Abstract
Steady-state activation of cardiac beta-adrenergic receptors leads
to an intracellular compartmentation of cAMP resulting from localized
cyclic nucleotide phosphodiesterase (PDE) activity. To evaluate the
time course of the cAMP changes in the different compartments, brief
(15 seconds) pulses of isoprenaline (100 nmol/L) were applied to
adult rat ventricular myocytes (ARVMs) while monitoring cAMP changes
beneath the membrane using engineered cyclic nucleotide-gated channels
and within the cytosol with the fluorescence resonance energy transfer-based
sensor, Epac2-camps. cAMP kinetics in the two compartments were compared
to the time course of the L-type Ca(2+) channel current (I(Ca,L))
amplitude. The onset and recovery of cAMP transients were, respectively,
30% and 50% faster at the plasma membrane than in the cytosol, in
agreement with a rapid production and degradation of the second messenger
at the plasma membrane and a restricted diffusion of cAMP to the
cytosol. I(Ca,L) amplitude increased twice slower than cAMP at the
membrane, and the current remained elevated for approximately 5 minutes
after cAMP had already returned to basal level, indicating that cAMP
changes are not rate-limiting in channel phosphorylation/dephosphorylation.
Inhibition of PDE4 (with 10 micromol/L Ro 20-1724) increased the
amplitude and dramatically slowed down the onset and recovery of
cAMP signals, whereas PDE3 blockade (with 1 micromol/L cilostamide)
had a minor effect only on subsarcolemmal cAMP. However, when both
PDE3 and PDE4 were inhibited, or when all PDEs were blocked using
3-isobutyl-l-methylxanthine (300 micromol/L), cAMP signals and I(Ca,L)
declined with a time constant >10 minutes. cAMP-dependent protein
kinase inhibition with protein kinase inhibitor produced a similar
effect as a partial inhibition of PDE4 on the cytosolic cAMP transient.
Consistently, cAMP-PDE assay on ARVMs briefly (15 seconds) exposed
to isoprenaline showed a pronounced (up to approximately 50%) dose-dependent
increase in total PDE activity, which was mainly attributable to
activation of PDE4. These results reveal temporally distinct beta-adrenergic
receptor cAMP compartments in ARVMs and shed new light on the intricate
roles of PDE3 and PDE4.
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