Abstract
4-phenylbutyrate (4-PB) has been shown to increase the protein content in a number of cells types. One such protein is Connexin43 (Cx43). We show here that 4-phenylbutyrate exposure results in significantly elevated cell to cell coupling, as determined by dual whole cell patch clamp. Incubation with 5 mM 4PB for 24 h or more nearly doubles junctional conductance. Interestingly, mRNA levels for Cx43 declined with exposure to 4-PB while western blot analysis revealed not significant change in protein levels. These data are most consistent with stabilization of the existing Cx43 pool or alterations in the number of functional channels within an existing pool of active and silent channels. These data represent a baseline for testing the efficacy of increased connexin mediated coupling in a variety of multicellular functions including erectile function.
Highlights
Corpora Cavernosa smooth muscle cells form a functional syncytium as a consequence of connexin derived gap junction channels connecting cell interiors (Harris, 2001)
An alternative explanation is an increase in the number of functional channels arising out of a pool of intact but normally silent channels. These data demonstrate 4-PB exposure results in enhanced junctional conductance (∼2X) consistent with previous observations on other cell types (Asklund et al, 2004; Khan et al, 2007; Kaufman et al, 2013). It is clear from the gene expression data that post-transcriptional mechanisms dominate in determining the final level of functional Cx43 protein expression since mRNA levels are significantly reduced (Figure 2)
Up-regulation of Cx43 protein expression and function has been documented in several cell types, by our group and others (Ammerpohl et al, 2004; Asklund et al, 2004; Hattori et al, 2007; Khan et al, 2007; Kaufman et al, 2013)
Summary
Corpora Cavernosa smooth muscle cells form a functional syncytium as a consequence of connexin derived gap junction channels connecting cell interiors (Harris, 2001). A number of studies strongly suggest that Cx43 expression and gap junction mediated coupling are essential for relaxation of corpora smooth muscle cells and erectile response (Christ and Lue, 2004; Pointis, 2006; Fu et al, 2008; Suadicani et al, 2009). The antiarrhythmic peptide ZP123 (Rotigaptide) has been to shown to increase junctional conductance in cardiac myocyte pairs as much as 69% within minutes (Eloff et al, 2003; Xing et al, 2003; Axelsen et al, 2006) but it has not been shown to affect expression levels suggesting that it acts to recruit channels from a silent pool. The idea of a silent reservoir is based on the fact that active Cx43 channels in corpora have been shown to have open probabilities approaching between 0.5 and unity (Brink et al, 1996; Christ and Brink, 1999)
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