Cyclic guanosine monophosphate compartmentation in rat cardiac myocytes

Abstract

Background: Cyclic guanosine monophosphate (cGMP) is the common second messenger for the cardiovascular effects of nitric oxide (NO) and natriuretic peptides, such as atrial or brain natriuretic peptide, which activate the soluble and particulate forms of guanylyl cyclase, respectively. However, natriuretic peptides and NO donors exert different effects on cardiac and vascular smooth muscle function. We therefore tested whether these differences are due to an intracellular compartmentation of cGMP and evaluated the role of phosphodiesterase (PDE) subtypes in this process.

Methods and results: Subsarcolemmal cGMP signals were monitored in adult rat cardiomyocytes by expression of the rat olfactory cyclic nucleotide-gated (CNG) channel alpha-subunit and recording of the associated cGMP-gated current (ICNG). Atrial natriuretic peptide (10 nmol/L) or brain natriuretic peptide (10 nmol/L) induced a clear activation of ICNG, whereas NO donors (S-nitroso-N-acetyl-penicillamine, diethylamine NONOate, 3-morpholinosydnonimine, and spermine NO, all at 100 micromol/L) had little effect. The ICNG current was strongly potentiated by nonselective PDE inhibition with isobutyl methylxanthine (100 micromol/L) and by the PDE2 inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (10 micromol/L) and Bay 60-7550 (50 nmol/L). Surprisingly, sildenafil, a PDE5 inhibitor, produced a dose-dependent increase of I(CNG) activated by NO donors but had no effect (at 100 nmol/L) on the current elicited by atrial natriuretic peptide.

Conclusions: These results indicate that in rat cardiomyocytes (1) the particulate cGMP pool is readily accessible at the plasma membrane, whereas the soluble pool is not; and (2) PDE5 controls the soluble but not the particulate pool, whereas the latter is under the exclusive control of PDE2. Differential spatiotemporal distributions of cGMP may therefore contribute to the specific effects of natriuretic peptides and NO donors on cardiac function.

Figure 1

Functional expression of CNGA2 channels in ARVMs. A, individual current traces of I_CNG obtained during 200 ms voltage pulse at −50 mV in infected (Ad-CNGA2) or non infected (NI) myocytes. Each trace was recorded in the presence of control solution supplemented (solid lines) or not (dotted lines) with the cGMP analog Sp-8 (100 μM). B, time course of I_CNG amplitude at −50 mV in an Ad-CNGA2 cell. The cell was initially superfused with control solution and then exposed to Sp-8 (100 μM). The Sp-8 activated current was blocked in a reversible manner by the presence of Mg²⁺ (10 mM). C, current-voltage relationships of I_CNG obtained with control solution (■) in the presence of 100 μM Sp-8 alone (▲) or with 10 mM Mg²⁺ (●). D, density of I_CNG obtained in Ad-CNGA2 or NI myocytes challenged with Sp-8 (10 μM or 100 μM). ***, p<0.005 vs. NI cells.

Figure 2

cGMP signals elicited by activation of soluble guanylyl cyclase (sGC). Typical experiment (A) and summary (B) of the effects of sGC activators on I_CNG in Ad-CNGA2 myocytes. I_CNG was measured as in Fig. 1. Specific activation of sGC was achieved by a direct agonist HMR1766 (HMR, 10 μM) or several NO-donors (SNAP, SIN-1, DEANO, SPNO, all at 100 μM concentration) and non selective PDE inhibition by IBMX (100 μM). At the end of each experiment the cell was challenged with Sp-8 (100 μM) as an internal control for WT-CNGA2 channel expression. ***, p<0.005 vs. IBMX; #, p<0.05 vs. basal; ###, p<0.005 vs. basal.

Figure 3

cGMP signals elicited by activation of particulate guanylyl cyclase (pGC). Typical experiment (A) and summary (B) of the effects of pGC activators on I_CNG in Ad-CNGA2 myocytes. Specific activation of pGC was achieved by ANP (10 nM) or BNP (10 nM) and non selective PDE inhibition by IBMX (100 μM). The effect of Sp-8 (100 μM) serves as an internal control for WT-CNGA2 channel expression. C, Concentration-dependent effects of ANP in Ad-CNGA2 myocytes. D, Non additive effect of both SNAP (100 μM) or BNP (10 nM) in ANP activated I_CNG. ^*, p<0.05 vs. IBMX; ^###, p<0.005 vs. basal; ns, non significant.

Figure 4

Role of PDE2 and PDE5 in cGMP signals generated by sGC. Typical experiment (A) and summary (B) of the effects of SNAP (100 μM) alone or in the presence of PDE inhibitors: EHNA (10 μM), sildenafil (Sil, 100 nM), Bay 60–750 (BAY, 50 nM), IBMX (100 μM). The effect of Sp-8 (100 μM) serves as an internal control for WT-CNGA2 channel expression. C, concentration-dependent effects of Sil in Ad-CNGA2 myocytes in the presence of SNAP. ^*, p<0.05, ^**, p<0.01 and ^***, p<0.005 vs. SNAP; ^###, p<0.005 vs. basal I_CNG.

Figure 5

Role of PDE2 and PDE5 in cGMP signals generated by pGC. Typical experiment (A) and summary (B) of the effects of ANP (10 nM) alone or in the presence of PDE inhibitors: EHNA (10 μM), sildenafil (Sil, 100 nM), IBMX (100 μM). The effect of Sp-8 (100 μM) serves as an internal control for WT-CNGA2 channel expression. ^***, p<0.005 vs. SNAP; ^###, p<0.005 vs. basal; ns, non significant.