Korean J Physiol Pharmacol.  2011 Dec;15(6):431-436. 10.4196/kjpp.2011.15.6.431.

Alteration of Ryanodine-receptors in Cultured Rat Aortic Smooth Muscle Cells

• Kim EJ
• Kim DK
• Kim SH
• Lee KM
• Park HS
• Kim SH

• KMID: 2285431
• DOI: http://doi.org/10.4196/kjpp.2011.15.6.431

Abstract

Vascular smooth muscle cells can obtain a proliferative function in environments such as atherosclerosis in vivo or primary culture in vitro. Proliferation of vascular smooth muscle cells is accompanied by changes in ryanodine receptors (RyRs). In several studies, the cytosolic Ca2+ response to caffeine is decreased during smooth muscle cell culture. Although caffeine is commonly used to investigate RyR function because it is difficult to measure Ca2+ release from the sarcoplasmic reticulum (SR) directly, caffeine has additional off-target effects, including blocking inositol trisphosphate receptors and store-operated Ca2+ entry. Using freshly dissociated rat aortic smooth muscle cells (RASMCs) and cultured RASMCs, we sought to provide direct evidence for the operation of RyRs through the Ca2+- induced Ca2+-release pathway by directly measuring Ca2+ release from SR in permeabilized cells. An additional goal was to elucidate alterations of RyRs that occurred during culture. Perfusion of permeabilized, freshly dissociated RASMCs with Ca2+ stimulated Ca2+ release from the SR. Caffeine and ryanodine also induced Ca2+ release from the SR in dissociated RASMCs. In contrast, ryanodine, caffeine and Ca2+ failed to trigger Ca2+ release in cultured RASMCs. These results are consistent with results obtained by immunocytochemistry, which showed that RyRs were expressed in dissociated RASMCs, but not in cultured RASMCs. This study is the first to demonstrate Ca2+ release from the SR by cytosolic Ca2+ elevation in vascular smooth muscle cells, and also supports previous studies on the alterations of RyRs in vascular smooth muscle cells associated with culture.

Keyword

Cell culture; Ca2+-induced Ca2+ release; Ryanodine receptor; Rat aortic smooth muscle

MeSH Terms

Animals
Atherosclerosis
Caffeine
Cell Culture Techniques
Cytosol
Immunohistochemistry
Inositol
Muscle, Smooth
Muscle, Smooth, Vascular
Myocytes, Smooth Muscle
Perfusion
Rats
Ryanodine
Ryanodine Receptor Calcium Release Channel
Sarcoplasmic Reticulum
Caffeine
Inositol
Ryanodine
Ryanodine Receptor Calcium Release Channel

Figure

• 

  Fig. 1 Effects of caffeine on RASMCs. Representative traces of caffeine-induced cytosolic Ca2+ responses in freshly dissociated RASMCs (A) and cultured RASMCs (B). (C) The application of 20 mM caffeine in Ca2+-free HEPES-PSS buffer induced a rapid and large cytosolic Ca2+ increase only in freshly dissociated RASMCs. Effects of caffeine on Ca2+ release from intracellular Ca2+ stores in permeabilized freshly dissociated and cultured RASMCs. The data were obtained from four experiments. The arrows indicate the starting points of drug perfusion. Caffeine (10 mM) induced Ca2+ release from the SR in freshly dissociated RASMCs (■), but not in cultured RASMCs (□).

• 

  Fig. 2 CICR in permeabilized RASMCs. Representative traces of CICR in freshly dissociated RASMCs (A) and cultured RASMCs (B). The fine line indicates Ca2+-free solution. Ca2+ (200 nM) induced Ca2+ release only in freshly dissociated RASMCs. (C) Effects of Ca2+ on Ca2+ release in permeabilized freshly dissociated and cultured RASMCs. The data, normalized to the initial 10s period prior to 200 nM Ca2+ perfusion, were obtained from four experiments. The arrows indicate the starting points of 200 nM Ca2+ perfusion. Ca2+ (200 nM) triggered Ca2+ release from the SR in freshly dissociated RASMCs (■), but had no effect in cultured RASMCs (□).

• 

  Fig. 3 Ryanodine-induced Ca2+ release in permeabilized RASMCs. Representative traces of ryanodine-induced Ca2+ release in freshly dissociated RASMCs (A) and cultured RASMCs (B). (C) Effects of ryanodine on Ca2+ release in permeabilized freshly dissociated RASMCs and cultured RASMCs. The data, normalized to the initial 10s period prior to ryanodine application, were obtained from four experiments. The arrow indicates the starting points of ryanodine perfusion. Ryanodine (10µM) induced Ca2+ release from the SR in freshly dissociated RASMCs (■), but not in cultured RASMCs (□).

• 

  Fig. 4 Expression of RyRs and IP3Rs in RASMCs. Expression of RyRs in freshly dissociated RASMCs (A) and cultured RASMCs (B). Expression of IP3Rs in freshly dissociated RASMCs (C) and cultured RASMCs (D). (a) Immunocytochemistry; (b) interference contrast micrographs; (c) merged images. Immunocytochemistry was performed using primary anti-RyRs and anti-IP3Rs antibodies, as described in Methods. The data show that RyRs are only present in freshly dissociated RASMCs (n=5), whereas IP3Rs are present in both freshly dissociated RASMCs and cultured RASMCs (n=5). Scale bars, 5µm (A, C) and 10µm (B, D).

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