ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells

Todd A. Wyatt, Allen J. Naftilan, Sharron H. Francis, Jackie D. Corbin

Research output: Contribution to journalArticlepeer-review

102 Scopus citations

Abstract

Guanosine 3′,5′-cyclic monophosphate (cGMP)-binding, cGMP-specific phosphodiesterase (PDE5) is abundant in vascular smooth muscle, and this enzyme is a potent substrate for cGMP-dependent protein kinase (PKG) in vitro. Binding of cGMP to the allosteric sites of PDE5 is required for this phosphorylation to occur. Vascular smooth muscle cells (VSMC) were used to determine if PDE5 is phosphorylated in intact cells when cGMP is increased. With the use of anti-PDE5 antibodies, a phosphorylated 93-kDa protein band was immunoprecipitated from early passaged primary cultures of VSMC that had been preincubated with 32Pi to label cellular ATP and then treated with atrial natriuretic factor (ANF). In the absence of ANF, there was no detectable incorporation of radiolabeled phosphate into this band. Phosphorylation of the 93-kDa protein was augmented by pretreating cells with 8-bromoguanosine 3′,5′-cyclic monophosphate (8-BrcGMP) to activate PKG before addition of ANF. 8-BrcGMP, which interacts poorly with the allosteric sites of PDE5, had no effect on PDE5 phosphorylation in the absence of ANF. Phosphorylation of PDE5 in response to treatment of cells with ANF was associated with a two- to fourfold increase in PDE activity in immunoprecipitates. Multiple-passaged VSMC, which are deficient in PKG but retain PDE5, demonstrated no ANF-dependent increase in phosphorylation or catalytic activity of PDE5. However, incubation of immunoprecipitated PDE5 from these cells with purified PKG, cGMP, and a phosphorylation mixture containing [γ-32P]ATP resulted in 32Pi incorporation into PDE5 that was correlated with increased catalytic activity. These studies are the first to demonstrate phosphorylation of PDE5 in intact cells, thus suggesting a physiological role for this enzyme in smooth muscle regulation.

Original languageEnglish (US)
Pages (from-to)H448-H455
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume274
Issue number2 43-2
DOIs
StatePublished - Feb 1998

Keywords

  • Cyclic nucleotides
  • Protein kinases
  • Protein phosphorylation
  • Smooth muscle relaxation

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

Fingerprint Dive into the research topics of 'ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells'. Together they form a unique fingerprint.

Cite this