¹Department of Cell and Molecular Physiology, School of Medicine, University of North Carolina at Chapel Hilli, Chapel Hill, North Carolina.

Correspondence: William J. Arendshorst, 6341-B Department of Cell and Molecular Physiology, CB #7545 School of Medicine, University of North Carolina at Chapel Hilli, Chapel Hill, NC 27599-7545 E-mail: arends@med.unc.edu

^*Supported by Research Grant HL-02334 from the Heart, Lung and Blood Institute of the National Institutes of Health, Bethesda, Maryland.

Received 18 September 2006; Revised 11 December 2006; Accepted 21 December 2006.

Abstract

Background: Phospholipase C (PLC)-₁ and -₁, but not -₁, protein expressions in fresh renal arterioles and aorta are greater in 6-week-old, spontaneously hypertensive rats (SHRs) versus normotensive Wistar-Kyoto rats (WKYs). This PLC activity is also greater in both vessels of SHRs. In the present study, we tested whether cultured vascular smooth muscle cells (VSMCs) of preglomerular arterioles and aorta accurately reflect strain differences observed in fresh vessels, with VSMCs of SHRs predicted to have higher levels of PLC isozymes and enzyme activity. We assessed the stability of variables over passages 3 to 11.

Methods: The VSMCs were isolated and cultured using standard techniques. The PLC-isozyme protein levels and catalytic activity were determined by Western blot analysis and inositol 1,4,5-trisphosphate (IP₃) production, respectively.

Results: Immunoblots showed expression of PLC-₁ and -₁, but not PLC-₁, in VSMCs from both vessels. Arteriolar VSMCs of SHRs had three-to-fivefold higher levels of PLC-₁ and -₁ during passages 3 to 8. Enzymatic activity in these VSMCs was higher in SHRs versus WKYs, especially during passages 6 to 11. In contrast, cultured aortic VSMCs of SHRs had two-to-threefold lower densities of PLC-₁ and -₁ protein.

Conclusions: Compared with fresh resistance arterioles and aorta, cultured VSMCs exhibit changes in PLC-isozyme protein levels and enzyme activity that vary with passage. The differences between cultured VSMCs of SHRs and WKYs do not accurately reflect those in fresh resistance and conduit vessels, either qualitatively or quantitatively. The results of VSMC culture studies should be interpreted with caution and should ideally be compared with more physiologically relevant fresh preparations.