Calmidazolium Chloride Research Articles

A SOX9 Defect of Calmodulin-dependent Nuclear Import in Campomelic Dysplasia/Autosomal Sex Reversal

During mammalian sex determination, SOX9 is translocated into the nuclei of Sertoli cells within the developing XY gonad. The N-terminal nuclear localization signal (NLS) is contained within a SOX consensus calmodulin (CaM) binding region, thereby implicating CaM in nuclear import of SOX9. By fluorescence spectroscopy and glutaraldehyde cross-linking, we show that the SOX9 HMG domain and CaM interact in vitro. The formation of a SOX9.CaM binary complex is calcium-dependent and is accompanied by a conformational change in SOX9. A CaM antagonist, calmidazolium chloride (CDZ), was observed to block CaM recognition of SOX9 in vitro and inhibit both nuclear import and consequent transcriptional activity of SOX9 in treated cells. The significance of the SOX9-CaM interaction was highlighted by analysis of a missense SOX9 mutation, A158T, identified from a XY female with campomelic dysplasia/autosomal sex reversal (CD/SRA). This mutant binds importin beta normally despite defective nuclear import. Fluorescence and quenching studies indicate that in the unbound state, the A158T mutant shows a similar conformation to that of the WT SOX9, but in the presence of CaM, the mutant undergoes unusual conformational changes. Furthermore, SOX9-mediated transcriptional activation by cells expressing the A158T mutant is more sensitive to CDZ than cells expressing WT SOX9. These results suggest first that CaM is involved in the nuclear transport of SOX9 in a process likely to involve direct interaction and second, that CD/SRA can arise, at least in part, from a defect in CaM recognition, ultimately leading to reduced ability of SOX9 to activate transcription of cartilage and testes-forming genes.

Synergistic stimulation of CCK secretion from STC-1 cells by sodium oleate and amino acids

A mixed meal is a potent stimulant for the release of gastrointestinal peptides. Fatty acids and amino acids (AA)are digested products of food that stimulate CCK secretion. The postprandial concentration of these nutrients in the intestinal lumen may not be comparable to those during intraduodenal infusion to demonstrate their secretagogue activity. Sodium oleate (SO) and AA have been shown to stimulate STC-l cells to secrete CCK. We hypothesized that these nutrients may act synergistically on CCK cells at subthreshold concentrations. Purpose: To investigate whether or not low doses of SO and AA (L-Trp and L-Phe)can act synergistically on CCK release from STC-l cells. Methods: STC-I cells were incubated without or with SO, L-Trp or L-Phe, or combination of SO with either AA in the absence or presence of different protein kinase inhibitors. The release of CCK in the medium was assessed by a specific RIA. The effect of SO and amino acid on intracellular Ca + concentration ([Ca +L) was determined spectrafluorometrically in Fura 2 AM-preloaded cells. Results: There was a significant synergistic stimulation of CCK release by SO and TrplPhe as demonstrated by the difference between their combinational stimulation and the sum of their individual effects shown in the Table below. The synergism was also timeand dose-dependent. There was no synergistic increase in [Ca+L level between SO (0.14 mM) and Phe (10 mM). The PKC inhibitor, staurosporine (10 nM)did not significantly inhibit each amino acid stimulated CCK release but significantly reduced their synergism with SO. On the other hand, the calmodulin antagonist, calmidazolium chloride did not significantly affect the synergistic effect between SO and amino acids. Conclusion: Combination of SO and TrplPhe synergistically stimulated CCK release from STC-I cells timeand dose-dependently. The synergism appears to be mediated by PKC rather than by Ca+{calmodulin-dependent protein kinase II nor the elevation of [Ca2+L known to mediate stimulation by SO. 599

Activation of adenylate cyclase results in down-regulation of c-jun mRNA expression in rat C6 glioma cells

To investigate the possible mechanisms involved in forskolin-induced c-jun mRNA decrease in rat C6 glioma cells, we examined effects of a PKA inhibitor (H-89), a l-type Ca 2+ channel blocker (nimodipine), a calmodulin activation inhibitor (calmidazolium chloride) and a Ca 2+/calmodulin-dependent protein kinase II inhibitor (KN-62) on forskolin-induced c-jun mRNA down-regulation. H-89 caused a reversal of forskolin-induced c-jun mRNA decrease. Furthermore, nimodipine, KN-62 and calmidazolium chloride partially blocked forskolin-induced c-jun mRNA down-regulation. Our results suggest that activation of adenylate cyclase appears to be involved in a down-regulation of c-jun mRNA expression through a PKA pathway. In addition, l-type calcium channels, calmodulin and Ca 2+/calmodulin-dependent protein kinase II may be partially involved in c-jun mRNA down-regulation induced by forskolin.

