NO donors potentiate the beta-adrenergic stimulation of I(Ca,L) and the muscarinic activation of I(K,ACh) in rat cardiac myocytes

Abstract

The effects of nitric oxide (NO) donors on the L-type Ca(2+) current (I(Ca,L)) and the muscarinic activated K(+) current (I(K,ACh)) were studied in isolated rat cardiac myocytes. The nitrosothiol S-nitroso-N-acetyl-D,L-penicillamine (SNAP, 1 pM-1 microM) strongly potentiated the stimulation of the I(Ca,L) elicited by subthreshold concentrations of isoprenaline (Iso, 0.1-0.5 nM) in ventricular myocytes. The effect of SNAP was mimicked by 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO, 1 pM-1 nM), a NONOate that spontaneously releases NO in a pH-controlled manner, and was blunted by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (100 microM), a NO trap. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxaline-1-one (10 microM), a guanylyl cyclase inhibitor, did not alter the effect of SNAP. SNAP (1 pM-1 microM) did not modify the effect of L858051 (0.1-0.3 microM), a forskolin analogue that activates adenylyl cyclase, on I(Ca,L) and did not enhance the basal I(Ca,L) in the presence of rolipram (1 microM), a phosphodiesterase type 4 inhibitor. Superfusion with Rp-CPT-cAMPS (500 microM), or internal dialysis with cAMP-dependent protein kinase (cA-PK) inhibitory peptide (PKI; 20 microM), inhibitors of the cA-PK, blunted the effect of SNAP (1 nM and 1 microM) on the Iso-stimulated (1-100 pM) I(Ca,L). SNAP (1 nM and 1 microM) potentiated the threshold stimulation of I(Ca,L) elicited by internal GTP-gammaS (10 microM), a non-hydrolysable analogue of GTP. SNAP (1 pM-1 microM) and DEANO (1 microM) potentiated the stimulation of I(K,ACh) elicited by low concentrations of ACh (1-2 nM) in rat atrial myocytes. The threshold stimulation of I(K,ACh) elicited by internal 5'-guanylylimidodiphosphate (10 microM) was also potentiated by NO donors. SNAP (1 microM) did not modify I(K,ACh) reconstituted in human embryonic kidney 293 cells, in the absence or in the presence of ACh (1 or 10 nM). Taken together, these data suggest that NO is a cGMP-independent modulator of G-protein-coupled muscarinic and beta-adrenergic receptor actions on cardiac ion channels. Although this action of NO seemed to occur at the level of G proteins, it appeared to require a component distinct from receptors, G proteins or their effectors.

Figure 1. The nitric oxide (NO) donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) modulates the threshold β-adrenergic stimulation of the L-type Ca²⁺ current (I_Ca,L) in rat ventricular myocytes

A, a myocyte was first exposed to external and internal control solutions. I_Ca,L (▪) was elicited at 0 mV from a holding potential of −50 mV. Superfusion of the myocyte with 0.1 nm isoprenaline (Iso) alone or in combination with SNAP (1 pm, 1 nm, or 1 μm) is indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graph (a–g). 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 in the presence of Iso 0.1 nm alone (squares) or in combination with SNAP (1 nm, triangles; 1 μm, circles).

Figure 2. Summary of the effects of NO donors on the threshold β-adrenergic stimulation of I_Ca,L in rat ventricular myocytes

A, mean stimulatory effects of various low concentrations of Iso on I_Ca,L, in the absence (filled bars) and in the presence of 1 nm SNAP (open bars). B, mean stimulatory effects of Iso (0.1–0.5 nm) in the absence (filled bars) and in the presence of different concentrations of SNAP or 2-(N,N-diethylamino)-diazenolate-2-oxide (DEANO; open bars). The amplitude of I_Ca,L in the presence of agonists was normalised to the amplitude of the basal I_Ca,L in the absence of agonists. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the Iso level: *P≤ 0.05; **P≤ 0.01; ***P≤ 0.005.

