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Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes.

Wang YG, Dedkova EN, Steinberg SF, Blatter LA, Lipsius SL - J. Gen. Physiol. (2002)

Bottom Line: Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i).We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling.The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Stritch School of Medicine, Department of Physiology, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA.

ABSTRACT
In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.

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Current-voltage relationships showing the effects of 0.1 μM ISO (A–C) and 1 μM zinterol (D) on ACh2-induced K+ conductance and ICa,L (insets). (A) ISO increased ICa,L and mediated a potentiated increase in ACh2-induced K+ conductance compared with ACh1. (B) In the presence of 0.01 μM ICI 118,551, a β2-AR antagonist, ISO increased ICa,L, but failed to potentiate ACh2-induced K+ conductance. (C) In the presence of 0.01 μM atenolol, a β1-AR antagonist, ISO increased ICa,L and potentiated ACh2-induced K+ conductance. (D) Zinterol increased ICa,L and potentiated ACh2-induced K+ conductance. c; control K+ conductance before ACh1. r; recovery after washout of ACh2. (insets) ICa,L calibration bars indicate 250 pA, 100 ms.
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Figure 1: Current-voltage relationships showing the effects of 0.1 μM ISO (A–C) and 1 μM zinterol (D) on ACh2-induced K+ conductance and ICa,L (insets). (A) ISO increased ICa,L and mediated a potentiated increase in ACh2-induced K+ conductance compared with ACh1. (B) In the presence of 0.01 μM ICI 118,551, a β2-AR antagonist, ISO increased ICa,L, but failed to potentiate ACh2-induced K+ conductance. (C) In the presence of 0.01 μM atenolol, a β1-AR antagonist, ISO increased ICa,L and potentiated ACh2-induced K+ conductance. (D) Zinterol increased ICa,L and potentiated ACh2-induced K+ conductance. c; control K+ conductance before ACh1. r; recovery after washout of ACh2. (insets) ICa,L calibration bars indicate 250 pA, 100 ms.

Mentions: Atrial myocytes were dispersed from adult cat atria using Langendorff perfusion and collagenase (type II; Worthington Biochemical) digestion as previously reported (Wu et al. 1991). No discernible differences were noted between left and right atrial myocytes. Cells used for electrophysiological studies were transferred to a small tissue bath (0.3 ml) on the stage of an inverted microscope (Nikon Diaphot) and superfused with a HEPES-buffered modified Tyrode solution containing the following (in mM): 145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 5 HEPES, and 11 glucose, and titrated with NaOH to a pH of 7.4. Solutions were perfused by gravity and heated to 35 ± 1°C. Atrial myocytes selected for study were elongated and quiescent. Voltage and ionic currents were recorded using a nystatin (150 μg/ml)-perforated patch (Horn and Marty 1988) whole-cell recording method (Hamill et al. 1981). This method minimizes dialysis of intracellular constituents with the internal pipette solution, and thereby preserves physiological milieu and second messenger signaling pathways. The internal pipette solution contained the following (in mM): 100 potassium glutamate, 40 KCl, 1.0 MgCl2, 4 Na2-ATP, 0.5 EGTA, and 5 HEPES, and titrated with KOH to pH 7.2. A single suction pipette was used to record voltage (bridge mode) or ionic currents (discontinuous voltage-clamp mode) using an Axoclamp 2A amplifier (Axon Instruments, Inc.). Computer software (Pclamp; Axon Instruments, Inc.) was used to deliver voltage protocols, acquire, and analyze data. The effects of ACh on K+ conductance were studied as previously described (Wang and Lipsius 1995). In brief, an atrial cell was treated with two consecutive exposures to ACh (ACh1 and ACh2) separated by a 6-min recovery period in ACh-free Tyrode solution (see Fig. 1 A). Changes in total membrane conductance were assessed by imposing voltage-clamp ramps (40 mV/s) between −130 and +30 mV before, during, and after each ACh exposure. Voltage ramps offer the advantage of a rapid method for measuring peak ACh-induced currents throughout the voltage range. In general, experimental interventions, such as exposure to ISO, zinterol, or spermine-NO were imposed during the recovery period between ACh1 and ACh2. In this way, we determined the effect of each intervention on ACh-induced K+ conductances by comparing the response to ACh2 in relation to ACh1. Measurements of K+ conductance were obtained at −130 and +30 mV. The effects of each ACh exposure on K+ conductance was fully reversible (see Fig. 1 A). Previous work indicates that the control currents do not affect the measurement of relative changes in K+ conductances induced by ACh2 in relation to ACh1 (Wang and Lipsius 1995). Therefore, in this study, ACh-induced K+ currents were measured without subtraction of control currents. Control experiments indicate that an initial 30-s exposure to ACh followed by a 6-min recovery period has no effect on the response to a second ACh exposure (Wang and Lipsius 1995). Therefore, any changes in K+ conductance elicited by ACh2 in relation to ACh1 are attributed to the experimental intervention imposed during the recovery interval. Previous work (Wang and Lipsius 1995) indicates that Ca2+ influx via ICa,L during β-AR stimulation enhances ACh-induced activation of IK,ATP. Therefore, ICa,L was activated during the interval between ACh exposures by depolarizing voltage pulses from a holding potential of −40 to 0 mV for 200 ms every 10 s. In some experiments, ICa,L was studied alone by replacing potassium glutamate with cesium glutamate in the pipette solution and adding 5 mM CsCl to the external solutions to block K+ conductances. In other experiments, ICa,L was recorded using a ruptured patch recording method to dialyze the cell interior with PKA inhibitors. PKA inhibitors were allowed to diffuse into the cell for ∼5 min before recordings were performed. In these experiments, the internal pipette solution contained the following (mM): 100 cesium glutamate, 40 CsCl, 1 MgCl2, 4 NaATP, 0.5 EGTA, 10 HEPES, and titrated with CsOH to pH 7.2. Unless stated otherwise, zinterol was tested in the presence of 0.01–0.1 μM atenolol to ensure β2-AR stimulation. Cells were exposed to receptor antagonists for ∼4 min before exposure to agonists. Inhibition of Gi-protein was achieved by incubating cells in pertussis toxin (PTX; 3.4 μg/ml; ≥3 h; 36°C) and confirmed by inhibition of ACh-activated IK,ACh.


Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes.

Wang YG, Dedkova EN, Steinberg SF, Blatter LA, Lipsius SL - J. Gen. Physiol. (2002)

Current-voltage relationships showing the effects of 0.1 μM ISO (A–C) and 1 μM zinterol (D) on ACh2-induced K+ conductance and ICa,L (insets). (A) ISO increased ICa,L and mediated a potentiated increase in ACh2-induced K+ conductance compared with ACh1. (B) In the presence of 0.01 μM ICI 118,551, a β2-AR antagonist, ISO increased ICa,L, but failed to potentiate ACh2-induced K+ conductance. (C) In the presence of 0.01 μM atenolol, a β1-AR antagonist, ISO increased ICa,L and potentiated ACh2-induced K+ conductance. (D) Zinterol increased ICa,L and potentiated ACh2-induced K+ conductance. c; control K+ conductance before ACh1. r; recovery after washout of ACh2. (insets) ICa,L calibration bars indicate 250 pA, 100 ms.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233856&req=5

Figure 1: Current-voltage relationships showing the effects of 0.1 μM ISO (A–C) and 1 μM zinterol (D) on ACh2-induced K+ conductance and ICa,L (insets). (A) ISO increased ICa,L and mediated a potentiated increase in ACh2-induced K+ conductance compared with ACh1. (B) In the presence of 0.01 μM ICI 118,551, a β2-AR antagonist, ISO increased ICa,L, but failed to potentiate ACh2-induced K+ conductance. (C) In the presence of 0.01 μM atenolol, a β1-AR antagonist, ISO increased ICa,L and potentiated ACh2-induced K+ conductance. (D) Zinterol increased ICa,L and potentiated ACh2-induced K+ conductance. c; control K+ conductance before ACh1. r; recovery after washout of ACh2. (insets) ICa,L calibration bars indicate 250 pA, 100 ms.
Mentions: Atrial myocytes were dispersed from adult cat atria using Langendorff perfusion and collagenase (type II; Worthington Biochemical) digestion as previously reported (Wu et al. 1991). No discernible differences were noted between left and right atrial myocytes. Cells used for electrophysiological studies were transferred to a small tissue bath (0.3 ml) on the stage of an inverted microscope (Nikon Diaphot) and superfused with a HEPES-buffered modified Tyrode solution containing the following (in mM): 145 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 5 HEPES, and 11 glucose, and titrated with NaOH to a pH of 7.4. Solutions were perfused by gravity and heated to 35 ± 1°C. Atrial myocytes selected for study were elongated and quiescent. Voltage and ionic currents were recorded using a nystatin (150 μg/ml)-perforated patch (Horn and Marty 1988) whole-cell recording method (Hamill et al. 1981). This method minimizes dialysis of intracellular constituents with the internal pipette solution, and thereby preserves physiological milieu and second messenger signaling pathways. The internal pipette solution contained the following (in mM): 100 potassium glutamate, 40 KCl, 1.0 MgCl2, 4 Na2-ATP, 0.5 EGTA, and 5 HEPES, and titrated with KOH to pH 7.2. A single suction pipette was used to record voltage (bridge mode) or ionic currents (discontinuous voltage-clamp mode) using an Axoclamp 2A amplifier (Axon Instruments, Inc.). Computer software (Pclamp; Axon Instruments, Inc.) was used to deliver voltage protocols, acquire, and analyze data. The effects of ACh on K+ conductance