kn-93 and 2-aminoethoxydiphenyl-borate

Endothelin (ET-1) is a peptide hormone mediating a wide variety of biological processes and is associated with development of cardiac dysfunction. Generally, ET-1 is regarded as a molecular marker released only in correlation with the observation of a hypertrophic response or in conjunction with other hypertrophic stress. Although the cardiac hypertrophic effect of ET-1 is demonstrated, inotropic properties of cardiac muscle during chronic ET-1-induced hypertrophy remain largely unclear. Through the use of a novel in vitro multicellular culture system, changes in contractile force and kinetics of rabbit cardiac trabeculae in response to 1 nM ET-1 for 24 hours can be observed. Compared to the initial force at t = 0 hours, ET-1 treated muscles showed a ~2.5 fold increase in developed force after 24 hours without any effect on time to peak contraction or time to 90% relaxation. ET-1 increased muscle diameter by 12.5 ± 3.2% from the initial size, due to increased cell width compared to non-ET-1 treated muscles. Using specific signaling antagonists, inhibition of NCX, CaMKII, MAPKK, and IP3 could attenuate the effect of ET-1 on increased developed force. However, among these inhibitions only IP3 receptor blocker could not prevent the increase muscle size by ET-1. Interestingly, though calcineurin-NFAT inhibition could not suppress the effect of ET-1 on force development, it did prevent muscle hypertrophy. These findings suggest that ET-1 provokes both inotropic and hypertrophic activations on myocardium in which both activations share the same signaling pathway through MAPK and CaMKII in associated with NCX activity.

Topics: Analysis of Variance; Animals; Benzylamines; Boron Compounds; Calcineurin; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cardiomegaly; Cyclosporine; Endothelins; Immunoblotting; In Vitro Techniques; Indoles; Kinetics; Maleimides; Mitogen-Activated Protein Kinases; Myocardial Contraction; NFATC Transcription Factors; Rabbits; Signal Transduction; Sulfonamides; Thiourea; Time Factors

Although understood to be ubiquitously expressed, the functional identification and significance of Mg(2+)-inhibited, nonspecific cation currents has been established in only a few cell types. Here we identified an outwardly rectifying nonspecific cation current in quiescent rat hepatocytes and the proliferating and polarized rat hepatoma, WIF-B. Under whole cell recording conditions in which cells were bathed and dialyzed with Na-gluconate solutions, the latter Ca(2+) and Mg(2+) free, current reversed close to 0 mV, was time independent, and was greater than 10 times higher at +120 mV compared with -120 mV. Outward current at -120 mV developed slowly, from 17.7 +/- 10.3 pA/pF at patch rupture to 106.6 +/- 15.6 pA/pF at 12 min in WIF-B cells, and 4.9 +/- 2.7 to 20.6 +/- 5.6 pA/pF in rat hepatocytes. The nonspecific TRP channel inhibitor, 2-aminoethoxyphenylborate (2-APB), inhibited current (IC(50) = 72 +/- 13 microM) and caused apoptotic cell death in WIF-B cells. Rat hepatocyte survival was more resistant to 2-APB. Dialysis of WIF-B cells with physiological concentrations of Mg(2+) and Mg-ATP, but not ATP alone, inhibited current development, suggesting that Trpm7 rather than Trpm6 underlies this current. RT-PCR demonstrated that both Trpm6 and Trpm7 are expressed at similar levels in both cell types, suggesting that the functional differences noted are not transcript dependent. Intracellular Ca(2+) (IC(50) = 125 +/- 35 nM) also inhibited current development, and this could be partially relieved by the calmodulin and Ca(2+)/calmodulin-dependent kinase inhibitors W-7, staurosporine, KN-93, or calmodulin kinase II (CaMKII) inhibitory peptide. To summarize, our results show that in addition to their established Mg(2+) sensitivity, Trpm7-like channels are inhibited by cytosolic Ca(2+) in a CaMKII-dependent manner and may support hepatocellular survival during proliferation.

Topics: Adenosine Triphosphate; Animals; Apoptosis; Benzylamines; Boron Compounds; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calmodulin; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Polarity; Cell Proliferation; Cell Survival; Dialysis; Dose-Response Relationship, Drug; Female; Hepatocytes; Humans; Liver Neoplasms; Magnesium; Membrane Potentials; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Staurosporine; Sulfonamides; Time Factors; TRPM Cation Channels

This study was designed to analyze the effects of the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609 (7-oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid) toward the aryl hydrocarbon receptor (AhR) pathway because Ca2+/calmodulin-dependent protein kinase (CaMK) Ialpha, known as a downstream CaMKK effector, has been recently shown to contribute to the AhR cascade. STO-609 failed to alter up-regulation of the AhR target CYP1A1 in response to the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in MCF-7 cells. STO-609, used at a 25 muM concentration known to fully inhibit CaMKK activity, was surprisingly found to markedly induce CYP1A1 expression and activity by itself in MCF-7 cells; it similarly up-regulated various other AhR target genes in human macrophages. STO-609-related CYP1A1 induction was prevented by chemical inhibition or small interfering RNA-mediated knockdown expression of AhR. Moreover, STO-609 was demonstrated to physically interact with the ligand-binding domain of AhR, as assessed by TCDD binding competition assay, and to induce AhR translocation to the nucleus. As already reported for AhR agonists, STO-609 triggered the increase of [Ca2+](i) and activation of CaMKIalpha, whose inhibition through the use of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester or the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine), respectively, prevented STO-609-mediated CYP1A1 activity induction. Taken together, these results demonstrate that the CaMKK inhibitor STO-609 can act as an AhR ligand and, in this way, fully activates the Ca2+/CaMKIalpha/AhR cascade. Such data, therefore, make unlikely any contribution of CaMKK activity to the AhR pathway and, moreover, suggest that caution may be required when using STO-609 as a specific inhibitor of CaMKKs.

