invasiveness of prostate cancer (PC) cells via activation of protein kinase A. Since the role of urokinase-type plasminogen activator (uPA) in invasion of PC has been established, we tested the hypothesis that CT increases invasion of PC cells by stimulating uPA secretion from PC cells. Exogenously added CT stimulated the secretion of uPA from PC-3M cells in a dose-dependent manner, which was blocked by Rp.cAMP, a competitive inhibitor of protein kinase A. CT stimulated the secretion of MMP-2 and MMP-9 from PC-3M cells, and also increased their invasiveness. Both these actions of CT were blocked by uPA-neutralizing antibodies. Immunofluorescence studies with PC-3M cells suggest that CT stimulated redistribution of cellular uPA to focal adhesion sites, which was further confirmed by co-immunoprecipitation of uPA with focal adhesion kinase (FAK) in response to CT. These results suggest that CT increases invasiveness of PC cells by stimulating PKA-mediated uPA secretion and by redirecting the secreted uPA to focal adhesion sites. The results also suggest that uPA may, at least in part, mediate proinvasive actions of CT on PC cells by stimulating the secretion of gelatinases and degradation of focal adhesion sites.

A Liquid Chromatography/Mass Spectrometry-Based Method for the Selection of ATP Competitive Kinase Inhibitors

The currently approved kinase inhibitors for therapeutic uses and a number of kinase inhibitors that are undergoing clinical trials are directed toward the adenosine triphosphate (ATP) binding site of protein kinases. The 5'-fluorosulfonylbenzoyl 5'-adenosine (FSBA) is an ATP-affinity reagent that covalently modifies a conserved lysine present in the nucleotide-binding site of most kinases. The authors have developed a liquid chromatography/mass spectrometry-based method to monitor binding of ATP competitive protein kinase inhibitors using FSBA as a nonselective activity-based probe for protein kinases. Their method provides a general, rapid, and reproducible means to screen and validate selective ATP competitive inhibitors of protein kinases.

Pituitary adenylate cyclase-activating polypeptide promotes differentiation of mouse neural stem cells into astrocytes

We have found that pituitary adenylate cyclase-activating polypeptide (PACAP) employed at the physiological concentrations induces the differentiation of mouse neural stem cells into astrocytes. The differentiation process was not affected by cAMP analogues such as dibutylic cAMP (db-cAMP) or 8Br-cAMP or by the specific competitive inhibitor of protein kinase A, Rp-adenosine-3′,5′-cyclic monophosphothioate triethylamine salt (Rp-cAMP). Expression of the PACAP receptor (PAC1) in neural stem cells was detected by both RT–PCR and immunoblot using an affinity-purified antibody. The PACAP selective antagonist, PACAP 6-38, had an inhibitory effect on the PACAP-induced differentiation of neural stem cells into astrocytes. These results indicate that PACAP acts on the PAC1 receptor on the plasma membrane of mouse neural stem cells, with the signal then transmitted intracellularly via a PAC1-coupled G protein, does not involve Gs. This signaling mechanism may thus play a crucial role in the differentiation of neural stem cells into astrocytes.

