Calcineurin (CaN), the Ca2+-dependent protein phosphatase (PP-2B) is a ubiquitously expressed heterodimer comprised of a catalytic subunit (CnA) and a regulatory subunit (CnB). Two isoforms of CnA exist, CnAα and CnAβ, although their respective functions are largely unknown. We recently reported that CaN mediates the Ca2+-dependent inhibition of ATP-sensitive K+ (KATP) channel in rat aortic smooth muscle (RASM; Wilson et al. 2000). However, the molecular identity of CaN isoforms in this tissue is presently undetermined. The main objectives of the present study were to: (1) determine which CnA isoforms were expressed in RASM, (2) assess the distribution of isoforms in cultured RASM cells, and (3) measure RASM calcineurin activity.
Male Sprague-Dawley rats (250-300 g) were humanely killed and the thoracic aorta was removed, dissected and denuded of endothelial and adventitial layers and subsequently snap-frozen in liquid N2. Using conventional techniques, mRNA was extracted from RASM. For western blotting and phosphatase activity, RASM were homogenised and cells lysed using a buffer containing (mM): Tris-HCl 50, EGTA 2, DTT 0.5, phenylmethylsulfonyl flouride 1, and a protease inhibitor cocktail.
Using reverse transcriptase-polymerase chain reaction (RT-PCR) we amplified mRNA from RASM for both CnAα and β isoforms. Western blotting of RASM homogenates detected the presence of the α isoform, although the β isoform was virtually undetectable. Confocal immunoflourescence staining for both CnA isoforms in cultured RASM cells using specific anti-CnAα and β antibodies, revealed a distinct cellular distribution of the isoforms. CnAα isoform was mainly present in the nucleus, whereas the β isoform was restricted to the cytosol.
Calcineurin activity in RASM lysates was measured using a colourimetric phosphatase assay (Biomol Green Assay). Activity was assessed using the highly selective CaN inhibitor, CaN autoinhibitory peptide (CAP; 100 µM). CAP caused a 13.13 ± 5.75 % (mean ± S.E.M., n = 3) inhibition of calmodulin/Mn2+-stimulated phosphatase activity. In addition, okadaic acid (OA, 1 µM; inhibitor of PP1 and PP2A) caused a 17.12 ± 8.13 % (n = 3) inhibition.
These results demonstrate the presence of both CnA α and β isoforms in RASM, which show distinct cellular distribution, suggesting possible differential cellular function. We can also confirm that the enzyme is functional, representing approximately 10 % of the total phosphatase activity in this tissue, as found in other cell types (Perrino & Soderling, 1998).This work was supported by MRC and NIH .
- Perrino, B.A. & Soderling, T.R. (1998). Calmodulin and Signal Transduction (book), pp. 169-236.
Wilson, A.J., Jabr, R.I. & Clapp, L.H. (2000). Circ. Res. 87, 1019-1025.