These results are in agreement with a number of biochemical studies showing a much higher specific activity of PDE3 and PDE4 compared with PDE1 and PDE2 in rat heart (Shahid & Nicholson, 1990; Bode em et al /em ., 1991; Dubois em et al /em ., 1993; Picq em et al /em ., 1996). room temperature. For enzymatic dissociation, 1?mg?ml?1 collagenase A (Boehringer Mannhein, Mannhein, Germany) and 300?M EGTA were added to the Ca2+-free Ringer solution, so that the free Ca2+ concentration was adjusted to 20?M. The hearts were perfused retrogradedly at a constant flow of 6?ml?min?1 and at 37C by Ca2+-free solution during 5?min followed by 1?h of perfusion at 4?ml?min?1 with the same solution containing collagenase. The ventricles were then separated from atria, chopped finely and agitated gently to dissociate individual cells. The resulting cell suspension was filtered on a gauze and the TSHR cells were allowed to settle down. The supernatant was discarded BRL 44408 maleate and cells resuspended four more times in Ca2+-free solution containing a progressively increasing calcium concentration. The cells were maintained at 37C until use. Measurement of rat PDE mRNA expression by reverse transcriptase polymerase chain reaction (RTCPCR) Total RNA was prepared from rat isolated ventricular myocytes and rat ventricular tissue using the Trizol RNA purification system (Gibco BRL, Cergy-Pontoise, France). All RNAs were checked in 1% formaldehyde agarose gel. cDNA was prepared from mRNA with random hexanucleotide primers (20?pmol?g?1 RNA) using MMLV reverse transcriptase and the conditions recommended by the manufacturer (Clontech, Palo Alto, CA, U.S.A.). Fifty ng of cDNA were amplified in 50?l PCR reaction mixture (200?M dNTPs final concentrations) containing 2.5?U of Taq BRL 44408 maleate polymerase in the buffer supplied by the manufacturer BRL 44408 maleate (Bioprobe Sytems, Montreuil, France), and 1?M primers. Oligonucleotide primers designed against the C-terminal region of each PDE sub-type were as follows: PDE1C (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”L41045″,”term_id”:”871804″L41045): Forward primer 5-TTTTCTCCTCTGTGTGACCG-3, reverse primer 5-GTGTTCCGTTGACTTGACCT-3, fragment size 505?bp; PDE2A2 (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”U21101″,”term_id”:”706929″U21101): Forward primer 5-GAAGGACTATCAGCGAATGC-3, reverse primer 5-GGATGGTGAACTTGTGGGAC-3, fragment size 461?bp; PDE3B1 (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”Z22867″,”term_id”:”406269″Z22867): Forward primer 5-CACCCAGGAAGAACAAATGC-3, reverse primer 5-AAGCCAGCAGCATCATAGGA-3, fragment size 551?bp; PDE4A5 (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”L27057″,”term_id”:”3334904″L27057): Forward primer 5-TACAGTGGTGGAAGTGGCAG-3, reverse primer 5-GAGCAGAGATGATGGCAGAA-3, fragment size 278?bp; PDE4 B2 (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”L27058″,”term_id”:”950096″L27058): Forward primer 5-GTTCTCCTCTCTACGCCAGCA-3, reverse primer 5-ACTTGGTAGGGTTGCTCAGGTC-3, fragment size 454?bp; PDE4 D3 (Genbank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”U09457″,”term_id”:”2564961″U09457): Forward primer 5-GCGTCCTCCTCCTTGATAACTATT-3, reverse primer 5-CTGACTCGCCATCTTCCTCTAA-3, fragment size 447?bp. The PCR products were separated on 1.7% agarose gel containing 0.01% ethidium bromide and photographed under UV irradiation at 320?nm. To assess relative quantities of cDNA from each sample, a second PCR BRL 44408 maleate amplification was conducted with primers directed to the rat housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as previously described (Grimaldi em et al /em ., 1998). All PCR procedures were performed as follows: 40 cycles and 35 cycles for isolated myocytes and ventricular tissue respectively (45?s at 94C, 50?s at 50C and 1?min at 72C) and a final elongation (7?min at 72C). Cyclic AMP radioimmunoassay Freshly isolated ventricular myocytes were prepared as described above from hearts of adult male Wistar rats weighting 200C250?g. Rod-shaped myocytes were counted using a Mallassez cell and resuspended in an adequate volume of the Ca2+-free Ringer solution supplemented with 1?mM Ca2+, in order to obtain a density of 105 cell?ml?1. Each incubation tube contained 500?l of this cell suspension to which was added successively 5?l of a 200 stock solution of the appropriate drug and 500?l of Ringer solution. Each condition including control (i.e. in absence of any drug) was tested in triplicate. Incubation was carried out at room temperature (20C24C) and lasted 15?min, allowing the cells to settle down. After this period, the supernatant was discarded and replaced by 500?l of cold (4C) ethanol to stop the reaction. Ethanol was BRL 44408 maleate evaporated (1?h at 37C and low pressure) and the pellet was homogenized in 200?l of buffer for cyclic AMP assay (cyclic AMP radioimmunoassay kit, Immunotech, Marseille, France). After centrifugation at 4500?r.p.m. for 5?min, the supernatants were diluted ten times in the same buffer and cyclic AMP was assayed according to the instructions provided by the manufacturer. Electrophysiological experiments The whole cell configuration of the patch-clamp technique was used to record the high-threshold L-type calcium current ( em I /em Ca) on Ca2+-tolerant rat ventricular myocytes. The cell was routinely depolarized every 8?s from ?50 to 0?mV for 400?ms. The use of a holding potential of ?50?mV allowed the elimination of fast sodium currents. Potassium currents were blocked by replacing all K+ ions with intracellular and extracellular Cs+. For the determination of current-voltage relationships for em I /em Ca (see Figure 4A) and em I /em Ca inactivation curve (see Figure 4B), a double pulse voltage clamp protocol was used (Kirstein em et al /em ., 1995). Briefly, every 4?s, the membrane potential of the cell, which was normally maintained at.