sq-23377 and calmidazolium

Glutamate-stimulated, astrocyte-derived carbon monoxide (CO) causes cerebral arteriole dilation by activating smooth muscle cell large-conductance Ca(2+)-activated K(+) channels. Here, we examined the hypothesis that glutamate activates heme oxygenase (HO)-2 and CO production via the intracellular Ca(2+) concentration ([Ca(2+)](i))/Ca(2+)-calmodulin signaling pathway in newborn pig astrocytes. The major findings are: 1) glutamate stimulated Ca(2+) transients and increased steady-state [Ca(2+)](i) in cerebral cortical astrocytes in primary culture, 2) in astrocytes permeabilized with ionomycin, elevation of [Ca(2+)](i) concentration-dependently increased CO production, 3) glutamate did not affect CO production at any [Ca(2+)](i) when the [Ca(2+)](i) was held constant, 4) thapsigargin, a sarco/endoplasmic reticulum Ca(2+)-ATPase blocker, decreased basal CO production and blocked glutamate-induced increases in CO, and 5) calmidazolium, a calmodulin inhibitor, blocked CO production induced by glutamate and by [Ca(2+)](i) elevation. Taken together, our data are consistent with the hypothesis that glutamate elevates [Ca(2+)](i) in astrocytes, leading to Ca(2+)- and calmodulin-dependent HO-2 activation, and CO production.

Topics: Analysis of Variance; Animals; Animals, Newborn; Astrocytes; Calcium Signaling; Calmodulin; Carbon Monoxide; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Glutamic Acid; Heme Oxygenase (Decyclizing); Imidazoles; Ionomycin; Ionophores; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Swine; Thapsigargin; Time Factors; Up-Regulation

The chronic maintenance of aldosterone production in the adrenal zona glomerulosa is associated with increased expression of aldosterone synthase (P450aldo), the enzyme responsible for the conversion of 11-deoxycorticosterone to aldosterone. The major physiologic regulators of aldosterone production are angiotensin II (ANG II) and (K+) which act in part through increasing intracellular calcium ([Ca2+]i). Recently we demonstrated that increased [Ca2+]i is associated with K+ induction of P450aldo expression. To determine whether Ca2+ regulation of P450aldo is mediated through calmodulin or calmodulin-dependent kinases (CaMK), we investigated the actions of calmidazolium (a calmodulin inhibitor) and KN93 (an inhibitor of CaMK) on expression of P450aldo in human adrenocortical H295R cell line. Treatment with either calmidazolium or KN93 completely inhibited K(+)-stimulated expression of P450aldo mRNA with little effect on ANG II or dibutyryl cyclic AMP-stimulated induction of this transcript. Cellular calcium levels were also increased using the calcium ionophore ionomycin and calcium channel agonist Bay K 8644. These compounds increased P450aldo mRNA and this calcium induction was inhibited by calmidazolium and KN93. These data show that K(+)-stimulated expression of P450aldo mRNA is regulated in a Ca2+ sensitive manner through mechanisms involving calmodulin and CaMK.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Benzylamines; Calcium; Calcium Channel Agonists; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Cytochrome P-450 CYP11B2; Enzyme Inhibitors; Gene Expression Regulation; Humans; Imidazoles; Ionomycin; Potassium; RNA, Messenger; Sulfonamides

Tissue factor (TF) procoagulant activity (PCA) on the surface of intact HL-60 cells is encrypted. This latent TF PCA was activated by exposing the cells to ionomycin, a calcium ionophore. Within seconds an increase in TF PCA of greater than 100-fold was observed. The ionomycin effect was blocked by pretreating the cells with calmidazolium, a calmodulin inhibitor. Changes in TF structure and function, coincident with the ionophore-induced increase in TF PCA, were identified. TF-factor VIIa complexes formed on both untreated and ionophore-treated cells, but pseudosubstrate inhibitors only bound to TF-factor VIIa on the ionophore-treated cells. TF PCA was inhibited by reacting cells with sulfosuccinimidyl-6-(biotinamido)hexanoate, and the rate of this reaction increased twofold after cells were exposed to ionomycin. When proteins on the surface of untreated cells, expressing minimal TF PCA, were cross-linked with 3-3'-dithiobis(sulfosuccinimidylpropionate), cross-linked TF dimers were produced. TF cross-linking was prevented by first treating the cells with ionomycin. These results suggest a mechanism for the ionomycin-induced increase in TF PCA. TF activation appears to be a calmodulin-dependent process, which exposes an essential macromolecular substrate binding site on TF, possibly as the result of a change in TF quaternary structure.

