Phenylephrine acts via IP3-dependent intracellular NO release to stimulate L-type Ca2+ current in cat atrial myocytes

Abstract

This study determined the effects of alpha1-adrenergic receptor (alpha1-AR) stimulation by phenylephrine (PE) on L-type Ca2+ current (I(Ca,L)) in cat atrial myocytes. PE (10 microm) reversibly increased I(Ca,L) (51.3%; n = 40) and shifted peak I(Ca,L) activation voltage by -10 mV. PE-induced stimulation of I(Ca,L) was blocked by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm ODQ, 2 microm H-89 or 10 microm LY294002, and was unaffected by 10 microm chelerythrine or incubating cells in pertussis toxin (PTX). PE-induced stimulation of I(Ca,L) also was inhibited by each of 10 microm ryanodine or 5 microm thapsigargin, by blocking IP3 receptors with 2 microm 2-APB or 10 microm xestospongin C or by intracellular dialysis of heparin. In field-stimulated cells, PE increased intracellular NO (NOi) production. PE-induced NOi release was inhibited by each of 1 microm prazocin, 10 microm L-NIO, 10 microm W-7, 10 microm LY294002, 2 microm H-89, 10 microm ryanodine, 5 microm thapsigargin, 2 microm 2-APB or 10 microm xestospongin C, and unchanged by PTX. PE (10 microm) increased phosphorylation of Akt, which was inhibited by LY294002. Confocal microscopy showed that PE stimulated NOi release from subsarcolemmal sites and this was prevented by 2 mm methyl-beta-cyclodextrin, an agent that disrupts caveolae formation. PE also increased local, subsarcolemmal SR Ca2+ release via IP3-dependent signalling. Electron micrographs of atrial myocytes show peripheral SR cisternae in close proximity to clusters of caveolae. We conclude that in cat atrial myocytes PE acts via alpha1-ARs coupled to PTX-insensitive G-protein to release NOi, which in turn stimulates I(Ca,L). PE-induced NOi release requires stimulation of both PI-3K/Akt and IP3-dependent Ca2+ signalling. NO stimulates I(Ca,L) via cGMP-mediated cAMP-dependent PKA signalling. IP3-dependent Ca2+ signalling may enhance local SR Ca2+ release required to activate Ca2+-dependent eNOS/NOi production from subsarcolemmal caveolae sites.

Figure 1. Effects of phenylephrine (PE; 10 μm) on I_Ca,L

A, PE reversibly increased peak I_Ca,L. B, consecutive measurements of peak I_Ca,L amplitude before, during and after exposure to PE shows the time course of PE-induced stimulation of I_Ca,L. C, the current–voltage relationship shows that PE reversibly increased I_Ca,L from −30 to +40 mV and shifted the voltage of maximum I_Ca,L activation by −10 mV without affecting the reversible potential. D, dose–response relationship of PE (1–500 μm) to stimulate I_Ca,L. The inset shows a sigmoidal dose–response relationship fitted with a Boltzmann equation. The numbers in parentheses indicate the number of cells tested in each experiment. *P < 0.05.

Figure 2. Pharmacological analysis of the signalling mechanisms responsible for PE-induced stimulation of I_Ca,L

Each experiment was performed by testing PE in the absence (control) and presence (test) of each drug in myocytes isolated from the same hearts. The control values from each experiment (n = 40) are grouped together for clarity. Compared with control (open bar), prazocin (1 μm), l-NIO (10 μm), W-7 (10 μm), ODQ (10 μm), H-89 (2 μm), LY294002 (10 μm) and ryanodine (10 μm) each significantly inhibited PE-induced stimulation of I_Ca,L. Incubation of cells in PTX or exposure to chelerythrine (4 μm) failed to affect PE-induced stimulation of I_Ca,L. The numbers in parentheses indicate the number of cells tested in each experiment. *P < 0.05.

Figure 3. Inhibition of IP₃ signalling inhibits PE-induced stimulation of I_Ca,L

A, original records showing the effects of PE to stimulate I_Ca,L recorded with a ruptured patch method. PE elicited a typical increase in I_Ca,L amplitude. B, another atrial myocyte in which I_Ca,L was recorded during intracellular dialysis of heparin (1 mg ml⁻¹) contained within the pipette solution. PE failed to increase I_Ca,L amplitude. In two additional series of experiments, PE was tested in the absence and presence of 2 μm 2-APB or 10 μm xestospongin C (incubation 2 h) using a perforated patch recording method. C, summary of the inhibitory effects of heparin, 2 μm 2-APB or 10 μm xestospongin C. Each IP₃ receptor blocking agent significantly inhibited PE-induced stimulation of I_Ca,L. Numbers in parentheses indicate the number of cells tested in each experiment. *P < 0.05.

Figure 4. Effects of PE to stimulate NO_i release in atrial myocytes

A; PE (10 μm) is unable to stimulate NO_i release in a quiescent atrial myocyte. Exposure to 300 μm spermine/NO (an NO donor) increases NO_i. B; PE (10 μm) stimulates NO_i release in an atrial myocyte field stimulated at 1 Hz. C; PE (1, 10, 100 μm) elicits a dose-dependent increase in NO_i release. D; summary of dose-dependent PE-induced stimulation of NO_i. The numbers in parentheses indicate the number of cells tested in each experiment.

