Protein Kinase G Regulates Potassium Chloride Cotransporter-3 Expression in Primary Cultures of Rat Vascular Smooth Muscle Cells

Mauricio Di Fulvio,Thomas M Lincoln,Peter K Lauf,Norma C Adragna

doi:10.1074/jbc.m100901200

Mauricio Di Fulvio, Thomas M Lincoln + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m100901200

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Abstract

K-Cl cotransport (KCC) is activated by nitric oxide donors and appears to be regulated by the cGMP signaling pathway. Expression of KCC mRNAs (KCC1-KCC4) in rat vascular smooth muscle cells (VSMCs) is unknown. We have reported the presence of KCC1 and KCC3 mRNAs in primary cultures of VSMCs by specific reverse transcription-polymerase chain reaction. KCC2 mRNA appeared at extremely low levels. KCC4 mRNA was undetectable. Semiquantitative reverse transcription-polymerase chain reaction revealed a 2:1 KCC1/KCC3 mRNA ratio in VSMCs. Depletion of protein kinase G (PKG)-1 from VSMCs did not change KCC3 mRNA expression. Analogous results were obtained with PKG-1-catalytic domain- and vector only-transfected VSMCs lacking endogenous PKG, suggesting no involvement of PKG-1 in the maintenance of basal KCC3 mRNA expression. However, 8-bromo-cGMP, a PKG stimulator, acutely increased KCC3 mRNA expression in a concentration- and time-dependent fashion; this effect was blocked by the PKG inhibitor KT5823 but not by actinomycin D. These findings show that VSMCs express mainly two mRNA isoforms, KCC1 and KCC3, and suggest that PKG participates post-transcriptionally in the acute KCC3 mRNA regulation. The role of KCC3 on cell volume and electrolyte homeostasis in response to PKG modulators remains to be determined.

Highlights

K-Cl cotransport (KCC),1 the coupled movement of potassium and chloride, is involved in cell volume maintenance, and its activity is highly regulated [1]
We recently found that KCC activity is present in vascular smooth muscle cells (VSMCs) and that it is activated by NO donors, an effect prevented by inhibitors of the cGMP pathway, protein phosphatases and tyrosine kinases [1, 2]
We performed simple RT-PCR reactions with KCC isoform-specific primers to obtain some information about the expression of KCC mRNAs in these cells, as well as in PKGϪ and PKGϩ VSMCs

Summary

Introduction

K-Cl cotransport (KCC),1 the coupled movement of potassium and chloride, is involved in cell volume maintenance, and its activity is highly regulated [1]. We report the mRNA expression pattern and relative abundance of the KCC isoforms in low passaged VSMCs and the regulation of KCC3 at the mRNA level by PKG. PKGϪ VSMCs. in all cell types, KCC3 mRNA was expressed at lower levels than KCC1 mRNA, and PKG appeared to be involved in the regulation of the KCC3 mRNA isoform.

Results

Conclusion