Topics: Anticoagulants; Calmodulin; Cell Membrane; Cross-Linking Reagents; Enzyme Inhibitors; Factor VIIa; Factor Xa; Gene Expression; HL-60 Cells; Humans; Imidazoles; Ionomycin; Kinetics; Lipoproteins; Macromolecular Substances; Succinimides; Tetradecanoylphorbol Acetate; Thromboplastin

Understanding the mechanisms involved in the biogenesis of N-arachidonoylethanolamine (anandamide) and N-palmitoylethanolamine is important in view of the possible role of these lipids as endogenous cannabinoid substances. Anandamide (which activates cannabinoid CB1 receptors) and N-palmitoylethanolamine (which activates a CB2-like receptor subtype in mast cells) may both derive from cleavage of precursor phospholipid, N-acylphosphatidylethanolamine (NAPE), catalyzed by Ca(2+)-activated D-type phosphodiesterase activity. We report here that the de novo biosynthesis of NAPE is enhanced in a Ca(2+)-dependent manner when rat cortical neurons are stimulated with the Ca(2+)-ionophore ionomycin or with membrane-depolarizing agents such as veratridine and kainate. This reaction is likely to be mediated by a neuronal N-acyltransferase activity, which catalyzes the transfer of an acyl group from phosphatidylcholine to the ethanolamine moiety of phosphatidylethanolamine. In addition, we show that Ca2+-dependent NAPE biosynthesis is potentiated by agents that increase cAMP levels, including forskolin and vasoactive intestinal peptide. Our results thus indicate that NAPE levels in cortical neurons are controlled by Ca2+ ions and cAMP. Such regulatory effect may participate in maintaining a supply of cannabimimetic N-acylethanolamines during synaptic activity, and prime target neurons for release of these bioactive lipids.

Topics: Amides; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acids; Arylamine N-Acetyltransferase; Astrocytes; Calcium; Calcium Channel Blockers; Calmodulin; Cannabinoids; Carbachol; Cyclic AMP; Endocannabinoids; Enzyme Inhibitors; Ethanolamine; Ethanolamines; Imidazoles; Ionomycin; Ionophores; Neurons; Nicotinic Agonists; Palmitic Acids; Phosphatidylethanolamines; Polyunsaturated Alkamides; Rats; Sodium Channel Agonists; Tritium; Vasoactive Intestinal Peptide; Veratridine

The possible role of intracellular Ca2+ level on Trypanosoma cruzi differentiation was explored. The addition to epimastigotes of a Triatoma infestans intestinal homogenate, which that triggers off the differentiation to the infective metacyclic form, induced a sudden rise in [Ca2+]i from the basal value, 94 +/- 28 to 584 +/- 43 nmole/liter. This increase was not affected by the presence of EGTA in the medium. Trypsin-treated intestinal homogenate did not alter the [Ca2+]i of epimastigotes. Calmodulin inhibitors (Calmidazolium, Trifluoperazine, and Chlorpromazine) blocked differentiation. Although the calcium ionophore ionomycin increased [Ca2+]i to 342 +/- 29 nmole/liter, it was unable to induce differentiation by itself. BAY K8644 and Methoxyverapamil (agonist and antagonist of Ca2+ channels, respectively) were unable to affect [Ca2+]i by themselves, or when added to stimulated parasites, and did not exert a stimulatory or inhibitory effect on morphogenesis. BAPTA/AM, a Ca2+ chelator, partially blocked the rise in [Ca2+]i and morphogenesis; this effect was reversed by ionomycin. The requirement of intracellular Ca2+ on epimastigote multiplication was also evaluated. The addition of EGTA to the culture medium led to a decrease in epimastigote multiplication till it practically ceased in the sixth passage. When such parasites were transferred to LIT they partially recovered the growth rate. Parasites from passages III, IV, and V in the Ca(2+)-depleted medium maintained their basal [Ca2+]i, but when treated with the intestinal homogenate, the rise in [Ca2+]i was abrogated. Accordingly, the differentiation percentages of such parasites dropped significantly compared with controls.

Topics: 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester; Animals; Calcimycin; Calcium; Calcium Channel Agonists; Calcium Channel Blockers; Chelating Agents; Chlorpromazine; Dopamine Antagonists; Egtazic Acid; Enzyme Inhibitors; Gallic Acid; Gallopamil; Imidazoles; Ionomycin; Ionophores; Morphogenesis; Triatoma; Trifluoperazine; Trypanosoma cruzi