Dynamic regulation of [Ca2+]iby plasma membrane Ca2+-ATPase and Na+/Ca2+exchange during capacitative Ca2+entry in bovine vascular endothelial cells

The dynamic regulation of Ca2+extrusion by the plasma membrane Ca2+-ATPase (PMCA) and Na+/Ca2+exchange (NCX) was investigated in single cultured calf pulmonary artery endothelial (CPAE) cells using indo-1 microfluorimetry to measure cytoplasmic Ca2+concentration ([Ca2+]i). The quantitative analysis of the recovery from an increase of [Ca2+]ielicited by activation of capacitative Ca2+entry (CCE) served to characterize kinetic parameters of these Ca2+extrusion systems in the intact cell. In CPAE cells the PMCA is activated in a Ca2+- and time-dependent manner. Full activation of the pump occurs only after [Ca2+]ihas been elevated for at least 1 min which results in an increase of the affinity of the pump for Ca2+and an increase in the apparent maximal extrusion rate (Vmax). Application of calmodulin antagonists W-7 and calmidazolium chloride (compound R 24571) revealed that calmodulin is a major regulator of PMCA activity in vivo. Sequential and simultaneous inhibition of PMCA and NCX suggested that both contribute to Ca2+extrusion in a non-additive fashion. The activity of one system is dynamically adjusted to compensate for changes in the extrusion rate by the alternative transporter. It was concluded that in vascular endothelial cells, the PMCA functions as a calmodulin-regulated, high-affinity Ca2+removal system. The contribution by the low-affinity NCX to Ca2+clearance became apparent at [Ca2+]i>~150 nM under conditions of submaximal activation of the PMCA.

Hydrogen peroxide stimulates extracellular signal-regulated protein kinases in pulmonary arterial smooth muscle cells.

Hydrogen peroxide (H2O2) has emerged as an important intracellular signaling molecule and has been shown to stimulate the growth of vascular smooth muscle cells. Activation of p44 and p42 extracellular signal-regulated protein kinases (ERK1 and ERK2) is an important step in the cascade leading to cell growth and proliferation. In the present study, we investigated the effects and mechanisms of H2O2 on activation of ERK1 and ERK2 in pulmonary arterial smooth muscle cells (PASMC). Assays of immune-complex kinase activity revealed that exposure of PASMC to H2O2 stimulated myelin basic protein (MBP) phosphorylation in a concentration- and time-dependent manner. Western blot analysis done with phospho-specific mitogen-activated protein (MAP) kinase antibodies demonstrated that H2O2 stimulated the phosphorylation of p42, p44, p46, and p38 MAP kinases. H2O2 also increased the expression of the early immediate genes c-jun and fra-1. Activation of ERK1 and ERK2 by H2O2 was significantly reduced by downregulation of protein kinase C (PKC) with phorbol-12-myristate-13-acetate (PMA) or by a PKC inhibitor, calphostin C. In addition, removal of extracellular Ca2+, depletion of the intracellular Ca2+ pool by thapsigargin, or pretreatment of PASMC with the calmodulin antagonist N-(6 aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or with calmidazolium chloride also decreased H2O2-induced ERK1 and ERK2 activation. Furthermore, stimulation of ERK1 and ERK2 activity by H2O2 was partly attenuated by genistein, a tyrosine kinase inhibitor. Taken together, these data suggest that H2O2 activates ERK1, ERK2, p46 JNK, and p38 MAP kinases in PASMC. The activation of ERK1 and ERK2 appears to be primarily dependent on PKC, and to be partly modulated by Ca2+/calmodulin and by activation of tyrosine kinases.

Calcium/Calmodulin-dependent Conversion of 5-Oxoeicosanoids to 6,7-Dihydro Metabolites by a Cytosolic Olefin Reductase in Human Neutrophils

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.

Constitutive calcium-dependent isoform of nitric oxide synthase in the human placental villous vascular tree

We have characterized the NO synthase enzyme in the villous vasculature of the human placenta as part of our ongoing studies of the regulation of NO synthesis in this circulation. NO synthase activity was determined by conversion of 3H L-arginine to 3H L-citrulline in cellular homogenate, cytosolic and particulate fractions. Optimal NO synthase activity was measured in all fractions in the presence of 1 mM NADPH, 10 microM tetrahydrobiopterin, 2 microM FAD, 100 microM free calcium and 50 U/ml calmodulin. The calmodulin inhibitor calmidazolium (50 microM) and FAD inhibitor diphenyliodonium chloride (1 microM) significantly reduced enzyme activity. The EC50 for calcium was 0.1 microM and Km for L-arginine 2.00 +/- 0.49 microM with Vmax 55.8 +/- 28.3 pmoles/mg protein/min. Enzyme activity was inhibited in both cytosolic and particulate fractions by ng-nitro-L-arginine and ng-monomethyl-L-arginine in a concentration-dependent manner (10(-8)-10(-4) M). A calcium-independent NO synthase activity was also determined, but only constituted between 5-6 per cent of total activity. On Western blotting, a single 135 kda species was identified in each fraction with a monoclonal antibody raised against bovine aortic endothelial NO synthase. The NO synthase enzyme of the villous vasculature appears to correspond to the type III calcium-calmodulin dependent endothelial isoform.

