G protein-mediated inhibitory effect of a nitric oxide donor on the L-type Ca2+ current in rat ventricular myocytes

Abstract

1. The role of the cGMP pathway in the modulation of the cardiac L-type Ca2+ current (ICa,L) by nitric oxide (NO) was examined in rat ventricular myocytes. 2. The NO donors DEANO, SIN-1, SNP, SNAP and GSNO had no significant effects on basal ICa,L. However, DEANO (100 microM) inhibited ICa,L after the current had been previously stimulated by either isoprenaline (Iso, 1-10 nM), a beta-adrenergic agonist, or isobutylmethyl-xanthine (IBMX, 10-80 microM), a wide spectrum phosphodiesterase (PDE) inhibitor. 3. The anti-adrenergic effect of DEANO on ICa,L was not mimicked by other NO donors (SIN-1, SNAP and SPNO). 4. The NO-sensitive guanylyl cyclase inhibitor ODQ (10 microM), antagonized the inhibitory effect of DEANO on ICa,L. Likewise, inhibitors of the cGMP-dependent protein kinase (cG-PK), Rp-8-chloro-phenylthio-cGMP (10 microM) and KT5823 (0.1 and 0.3 microM), also abolished the inhibitory effect of DEANO on Iso (1-10 nM)-stimulated ICa,L. 5. Intracellular dialysis with exogenous cAMP (10-100 microM) blunted the inhibitory effect of DEANO (10 and 100 microM) on ICa,L. SNAP and SNP also had no effect on the cAMP-stimulated ICa,L. 6. Pre-treatment of the myocytes with pertussis toxin (0.5 microg ml-1, 4-6 h at 37 degrees C) eliminated the inhibitory effect of DEANO (100 microM) on ICa,L, in the presence of either Iso (0.01 and 1 nM) or IBMX (10-80 microM). 7. These results demonstrate that DEANO produces anti-adrenergic effects in rat ventricular myocytes. This effect of DEANO occurs in a cGMP-dependent manner, and involves activation of cG-PK and regulation of a pertussis toxin-sensitive G protein.

Figure 2. DEANO inhibits the β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of isoprenaline (Iso, 10 nM) and DEANO (100 μM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B (same experiment as in A), current-voltage relationships of I_Ca,L (filled symbols) and of the steady-state current at the end of the pulse (open symbols) obtained under basal conditions, and in the presence of Iso (10 nM) with or without DEANO (100 μM).

Figure 1. NO donors do not regulate basal I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control intracellular and extracellular control solutions. I_Ca,L (□) was elicited at 0 mV from a holding potential of −50 mV. Superfusion of the myocyte with 100 μM DEANO is indicated by the horizontal line. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO (100 μM), GSNO (1 mM), SNAP (1 mM), SNP (500 μM) and SIN-1 (1 mM) on basal I_Ca,L amplitude. The amplitude of I_Ca,L in the presence of NO donors was normalized to the amplitude of basal I_Ca,L in control conditions (set to 100 %). Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars.

Figure 3. Summary of the effects of DEANO on the β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

The amplitude of I_Ca,L in the presence of Iso (1, 3 or 10 nM), without (□) or with DEANO (100 μM, ▪), was normalized to the basal I_Ca,L amplitude (set to 100 %). Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from basal (*) or Iso levels (†) are indicated as: *P < 0.001; **, ‡P < 0.0001.

Figure 4. Other NO donors do not inhibit the β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (10 nM) and SIN-1 (100 μM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of three NO donors (SNAP, SIN-1, SPNO, at 100 μM) on I_Ca,L in the presence of either 1 nM (▪) or 10 nM Iso (□). The effects of NO donors on I_Ca,L are presented as percentage variations from the amplitude of the Iso-stimulated I_Ca,L. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars.

