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

Abstract

1 The e ects 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 di erent cardiac PDE subtypes was con®rmed by RT ± PCR. 2 IBMX (100 mM), a broad-spectrum PDE inhibitor, increased basal I Ca by 120% and [cAMP] i by 70%, similarly to a saturating concentration of the b-adrenoceptor agonist isoprenaline (1 mM). However, MIMX (1 mM), a PDE1 inhibitor, EHNA (10 mM), a PDE2 inhibitor, cilostamide (0.1 mM), a PDE3 inhibitor, or Ro 20-1724 (0.1 mM), a PDE4 inhibitor, had no e ect on basal I Ca and little stimulatory e ects 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 e ect on I Ca or [cAMP] i . While all combinations tested signi®cantly increased [cAMP] i (40 ± 50%), only cilostamide (0.1 mM)+Ro20-1724 (0.1 mM) produced a signi®cant stimulation of I Ca (50%). Addition of EHNA (10 mM) 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 mM) or isoprenaline (1 mM). 4 When tested on top of a sub-maximal concentration of isoprenaline (1 nM), which increased I Ca by (&40% and had negligible e ect on [cAMP] i , each selective PDE inhibitor induced a clear stimulation of [cAMP] i and an additional increase in I Ca . Maximal e ects on I Ca were &8% for MIMX (3 mM), &20% for EHNA (1 ± 3 mM), &30% for cilostamide (0.3 ± 1 mM) and &50% for Ro20-1724 (0.1 mM). 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 I Ca , all four PDE subtypes determine the response of I Ca to a stimulus activating cyclic AMP production, with the rank order of potency PDE44PDE34PDE24PDE1.

Increasing Ryanodine Receptor Open Probability Alone Does Not Produce Arrhythmogenic Calcium Waves

“…Furthermore, the effects of caffeine occur and disappear within 1 or 2 stimuli a time course, which is much faster than that of cAMP-dependent changes produced by adding or removing phosphodiesterase inhibitors. 2,31 Caffeine also changes the relationship between [Ca 2ϩ ] i and contraction, 32,33 but this does not occur at concentrations of 1 mmol/L and below, and it is also not obvious how such an effect could affect diastolic Ca 2ϩ release.…”

Negative Feedback Exerted by cAMP-dependent Protein Kinase and cAMP Phosphodiesterase on Subsarcolemmal cAMP Signals in Intact Cardiac Myocytes

“…cardiac myocytes (20,28) to the attenuation of the ␤-adrenergic response. In the experiment shown in Fig.…”

“…Male Wistar rats (160 -180 g) were subjected to anesthesia by intraperitoneal injection of pentothal (0.1 mg/g), and hearts were excised rapidly. Individual ventricular myocytes were obtained by retrograde perfusion of the heart as previously described (20). Freshly isolated cells were suspended in minimal essential medium (MEM: M 4780; Sigma) containing 1.2 mM Ca 2ϩ , 2.5% fetal bovine serum (FBS, Invitrogen, Cergy-Pontoise, France), 1% penicillin-streptomycin and 2% HEPES (pH 7.6) and plated on laminin-coated culture dishes (10 g/ml laminin, 2 h) at a density of 10 4 cells per dish.…”

Rôle des phosphodiestérases des nucléotides cycliques de types 3 et 4 dans le couplage excitation-contraction et les arythmies cardiaques

“…Lors d'une stimulation -AR non maximale, l'inhibition de la PDE3 potentialise l'amplitude de I Ca,L , des transitoires calciques et de la contraction. Cela s'accompagne d'une augmentation de la phosphorylation dépen-dante de la PKA des LTCC, de PLB, et de RyR2 [21] (D. Mika, J. Leroy, R. Fischmeister, G. Vandecasteele ; résultats non publiés). Une situation similaire est retrouvée chez la souris, espèce dans laquelle l'inhibition de la PDE3 augmente les transitoires calciques et la contraction cardiaque basales, ainsi que le taux de phosphorylation par la PKA du PLB et des RyR2, sans augmenter l'amplitude d'I Ca,L [22,23].…”

“…Dans la plupart des espèces, l'inhibition des PDE4 n'exerce peu ou pas d'effet inotrope positif direct, mais devient importante lorsque les taux d'AMPc sont augmentés [16,27]. Par exemple, chez le rat, l'inhibition des PDE4 n'a aucun effet sur l'AMPc, I Ca,L et le CEC en conditions basales, mais devient prédominante pour contrôler ces paramètres lors d'une stimulation -AR ou lors de l'inhibition concomitante de la PDE3 [21,28] (D. Mika, J. Leroy, R. Fischmeister, G. Vandecasteele ; résultats non publiés). Chez la souris, l'inhibition des PDE4 n'a pas d'effet sur I Ca,L en conditions basales, mais potentialise fortement les effets d'une stimulation -AR sur I Ca,L et sur la contraction [29].…”