Sustained Ca2+ influx follows discharge of intracellularly stored Ca2+ in a variety of cell types previously equilibrated in Ca(2+)-free media, including cultured human mesangial cells. This Ca2+ influx pathway has been referred to as capacitative Ca2+ entry or Ca2+ release-activated Ca2+ influx (iCRAC). This study investigated two cellular mechanisms potentially controlling iCRAC in human mesangial cells, protein kinase C (PKC), a key signalling kinase activated by vasoconstrictors that release Ca2+ from internal stores, and calmodulin, a Ca(2+)-binding protein that may couple Ca2+ release to the putative channel(s). The PKC activator phorbol myristate acetate (PMA) dose-dependently inhibited both Ca2+ influx in resting cells and iCRAC, assessed by microfluorometry in fura-2-loaded monolayers, when added before or after 1 uM angiotensin II (AngII) (Ca2+ influx at 1 mM (Ca2+)e +278 +/- 56%/+80 +/- 8%, at 10 mM + 473 +/- 59%/+250 +/- 24% (Ca2+)e, -/+ PMA, respectively, P < 0.05). PMA did not affect 5 uM ionomycin-induced iCRAC, possibly because it downregulated Ca2+ release by AngII but not by ionomycin, suggesting a key role of released Ca2+ in triggering subsequent Ca2+ influx. This was confirmed by buffering the (Ca2+)i elevation induced by AngII with intracellularly trapped 1,2-bis-(0-Aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA), which abolished any subsequent iCRAC. Moreover, the calmodulin inhibitors calmidazolium (10 uM), trifluoperazine (0.1 mM), or W-7 (0.1 mM) significantly inhibited AngII- or ionomycin-activated iCRAC (+106 +/- 38/229 +/- 53, +58 +/- 9/195 +/- 29, +161 +/- 38/180 +/- 40% at 1/10 mM (Ca2+)e, all P < 0.05), but did not affect basal Ca2+ entry, consistent with a direct role of cytoplasmic Ca2+ in the regulation of ion gating. These results indicate that iCRAC is under the control of both PKC and calmodulin, and that the site of regulation is distal to the emptying of Ca2+ stores. iCRAC may represent a key mechanism for the control of Ca(2+)-regulated mesangial functions.

Topics: Angiotensin II; Biological Transport; Calcium; Calmodulin; Cells, Cultured; Egtazic Acid; Enzyme Inhibitors; Glomerular Mesangium; Humans; Imidazoles; Ionomycin; Phosphorylation; Protein Kinase C; Protein Processing, Post-Translational; Signal Transduction; Sulfonamides; Tetradecanoylphorbol Acetate; Trifluoperazine

Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV), a member of the CaM-kinase family involved in transcriptional regulation, is stimulated by Ca2+/CaM but also requires phosphorylation by a CaM-kinase kinase for full activation. In this study we investigated the physiological role of a CaM-kinase cascade in Jurkat T human lymphocytes through antigen receptor (CD3) signaling. Total and Ca(2+)-independent CaM-kinase IV activities were increased 8-14-fold by anti-CD3 antibody. This CD3-mediated activation involved phosphorylation since the immunoprecipitated CaM-kinase IV from stimulated Jurkat cells could be subsequently inactivated in vitro by protein phosphatase 2A. CaM-kinase IV immunoprecipitated from unstimulated Jurkat cells or CD3-negative mutant Jurkat cells could be activated in vitro 10-40-fold by CaM-kinase kinase purified from rat brain or thymus, whereas CaM-kinase IV from CD3-stimulated wild-type Jurkat cells was only activated to 2-3-fold by exogenous CaM-kinase kinase. CaM-kinase IV activation was triggered by Ca2+ acting through calmodulin since activation could also be elicited by ionomycin treatment, and CD3-mediated activation was blocked by the calmodulin antagonist calmidazolium. These data are consistent with a CaM-kinase cascade in which CaM-kinase IV is activated by a CaM-kinase kinase cascade triggered by elevated intracellular calcium in Jurkat cells.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Blotting, Western; Brain; Calcium-Calmodulin-Dependent Protein Kinase Kinase; Calcium-Calmodulin-Dependent Protein Kinase Type 4; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Enzyme Activation; Enzyme Inhibitors; Humans; Imidazoles; Ionomycin; Kinetics; Mitogen-Activated Protein Kinase Kinases; Molecular Sequence Data; Peptide Fragments; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinases; Protein Phosphatase 2; Protein Serine-Threonine Kinases; Rats; Receptor-CD3 Complex, Antigen, T-Cell; T-Lymphocytes

