8-pCPT-cGMP stimulates alpha beta gamma-ENaC activity in oocytes as an external ligand requiring specific nucleotide moieties

Department of Biochemistry, University of Texas Health Science Center at Tyler, Tyler, Texas, USA.
AJP Renal Physiology (Impact Factor: 4.42). 12/2009; 298(2):F323-34. DOI: 10.1152/ajprenal.00307.2009
Source: PubMed

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.

  • [Show abstract] [Hide abstract]
    ABSTRACT: The amiloride-sensitive epithelial sodium channel (ENaC) is a major contributor to intracellular sodium homeostasis. In addition to epithelial cells, osteoblasts (Obs) express functional ENaCs. Moreover, a correlation between bone Na content and bone disease has been reported, suggesting that ENaC-mediated Na(+) regulation may influence osteogenesis. Obs were isolated and cultured by enzyme digestion. Cell proliferation and differentiation were evaluated by WST-8 assay kit and AKP assay kit respectively. PKGII expression was silenced by siRNA. The mRNA expression was investigated by semi-quantitative PCR and the protein expression was determined by Western-blot. The cell-permeable cGMP analog 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP) increased α-ENaC channel expression in primary rat Obs as indicated by RT-PCR. In addition, 8-pCPT-cGMP stimulation enhanced expression of the mRNA encoding cGMP-dependent protein kinases II (PKGII). The cGMP analog also promoted osteoblast proliferation, differentiation and induced the expression of several osteogenic genes, including core binding factor al, osteocalcin, alkaline phosphatase, collagen type I, and osteopontin. Furthermore, the expression of α-ENaC, the main functional subunit of ENaC, was reduced when a small interfering RNA specific for PKGII was introduced into Obs. Treatment with 8-pCPT-cGMP in cells transfected with the siRNA for PKGII partially reversed downregulated α-ENaC mRNA expression. Our results suggest that 8-pCPT-cGMP stimulates proliferation, differentiation, and osteogenic gene expression in Obs through cGMP/PKGII-dependent regulation of ENaC channel expression. The cGMP/PKGII signaling pathway is a potential target for pharmaceutical interventions to treat metabolic bone diseases.
    Molecular Biology Reports 01/2014; 41(4). DOI:10.1007/s11033-014-3065-1 · 1.96 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Epithelial sodium channels (ENaCs) located at the apical membrane of polarized epithelial cells are regulated by the second messenger cGMP. The mechanism for this regulation has not been completely characterized. Guanylyl cyclases synthesize cGMP in response to various intracellular and extracellular signals. We investigated the regulation of ENaC activity by natriuretic peptide-dependent activation of guanylyl cyclases in Xenopus 2F3 cells. Confocal microscopy studies show natriuretic peptide receptors including those coupled to guanylyl cyclases are expressed at the apical membrane of Xenopus 2F3 cells. Single-channel patch clamp studies using Xenopus 2F3 cells revealed that ANP or 8-pCPT-cGMP, but not CNP or cANF decreased the open probability of ENaC. This suggests that NPR-A, but not NPR-B or NPR-C is involved in the natriuretic peptide mediated regulation of ENaC activity. Also, it is likely that a signaling pathway involving cGMP and NO are involved in this mechanism since inhibitors of soluble Guanylyl Cyclase, Protein Kinase G, iNOS, or an NO scavenger blocked or reduced the effect of ANP on ENaC activity.
    AJP Renal Physiology 01/2013; 304(7). DOI:10.1152/ajprenal.00638.2012 · 4.42 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and non-epithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathologic conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.
    AJP Lung Cellular and Molecular Physiology 09/2012; 303(12). DOI:10.1152/ajplung.00206.2012 · 4.04 Impact Factor