SERCA2A and SERCA2B exhibit discrete spatial distributions in rat pulmonary arterial smooth muscle cells

J.H. Clark1, F. Wuytack2 and A.M. Evans1(D. J. MacEwan) 1Division of Biomedical Sciences, School of Biology, Bute Building, Westburn Lane, St Andrews, Fife, KY16 9TS. 2Laboratory of Physiology, O/N Gasthuisberg, K.U. Leuven, Herestraat 49, B-3000 Leuven, Belgium.

β-adrenoceptor activation mediates pulmonary artery dilation, in part, via Ca2+ release from a cyclopiazonic acid (CPA)-sensitive sarcoplasmic reticulum (SR) store whilst hypoxia elicits pulmonary artery constriction, in part, via Ca2+ release from a CPA-insensitive SR store (Evans et al., 2002; Boittin et al., 2003). The aim of this study was to determine whether multiple isoforms of the SR Ca2+ ATPase (SERCA) are expressed in pulmonary artery smooth muscle cells (PASMCs).

Adult male Wistar rats (~200g) were humanely killed and the 2nd order branches of the pulmonary artery removed. The arteries were either snap frozen in liquid nitrogen or enzymically digested to isolate PASMCs (Boittin et al., 2003). Tissue lysates were prepared from the arteries and assayed by Western blotting using sequence-specific antibodies against identified SERCA isoforms. Immunocytochemical studies were performed on isolated PASMCs as previously described (Evans et al., 2005) using the SERCA antibodies along with a secondary antibody conjugated to the fluorophore Texas Red. Fluorescent labelling was visualised using a Deltavision microscope system (Applied Precision) and analysed using Volocity analysis software (Improvision).

Western blotting identified specific bands of approximately 110kDa for SERCA2a and SERCA2b, but not for SERCA1 or SERCA3, in pulmonary artery smooth muscle lysates. In addition, immunocytochemistry on acutely isolated PASMCs identified discrete patterns of labelling for SERCA2a and SERCA2b. The spatial distribution of each SERCA subtype was analysed volumetrically by sectioning the cell into perinuclear, extraperinulcear and subplasmalemmal regions. The fluorescent labelling was measured as the mean volume of labelling per µm3 of the volume of the cellular region ± S.E.M. The majority of SERCA2a fluorescent labelling was found within the perinuclear region of the cell with 0.125 ± 0.019 µm3 of SERCA2a labelling per µm3 compared to 0.003 ± 0.001µm3 per µm3 and 0.003 ± 0.002 µm3 of per µm3 for the extraperinuclear and subplasmalemmal regions respectively (n=12). In contrast SERCA2b labelling was primarily found in close proximity to the plasma membrane with 0.106 ± 0.020 µm3 of SERCA2b labelling per µm3 compared to 0.037 ± 0.012 µm3 per µm3 and 0.009 ± 0.003 µm3 of per µm3 for the perinuclear and extraperinuclear regions respectively (n=10).

We conclude that two SERCA isoforms, SERCA2a and SERCA2b, are present within PASMCs of the 2nd order branches of the rat pulmonary arterial tree and that each of these SERCA has it’s own unique spatial distribution. These findings lend further support to our proposal that there may be at least two spatially and functionally segregated SR Ca2+ stores within pulmonary arterial smooth muscle cells, each served by a different SERCA pump.

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Evans, A. M. et al. (2002). Respir. Physiol. Neurobiol. 132, 3-15.
Evans, A. M. et al. (2005). J. Biol. Chem. 280, 41504-41511.

This work was supported by the British Heart Foundation.