Diethyl pyrocarbonate, a histidine-modifying agent, directly stimulates activity of ATP-sensitive potassium channels in pituitary GH(3) cells.
The ATP-sensitive K(+) (K(ATP)) channels are composed of sulfonylurea receptor and inwardly rectifying K(+) channel (Kir6.2) subunit. These channels are regulated by intracellular ADP/ATP ratio and play a role in cellular metabolism. Diethyl pyrocarbonate (DEPC), a histidine-specific alkylating reagent, is known to modify the histidine residues of the structure of proteins. The objective of this study was to determine whether DEPC modifies K(ATP)-channel activity in pituitary GH(3) cells. Steady-state fluctuation analyses of macroscopic K(+) current at -120 mV produced power spectra that could be fitted with a single Lorentzian curve in these cells. The time constants in the presence of DEPC were increased. Consistent with fluctuation analyses, the mean open time of K(ATP)-channels was significantly increased during exposure to DEPC. However, DEPC produced no change in single-channel conductance, despite the ability of this compound to enhance K(ATP)-channel activity in a concentration-dependent manner with an EC(50) value of 16 microM. DEPC-stimulated K(ATP)-channel activity was attenuated by pretreatment with glibenclamide. In current-clamp configuration, DEPC decreased the firing of action potentials in GH(3) cells. A further application of glibenclamide reversed DEPC-induced inhibition of spontaneous action potentials. Intracellullar Ca(2+) measurements revealed the ability of DEPC to decrease Ca(2+) oscillations in GH(3) cells. Simulation studies also demonstrated that the increased conductance of K(ATP)-channels used to mimic DEPC actions reduced the frequency of spontaneous action potentials and fluctuation of intracellular Ca(2+). The results indicate that chemical modification with DEPC enhances K(ATP)-channel activity and influences functional activities of pituitary GH(3) cells.
Wu
Sheng-Nan
SN
Department of Physiology, National Cheng Kung University Medical College, No 1, University Road, Tainan 70101, Taiwan. snwu@mail.ncku.edu.tw
Chang
Han-Dong
HD
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D001692:Biological Transport; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D006801:Humans; D008077:Lipoproteins, LDL / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D010902:Pituitary Gland / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D015221:Potassium Channels / Q000819:agonists*
D008077:Lipoproteins, LDL; D015221:Potassium Channels; D000255:Adenosine Triphosphate; D002784:Cholesterol; D004047:Diethyl Pyrocarbonate