Ethanol stimulates ciliary beating by dual cyclic nucleotide kinase activation in bovine bronchial epithelial cells.
Previously, we have shown that ethanol (EtOH) stimulates a rapid increase in the ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBECs) via the activation of PKA. We have also shown that inhibitors of nitric oxide synthase block EtOH-stimulated increases in CBF. We hypothesize that EtOH acutely stimulates CBF via the activation of both PKA and PKG pathways. Using chemiluminescence detection of nitric oxide (NO), we directly measured increases in NO production in BBECs treated with 100 mmol/L of EtOH beginning at 25 minutes. Pretreatment of BBECs with guanylyl cyclase inhibitors, ODQ or LY83583, resulted in the inhibition of EtOH-stimulated CBF. Low concentrations (1 nmol/L) of cyclic nucleotide analogues do not stimulate CBF increases. However, a combination of both 1 nmol/L of 8Br-cAMP and 8Br-cGMP stimulates a significant increase over baseline CBF. This effect could be blocked by pretreating BBECs with inhibitors of either PKA or PKG. Very high concentrations of either 8Br-cAMP or 8Br-cGMP (> or =100 micromol/L) were required to cross-activate both PKA and PKG. This suggests that cross-activation of PKA by cGMP is not occurring at the concentrations (1 nmol/L) capable of stimulating CBF. 8-pCPT-cGMPS, an antagonist analogue to cGMP, blocked EtOH-stimulated PKA activity increases. These data support that EtOH-stimulated increases in CBF require the dual activation of both PKA (via cAMP) and PKG (via NO).
Wyatt
Todd A
TA
Research Service, Department of Veterans Affairs Medical Center, Omaha, Nebraska 68198, USA. twyatt@unmc.edu
Forgèt
Mary A
MA
Sisson
Joseph H
JH
D000818:Animals; D001980:Bronchi / Q000187:drug effects* / Q000502:physiology*; D002417:Cattle; D002478:Cells, Cultured; D002923:Cilia / Q000187:drug effects / Q000502:physiology; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism / Q000502:physiology; D000431:Ethanol / Q000494:pharmacology*; D006162:Guanylate Cyclase / Q000037:antagonists & inhibitors; D009569:Nitric Oxide / Q000378:metabolism; D010069:Oxadiazoles / Q000494:pharmacology; D011810:Quinoxalines / Q000494:pharmacology; D013873:Thionucleotides / Q000494:pharmacology
C095284:1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one; D004791:Enzyme Inhibitors; D010069:Oxadiazoles; D011810:Quinoxalines; D013873:Thionucleotides; D009569:Nitric Oxide; D000431:Ethanol; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006162:Guanylate Cyclase; D006152:Cyclic GMP