Figure 3. The potentiating effect of NO donors on the Iso-stimulated I_Ca,L is cGMP-independent in rat ventricular myocytes

A, a myocyte was first exposed to control solutions. Superfusion of the myocyte with 0.2 nm Iso alone or in combination with SNAP (1 nm or 1 μm) or 1H-[1, 2, 4]oxadiazolo[4, 3-a]quinoxaline-1-one (ODQ) (10 μm) is indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graph (a-h). The dotted line indicates the zero-current level. B, mean stimulatory effects of Iso (0.1 and 0.2 nm) on I_Ca,L, in the presence (filled bars) and in the absence of 10 μm ODQ (open bars), with or without SNAP (1 nm or 1 μm), as indicated. The amplitude of I_Ca,L in the presence of agonists was normalised to the amplitude of the basal I_Ca,L in the absence of agonists. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the Iso level: *P≤ 0.05; ***P≤ 0.005.

Figure 4. Effect of SNAP on the L858051-stimulated I_Ca,L in rat ventricular myocytes

A, a myocyte was first exposed to control solutions. Applications of L858051 (0.2 μm), SNAP (1 nm) and cilostamide (Cil, 0.3 μm) are indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graph (a-e). The dotted line indicates the zero-current level. B, summary of the effects of L858051 alone (open bar) or in the presence (filled bars) of SNAP (1 pm, 1 nm, 1 μm), or cilostamide (0.3 μm). The amplitude of I_Ca,L in the presence of agonists was normalised to the amplitude of the basal I_Ca,L in the absence of agonists. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from L858051 level: ***P≤ 0.005.

Figure 5. cAMP-dependent protein kinase (cA-PK) inhibitors antagonise the effects of NO donors on the Iso-stimulated I_Ca,L in rat ventricular myocytes

A, a myocyte was first dialysed with cA-PK inhibitory peptide (PKI; 20 μm) and exposed to external control solution. Applications of Iso (100 pM, 10 nm) and SNAP (1 nm, 1 μm) are indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graph (a-h). The dotted line indicates the zero-current level. B, summary of the effects of Iso (100 pm), SNAP (1 nm, 1 μm), in myocytes dialysed with PKI (20 μm). C, overview of the effects of Iso (1–100 pm), SNAP (1 nm), in the absence or in the presence of external adenosine-3′,5′-cyclic monophosphothioate, Rp-isomer (Rp-cA 300 μm). The amplitude of I_Ca,L in the presence of agonists was normalised to the amplitude of the basal I_Ca,L in the absence of agonists. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the Iso (*) or the Iso plus SNAP (#) levels: *P≤ 0.05; #P≤ 0.01.

Figure 6. Effect of SNAP on I_Ca,L in the presence of GTP-γS in rat ventricular myocytes

A, a myocyte was first dialysed with 10 μm GTP-γS, a non-hydrolysable analogue of GTP, and exposed to control external solution. The lines indicate applications of SNAP (1 nm, 1 μm). Traces were recorded at the times indicated by the corresponding letters on the main graph (a-e). The dotted line indicates the zero-current level. B, summary of the effects of SNAP (1 nm, 1 μm) in 10 μm GTP-γS-dialysed myocytes. The amplitude of I_Ca,L in the presence of SNAP was normalised to the amplitude of the basal I_Ca,L in the absence of SNAP. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the GTP-γS level are: *P≤ 0.05; ***P≤ 0.005.

Figure 7. SNAP potentiates the muscarinic activation of the muscarinic-activated K⁺ current, I_K,ACh, in rat atrial myocytes

A, an atrial myocyte was first exposed to control potassium-rich solutions, and superfusion with SNAP (1 nm, 1 μm) or ACh (3 μm) was performed as indicated by the lines. I_K,ACh was measured at 0 mV (•) or −120 mV (▪) from a holding potential of −50 mV. B, same as in A, except that SNAP (1 pm, 1 nm, 1 μm) was applied in the presence of ACh (2 nm). Traces were recorded at the times indicated by the corresponding letters on the main graphs. The dotted lines indicate the zero-current level.

