Citations

ATP-sensitive K+ (KATP) channels are present at high density in membranes of cardiac cells where they regulate cardiac function during cellular metabolic impairment. KATP channels have been implicated in the shortening of the action potential duration and the cellular loss of K+ that occurs during metabolic inhibition. KATP channels have been associated with the cardioprotective mechanism of ischemia-related preconditioning. Intracellular ATP (ATPi) is the main regulator of KATP channels. ATPi has two functions: 1) to close the channel (ligand function) and 2) in the presence of Mg2+, to maintain the activity of KATP channels (presumably through an enzymatic reaction). KATP channel activity is modulated by intracellular nucleoside diphosphates that antagonize the ATPi-induced inhibition of channel opening or induce KATP channels to open. How nucleotides will affect KATP channels depends on the state of the channel. K+ channel-opening drugs are pharmacological agents that enhance KATP channel activity through different mechanisms and have great potential in the management of cardiovascular conditions. KATP channel activity is also modulated by neurohormones. Adenosine, through the activation of a GTP-binding protein, antagonizes the ATPi-induced channel closure. Understanding the molecular mechanisms that underlie KATP channel regulation should prove essential to further define the function of KATP channels and to elucidate the pharmacological regulation of this channel protein. Since the molecular structure of the KATP channel has now become available, it is anticipated that major progress in the KATP channel field will be achieved.

Abstract The Yin Yang 1 (YY1) transcription factor is a member of Polycomb protein family, with important roles in development, differentiation, replication and cell proliferation. While YY1 expression is correlated with survival in serous ovarian cancer, its role in chemotherapeutic agent-mediated cytotoxicity is not known. Immunoblot analysis revealed that YY1 is highly expressed in ovarian cancer cell lines, but not in immortalized ovarian surface epithelial (OSE) cell lines, suggesting a possible role of YY1 in ovarian carcinogenesis. shRNA mediated stable downregulation of YY1 resulted in inhibition of proliferation by CyQuant assay in SKOV3 and SKOV3ip cells. While YY1 downregulation increased the sensitivity against paclitaxel in both cell lines compared to nontargeted cells (NTC) as determined by MTT and clonogenic assays, it had no effect on sensitivity to cisplatin-mediated cytotoxicity. YY1 downregulated clones treated with paclitaxel resulted in an increase in cleavage of poly (ADP-ribose) polymerase and caspase-3/7 activity compared to NTC clones treated with it in SKOV3 and SKOV3ip1 cells. To determine the mechanism of paclitaxel sensitivity modulated by YY1, we performed cell cycle analysis and determined the expression of microtubules at protein level. Cell cycle analysis showed that downregulation of YY1 decreased the proportion of G0/G1 phase with corresponding increase in cells accumulating both S and G2/M phases in SKOV3 and SKOV3ip1 cells. Using nocodazole which synchronizes cells into mitotic phase, we found that blocking mitotic exit by YY1 knockdown slowed the cyclin B1 proteolysis, thus allowing more time for death initiation by paclitaxel. Immunoblot analysis showed that downregulation of YY1 increased the acetylation of alpha-tubulin and attenuated betaIII-tubulin expression compared to NTC cells. Immunoprecipitation and immunofluorescence assays revealed that YY1 interacts with HDAC6 which deacetylates alpha-tubulin and these proteins co-localized in mitotic phase, suggesting that dysfunction of HDAC6 by downregulation of YY1 could lead to microtubules stabilization, resulting in the paclitaxel-induced apoptosis by blocking mitotic exit. Moreover, nude mice bearing xenografts with stable downregulation of YY1 in SKOV3ip1 were more sensitive to paclitaxel-induced cytotoxicity compared to xenografts of NTC cells. Additionally immunohistchemical staining showed a decrease in betaIII-tubulin expression in tumor of YY1 downregulated clones compared to that of NTC clones. Collectively, these results indicate that YY1 acts as a modulator of microtubules expression and might be therapeutically targeted against epithelial ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4960. doi:1538-7445.AM2012-4960

Effects of ER-001533 (ER), a newly synthesized vasorelaxant, on the membrane currents were examined in single ventricular cells of guinea pigs. The patch-clamp technique was used in the "whole-cell" and "inside-out" patch configurations. In the whole-cell clamp condition, ER induced a time-independent K(+)-dominant current, which was inhibited by glibenclamide (1-3 microM), suggesting that ER activated the cardiac ATP-sensitive K+ channel (KATP). To elucidate the mechanism of ER-mediated KATP channel activation, the drug was applied to the inside-out patches before and after channel "run-down." Since nucleotide diphosphates could induce the channel openings after complete run-down, effects of the drug on the nucleotide diphosphate-induced channel openings were also examined. Before run-down, ER activated the KATP channel only in the presence of ATP. ER shifted the relation between [ATP]i and the channel activity to the right in a concentration-dependent fashion without a significant alteration of the slope. After channel run-down, ER did not affect the channel openings either in the absence or in the presence of UDP. However, ER could relieve the ATP-gamma-S inhibition of the UDP-induced channel openings. Thus, we conclude that ER antagonizes the inhibitory effect of ATP on the KATP channel in a competitive manner, thereby enhancing the channel openings.

Vasorelaxant agents such as pinacidil, lemakalim, and nicorandil, known as K+ channel openers, can activate the ATP-sensitive K+ channel (KATP channel) in cardiac myocytes. The aim of this study was to elucidate the molecular mechanisms underlying the K+ channel opener-mediated cardiac KATP channel activation by using the inside-out patch-clamp technique in guinea pig ventricular myocytes. Effects of pinacidil, lemakalim, and nicorandil on the KATP channel were examined both before and after channel "run-down". Since nucleotide diphosphates (NDPs) could activate the channel after complete run-down, effects of the drugs on the NDP-induced channel openings were also examined. We made the following observations: 1) Pinacidil (10-100 microM) and lemakalim (300 microM) activated the KATP channel before run-down and after run-down when NDPs were present. 2) Nicorandil (30 microM-1 mM) activated the KATP channel only in the presence of NDPs regardless of the condition of the channel with respect to run-down. 3) None of these K+ channel openers activated the channel after run-down in the absence of NDPs. These observations suggest that NDP binding is essential for nicorandil-mediated activation of the KATP channel and indicate that the molecular mechanisms underlying nicorandil activation are distinct from those of pinacidil and lemakalim activation of the KATP channel. We discuss the possible interactions between the drugs and the KATP channel based on a functional channel model.