Cyclophilins (CyP) are a family of cyclosporin (CSA) binding proteins with ubiquitous distribution. Animal cyclophilins contain several isoforms. in particular CyP-A (cytosol), CyP-B and CyPC (endoplasmic reticulum) and CyP-D (mitochondria). All cyclophilins catalyse rotation of accessible peptidyl-prolyl bonds in proteins and are believed to play a role in folding of nascent proteins in their respective compartments [I]. The peptidylprolylcis-trans isomerase activity (PPIase) is blocked by CSA. Other actions of cyclophilins are unrelated to PPIase activity, Thus the CyP-A/CSA complex interacts with and inhibits the Ca+*/calmodulin-dependent serinelthreonine phosphatase calcineurin [2]. Interest in the possible role of cyclophilins in cell death has arisen from the observed effects of CSA. CSA offers some protection against anoxiahoxygenation induced necrosis of heart and liver cells [3,5]. In some studies CSA inhibited early events in apoptosis [6,7]. However, deductions about the molecular events involving CSA may be. fraught with problems in view of the presence of several CyP isoforms in different cellular compartments clearly performing different tasks. We are using an alternative approach based on the selective suppression of CyP isoforms using antisense oligodeoxynucleotides (AS-ODN). Here we report on the suppression of CyP-A in rat cardiomyocytes transfected with the antisense: 3' CAT GGC I T C CAC AAT CiCT 5' . Primary cultures of rat cardiomyocytes were prepared by the method of Fluri et a1 [9], with minor modifications. Four two-week old rat pups were anaesthetised with ether, anticoagulated with heparin and sacrificed ten minutes later. The hearts were removed and the cells were enzymatically isolated by the following dissociation procedure. Initially, tissue fragments were incubated in digest medium (Hanks balanced salt solution containing 0.8 mg/ml collagenase, 20 pg/ml DNase, 20 d m l penicillin, 20 pg/ml streptomycin, 1 mM taurine and 40 pM CaCI2. The cell digest was stirred for 15 minutes at 35'C. The solution was replaced with fresh dissociation medium and the incubation procedure repeated three times. The initial solution was discarded and the subsequent solutions were collected for centrifugation. The resulting cell pellets were resuspended in culture medium (M 199 containing 20 d m l penicillin, 20 @ml streptomycin. 2 pg/ml Vit B,2 and 10% Foetal calf serum). Total cell yield was recorded using a haemocytometer (1-1.5 x 106 cells/animal). Cells were seeded onto laminin treated glass coverslips at a density of 5 x 10-'/0.5ml and incubated in an atmosphere of 5%C02/95%02 at 37°C for three hours. A further 0.51111 of culture medium was added at this point and incubation continued for three hours. Cytosine arabinoside was then added to the cells to a final concentration of 1 pM. The cells were incubated overnight, after which the non attached cells were removed. The cell layer was washed with saline, fresh culture medium added, and the cells transfected with AS-ODN. After 24h the medium was removed and the cells washed in 150 mM NaCI, 10 mM Hepes, pH 7.4. The cells were scraped off the coverslips in a final volume of 300 pl of medium containing 200 mM mannitol. 70 mM sucrose, 10 mM Tris. 1 mM EGTA, 50 mM KCI and protease inhibitors (antipain, chymostatin, pepstatin, leupeptin and aprotinin at a final concentration of 1 pg/ml). The cells were homogenised on ice and ultracentrifuged. The supernatant was taken as the cytosolic fraction. The PPIase activity attributable to CyP-A in the cytosolic fraction was assayed in the presence of FK506 to inhibit noncyclophilin PPIase. PPIases are conventionally assayed spectrophotometrically by exploiting the specificity of chymotrypsin for the trans Xpro isomer in substrates of the type X-pro-phe-Y, where Y is a chromogen. The pre-existing trans isomer is hydrolysed instantaneously by high concentrations of chymotrypsin, but subsequent hydrolysis requires cis-trans isomerisation. The technique is limited by the high rate of uncatalysed cis-trans isomerisation and the low initial (equilibrium) proportion of the cis isomer in H20. We have used the technique of Kofron et al, [8]. in which the peptide was dissolved in H20-free 400 mM LiCl in trifluorethanol; this increased the proportion of the cis isomer to about 50% (compared to 10% in 95% ethanol). T h e s modifications allowed us to conduct microassays with a rapid mixing device in 5 pl reaction volume on the stage of a fluorescence microscope. This enabled PPIase to be assayed in extracts containing 10.' cells.