Nitric Oxide Signaling Pathway Regulates Potassium Chloride Cotransporter-1 mRNA Expression in Vascular Smooth Muscle Cells

Mauricio Di Fulvio,Peter K Lauf,Norma C Adragna

doi:10.1074/jbc.m104899200

Mauricio Di Fulvio, Peter K Lauf + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m104899200

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Abstract

Rat vascular smooth muscle cells (VSMCs) express at least two mRNAs for K-Cl cotransporters (KCC): KCC1 and KCC3. cGMP-dependent protein kinase I regulates KCC3 mRNA expression in these cells. Here, we show evidence implicating the nitric oxide (NO)/cGMP signaling pathway in the expression of KCC1 mRNA, considered to be the major cell volume regulator. VSMCs, expressing soluble guanylyl cyclase (sGC) and PKG-I isoforms showed a time- and concentration-dependent increase in KCC1 mRNA levels after treatment with sodium nitroprusside as demonstrated by semiquantitative RT-PCR. sGC-dependent regulation of KCC1 mRNA expression was confirmed using YC-1, a NO-independent sGC stimulator. The sGC inhibitor LY83583 blocked the effects of sodium nitroprusside and YC-1. Moreover, 8-Br-cGMP increased KCC1 mRNA expression in a concentration- and time-dependent fashion. The 8-Br-cGMP effect was partially blocked by KT5823 but not by actinomycin D. However, actinomycin D and cycloheximide increased basal KCC1 mRNA in an additive manner, suggesting different mechanisms of action for both drugs. These findings suggest that in VSMCs, the NO/cGMP-signaling pathway participates in KCC1 mRNA regulation at the post-transcriptional level.

Highlights

Most cells are freely permeable to water, and the movement of water correlates with the osmotic pressure gradient between the intracellular and extracellular space
Expression of the mRNA Components of the nitric oxide (NO)/cGMP Signaling Pathway in Rat vascular smooth muscle cells (VSMCs)—To ensure that primary cultures of VSMCs at passage 1 express the main components of the NO/cGMP signaling pathway under our experimental conditions, we performed RT-PCR with a specific set of primers to test whether both isoforms of the soluble guanylyl cyclase (sGC) [36], as well as the two different isoforms of PKG-I [23, 24], were present
These results indicate that the major components of the NO/ cGMP signaling cascade are present at the mRNA level and show optimal conditions for studying the actions of NO donors and cGMP analogs in VSMCs

Summary

Introduction

Most cells are freely permeable to water, and the movement of water correlates with the osmotic pressure gradient between the intracellular and extracellular space. We used primary cultures of freshly isolated rat VSMCs expressing sGC (␣1/␤1) mRNAs and the two type I versions of PKG (␣/␤) to determine whether the NO/cGMP signal transduction pathway is involved in the regulation of KCC1 mRNA expression, as well as the role of de novo transcription and

Results

Conclusion