Topics: Active Transport, Cell Nucleus; AMP-Activated Protein Kinases; Aryl Hydrocarbon Hydroxylases; Benzimidazoles; Benzoflavones; Benzylamines; Boron Compounds; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 1; Cell Line, Tumor; Chelating Agents; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1B1; Cytochrome P-450 Enzyme System; Egtazic Acid; Enzyme Inhibitors; Gene Expression; Humans; Integrin beta Chains; Interleukin-8; Ionomycin; Macrophages; Naphthalimides; Phosphorylation; Polychlorinated Dibenzodioxins; Receptors, Aryl Hydrocarbon; RNA, Small Interfering; Sulfonamides

The role of glycolytically generated ATP in Ca(2+)/calmodulin-dependent kinase II (CaMKII)-mediated regulation of intracellular Ca(2+) signaling was examined in cultured calf pulmonary artery endothelial (CPAE) cells. Exposure of cells (extracellular Ca(2+) concentration = 2 mM) to glycolytic inhibitors 2-deoxy-D-glucose (2-DG), pyruvate (pyr) + beta-hydroxybutyrate (beta-HB), or iodoacetic acid (IAA) caused an increase of intracellular Ca(2+) concentration ([Ca(2+)](i)). CaMKII inhibitors (KN-93, W-7) triggered a similar increase of [Ca(2+)](i). The rise of [Ca(2+)](i) was characterized by a transient spike followed by a small sustained plateau of elevated [Ca(2+)](i). In the absence of extracellular Ca(2+) 2-DG caused an increase in [Ca(2+)](i), suggesting that inhibition of glycolysis directly triggered release of Ca(2+) from intracellular endoplasmic reticulum (ER) Ca(2+) stores. The inositol-1,4,5-trisphosphate receptor (IP(3)R) inhibitor 2-aminoethoxydiphenyl borate abolished the KN-93- and 2-DG-induced Ca(2+) response. Ca(2+) release was initiated in peripheral cytoplasmic processes from which activation propagated as a [Ca(2+)](i) wave toward the central region of the cell. Focal application of 2-DG resulted in spatially confined elevations of [Ca(2+)](i). Propagating [Ca(2+)](i) waves were preceded by [Ca(2+)](i) oscillations and small, highly localized elevations of [Ca(2+)](i) (Ca(2+) puffs). Inhibition of glycolysis with 2-DG reduced the KN-93-induced Ca(2+) response, and vice versa during inhibition of CaMKII 2-DG-induced Ca(2+) release was attenuated. Similar results were obtained with pyr + beta-HB and W-7. Furthermore, 2-DG and IAA caused a rapid increase of intracellular Mg(2+) concentration, indicating a concomitant drop of cellular ATP levels. In conclusion, CaMKII exerts a profound inhibition of ER Ca(2+) release in CPAE cells, which is mediated by glycolytically generated ATP, possibly through ATP-dependent phosphorylation of the IP(3)R.

Topics: 3-Hydroxybutyric Acid; Adenosine Triphosphate; Animals; Antimetabolites; Benzylamines; Boron Compounds; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cattle; Cells, Cultured; Cytoplasm; Deoxyglucose; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endothelial Cells; Enzyme Inhibitors; Extracellular Fluid; Glycolysis; Inositol 1,4,5-Trisphosphate Receptors; Iodoacetic Acid; Kinetics; Magnesium; Phosphorylation; Pulmonary Artery; Pyruvic Acid; Sulfonamides

The effects of inhibitors of CaMKII on intracellular Ca2+ signaling were examined in single calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorometry to measure cytoplasmic Ca2+ concentration ([Ca2+]i). The three CaMKII inhibitors, KN-93, KN-62, and autocamtide-2-related inhibitory peptide (AIP), all reduced the plateau phase of the [Ca2+]i transient evoked by stimulation with extracellular ATP. Exposure to KN-93 or AIP alone in the presence of 2 mM extracellular Ca2+ resulted in a dose-dependent increase of [Ca2+]i consisting of a rapid and transient Ca2+ spike followed by a small sustained plateau phase of elevated [Ca2+]i. Exposure to KN-93 in the absence of extracellular Ca2+ caused a transient rise of [Ca2+]i, suggesting that exposure to CaMKII inhibitors directly triggered release of Ca2+ from intracellular endoplasmic reticulum (ER) Ca2+ stores. Repetitive stimulation with KN-93 and ATP, respectively, revealed that both components released Ca2+ largely from the same store. Pretreatment of CPAE cells with the membrane-permeable inositol 1,4,5-trisphosphate (IP3) receptor blocker 2-aminoethoxydiphenyl borate caused a significant inhibition of the KN-93-induced Ca2+ response, suggesting that exposure to KN-93 affects Ca2+ release from an IP3-sensitive store. Depletion of Ca2+ stores by exposure to ATP or to the ER Ca2+ pump inhibitor thapsigargin triggered robust capacitative Ca2+ entry (CCE) signals in CPAE cells that could be blocked effectively with KN-93. The data suggest that in CPAE cells, CaMKII modulates Ca2+ handling at different levels. The use of CaMKII inhibitors revealed that in CPAE cells, the most profound effects of CaMKII are inhibition of release of Ca2+ from intracellular stores and activation of CCE.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Adenosine Triphosphate; Animals; Benzylamines; Biological Transport, Active; Boron Compounds; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Cattle; Cell Line; Endothelial Cells; Endothelium, Vascular; Enzyme Activation; Peptides; Sulfonamides