sq-23377 and 1-ethyl-2-benzimidazolinone

Temozolomide (TMZ), an oral alkylator of the imidazotetrazine family, is used to treat glioma. Whether this drug has any ionic effects in glioma cells remains largely unclear.. With the aid of patch-clamp technology, we investigated the effects of TMZ on the ionic currents in U373 glioma cells. The mRNA expression of KCNN4 (KCa3.1) in U373 glioma cells and TMZ's effect on K+ currents in these KCNN4 siRNA-transfected U373 cells were investigated.. In whole-cell recordings, TMZ decreased the amplitude of voltage-dependent K+ currents (IK) in U373 cells. TMZ-induced IK inhibition was reversed by ionomycin or 1-ethyl-2-benzimidazolinone (1-EBIO). In cell-attached configuration, TMZ concentration-dependently reduced the activity of intermediate-conductance Ca2+-activated K+ (IKCa) channels with an IC50 value of 9.2 µM. Chlorzoxazone or 1-EBIO counteracted the TMZ-induced inhibition of IKCa channels. Although TMZ was unable to modify single-channel conductance, its inhibition of IKCa channels was weakly voltage-dependent and accompanied by a significant prolongation in the slow component of mean closed time. However, neitherlarge-conductance Ca2+-activated (BKCa) nor inwardly rectifying K+ (Kir) channels were affected by TMZ. In current-clamp mode, TMZ depolarized the cell membrane and 1-EBIO reversed TMZ-induced depolarization. TMZ had no effect on IK in KCNN4 siRNA-transfected U373 cells.. In addition to the DNA damage it does, its inhibitory effect on IKCa channels accompanied by membrane depolarization could be an important mechanism underlying TMZ-induced antineoplastic actions.

Topics: Alkylating Agents; Benzimidazoles; Cell Line, Tumor; Dacarbazine; DNA Damage; Glioma; Humans; Intermediate-Conductance Calcium-Activated Potassium Channels; Ionomycin; Large-Conductance Calcium-Activated Potassium Channels; Membrane Potentials; Patch-Clamp Techniques; Potassium Channels, Inwardly Rectifying; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Temozolomide

This study was undertaken to determine whether long-term in vivo administration of nitroglycerine (NTG) downregulates the hyperpolarization induced by acetylcholine (ACh) in aortic valve endothelial cells (AVECs) of the rabbit and, if so, whether antioxidant agents can normalize this downregulated hyperpolarization. ACh (0.03-3 microM) induced a hyperpolarization through activations of both apamin- and charybdotoxin-sensitive Ca2+-activated K+ channels (K(Ca)) in rabbit AVECs. The intermediate-conductance K(Ca) channel (IK(Ca)) activator 1-ethyl-2-benzimidazolinone (1-EBIO, 0.3 mM) induced a hyperpolarization of the same magnitude as ACh (3 microM). The ACh-induced hyperpolarization was significantly weaker, although the ACh-induced [Ca2+]i increase was unchanged, in NTG-treated rabbits (versus NTG-untreated control rabbits). The hyperpolarization induced by 1-EBIO was also weaker in NTG-treated rabbits. The reduced ACh-induced hyperpolarization seen in NTG-treated rabbits was not modified by in vitro application of the superoxide scavengers Mn-TBAP, tiron or ascorbate, but it was normalized when ascorbate was coadministered with NTG in vivo. Superoxide production within the endothelial cell (estimated by ethidium fluorescence) was increased in NTG-treated rabbits and this increased production was normalized by in vivo coadministration of ascorbate with the NTG. It is suggested that long-term in vivo administration of NTG downregulates the ACh-induced hyperpolarization in rabbit AVECs, possibly through chronic actions mediated by superoxide.

