Negative feedback exerted by cAMP-dependent protein kinase and cAMP phosphodiesterase on subsarcolemmal cAMP signals in intact cardiac myocytes: an in vivo study using adenovirus-mediated expression of CNG channels.
Intracardiac cAMP levels are modulated by hormones and neuromediators with specific effects on contractility and metabolism. To understand how the same second messenger conveys different information, mutants of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit CNGA2, encoded into adenoviruses, were used to monitor cAMP in adult rat ventricular myocytes. CNGA2 was not found in native myocytes but was strongly expressed in infected cells. In whole cell patch-clamp experiments, the forskolin analogue L-858051 (L-85) elicited a non-selective, Mg2+ -sensitive current observed only in infected cells, which was thus identified as the CNG current (ICNG). The beta-adrenergic agonist isoprenaline (ISO) also activated ICNG, although the maximal efficiency was approximately 5 times lower than with L-85. However, ISO and L-85 exerted a similar maximal increase of the L-type Ca2+ current. The use of a CNGA2 mutant with a higher sensitivity for cAMP indicated that this difference is caused by the activation of a localized fraction of CNG channels by ISO. cAMP-dependent protein kinase (PKA) blockade with H89 or PKI, or phosphodiesterase (PDE) inhibition with IBMX, dramatically potentiated ISO- and L-85-stimulated ICNG. A similar potentiation of beta-adrenergic stimulation occurred when PDE4 was blocked, whereas PDE3 inhibition had a smaller effect (by 2-fold). ISO and L-85 increased total PDE3 and PDE4 activities in cardiomyocytes, although this effect was insensitive to H89. However, in the presence of IBMX, H89 had no effect on ISO stimulation of ICNG. This study demonstrates that subsarcolemmal cAMP levels are dynamically regulated by a negative feedback involving PKA stimulation of subsarcolemmal cAMP-PDE.
Laboratoire de Cardiologie Cellulaire et Moléculaire, INSERM U-446, Université Paris-Sud, Faculté de Pharmacie, F-92296 Chātenay-Malabry, France.
Francesca
F
Rochais
Grégoire
G
Vandecasteele
Florence
F
Lefebvre
Claire
C
Lugnier
Hazel
H
Lum
Jean-Luc
JL
Mazet
Dermot M F
DM
Cooper
Rodolphe
R
Fischmeister
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000031:analogs & derivatives* / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D054815:Cyclic Nucleotide-Gated Cation Channels; D004224:Diterpenes; D005246:Feedback; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects
D054815:Cyclic Nucleotide-Gated Cation Channels; D004224:Diterpenes; D007473:Ion Channels; D011994:Recombinant Proteins; C055627:L 858051; D005576:Colforsin; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D007545:Isoproterenol; D015056:1-Methyl-3-isobutylxanthine