PDGF-BB REGULATES IGF-MEDIATED IGFBP-4 PROTEOLYSIS IN FETAL LUNG FIBROBLASTS

Insulin-like growth factor (IGF)-stimulated lung fibroblast proliferation may be regulated by locally produced IGF-binding proteins (IGFBPs) during lung development. Recent evidence has shown that many growth factors participate in the regulation of cell proliferation by regulating IGFBPs. Because platelet-derived growth factor-BB (PDGF-BB)is highly expressed during lung development and is known to regulate IGFBP-4 production bylung cells, we examined the mechanisms by which PDGF-BB regulates IGFBP-4 production using primary cultures of 19-day gestation rat lung fibroblasts. Exposure of fetal rat lung fibroblasts to PDGF-BB increased IGFBP-4 mRNA transcript abundance by 3.6- and 2.4-fold at 18 and 40 hours, respectively. Addition of Rp-adenosine 3'-5'-cyclic monophosphothioa tetriethylamine (rp-cAMPS),a competitive inhibitor of protein kinase A, blunted the PDGF-BB-stimulated increase in conditioned medium (CM)IGFBP-4 and the increase in IGFBP-4 mRNA. Proteolysis of IGFBP-4 was detected in aliquots of cell-free CM from cells exposed to SFMfor 48 hours. IGFBP-4 proteolysis was inhibited by EDTA and 1,10-phenanthroline and was accentuated by the addition of IGF-I and IGF-IIand, to a lesser extent, by des(1-3)IGF-I. Exposure of cells to PDGF-BB for 48 hours resulted in an inhibition of IGFBP-4 proteolysis that was associated with a decrease in the concentration of IGF-I in CM. These studies demonstrate that PDGF-BB increases the accumulation of IGFBP-4 in fetal rat lung fibroblasts CM through increased production and by inhibiting IGF-mediated IGFBP-4 proteolysis.

Effects of vesnarinone on nitric oxide synthesis in rat cardiac myocytes.

The purpose of this study was to investigate the effects of vesnarinone on nitric oxide (NO) synthesis in cardiac myocytes. We measured the accumulation of nitrite, a stable oxidation product of NO synthase (iNOS) protein in cultured neonatal rat cardiac myocytes. Incubation of the cultures with interleukin-1 beta (IL-1 beta; 10 ng/ml) and tumor necrosis factor alpha (TNF alpha; 10 ng/ml) caused a marked increase in nitrite production. Although vesnarinone by itself showed no effect on nitrite accumulation, it enhanced cytokine-induced nitrite production by cardiac myocytes in a dose-dependent manner. The effect of vesnarinone was completely abolished in the presence of NG-monomethyl-L-arginine or actinomycin D. The vesnarinone-induced nitrite production was accompanied by increased iNOS protein expression. In the presence of dibutyryl-cAMP, cytokine-induced nitrite accumulation was further increased, but the stimulatory effect on vesnarinone on nitrite accumulation was diminished. The effect of vesnarinone was also inhibited by Rp-8-Br-cAMPS, a competitive inhibitor of protein kinase A, in a dose-dependent manner. These findings indicate that vesnarinone increases NO synthesis in cytokine-stimulated cardiac myocytes, at least partially through a cAMP-dependent pathway.

GH signalling in pancreatic beta-cells.

GH and its related peptide PRL are known to stimulate proliferation and insulin biosynthesis in pancreatic beta-cells, and assumed to be involved in their functional maturation. We investigated signal transduction of GH and PRL in insulin-secreting cells using the differentiated rat insulinoma cell line, INS-1. In these cells, both hormones stimulated proliferation and DNA synthesis, increased viability, cellular metabolism and insulin content. GH induced cytosolic Ca2+ ([Ca2+]i) rises, which appear to be due to Ca2+-influx through voltage-gated Ca2+-channels. GH also promoted tyrosine phosphorylation of several proteins in INS-1 cells, one of which was identified as JAK2 tyrosine kinase. Moreover, GH caused changes in DNA binding of nuclear proteins to some interferon-gamma-activated sites. Verapamil inhibited neither DNA synthesis nor JAK2 phosphorylation stimulated by GH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is also suggested because Rp-cAMPS, a competitive inhibitor of protein kinase A, abolished both [Ca2+]i rises and DNA synthesis stimulated by GH. The effects of GH and PRL on [Ca2+]i, JAK2 phosphorylation and DNA binding of the STATs were virtually identical in INS-1 cells. Since both hormones failed to activate MAP kinase in these cells, it is strongly suggested that activation of the JAK-STAT pathway is the major signalling event for the mitogenic effects of GH and PRL in beta-cells. It remains to be clarified whether the [Ca2+]i rise mediates other effects of these hormones.

Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells

We investigated the effects of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide (NO) synthesis in cultured rat vascular smooth muscle cells. Incubation of the cultures with interleukin-1β (10 ng/ml) for 24 h caused a significant increase in the accumulation of nitrite, a stable metabolite of NO. Although cilostazol by itself showed no effect on nitrite accumulation, it stimulated interleukin-1β-induced nitrite accumulation in a concentration-dependent manner (10 −8–10 −5 M). This effect of cilostazol was completely abolished in the presence of N G-monomethyl- l-arginine, actinomycin D or dexamethasone. The cilostazol-induced nitrite production was accompanied by increased inducible NO synthase protein expression. In the presence of dibutyryl-cAMP, interleukin-1β-induced nitrite accumulation was further increased, but the stimulatory effect of cilostazol on nitrite accumulation was blunted. The effect of cilostazol was also abolished in the presence of Rp-8-bromoadenosine-3′,5′-cyclic monophosphorothioate, a competitive inhibitor of protein kinase A. Addition of cilostazol to the cultures significantly increased intracellular cAMP levels of vascular smooth muscle cells. These results indicate that cilostazol increases NO synthesis in interleukin-1β-stimulated vascular smooth muscle cells, at least partially through a cAMP-dependent pathway.

Postreceptor signalling of growth hormone and prolactin and their effects in the differentiated insulin-secreting cell line, INS-1.

Signal transduction of two mitogens for pancreatic beta-cells, GH and PRL, was investigated using the differentiated insulin-secreting cell line, INS-1. Addition of human GH (hGH) or ovine PRL in a serum-substitute medium increased growth, insulin content, and nutrient metabolism evaluated by tetrazolium salt reduction. hGH, bovine GH (bGH), and PRL also stimulated [3H]thymidine incorporation in a dose-dependent manner (1 pM - 1 nM). hGH induced cytosolic Ca2+ ([Ca2+]i) rises, which were transient, dependent on the presence of extracellular Ca2+, blocked by verapamil, calciseptine, and the hyperpolarizing agent diazoxide, suggesting that hGH stimulates Ca(2+)-influx through L-type Ca(2+)-channels. Similar effects on [Ca2+]i were observed with bGH or PRL. hGH caused membrane depolarization in a small proportion of the cells ( < 25%) as detected by cell-attached patch-clamp analysis. However, hGH failed to stimulate acute insulin secretion. hGH, bGH, and PRL promoted tyrosine phosphorylation of JAK2 tyrosine kinase. Verapamil inhibited neither [3H]thymidine incorporation nor JAK2 phosphorylation stimulated by hGH, whereas a tyrosine kinase inhibitor, lavendustin A, blocked the mitogenic effect. Involvement of cAMP is suggested because Rp-cyclic adenosine-3', 5'-monophosphorothioate, a competitive inhibitor of protein kinase A, abolished hGH-induced [Ca2+]i rises and DNA synthesis. cAMP appears to play a permissive role, although hGH failed to raise cellular cAMP levels. These results support the idea that activation of JAK2 is a major signaling event, whereas the [CA2+]i rise is not a prerequisite, for the mitogenic effects of GH and PRL in insulin-secreting cells.

Ginsenoside-Rg 1 Regulates the Induction of Tyrosine Aminotransferase Gene Transcription in Rat Hepatocyte Cultures

Ginsenosides present in the roots of Panax ginseng C. A. Meyer have been shown to induce a number of hepatocyte gene expression. We have recently demonstrated that ginsenoside-Rg 1 (G-Rg 1) stimulated the enzyme activity of tyrosine aminotransferase (TAT), a hepatocyte specific enzyme, of which enzyme activity was dose-dependently inhibited by RU486, a specific glucocorticoid antagonist. This study was therefore designed to determine whether G-Rg 1 induces the transcriptional activity of TAT gene and to investigate whether G-Rg 1 induces the gene transcription by glucocorticoid receptor- or cAMP-mediated induction mechanism. The slot blot hybridization analysis revealed that the TAT-mRNA level was increased by 9.3-fold in hepatocyte cultures in response to G-Rg 1 stimulation. In contrast, the inductive effect of G-Rg 1 was almost equally inhibited, that is, by 49% or 50% respectively in the presence of RU486 or Rp-cAMPS, a specific competitive inhibitor of protein kinase A. These results in hepatocyte cultures suggest that G-Rg 1 modulates the TAT gene transcription through its influence on a functional or cooperative interaction between glucocorticoid receptor- and cAMP-mediated induction mechanism.

