Pflügers Archiv - European Journal of Physiology

, Volume 465, Issue 2, pp 221–232

Involvement of large conductance Ca2+-activated K+ channel in laminar shear stress-induced inhibition of vascular smooth muscle cell proliferation

  • Xiaoling Jia
  • Jingyun Yang
  • Wei Song
  • Ping Li
  • Xia Wang
  • Changdong Guan
  • Liu Yang
  • Yan Huang
  • Xianghui Gong
  • Meili Liu
  • Lisha Zheng
  • Yubo Fan
Ion Channels, Receptors and Transporters

DOI: 10.1007/s00424-012-1182-z

Cite this article as:
Jia, X., Yang, J., Song, W. et al. Pflugers Arch - Eur J Physiol (2013) 465: 221. doi:10.1007/s00424-012-1182-z

Abstract

The large conductance Ca2+-activated K+ (BKCa) channel in vascular smooth muscle cell (VSMC) is an important potassium channel that can regulate vascular tone. Recent work has demonstrated that abnormalities in BKCa channel function are associated with changes in cell proliferation and the onset of vascular disease. However, until today there are rare reports to show whether this channel is involved in VSMC proliferation in response to fluid shear stress (SS). Here we investigated a possible role of BKCa channel in VSMC proliferation under laminar SS. Rat aortic VSMCs were plated in parallel-plate flow chambers and exposed to laminar SS with varied durations and magnitudes. VSMC proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and DNA synthesis. BKCa protein and gene expression was determined by flow cytometery and RT-PCR. The involvement of BKCa in SS-induced inhibition of proliferation was examined by BKCa inhibition using a BKCa specific blocker, iberiotoxin (IBTX), and by BKCa transfection in BKCa non-expressing CHO cells. The changes in [Ca2+]i were determined using a calcium-sensitive dye, fluo 3-AM. Membrane potential changes were detected with a potential-sensitive dye, DiBAC4(3). We found that laminar SS inhibited VSMC proliferation and stimulated BKCa channel expression. Furthermore, laminar SS induced an increase in [Ca2+]i and membrane hyperpolarization. Besides in VSMC, the inhibitory effect of BKCa channel activity on cell proliferation in response to SS was also confirmed in BKCa-transfected CHO cells showing a decline in proliferation. Blocking BKCa channel reversed its inhibitory effect, providing additional support for the involvement of BKCa in SS-induced proliferation reduction. Our results suggest, for the first time, that BKCa channel mediates laminar SS-induced inhibition of VSMC proliferation. This finding is important for understanding the mechanism by which SS regulates VSMC proliferation, and should be helpful in developing strategies to prevent flow-initiated vascular disease formation.

Keywords

Laminar shear stressBKCa channelVSMCProliferation

Supplementary material

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Supplement Figure 1

A B: The acquired VSMC is positively α-SMA-stained (A) or SM-MHC-1-stained (B). Green, α-SMA positive stain; Red, SM-MHC-1 positive stain; Blue, nuclear stain. (JPEG 16 kb)

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Supplementary Figure 2

A B C D. BKCa transfections in CHO cells were observed by laser confoco microscopy (upper panel) and quantified by flow cytometry (bottom panel). Both α-subunit (A) and β1-subunit (B) were expressed in transfected CHO cells. Green, α-subunit positive stain; Red, β1-subunit positive stain; Blue, nuclear stain. The transfection rate was 98.7 % for α-subunit (C) and 99.8 % for β1-subunit (D). (JPEG 31 kb)

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Supplementary Figure 3

Schematic drawing of the shear apparatus. Culture medium pumped into upper reservoir. The liquid surface of upper reservoir was steady. Therefore, the height of upper reservoir controlled the fluid flow rate, further controlled the magnitude of wall shear stress flown over VSMCs monolayer. (JPEG 16 kb)

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Supplementary Figure 4

A B. Blockade of BKCa channel increases the PCNA fluorescence intensity of VSMCs in shear cases, but has no effect in static cases. A. Shear cases. B. Static cases. Green, FITC stained PCNA; Blue, DAPI-stained nuclear. Representative laser confoco microscope graphs for static cases. Green, FITC stained PCNA; Blue, DAPI-stained nuclear. (JPEG 189 kb)

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Supplementary Figure 4

C D. Blockade of BKCa channel promotes cell cycle progress of VSMCs subjected to the described laminar SS for 12 h (D) and 24 h (E). (JPEG 123 kb)

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Supplementary Figure 5

The expression of BKCa channel has no alteration after IBTX treatment. (A) α- subunit ; (B) β1-subunit. (JPEG 15 kb)

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Supplementary Figure 6

The PCNA decrease was effectively reversed by the IBTX treatment in shear cases, but had no effects in static cases. (A) Representative laser confoco microscope graphs. (B) Values of the PCNA fluorescent intensity with and without BKCa blockade. *P < 0.05, No blockade (α+/β1+) vs t blockade (α+/β1++ IBTX) (n = 3). (JPEG 102 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Xiaoling Jia
    • 1
  • Jingyun Yang
    • 1
  • Wei Song
    • 1
  • Ping Li
    • 1
  • Xia Wang
    • 1
  • Changdong Guan
    • 1
  • Liu Yang
    • 1
  • Yan Huang
    • 1
  • Xianghui Gong
    • 1
  • Meili Liu
    • 1
  • Lisha Zheng
    • 1
  • Yubo Fan
    • 1
  1. 1.Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Medical EngineeringBeihang UniversityBeijingChina