Decreased adenylyl cyclase protein and function in airway smooth muscle by chronic carbachol pretreatment

Abstract

Cellular levels of cAMP are an important determinant of airway smooth muscle tone. We have previously shown that chronic (18 h) but not acute (30 min or 2 h) pretreatment with the muscarinic receptor agonist carbachol resulted in decreased adenylyl cyclase activity in response to GTP, isoproterenol, or forskolin via a pathway blocked by the protein kinase C inhibitor staurosporine. The present study was designed to determine if carbachol-induced decreases in adenylyl cyclase activity were due to regulatory events at the level of either G(s)alpha or adenylyl cyclase. Detergent-solubilized G(s)alpha from control or carbachol-pretreated bovine airway smooth muscle had similar adenylyl cyclase activity in response to either NaF or guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) when reconstituted into S49 cyc(-) membranes that lack endogenous G(s)alpha (carbachol pretreated: GTPgammaS, 93 +/- 13% of control; NaF/AlCl(3), 99 +/- 8.6% of control; n = 4). Exogenous G(s)alpha solubilized from red blood cells failed to restore normal adenylyl cyclase activity when reconstituted into carbachol-pretreated bovine airway smooth muscle (carbachol pretreated: GTP, 36 +/- 10% of control; NaF/AlCl(3), 54 +/- 11% of control; n = 4). [(3)H]forskolin radioligand saturation binding assays revealed a decreased quantity of total adenylyl cyclase protein after carbachol pretreatment (maximal binding: 152 +/- 40 and 107 +/- 31 fmol/mg protein in control and carbachol-pretreated airway smooth muscle, respectively). These results suggest that chronic activation of muscarinic receptors downregulates the expression of adenylyl cyclase protein in bovine airway smooth muscle.

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“…Chronic treatment of canine ASM cultures with carbachol reduced basal and agonist-stimulated AC activity, an effect that was reversed by PKC inhibition [ 51 ]. Similar results were obtained in studies of bovine ASM [ 50 ]. In contrast, chronic treatment of human ASM cultures with carbachol (as well as numerous other agonists of Gi-coupled receptors) promoted AC sensitization but in a PKC-insensitive, pertussis-toxin sensitive manner [ 46 ].…”

“…However, a growing appreciation that GPCR signaling may be highly compartmentalized [ 17 ] suggests that even small changes in Gα subtype expression may regulate GPCR signaling. Consistent with this notion are observations that exposure of lung [ 110 - 112 ], ASM strips ex vivo [ 113 , 114 ], or ASM cultures [ 50 ] to various agents can elicit a loss of β 2 AR function that is associated with increased expression of specific Gαi isoforms or decreased expression of Gαs.…”

Selective coupling of type 6 adenylyl cyclase with type 2 IP3 receptors mediates direct sensitization of IP3 receptors by cAMP

“…Cells were lysed (Polytron, Inc.), and the lysates were used for equilibrium competition binding assays. Incubations, in a fi nal volume of 500 μ l, included 3 H-IP 3 (1.5 nM, 21 Ci/mmol; PerkinElmer), 3 H-DHA (0.165 nM, 97 Ci/mmol; GE Healthcare), or 3 H-FK (10 nM, 27 Ci/mmol; PerkinElmer; with 100 μ M p[NH]ppG, 10 μ M isoproterenol, and 40 μ M cytochalasin; Emala et al, 2000 ); each with appropriate concentrations of unlabeled competing ligand. After equilibrium had been attained (5 min on ice for 3 H-IP 3 , 90 min at 25 ° C with shaking for 3 H-DHA, and 10 min at 25 ° C for 3 H-FK), incubations were terminated by centrifugation (20,000 g for 5 min at 4 ° C) and the pellet was washed with cold buffer, then resuspended in 200 μ l of buffer for liquid scintillation counting.…”

Beta-1 selective adrenergic antagonist landiolol and esmolol can be safely used in patients with airway hyperreactivity

“…The reason for this is not known at present; however, the beta-2 adrenoceptor in airway smooth muscles could have been modified as well as the ACh receptor in this asthmatic model. 33,34 On the basis of the apparent safety of esmolol and landiolol in the animal study, we next chose to assess their safety in human patients with hyperreactive airways.…”