Simultaneous measurements of intracellular cAMP and L‐type Ca2+ current in single frog ventricular myocytes

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Abstract
1 The cAMP fluorescent probe FlCRhR was used to monitor changes in intracellular cAMP concentration ([cAMP]i) in isolated frog ventricular myocytes. The probe was introduced into the cell through a patch pipette which allowed simultaneous recording of the whole-cell L-type Ca2+ current (ICa). Ratiometric imaging was used to monitor [cAMP]i changes in response to the β-adrenergic agonist isoprenaline (ISO) or to the direct adenylyl cyclase activator forskolin (FSK). 2 FlCRhR fluorescence was distributed in the cytosol in a striated pattern, with high fluorescence in the I-bands and low fluorescence in the A-bands. This pattern of distribution was mimicked by fluorescein dextran, another high molecular weight fluorescent molecule, and was therefore likely to be due to anisotropic diffusion of the probe in the cytosol due to the hindrance generated by sarcomeric proteins in the A-bands. 3 Introduction of FlCRhR into the cell induced a small ≈70% stimulatory effect on basal ICa, attenuating about 2-fold a subsequent response of ICa to 1-10 μm ISO (from 400 to 200%). 4 Brief (10 s) application of a saturating concentration of ISO (1-20 μm) to the cell induced a transient increase in both ICa and [cAMP]i. However, the [cAMP]i transient was ≈2-fold shorter in duration than the ICa transient, i.e. ICa was still strongly enhanced when [cAMP]i had already returned to control level. This indicates that hydrolysis of cAMP by phosphodiesterases is not a rate limiting step in the recovery of ICa from ISO stimulation. 5 When the application of ISO was maintained, ICa and [cAMP]i responses followed a similar time course, with a half-maximal response at ≈60 s. This suggests that activation of Ca2+ channels by cAMP-dependent protein kinase occurs on a much faster time scale than the rise in [cAMP]i. 6 When the cells were exposed to FSK (13 μm), both responses of ICa and [cAMP]i were ≈2-fold slower than with ISO. This demonstrates that the slower response of ICa to FSK is due to a slower rise in [cAMP]i rather than to some inhibitory effect of FSK on ICa or to a direct or priming effect of the stimulatory G protein Gs on Ca2+ channels. 7 Simultaneous measurements of [cAMP]i and ICa changes in intact cardiac myocytes opens the way to dissect the temporal sequence of events in the cAMP cascade mediating the response of the heart to a large number of hormones and inotropic agents.