Injections of calmidazolium chloride into the ipsilateral medial vestibular nucleus or fourth ventricle reduce spontaneous ocular nystagmus following unilateral labyrinthectomy in guinea pigs.

The effects of three injections (0.5-4.5 h post-operation) of 1-[bis-(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4- dichlorobenzyloxy)phenethyl]-imidazolium chloride (calmidazolium chloride, R24571), into the ipsilateral medial vestibular nucleus or fourth ventricle, on vestibular compensation for unilateral labyrinthectomy was studied in guinea pigs. R24571, a calmodulin antagonist and inhibitor of several Ca(2+)-dependent enzymes, caused a significant reduction in the average frequency of spontaneous ocular nystagmus (spontaneous nystagmus) during the first 53 h following unilateral labyrinthectomy (n = 5), compared with vehicle-injected animals (n = 5). Although a statistical analysis was not performed on the yaw head tilt and roll head tilt data because of the large variability between animals over the 53-h period of compensation, most R24571-treated animals had less yaw head tilt (4/4 animals) and roll head tilt (4/5 animals) at 9-11 h post-labyrinthectomy than the average values for the vehicle groups at that time. The decrease in the frequency of spontaneous nystagmus following R24571 treatment was not associated with general ataxia or sedation. These results are consistent with recent biochemical studies in suggesting that intracellular pathways associated with Ca2+ may be involved in the neuronal mechanisms of vestibular compensation following unilateral labyrinthectomy.

Time-dependent inhibition of inositol-1,4,5-trisphosphate-5-phosphatase by calmidazolium chloride in rat GH 3 cells

The calmodulin inhibitor calmidazolium chloride inhibited the activity of soluble and particulate Ins(1,4,5)P 3-5-phosphatase from GH 3 cells, with an IC 50 value of ∼ 100 μM following a 10-min preincubation with enzyme. The inhibition was time-dependent and could not be reversed by washing of the particulate fraction. It is concluded that although the inhibitory effect of calmidazolium chloride cannot be related per se to inhibition of calmodulin function, effects of this compound unrelated to actions upon calmodulin function may be found when concentrations that are only moderately supramaximal are used.

Molecular mechanisms of brainstem plasticity. The vestibular compensation model.

Vestibular compensation is the process of behavioral recovery that occurs following unilateral deafferentation of the vestibular nerve fibers (unilateral labyrinthectomy, UL). Since UL results in a permanent loss of vestibular input from the ipsilateral vestibular (VIIIth) nerve, vestibular compensation is attributed to CNS plasticity and has been used as a general model of lesion-induced CNS plasticity. Behavioral recovery from the ocular motor and postural symptoms of UL is correlated with a partial return of resting activity to neurons in the vestibular nucleus (VN) on the deafferented side (the "deafferented VN"), and lesions to the deafferented VN prevent compensation; therefore, the regeneration of resting activity within the deafferented VN is believed to have a causal role in vestibular compensation. The biochemical mechanisms responsible for the adaptive neuronal changes within the deafferented VN are poorly understood. Neuropeptide hormone fragments, such as adrenocorticotrophic hormone (ACTH)-4-10, have been shown to accelerate vestibular compensation and can act directly on some VN neurons in vitro. Antagonists for the N-methyl-D-aspartate (NMDA) receptor have been shown to inhibit vestibular compensation if administered early in the compensation process. Biochemical studies in frog indicate marked alterations in the phosphorylation patterns of several proteins during compensation, and the in vitro phosphorylation of some of these proteins is modulated by ACTH-(1-24), calcium (Ca2+), and calmodulin or protein kinase C. It is therefore possible that ACTH fragments and NMDA antagonists (via their effects on NMDA receptor-mediated Ca2+ channels) modulate vestibular compensation through their action on Ca(2+)-dependent pathways within VN neurons. Recent studies have shown that some Ca2+ channel antagonists and the Ca(2+)-dependent enzyme inhibitor calmidazolium chloride facilitate vestibular compensation. How the regulation of Ca2+ may be related to the neuronal changes responsible for vestibular compensation is unclear at present.