Figure 5. The guanylyl cyclase inhibitor ODQ antagonizes the effect of DEANO on the β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (3 nM), DEANO (100 μM) and ODQ (10 μM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO without (▪) or with ODQ (10 μM, □), in the presence of 1, 3 or 10 nM Iso. DEANO was used at 10 μM (in the presence of Iso 10 nM) or 100 μM (in the presence of 1 and 3 nM Iso). The effects of DEANO on I_Ca,L are presented as percentage variations from the amplitude of the Iso-stimulated I_Ca,L. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from Iso (*) or Iso + DEANO (†) levels are indicated as: *, †P < 0.05; ‡P < 0.01; ***P < 0.005.

Figure 6. cG-PK inhibitors antagonize the effect of DEANO on the β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control intracellular and extracellular solutions. Applications of Iso (1 nM), DEANO (100 μM) and Rp8cG (10 μM) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B, summary of the effects of DEANO (100 μM) without (□) or with cG-PK inhibitors (▪; Rp8cG, 10 μM; KT5823, 0.1 or 0.3 μM), in the presence of 1 or 10 nM Iso. The effects of DEANO on I_Ca,L are presented as percentage variations from the amplitude of the Iso-stimulated I_Ca,L. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from Iso (*) or Iso + DEANO (†) levels are indicated as: *, †P < 0.05; ‡P < 0.01; ***P < 0.001.

Figure 7. DEANO does not inhibit the stimulation of I_Ca,L induced by exogenous cAMP

A, the pipette solution contained 30 μM cAMP, and cAMP dialysis started when the patch was ruptured (arrow). B, a myocyte was first dialysed with the control solution, and intracellular dialysis with 30 μM cAMP started at the time indicated by the arrow. Applications of DEANO (100 μM) in A or SNP (5, 50 and 500 μM) in B were performed as indicated by the horizontal lines. In A, current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. C, summary of the effects of DEANO, SNP and SNAP on the cAMP (10–100 μM)-stimulated I_Ca,L. Variations are given as percentage change over the amplitude of the cAMP-stimulated I_Ca,L. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars.

Figure 8. DEANO does not inhibit the β-adrenergic stimulation of I_Ca,L in PTX-treated rat ventricular myocytes

A, a myocyte was incubated with pertussis toxin (PTX, 0.5 μg ml⁻¹, 4 h, 37 °C) prior to the experiment. It was first exposed to control extracellular and intracellular solutions, and applications of Iso (1 nM), DEANO (100 μM) and acetylcholine (ACh, 1 μM) were performed as indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graph. The dotted line indicates the zero-current level. B and C, summary of the effects of DEANO (100 μM) and ACh (1 μM) on the Iso (0.01 and 1 nM)-stimulated I_Ca,L in PTX-treated myocytes. The amplitude of I_Ca,L is presented as percentage increase over basal amplitude (in B) or as percentage variations from the amplitude of the Iso-stimulated I_Ca,L (in C). Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from Iso level are indicated as: *P < 0.05.

Figure 9. Effects of DEANO and ACh on IBMX-stimulated I_Ca,L in rat ventricular myocytes

Untreated (A) and PTX-treated (B) myocytes were first exposed to control extracellular and intracellular solutions. Applications of IBMX (40 μM in A, 20 μM in B), DEANO (100 μM, in A and B) and ACh (1 μM, in B) are indicated by the horizontal lines. Current traces on top were recorded at the times indicated by the corresponding letters on the main graphs. The dotted lines indicate the zero-current level. C, summary of the effects of IBMX (10–80 μM) on I_Ca,L, used either alone (□) or in the presence of DEANO (100 μM) or ACh (1 μM) (▪) in untreated (left) or PTX-treated myocytes (right). The effects of IBMX on I_Ca,L are presented as percentage variations from the amplitude of basal I_Ca,L (set to 100 %). Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from basal (*) and IBMX (†) levels are indicated as: †P < 0.05; **, ‡P < 0.01; ***P < 0.005.