8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.
Epithelial sodium channels (ENaC) are regulated by protein kinase A, in addition to a broad spectrum of other protein kinases. It is not clear whether cGMP/PKG signaling might regulate ENaC activity. We examined the responses of alphabetagamma-ENaC channels expressed in Xenopus oocytes to 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP), a cell-permeable cGMP analog. This compound stimulated human alphabetagamma-ENaC activity in a dose-dependent fashion, but cell-impermeable cGMP had no effect. Similar stimulatory effects of cGMP were observed in oocytes expressing either mouse or rat alphabetagamma-ENaC channels. The identical ion selectivity and amiloride sensitivity of the 8-pCPT-cGMP-activated currents to those of alphabetagamma-ENaC channels suggest that the cGMP-activated currents are associated with expressed ENaC. The PKGI activator Sp isomer of beta-phenyl-1,N(2)-etheno-8-bromo-cGMP did not elicit a rise in ENaC current and that the 8-pCPT-cGMP-induced activation of ENaC channels was blocked by incubating oocytes with a PKG inhibitor, but not with other cGMP-sensitive kinase inactivators for PKA, MEK, MAP, and PKC. Surprisingly, both site-directed mutation of putative consensus PKG phosphorylation sites and truncation of entire cytosolic NH(2)- and COOH-terminal tails did not alter the response to 8-pCPT-cGMP. The ENaC activity was activated to the same extent by 8-pCPT-cGMP in cells in which PKGII expression was knocked down using small interfering RNA. Analog to 8-CPT-cAMP, 8-pCPT-cGMP was capable of activating ENaC in the identical manner in cell-free outside-out patches. We conclude that the rapid upregulation of human alphabetagamma-ENaC activity in oocytes by external 8-pCPT-cGMP and 4-chlorothiolphenol-cAMP depends on the para-chlorophenylthiol and the hydroxy groups, and 8-pCPT-cGMP may serve as a novel ENaC ligand in addition to activating PKG signal.
Nie
Hong-Guang
HG
Department of Biochemistry, University of Texas Health Science Center at Tyler, Tyler, Texas, USA.
Zhang
Wei
W
Han
Dong-Yun
DY
Li
Qing-Nan
QN
Li
Jun
J
Zhao
Run-Zhen
RZ
Su
Xue-Feng
XF
Peng
Ji-Bin
JB
Ji
Hong-Long
HL
D000818:Animals; D000242:Cyclic AMP / Q000031:analogs & derivatives / Q000494:pharmacology; D006152:Cyclic GMP / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004553:Electric Conductivity; D020536:Enzyme Activators / Q000494:pharmacology; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D005260:Female; D006801:Humans; D007527:Isoenzymes / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008094:Lithium / Q000494:pharmacology; D051379:Mice; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D020033:Protein Isoforms; D011494:Protein Kinases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D013873:Thionucleotides / Q000008:administration & dosage* / Q000494:pharmacology; D015854:Up-Regulation; D014982:Xenopus laevis
D020536:Enzyme Activators; D053503:Epithelial Sodium Channels; D007527:Isoenzymes; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D013873:Thionucleotides; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D008094:Lithium; D000242:Cyclic AMP; D011494:Protein Kinases; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP; D011188:Potassium