1. The spasmolytic and anti-spasmogenic activity of β-adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti-spasmogenic activity of isoprenaline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive 'antagonists' of PKA. 2. Anion-exchange chromatography of guinea-pig tracheae resolved two peaks of PKA activity that corresponded to the type I (∼5%) and type II (∼93%) isoenzymes. 3. Pre-treatment of tracheae with zardaverine (30 μM), vasoactive intestinal peptide (VIP) (1 μM) and the non-selective activator of PKA, Sp-8-CPT-cAMPS (10 μM), produced a non-parallel rightwards shift in the concentration-response curves that described acetylcholine (ACh)-induced tension generation. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 μM), abolished this effect. 4. Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 μM) produced a non-parallel rightwards shift of the concentration-response curves that described ACh-induced tension generation. The selective cyclic GMP-dependent protein kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 μM), abolished this effect. 5. Pre-treatment of tracheae with isoprenaline (1 μM) produced a 10 fold shift to the right of the ACh concentration-response curve by a mechanism that was unaffected by Rp-8-Br-cAMPS (300 μM, selective inhibitor of type I PKA), Rp-8-CPT-cAMPS (300 μM) and Rp-8-pCPT-cGMPS (300 μM). 6. We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardaverine and VIP in guinea-pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contractions by a mechanism(s) that is independent of PKA and PKG.

Evidence that the anti-spasmogenic effect of the β-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase

Spicuzza, Lucia;
2001

Abstract

1. The spasmolytic and anti-spasmogenic activity of β-adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti-spasmogenic activity of isoprenaline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive 'antagonists' of PKA. 2. Anion-exchange chromatography of guinea-pig tracheae resolved two peaks of PKA activity that corresponded to the type I (∼5%) and type II (∼93%) isoenzymes. 3. Pre-treatment of tracheae with zardaverine (30 μM), vasoactive intestinal peptide (VIP) (1 μM) and the non-selective activator of PKA, Sp-8-CPT-cAMPS (10 μM), produced a non-parallel rightwards shift in the concentration-response curves that described acetylcholine (ACh)-induced tension generation. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 μM), abolished this effect. 4. Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 μM) produced a non-parallel rightwards shift of the concentration-response curves that described ACh-induced tension generation. The selective cyclic GMP-dependent protein kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 μM), abolished this effect. 5. Pre-treatment of tracheae with isoprenaline (1 μM) produced a 10 fold shift to the right of the ACh concentration-response curve by a mechanism that was unaffected by Rp-8-Br-cAMPS (300 μM, selective inhibitor of type I PKA), Rp-8-CPT-cAMPS (300 μM) and Rp-8-pCPT-cGMPS (300 μM). 6. We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardaverine and VIP in guinea-pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contractions by a mechanism(s) that is independent of PKA and PKG.
Airways tone; Cyclic AMP; Isoprenaline; Protein kinase A; β-Adrenoceptor agonist; Acetylcholine; Adrenergic beta-Agonists; Animals; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Guinea Pigs; Isoenzymes; Isoproterenol; Muscle Contraction; Muscle Tonus; Muscle, Smooth; Pyridazines; Trachea; Pharmacology
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11769/363590
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