cAMP microdomains and L-type Ca2+ channel regulation in guinea-pig ventricular myocytes

Abstract

Many different receptors can stimulate cAMP synthesis in the heart, but not all elicit the same functional responses. For example, it has been recognized for some time that prostaglandins such as PGE1 increase cAMP production and activate PKA, but they do not elicit responses like those produced by beta-adrenergic receptor (betaAR) agonists such as isoproterenol (isoprenaline), even though both stimulate the same signalling pathway. In the present study, we confirm that isoproterenol, but not PGE1, is able to produce cAMP-dependent stimulation of the L-type Ca(2+) current in guinea pig ventricular myocytes. This is despite finding evidence that these cells express EP(4) prostaglandin receptors, which are known to activate G(s)-dependent signalling pathways. Using fluorescence resonance energy transfer-based biosensors that are either freely diffusible or bound to A kinase anchoring proteins, we demonstrate that the difference is due to the ability of isoproterenol to stimulate cAMP production in cytosolic and caveolar compartments of intact cardiac myocytes, while PGE1 only stimulates cAMP production in the cytosolic compartment. Unlike other receptor-mediated responses, compartmentation of PGE1 responses was not due to concurrent activation of a G(i)-dependent signalling pathway or phosphodiesterase activity. Instead, compartmentation of the PGE1 response in cardiac myocytes appears to be due to transient stimulation of cAMP in a microdomain that can communicate directly with the bulk cytosolic compartment but not the caveolar compartment associated with betaAR regulation of L-type Ca(2+) channel function.

Figure 1. Guinea pig ventricular myocytes express EP receptors, but PGE1 produces no detectable responses

A, message for EP₃ and EP₄ prostaglandin receptors identified by RT-PCR. Representative result from one of three preparations. B, time course of changes in amplitude of the L-type Ca²⁺ current and examples of current traces under control conditions (1), during exposure to 10 μm PGE1 (2), and during exposure to 1 μM Iso (3). C, average increase in Ca²⁺ current amplitude recorded in the presence of 10 μM PGE and following subsequent exposure to 1 μM Iso (n = 5). D, time course of changes in the CFP/YFP emission intensity ratio produced by the PKA-based biosensor during exposure of an isolated myocyte to 10 μm PGE1 and 1 μm Iso. E, average change in CFP/YFP emission intensity ratio produced by 10 μM PGE1 and subsequent exposure to 1 μM Iso in untreated cells (n = 5).

Figure 2. Blocking AKAP-interaction reveals PGE1-induced changes in cAMP activity detected by the PKA-based sensor

A, time course of changes in CFP/YFP emission intensity ratio during exposure of an Ht31-treated myocyte to 1 μM PGE1. The intensity ratio is an average of the values measured across the entire cell. B, average change in CFP/YFP emission intensity ratio produced by 1 μM PGE1 in cells treated with inactivate, Ht31P peptide (n = 4) or active Ht31 peptide (n = 4). C, pseudocolour images illustrating the CFP/YFP emission ratio in a cell treated with Ht31 peptide before (1) and during the peak (2) and steady-state response (3) to 1 μM PGE1. D, fluorescence intensity profile of CFP emission measured across a 3 μm × 14 μm region of a cell treated with control Ht31P (♦) or active Ht31 peptide (⋄). E, average peak amplitude of the CFP fluorescence measured at the Z-lines in a 3 μm × 14 μm region of cells treated with control Ht31P (n = 4) or active Ht31 peptide (n = 4).

Figure 3. PGE1 induced changes in cAMP detected by the Epac-based sensor

A, time course of changes in the CFP/YFP intensity ratio during exposure of an isolated myocyte to 1 μm Iso. B, time course of changes in CFP/YFP intensity ratio during exposure of an isolated myocyte to 10 μm PGE1. The intensity ratio is an average of the values measured across the entire cell. C, average change in CFP/YFP emission intensity ratio produced by exposure to 1 μM Iso (n = 8) and 10 μM PGE1 (n = 8) D, pseudocolour images illustrating the CFP/YFP emission ratio before (1) and during the (2) and steady-state response (3) to 10 μm PGE1.

Figure 4. PGE1 does not produce a G_i-dependent inhibitory effect on βAR stimulation of the L-type Ca²⁺ current

A, time course of changes in the amplitude of the L-type Ca²⁺ current and example of current traces recorded during exposure to 0.001 μm Iso (1), 10 μm PGE1 plus 0.001 μm Iso (2), and 1 μm Iso (3). B, average increase in amplitude of the L-type Ca²⁺ current recorded in the presence of 0.001 μm Iso and upon subsequent addition of 10 μm PGE1 (n = 5).

Figure 5. Pertussis toxin (PTX) inhibition of G_i does not reveal a PGE1 stimulatory effect

A, time course of changes in the amplitude of the L-type Ca²⁺ current and examples of current traces recorded before (1) and during exposure of a PTX-treated myocyte to 10 μm PGE1 (1) and a normally subthreshold concentration (0.0003 μm) of Iso (3). B, average increase in the amplitude of the L-type Ca²⁺ current produced by 10 μm PGE1 and subsequent exposure to a subthreshold concentration of Iso (0.0003 μm) in PTX-treated myocytes (n = 5). C, time course of changes in the CFP/YFP emission intensity ratio during exposure of a PTX-treated myocyte to 10 μm PGE1 and 0.0003 μm Iso. D, average change in CFP/YFP emission intensity ratio produced by 10 μm PGE1 and subsequent exposure to 0.0003 μm Iso in PTX-treated cells (n = 3).

Figure 6. Phosphodiesterase inhibition does not reveal a PGE1 stimulatory effect

A, time course of changes in the amplitude of the L-type Ca²⁺ current and examples of current traces recorded during exposure to 10 μm IBMX (1), IBMX plus 10 μm PGE1 (2), and IBMX plus a normally subthreshold concentration (0.0003 μm) of Iso. B, average increase in L-type Ca²⁺ current amplitude during exposure to 10 μm IBMX, IBMX plus 10 μm PGE1, and IBMX plus a subthreshold concentration of Iso (0.0003 μm; n = 8). C, time course of changes in the CFP/YFP emission intensity ratio during exposure to 10 μm IBMX, IBMX plus 10 μm PGE1, and IBMX plus 0.0003 μm Iso. D, average change in CFP/YFP emission intensity ratio during exposure to 10 μm IBMX, IBMX plus 10 μm PGE1, and IBMX plus 0.0003 μm Iso (n = 3).