Figure 8. Summary of the effects of NO donors on I_K,ACh in rat atrial myocytes

A, mean stimulatory effects of SNAP (1 μm) on I_K,ACh, in the presence of various concentrations of ACh. Inset (same experiments as in the main graph), the effects of ACh, in the absence (•) and in the presence of SNAP (□), were normalised to the amplitude of the end pulse current at 0 mV in the absence of agonist. The dose-response curve to ACh alone was fitted to the Michaelis-Menten equation (continuous line), as described in the text. The dose-response curve to ACh in the presence of SNAP was fitted by eye (dotted line). B, mean stimulatory effects of SNAP (1 pm, 1 nm, 1 μm) or DEANO (1 μm) on I_K,ACh, in the presence of ACh (1 and 2 nm). In A and B, the amplitude of I_K,ACh (the end pulse current at 0 mV) in the presence of ACh + NO donors was normalised to the amplitude of I_K,ACh in the presence of ACh alone. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the ACh level: *P≤ 0.05; **P≤ 0.01: ***P≤ 0.005.

Figure 9. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO) antagonises the effects of DEANO on the ACh-activated I_K,ACh in rat atrial myocytes

A, an atrial myocyte was first exposed to control potassium-rich solutions, and superfusion with DEANO (1 μm), cPTIO (100 μm) and ACh (10 pm, or 3 μm) was performed as indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graphs (a-h). The dotted line indicates the zero-current level. B, mean stimulatory effects of ACh (1 pm, 1 nm, 1 μm) on I_K,ACh, with or without DEANO (1 μm) in the absence or presence of cPTIO (100 μm). The amplitude of I_K,ACh (at 0 mV) in the presence of compounds was normalised to the amplitude of the end pulse current (at 0 mV) in the absence of compounds. Bars are the means and lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences (P≤ 0.05) from basal level (*), or the Iso level (#), or the Iso plus DEANO level ($) are indicated near the bars.

Figure 10. Effects of SNAP on I_K,ACh in the presence of 5′-guanylylimidodiphosphate (GppNHp) in rat atrial myocytes

A, an atrial myocyte was dialysed with GppNHp (10 μm) and exposed to external control K⁺-solutions. Superfusion with SNAP (1 nm, 1 μm) and ACh (3 μm) was performed as indicated by the lines. Traces were recorded at the times indicated by the corresponding letters on the main graph (a-f). The dotted line indicates the zero-current level. B, mean stimulatory effects of SNAP (1 nm, 1 μm) and ACh (3 μm) on I_K,ACh in GppNHp-dialysed (10 μm) rat atrial myocytes. The amplitude of I_K,ACh (at 0 mV) in the presence of agonists was normalised to the amplitude of the end pulse current (at 0 mV) in the presence of GppNHp alone. Bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the GppNHp level: *P≤ 0.05; **P≤ 0.01; ***P≤ 0.005.

Figure 11. Effects of SNAP on I_K,ACh in human embryonic kidney (HEK)293 cells

A, representative traces of the effects of Ba²⁺ ions (1 mm, •) or ACh (1 μm, ▪) in HEK293 cells. Control traces are unlabelled. The dotted line indicates the zero-current level. The voltage steps are indicated on the top of the panel. B, mean effects of Ba²⁺ ions (1 mm) and ACh (1 nm, 10 nm, 1 μm) on I_K,ACh in HEK293 cells. The amplitude of I_K,ACh (at −110 mV) in the presence of compounds was normalised to the amplitude of the end pulse current in the absence of compounds. C, representative traces of the effects of ACh alone (1 nm and 1 μm) and of ACh (1 nm) plus SNAP (1 μm) in HEK293 cells. A square symbol (▪) indicates the trace obtained in the presence of ACh 1 μm, and the other traces are labelled with a circle (•). The dotted lines indicate the zero-current level. D, mean effects of SNAP (1 μm) in the absence or in the presence of ACh (1 nm or 10 nm) on I_K,ACh in HEK293 cells. The amplitude of I_K,ACh (at −110 mV) in the presence of SNAP was normalised to the amplitude of the end pulse current in the corresponding experimental condition. In B and D, bars are the means and vertical lines are the s.e.m. of the number of experiments indicated near the bars. Significant differences from the basal level: *P≤ 0.05; ***P≤ 0.005.