were studied as previously described (Wang and Lipsius 1995). In brief, an atrial cell was treated with two consecutive exposures to ACh (ACh1 and ACh2) separated by a 6-min recovery period in ACh-free Tyrode solution (see Fig. 1 A). Changes in total membrane conductance were assessed by imposing voltage-clamp ramps (40 mV/s) between −130 and +30 mV before, during, and after each ACh exposure. Voltage ramps offer the advantage of a rapid method for measuring peak ACh-induced currents throughout the voltage range. In general, experimental interventions, such as exposure to ISO, zinterol, or spermine-NO were imposed during the recovery period between ACh1 and ACh2. In this way, we determined the effect of each intervention on ACh-induced K+ conductances by comparing the response to ACh2 in relation to ACh1. Measurements of K+ conductance were obtained at −130 and +30 mV. The effects of each ACh exposure on K+ conductance was fully reversible (see Fig. 1 A). Previous work indicates that the control currents do not affect the measurement of relative changes in K+ conductances induced by ACh2 in relation to ACh1 (Wang and Lipsius 1995). Therefore, in this study, ACh-induced K+ currents were measured without subtraction of control currents. Control experiments indicate that an initial 30-s exposure to ACh followed by a 6-min recovery period has no effect on the response to a second ACh exposure (Wang and Lipsius 1995). Therefore, any changes in K+ conductance elicited by ACh2 in relation to ACh1 are attributed to the experimental intervention imposed during the recovery interval. Previous work (Wang and Lipsius 1995) indicates that Ca2+ influx via ICa,L during β-AR stimulation enhances ACh-induced activation of IK,ATP. Therefore, ICa,L was activated during the interval between ACh exposures by depolarizing voltage pulses from a holding potential of −40 to 0 mV for 200 ms every 10 s. In some experiments, ICa,L was studied alone by replacing potassium glutamate with cesium glutamate in the pipette solution and adding 5 mM CsCl to the external solutions to block K+ conductances. In other experiments, ICa,L was recorded using a ruptured patch recording method to dialyze the cell interior with PKA inhibitors. PKA inhibitors were allowed to diffuse into the cell for ∼5 min before recordings were performed. In these experiments, the internal pipette solution contained the following (mM): 100 cesium glutamate, 40 CsCl, 1 MgCl2, 4 NaATP, 0.5 EGTA, 10 HEPES, and titrated with CsOH to pH 7.2. Unless stated otherwise, zinterol was tested in the presence of 0.01–0.1 μM atenolol to ensure β2-AR stimulation. Cells were exposed to receptor antagonists for ∼4 min before exposure to agonists. Inhibition of Gi-protein was achieved by incubating cells in pertussis toxin (PTX; 3.4 μg/ml; ≥3 h; 36°C) and confirmed by inhibition of ACh-activated IK,ACh.

Bottom Line: Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i).We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling.The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: Stritch School of Medicine, Department of Physiology, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA.

ABSTRACT
In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.

Show MeSH
Related in: MedlinePlus