Role of tyrosine kinase activity in alpha-adrenergic inhibition of the beta-adrenergically regulated L-type Ca(2+) current in guinea-pig ventricular myocytes

Abstract

1. The purpose of this study was to investigate the hypothesis that tyrosine kinase activity contributes to alpha(1)-adrenergic inhibition of beta-adrenergic responses in cardiac myocytes. We addressed this question by studying the pharmacological regulation of the L-type Ca(2+) current in acutely isolated adult guinea-pig ventricular myocytes using the whole-cell patch-clamp technique. 2. The selective alpha(1)-adrenergic receptor agonist methoxamine had no effect on the basal L-type Ca(2+) current. Methoxamine also had no effect on cAMP-dependent stimulation of the Ca(2+) current mediated by H(2) histamine receptor activation. However, methoxamine did inhibit cAMP-dependent stimulation of the Ca(2+) current mediated by beta-adrenergic receptor activation. The ability of methoxamine to inhibit beta-adrenergic regulation of the Ca(2+) current was significantly antagonized by the tyrosine kinase inhibitors genistein and lavendustin A. 3. The inhibitory effect of methoxamine was also mimicked by the phosphotyrosine phosphatase inhibitor pervanadate (PVN). PVN had no effect on basal Ca(2+) current or Ca(2+) current stimulated by histamine, but it did inhibit Ca(2+) current stimulated by beta-adrenergic receptor activation. Furthermore, the ability of PVN to inhibit beta-adrenergic stimulation of the Ca(2+) current was antagonized by lavendustin A. 4. These results are consistent with the conclusion that in guinea-pig ventricular myocytes alpha-adrenergic inhibition of beta-adrenergic responses involves a tyrosine kinase-dependent signalling pathway. The fact that methoxamine and PVN antagonized cAMP-dependent responses mediated by beta-adrenergic, but not H(2) histamine, receptor activation suggests that the inhibitory effect of alpha-adrenergic stimulation and tyrosine kinase activity is at the level of the beta-adrenergic receptor.

Figure 1. The α₁-AR agonist methoxamine selectively inhibits the L-type Ca²⁺ current recorded in the presence of the β-AR agonist isoprenaline (Iso)

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 30 nm Iso and Iso plus 30 μm methoxamine. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, cumulative results of experiments in which cells were exposed to 30 μm methoxamine alone (n = 8; mean basal current amplitude, 902 ± 144 pA) or 30 μm methoxamine following exposure to 30 nm isoprenaline (n = 24; mean basal current amplitude, 816 ± 85.3 pA). Responses were normalized to the magnitude of the baseline current recorded before exposure to any drug(s). For each experiment, the magnitude of the current recorded in the presence of methoxamine was compared to the magnitude of the current recorded in the absence of methoxamine in the same cell. Statistical significance was evaluated by a paired t test.

Figure 7. The phosphotyrosine phosphatase inhibitor pervanadate (PVN) selectively inhibits the L-type Ca²⁺ current recorded in the presence of the β-AR agonist isoprenaline (Iso)

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 30 nm Iso, Iso plus the vehicle for PVN (H₂O₂/catalase), and Iso plus 100 μm PVN. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, cumulative results of experiments in which cells were exposed to 100 μm PVN alone (n = 8; mean basal current amplitude, 830 ± 162 pA) or 100 μm PVN following exposure to 30 nm isoprenaline (n = 13; mean basal current amplitude, 1021 ± 88.84 pA). Responses were normalized to the magnitude of the baseline current recorded before exposure to any drug(s). For each experiment, the magnitude of the current recorded in the presence of PVN was compared to the magnitude of the current recorded in the absence of PVN in the same cell. Statistical significance was evaluated by a paired t test.

Figure 2. The α₁-AR agonist methoxamine decreases the sensitivity of the L-type Ca²⁺ current to β-AR stimulation

At each concentration of isoprenaline, the amplitude of the Ca²⁺ current was measured in the absence (▪) and presence (□) of 30 μm methoxamine. These values are presented as the percentage increase over the magnitude of the basal Ca²⁺ current measured in the same cell in the absence of any drug. The continuous lines represent the best fits of the respective data points to a logistic equation (see Methods). Control: E_max= 246 ± 8.32 %; EC₅₀= 2.5 ± 0.26 nm; n = 2.4± 0.54. Methoxamine: E_max= 243 ± 28.1 %; EC₅₀= 14 ± 5.6 nm; n = 1.1± 0.40. The numbers in parentheses indicate sample population size.