Topics: Acetylcholine; Animals; Antioxidants; Aortic Valve; Apamin; Ascorbic Acid; Benzimidazoles; Biological Factors; Calcium; Calcium Channel Agonists; Charybdotoxin; Dose-Response Relationship, Drug; Down-Regulation; Drug Tolerance; Endothelial Cells; Ionomycin; Ionophores; Male; Membrane Potentials; Nitroglycerin; Potassium Channels, Calcium-Activated; Rabbits; Superoxides; Time Factors; Vasodilator Agents

Airway mucociliary clearance is subject to the autocrine/paracrine regulation of extracellular nucleotides released from the airway epithelial cells. The present study was performed in pursuit of effective modulators of ATP release under physiologic conditions in polarized human airway epithelial cells (Calu-3). Neither isoproterenol, forskolin, nor ionomycin augmented extracellular ATP release detected by luciferase assay. However, direct activation of the human intermediate conductance, Ca(2+)-activated K(+) channel (hIK-1) by 1-ethyl-2-benzimdazolinone (1-EBIO, 1 mM) and chlorzoxazone (CZ, 1 mM) increased ATP release predominantly in the apical compartment. Measurement of fluo-3 signals revealed that 1-EBIO- and CZ-stimulated cytosolic Ca(2+) mobilization was suppressed by the presence of MRS-2179, a specific P2Y(1) receptor antagonist. The hIK-1-mediated ATP release was inhibited by a hIK-1 blocker (charybdotoxin), and an Na(+)-K(+)-2Cl(-) cotransport blocker (bumetanide) without interruption by GdCl(3), an inhibitor of stretch-activated nonselective cation (SA) channels, or glybenclamide, a blocker of the cystic fibrosis transmembrane conductance regulator (CFTR). These results suggest that a cell volume decrease via the hIK-1-mediated KCl loss and the resultant induction of a regulatory volume increase via the Na(+)-K(+)-2Cl(-) transporter may trigger release of ATP, which causes P2Y(1)-mediated Ca(2+) mobilization, through mechanisms unrelated to the CFTR and SA channels.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Benzimidazoles; Bumetanide; Calcium; Cell Size; Charybdotoxin; Chlorzoxazone; Colforsin; Cystic Fibrosis Transmembrane Conductance Regulator; Cytosol; Epithelial Cells; Glyburide; Humans; Ionomycin; Isoproterenol; Luciferases; Lung; Potassium Channels, Calcium-Activated; Potassium Chloride; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2Y1

The Ca(2+)-activated K(+) channel rSK4 is the rat homologue of the human SK4/IK1 (KCNN4) channel. In colonic mucosa rSK4 plays a key role during acetylcholin-induced secretion. This study was aimed to characterize the properties of the rat SK4 channel.. Electrophysiological measurements were performed on rSK4 expressing Xenopus laevis oocytes and rat colonic crypts. Intracellular Ca(2+) activity was assessed by Oregon Green fluorescence measurements.. The 10 pS rSK4 expressed in oocytes was Ca(2+)-sensitive and inhibited by calmodulin antagonists. 1-ethyl-2-benzimidazolinone (1-EBIO), a known activator of SK4/IK1 channels, also activated rSK4. 1-EBIO affected the current neither at saturating Ca(2+) activities nor under Ca(2+)-free conditions, but increased the Ca(2+) sensitivity of rSK4. rSK4 was strongly activated by cytosolic ATP. However, PKA itself, PKA inhibitors and mutation of the PKA phosphorylation site (S332A) did not affect channel activity. The PKC activator 1,2-dioctanoyl-sn-glycerol and the PKC inhibitor bisindolylmaleimide also failed to influence rSK4.. The Ca(2+)-sensitive rSK4 is activated by 1-EBIO probably via facilitation of the Ca(2+)-calmodulin-rSK4 interaction. The strong ATP-activation of rSK4 is likely to be caused by phosphorylation via a yet unknown kinase and might involve additional subunits.

Topics: 1-Methyl-3-isobutylxanthine; Adenosine Triphosphate; Animals; Benzimidazoles; Calcium; Calmodulin; Charybdotoxin; Colforsin; Colon; Cyclic AMP-Dependent Protein Kinases; Enzyme Activation; Intermediate-Conductance Calcium-Activated Potassium Channels; Intestinal Mucosa; Ionomycin; Oocytes; Patch-Clamp Techniques; Phosphorylation; Potassium; Potassium Channels; Potassium Channels, Calcium-Activated; Protein Kinase C; Rats; Recombinant Proteins; Sulfonamides; Xenopus laevis