Ethanol causes desensitization of receptor-mediated phospholipase C activation in isolated hepatocytes

The effect of ethanol on receptor-mediated phospholipase C-linked signal transduction processes was investigated in isolated rat hepatocytes. Pretreatment of the cells with ethanol (6-300 mM) markedly inhibited a subsequent stimulation of phospholipase C by vasopressin, angiotensin II, or epidermal growth factor. By contrast, the effects of the alpha 1-adrenergic agonist phenylephrine and of glucagon were not affected by ethanol pretreatment. Ethanol inhibited the agonist-induced decrease in polyphosphoinositides, the formation of inositol phosphates, and the increase in cytosolic free Ca2+ levels, as detected with the intracellular Ca2+ indicator indo-1. The effects of ethanol were concentration dependent and were pronounced at low concentrations of agonists but were not significant at saturating levels. Pretreatment of the cells with the protein kinase C inhibitor H7 partly prevented the inhibition by ethanol of vasopressin-induced phospholipase C activation. By contrast, pretreatment of the cells with (Rp)-adenosine cyclic 3':5'-phosphorothioate [Rp)-cAMP-S), a competitive inhibitor of protein kinase A, potentiated the inhibitory effect of ethanol on the Ca2+ mobilization by vasopressin. (Rp)-cAMP-S similarly potentiated the inhibition of phospholipase C by the protein kinase C-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The kinase A inhibitor also made the Ca2+ mobilization by phenylephrine sensitive to ethanol, indicating that the formation of cAMP in the cells played a role in suppressing the sensitivity to ethanol. Pretreatment of the cells with ethanol enhanced the inhibitory effects of TPA on the vasopressin-induced phospholipase C activation at all concentrations of the hormone; however, these synergistic effects were prevented when TPA was added prior to ethanol, a condition that prevents the activation of phospholipase C by ethanol. The data indicate that ethanol causes desensitization of the receptor-mediated phospholipase C secondary to the ethanol-induced activation of phospholipase C and activation of protein kinase C. Ethanol treatment also affects the sensitivity of the phospholipase C system to control by protein kinases A and C. The data indicate that ethanol can affect the control of intracellular signal transduction processes in liver cells under physiologically relevant conditions.

N-(2-Aminoethyl)-5-isoquinolinesulfonamide, a newly synthesized protein kinase inhibitor, functions as a ligand in affinity chromatography. Purification of Ca2+-activated, phospholipid-dependent and other protein kinases.

We designed a simple procedure for the purification of Ca2+-activated, phospholipid-dependent protein kinase (protein kinase C) from rabbit brain, using affinity chromatography with a new affinity adsorbent. The adsorbent was synthesized by attaching the amino residue of N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) to cyanogen bromide-activated Sepharose. H-9 is a potent competitive inhibitor of protein kinase C, cGMP-, and cAMP-dependent protein kinase with respect to ATP and exhibits inhibition constants of 18, 0.87, and 1.9 microM, respectively (Hidaka, H., Inagaki, M., Kawamoto, S., and Sasaki, Y. (1984) Biochemistry, 23, 5036). A 960-fold purification was achieved in the two-step procedure, which entailed DEAE-cellulose and the affinity chromatography. The resultant preparation was essentially homogeneous, as indicated by polyacrylamide gel electrophoresis under conditions of denaturation with sodium dodecyl sulfate. The affinity of protein kinase C for the H-9-Sepharose was high, and the enzyme could not be eluted either by a high concentration of sodium chloride or by 40% glycerol. The protein kinase C could be eluted from H-9-Sepharose by the buffer containing both 0.2 M NaCl and 20% glycerol, thereby suggesting that the binding between protein kinase C and H-9-Sepharose was due to both hydrophobic and electrostatic interactions. H-9 coupled to Sepharose retained both cyclic nucleotide-dependent protein kinases and protein kinase C, and these enzymes could be eluted separately by the buffer containing L-arginine, a potent inhibitor of these three kinases. The novel aspects of these three multifunctional protein kinases can thus be investigated using isoquinolinesulfonamide derivatives.