Figure 3. The α₁-AR agonist methoxamine does not inhibit the L-type Ca²⁺ current recorded in the presence of histamine

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 300 nm histamine and histamine plus 30 μm methoxamine. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, cumulative results of experiments in which cells were exposed to 30 μm methoxamine following exposure to 300 nm histamine (n = 12; mean basal current amplitude, 854 ± 138 pA). Responses were normalized to the magnitude of the baseline current recorded before exposure to histamine. For each experiment, the magnitude of the current recorded in the presence of methoxamine was compared to the magnitude of the current recorded in the absence of methoxamine in the same cell. Statistical significance was evaluated by a paired t test.

Figure 4. The α₁-AR agonist methoxamine has no effect on the sensitivity of the L-type Ca²⁺ current to histamine receptor activation

At each concentration of histamine, the amplitude of the Ca²⁺ current was measured in the absence (▪) and presence (□) of 30 μm methoxamine. These values are presented as the percentage increase over the magnitude of the basal Ca²⁺ current measured in the same cell in the absence of any drug. The continuous lines represent the best fits of the respective data points to a logistic equation (see Methods). Control: E_max= 225 ± 8.39 %; EC₅₀= 40 ± 2.6 nm; n = 3.8± 0.71. Methoxamine: E_max= 223 ± 19.3 %; EC₅₀= 38 ± 5.7 nm; n = 4.0± 1.9. The numbers in parenthesis indicate sample population size.

Figure 5. The tyrosine kinase inhibitor genistein attenuates the ability of the α₁-AR agonist methoxamine to inhibit the L-type Ca²⁺ current recorded in the presence of isoprenaline (Iso)

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 30 nm Iso and Iso plus 30 μm methoxamine in the presence of 50 μm genistein. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, magnitude of methoxamine-induced inhibition of Iso response in the absence (n = 24; mean basal current amplitude, 816 ± 85.3 pA) and presence (n = 7; mean basal current amplitude, 401 ± 45.6 pA) of 50 μm genistein. Statistical significance was evaluated by an unpaired t test.

Figure 6. The tyrosine kinase inhibitor lavendustin A attenuates the ability of the α₁-AR agonist methoxamine to inhibit the L-type Ca²⁺ current recorded in the presence of isoprenaline (Iso)

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 10 nm isoprenaline and isoprenaline plus 30 μm methoxamine in the presence of 5 μm lavendustin A. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, magnitude of methoxamine-induced inhibition of Iso response in the absence of any other drug (n = 9; mean basal current amplitude, 817 ± 114 pA), in the presence of 5 μm lavendustin A (n = 13; mean basal current amplitude, 951 ± 94.5 pA), and in the presence of the inactive analogue lavendustin B (n = 11; mean basal current amplitude, 853 ± 105 pA). Statistical significance was evaluated by one-way analysis of variance and a t test with Bonferroni correction.

Figure 8. The phosphotyrosine phosphatase inhibitor pervanadate (PVN) does not inhibit the L-type Ca²⁺ current recorded in the presence of histamine

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 300 nm histamine and histamine plus 100 μm PVN. Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, cumulative results of experiments in which cells were exposed to 100 μm PVN in the presence of 300 nm histamine (n = 6; mean basal current amplitude, 889 ± 140 pA). Responses were normalized to the magnitude of the baseline current recorded before exposure to any drug(s). Statistical significance was evaluated by a paired t test.

Figure 9. Pervanadate-induced increase in tyrosine phosphorylation of proteins in guinea-pig ventricular myocytes

Anti-phosphotyrosine antibody was used to immunoprecipitate (IP) tyrosine-phosphorylated proteins from cell lysates prepared from untreated (control) myocytes and myocytes exposed to 100 μm pervanadate for 5 min (PVN treated). Similar results were obtained in a total of 3 experiments. Detection of tyrosine-phosphorylated proteins in the control lane was minimized due to the brief ECL exposure time, which was used to minimize saturation of signals in the PVN-treated lane.

Figure 10. The tyrosine kinase inhibitor lavendustin A attenuates the ability of the phosphotyrosine phosphatase inhibitor pervanadate (PVN) to inhibit the L-type Ca²⁺ current recorded in the presence of isoprenaline (Iso)

A, representative time course of changes in magnitude of peak inward Ca²⁺ current following exposure to 30 nm Iso and Iso plus 100 μm PVN in the presence of lavendustin A (5 μm external; 0.5 μm internal). Currents were elicited by depolarizing test pulses to 0 mV applied once every 5 s. B, example of current traces recorded at time points indicated in the experiment illustrated in A. C, magnitude of PVN-induced inhibition of Iso response in the absence of any other drug (n = 13; mean basal current amplitude, 1021 ± 88.84 pA), in the presence of lavendustin A (5 μm external; 0.5 μm internal) (n = 10; mean basal current amplitude, 819 ± 50.3 pA), and in the presence of the inactive analogue lavendustin B (5 μm external; 0.5 μm internal) (n = 9; mean basal current amplitude, 940 ± 98.5 pA). Statistical significance was evaluated by one-way analysis of variance and a t test with Bonferroni correction.