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8-pCPT-cGMP stimulates alphabetagamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties.
- Source :
-
American journal of physiology. Renal physiology [Am J Physiol Renal Physiol] 2010 Feb; Vol. 298 (2), pp. F323-34. Date of Electronic Publication: 2009 Dec 09. - Publication Year :
- 2010
-
Abstract
- 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.
- Subjects :
- Animals
Cyclic AMP analogs & derivatives
Cyclic AMP pharmacology
Cyclic GMP administration & dosage
Cyclic GMP metabolism
Cyclic GMP-Dependent Protein Kinases drug effects
Cyclic GMP-Dependent Protein Kinases genetics
Cyclic GMP-Dependent Protein Kinases metabolism
Cytosol metabolism
Dose-Response Relationship, Drug
Electric Conductivity
Enzyme Activators pharmacology
Female
Humans
Isoenzymes drug effects
Isoenzymes genetics
Isoenzymes metabolism
Lithium pharmacology
Mice
Oocytes drug effects
Oocytes physiology
Phosphorylation
Potassium pharmacology
Protein Isoforms
Protein Kinases metabolism
Protein Structure, Tertiary
RNA, Small Interfering pharmacology
Rats
Thionucleotides pharmacology
Up-Regulation
Xenopus laevis
Cyclic GMP analogs & derivatives
Epithelial Sodium Channels metabolism
Oocytes metabolism
Thionucleotides administration & dosage
Subjects
Details
- Language :
- English
- ISSN :
- 1522-1466
- Volume :
- 298
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- American journal of physiology. Renal physiology
- Publication Type :
- Academic Journal
- Accession number :
- 20007351
- Full Text :
- https://doi.org/10.1152/ajprenal.00307.2009