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

1. The spasmolytic and anti-spasmogenic activity of beta -adrenoceptor agon ists on airways smooth muscle is thought to involve activation of the cycli c AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have teste d the hypothesis that PKA mediates the anti-spasmogenic activity of isopren aline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as com petitive 'antagonists' of PKA. 2 Anion-exchange chromatography of guinea-pig tracheae resolved two peaks o f PKA activity that corresponded to the type I (similar to 5%) and type II (similar to 93%) isoenzymes. 3 Pre-treatment of tracheae with zardaverine (30 muM), vasoactive intestina l peptide (VIP) (1 muM) and the non-selective activator of PKA, Sp-8-CPT-cA MPS (10 muM). produced a non-parallel rightwards shift in the concentration -response curves that described acetylcholine (ACh)-induced tension generat ion. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 muM), abolish ed this effect. 4 Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 muM) produced a non- parallel rightwards shift of the concentration-response curves that describ ed ACh-induced tension generation. The selective cyclic GMP-dependent prote in kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 muM), abolished this effect . 5 Pre-treatment of tracheae with isoprenaline (1 muM) produced a 10 fold sh ift to the right of the ACh concentration-response curve by a mechanism tha t was unaffected by Rp-8-Br-cAMPS (300 muM, selective inhibitor of type I P KA), Rp-8-CPT-cAMPS (300 pm) and Rp-8-pCPT-cGMPS (300 muM). 6 We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardave rine and VIP in guinea-pig trachea is attributable to activation of the cyc lic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contraction s by a mechanism(s) that is independent of PKA and PKG.