We used the secretory colonic cell line T84 to study the regulatory pathways controlling the Ca-stimulated Cl conductance [GCl(Ca)]. Under whole cell patch clamp, basal (unstimulated) current levels averaged 73 +/- 9 pA/20 pF (n = 93) and increased to 600 +/- 100 pA/20 pF (n = 53; at +100 mV) on exposure to 1-2 microM ionomycin. Bath application of the calmodulin (CaM) antagonists trifluoperazine, calmidazolium, or sphingosine (50 microM) reversibly inhibited GCl(Ca), whereas the protein kinase C antagonists H7 and phloretin (50 microM) were without effect. This suggests that increases in intracellular Ca stimulate GCl(Ca) via a CaM-dependent process rather than activating Cl channels directly. To assess the involvement of protein kinases in the Ca-dependent stimulation of Cl conductance, we employed pseudosubstrate peptide inhibitors of protein kinase C (PKC) and the Ca/CaM-dependent protein kinase II (CaMKII). Cellular concentrations of inhibitors during whole cell recording were estimated to be 4-20 times the inhibitory constant values for kinase inhibition observed in vitro. Pipette solutions containing the PKC peptide inhibitor PKC-(19-36) (7.5 microM) had no effect on GCl(Ca). In contrast, stimulation of GCl(Ca) by ionomycin was abolished when pipette solutions contained 10 microM CaMKII peptide inhibitor CaMKII-(273-302). The truncated peptide CaMKII-(284-302) (20 microM) lacks the CaMKII inhibitory domain and did not affect GCl(Ca). These data suggest that CaM, acting through the multifunctional CaMKII, mediates the Ca-dependent stimulation of Cl conductance in colonic secretory cells.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Cell Line; Chloride Channels; Colon; Imidazoles; Ion Channels; Ionomycin; Isoquinolines; Kinetics; Membrane Proteins; Peptide Fragments; Phloretin; Piperazines; Protein Kinase C; Protein Kinases; Sphingosine; Trifluoperazine

Kinetic analysis of transferrin receptor properties in 6-8 day rat reticulocytes showed the existence of a single class of high-affinity receptors (Kd 3-10 nM), of which 20-25% were located at the cell surface and the remainder within an intracellular pool. Total transferrin receptor cycling time was 3.9 min. These studies examined the effects of various inhibitors on receptor-mediated transferrin iron delivery in order to define critical steps and events necessary to maintain the functional integrity of the pathway. Dansylcadaverine inhibited iron uptake by blocking exocytic release of transferrin and return of receptors to the cell surface, but did not affect transferrin endocytosis; this action served to deplete the surface pool of transferrin receptors, leading to shutdown of iron uptake. Calmidazolium and other putative calmodulin antagonists exerted an identical action on iron uptake and receptor recycling. The inhibitory effects of these agents on receptor recycling were overcome by the timely addition of Ca2+/ionomycin. From correlative analyses of the effects of these and other inhibitors, it was concluded that: (1) dansylcadaverine and calmodulin antagonists inhibit iron uptake by suppression of receptor recycling and exocytic transferrin release, (2) protein kinase C, transglutaminase, protein synthesis and release of transferrin-bound iron are not necessary for the functional integrity of the iron delivery pathway, (3) exocytic transferrin release and concomitant receptor recycling in rat reticulocytes is dependent upon Ca2+/calmodulin, (4) dansylcadaverine, dimethyldansylcadaverine and calmidazolium act on iron uptake by interfering with calmodulin function, and (5) the endocytotic and exocytotic arms of the iron delivery pathway are under separate regulatory control.

Topics: Animals; Biological Transport; Cadaverine; Calcium; Calmodulin; Cell Membrane; Endocytosis; Exocytosis; Imidazoles; Ionomycin; Iron; Kinetics; Microscopy, Electron; Rats; Rats, Inbred Strains; Receptors, Transferrin; Reticulocytes

The effect of the Ca2+ ionophore ionomycin on neoplastic thymocytes in comparison to its effect on normal thymus cells was studied. Ionomycin increases intracellular Ca2+ in normal lymphocytes but fails to increase Ca2+ in neoplastic thymocytes. In these cells the ionophore causes a transient increase in cytosolic free Ca2+. The lack of effect of ionomycin reproduces that of A23187, but it does not depend on reduced availability of intracellular Mg2+ to exchange with Ca2+; it appears to depend on the strong activity of the plasma membrane Ca2+-extruding pump that counteracts ionomycin permeabilization and that can be partly inhibited by the calmodulin inhibitor R24571 (calmidazolium). Neoplastic thymocytes show a high content of magnesium, the intracellular binding of which is efficiently regulated by endogenous ATP. The data show also an interesting correlation between the regulation of energy metabolism (aerobic glycolysis) and cation homeostasis in the neoplastic cells studied.

Topics: Animals; Calcium; Ethers; Glycolysis; Imidazoles; In Vitro Techniques; Iodoacetates; Iodoacetic Acid; Ionomycin; Kinetics; Lymphoma; Magnesium; Mice; Mice, Inbred BALB C; Reference Values; Thymoma; Thymus Gland; Thymus Neoplasms