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 journalArticle

102 Citations (Scopus)

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)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume274
Issue number2 43-2
StatePublished - Feb 1 1998

Fingerprint

Phosphoric Diester Hydrolases
Atrial Natriuretic Factor
Vascular Smooth Muscle
Smooth Muscle Myocytes
Phosphorylation
Allosteric Site
Adenosine Triphosphate
Type 5 Cyclic Nucleotide Phosphodiesterases
Cyclic GMP-Dependent Protein Kinases
Guanosine
Enzymes
Smooth Muscle
Anti-Idiotypic Antibodies
Proteins
Phosphates

Keywords

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

ASJC Scopus subject areas

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

Cite this

ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells. / Wyatt, Todd A; Naftilan, Allen J.; Francis, Sharron H.; Corbin, Jackie D.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 274, No. 2 43-2, 01.02.1998.

Research output: Contribution to journalArticle

@article{63429f8ffaf540128f16422a5537d0ad,
title = "ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells",
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.",
keywords = "Cyclic nucleotides, Protein kinases, Protein phosphorylation, Smooth muscle relaxation",
author = "Wyatt, {Todd A} and Naftilan, {Allen J.} and Francis, {Sharron H.} and Corbin, {Jackie D.}",
year = "1998",
month = "2",
day = "1",
language = "English (US)",
volume = "274",
journal = "American Journal of Physiology - Renal Physiology",
issn = "0363-6127",
publisher = "American Physiological Society",
number = "2 43-2",

}

TY - JOUR

T1 - ANF elicits phosphorylation of the cGMP phosphodiesterase in vascular smooth muscle cells

AU - Wyatt, Todd A

AU - Naftilan, Allen J.

AU - Francis, Sharron H.

AU - Corbin, Jackie D.

PY - 1998/2/1

Y1 - 1998/2/1

N2 - 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.

AB - 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.

KW - Cyclic nucleotides

KW - Protein kinases

KW - Protein phosphorylation

KW - Smooth muscle relaxation

UR - http://www.scopus.com/inward/record.url?scp=0031938315&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0031938315&partnerID=8YFLogxK

M3 - Article

VL - 274

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 0363-6127

IS - 2 43-2

ER -