Figure 5. Pharmacological analysis of the signalling mechanisms responsible for PE-induced stimulation of NO_i release

Each experiment was performed by testing PE in the absence (control) and presence (test) of each drug on myocytes isolated from the same hearts. The control values from each experiment (n = 23) are grouped together for clarity. Compared with control (open bar) prazocin (1 μm), l-NIO (10 μm), W-7 (10 μm) and LY294002 (10 μm) each significantly inhibited PE-induced stimulation of NO_i release. H-89 (2 μm) blocked approximately 50% of PE-induced NO_i production. Incubation of cells in PTX had no effect on PE-induced NO_i production. The numbers in parentheses indicate the number of cells tested in each experiment. *P < 0.05.

Figure 6. PE acts via PI-3K-dependent signalling to phosphorylate Akt

The Western blots show phosphorylated (pAkt; Ser⁴⁷³) (upper) and total (lower) Akt in the absence and presence of 10 μm LY294002. Compared with control 10 μm PE significantly increased phosphorylation of Akt. Prior exposure to 10 μm LY294002 blocked PE-induced Akt phosphorylation. The graph summarizes the normalized data obtained in 9 separate experiments. *P < 0.05.

Figure 7. Inhibition of IP₃ receptor signalling or SR Ca²⁺ release inhibits PE-induced stimulation of NO_I

A and B, compared to control (cntrl) PE (10 μm)-induced stimulation of NO_i release was blocked by 2 μm 2-APB (A) and markedly inhibited by 2 h incubation in 10 μm xestospongin C (+ Xesto C) (B). C, prior exposure to 10 μm ryanodine blocked PE-induced stimulation of NO_i release. D, summary of the effects of 2-APB, xestospongin C, and ryanodine compared to control (filled bar) effects of PE to increase NO_i. The numbers in parentheses indicate that number of cells tested in each experiment. *P < 0.01; †P < 0.05.

Figure 8. 2-Dimensional surface plots obtained from atrial myocytes showing the spatial patterns of NO_i production induced by 10 μm PE

A, compared with control, after 3 min of exposure to PE, NO_i production increased at discrete sites primarily along the cell periphery. At 5 min of PE exposure NO_i production along the cell periphery was further increased at additional sites along the cell periphery with smaller increases of NO_i within the cell interior. B, another atrial myocyte was incubated (1 h) in 2 mm methyl-β-cyclodextrin (cyclodextrin) to disrupt caveolae formation. Exposure to 10 μm PE failed to increase NO_i.

Figure 9. Confocal laser linescan images of local SR Ca²⁺ release, i.e. Ca²⁺ sparks, recorded from quiescent atrial myocytes

Each panel shows confocal line scan images of spontaneous subsarcolemmal Ca²⁺ spark activity. The traces below each panel show local subcellular changes in [Ca²⁺]_i within the subsarcolemmal space that correspond with the arrows at the left margin of each panel. A, compared with control (a), PE (10 μm) increased Ca²⁺ spark activity (b). B, compared with control (a), 2 μm 2-APB had little effect on Ca²⁺ spark activity (b) but blocked PE-induced increases in Ca²⁺ sparks (c). C, normalized Ca²⁺ spark frequency (sparks s⁻¹ (100 μm)⁻¹); compared with control (Ctrl) PE significantly increased Ca²⁺ spark frequency (170%; n = 6). D, compared with control (Crtl), 2-APB blocked the effect of PE to increase Ca²⁺ spark frequency (n = 9). *P < 0.05.

Figure 10. Electron micrographs of an atrial myocyte cut parallel with the longitudinal axis of the cell

A, micrograph shows sarcomere units (Z lines) and an extensive network of longitudinal sarcoplasmic reticulum (lsr) throughout the cell interior. B, higher magnification of inset in A shows that the longitudinal sarcoplasmic reticulum is connected to electron dense regions at the cell periphery which are terminal SR (tsr) Ca²⁺ release sites. Between each terminal SR Ca²⁺ release site are grape-like clusters of caveolae (Cav). Calibration bars = 1.0 μm.

Figure 11. Proposed signalling mechanisms underlying PE-induced stimulation of I_Ca,L via NO signalling

PE acts via α₁-ARs coupled to G_q, presumably localized within caveolae, to activate both PLC – IP₃ and PI-3K/Akt signalling. IP₃ stimulates SR Ca²⁺ release via IP₃Rs. IP₃-mediated Ca²⁺ signalling enhances local SR Ca²⁺ release triggered by Ca²⁺ influx via I_Ca,L. In conjunction with receptor-mediated stimulation of PI-3K/Akt signalling, SR Ca²⁺ release activates CaM-dependent eNOS to stimulate NO_i production. NO acts via cGMP-induced inhibition of PDE III to stimulate endogenous cAMP-dependent PKA activity. cAMP/PKA stimulates Ca²⁺ influx via I_Ca,L which in turn enhances Ca²⁺-mediated stimulation of eNOS and NO_i production. Steady-state stimulation of NO_i production may be determined by the dose-dependent receptor-mediated stimulation of PI-3K/Akt/eNOS signalling.