Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current in rat ventricular myocytes

1 The effects of several phosphodiesterase (PDE) inhibitors on the L-type Ca current (I-Ca) and intracellular cyclic AMP concentration ([cAMP](i)) were examined in isolated rat ventricular myocytes. The presence of mRNA transcripts encoding for the different cardiac PDE subtypes was confirmed by RT-PCR. 2 IBMX (100 mu M), a broad-spectrum PDE inhibitor, increased basal I-Ca by 120% and [cAMP](i) by 70%, similarly to a saturating concentration of the beta-adrenoceptor agonist isoprenaline (1 mu M). However, MIMX (1 mu M), a PDE1 inhibitor, EHNA (10 mu M), a PDE2 inhibitor, cilostamide (0.1 mu M), a PDE3 inhibitor, or Ro 20-1724 (0.1 mu M), a PDE4 inhibitor, had no effect on basal I-Ca and little stimulatory effects on [cAMP](i) (20-30%). 3 Each selective PDE inhibitor was then tested in the presence of another inhibitor to examine whether a concomitant inhibition of two PDE subtypes had any effect on I-Ca or [cAMP](i). While all combinations tested significantly increased [cAMP](i) (40-50%), only cilostamide (0.1 mu M)+ Ro20-1724 (0.1 mu M) produced a significant stimulation of I-Ca (50%). Addition of EHNA (10 mu M) to this mix increased I-Ca to 110% and [cAMP](i) to 70% above basal, i.e, to similar levels as obtained with IBMX (100 mu M) or isoprenaline (1 mu M). 4 When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I-Ca by (approximate to 40%) and had negligible effect on [cAMP](i), each selective PDE inhibitor induced a clear stimulation of [cAMP](i) and an additional increase in I-Ca. Maximal effects on I-Ca were approximate to 8% for MIMX (3 mu M), approximate to 20% for EHNA (1-3 mu M), approximate to 30% for cilostamide (0.3-1 mu M) and approximate to 50% for Ro20-1724 (0.1 mu M). 5 Our results demonstrate that PDE1-4 subtypes regulate I-Ca in rat ventricular myocytes. While PDE3 and PDE4 are the dominant PDE subtypes involved in the regulation of basal Ic,, all four PDE subtypes determine the response of I-Ca to a stimulus activating cyclic AMP production, with the rank order of potency PDE4 >PDE3 > PDE2 > PDE1.