Impaired alveolar liquid clearance after 48-h isoproterenol infusion spontaneously recovers by 96 h of continuous infusion

We previously demonstrated that 48-h isoproterenol (Iso) infusion in rats impaired the ability of β-adrenoceptor (β-AR) agonists to increase alveolar liquid clearance (ALC). In this study, we determined whether this impairment persisted over longer time periods by infusing 400 μg·kg−1·h−1 Iso by osmotic minipump for 24–144 h ( n = 6–7/group). ALC in control rats was 19.0 ± 2.4 (SD)% of instilled volume absorbed per hour. In Iso-infused rats, ALC was elevated at 24 h (34.9 ± 2.4%) and decreased at 48 h (15.2 ± 4.4%) and had recovered to 24 h values at 96 h (37.3 ± 3.8%) and 144 h (35.2 ± 3.3%). Plasma Iso concentrations remained elevated at all Iso infusion times. Peripheral lung β2-AR expression exhibited a parallel time course, with a reduction in expression observed at 48 h, followed by an increase to 24 h values at 96 and 144 h. Propranolol prevented the increase in ALC observed at 96 and 144 h, indicating that the recovery in ALC was mediated by a recovery of β-AR function and β-AR signaling. ALC at 96 and 144 h could not be further increased by terbutaline, indicating that ALC was maximally stimulated. These data indicate that recovery of β-AR-stimulated ALC can occur in the continued presence of Iso and is mediated by a recovery of the ability of the distal lung epithelium to respond to β-AR stimulation.

PKA delivery to the distal lung air spaces increases alveolar liquid clearance after isoproterenol-induced alveolar epithelial PKA desensitization

Isoproterenol (Iso) infusion for 48 h in rats decreases the ability of β-adrenoceptor (β-AR) agonists to increase alveolar liquid clearance (ALC). An impairment in protein kinase A (PKA) function appears to be critical in producing the desensitized ALC response. To test this hypothesis, we used a novel protein delivery reagent (Chariot, Active Motif) to deliver either the PKA catalytic subunit or the PKA holoenzyme to the distal lung epithelium of Iso-infused rats (400 μg·kg−1·h−1, 48 h). After this infusion, ALC was measured by mass balance over 2 h. ALC in Iso-infused rats was 27.9% (SD 5.8) of instilled volume absorbed. Delivery of the catalytic PKA subunit to Iso-infused rats increased ALC to 47.7% (SD 8.9) ( P < 0.05). ALC in Iso-infused rats delivered the inactive PKA holoenzyme [29.6% (SD 2.5)] was not increased above baseline values. Subsequent holoenzyme activation by intravenous infusion of the stable cAMP analog Sp-8-Bromo-cAMPS increased ALC to 41.7% (SD 8.8) ( P < 0.05). Immunohistochemical localization of Chariot-delivered PKA revealed staining in the alveolar and distal airway epithelium. These data indicate that protein delivery reagents can be used to rapidly deliver biologically active proteins to the distal lung epithelium and that PKA desensitization may be an important rate-limiting event in the development of Iso-induced desensitization of the alveolar epithelial β-AR signaling pathway.

Postreceptor defects in alveolar epithelial β-adrenergic signaling after prolonged isoproterenol infusion

We previously found that prolonged isoproterenol (Iso) infusion in rats impaired the ability of β-adrenoceptor (β-AR) agonists to increase alveolar liquid clearance (ALC). Here, we determined if postreceptor defects in β-AR signaling contribute to this impairment. Iso was infused using subcutaneous miniosmotic pumps (4, 40, or 400 μg · kg-1 · h-1) in rats for 48 h. At this time, forskolin-stimulated ALC was measured by mass balance. Forskolin-stimulated ALC [33.4 ± 2.1%/h (mean ± SE) in vehicle-infused rats] was reduced by 25 and 38%, respectively, after the 40 and 400 μg · kg-1 · h-1 Iso infusions. The ability of forskolin to increase cAMP was reduced by 70% in alveolar type II (ATII) cells isolated from rats infused with 400 μg · kg-1 · h-1 Iso. Additionally, the ability of the stable cAMP analog 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer, to increase ALC (48.7 ± 3.0% in vehicle-infused rats) was reduced by 25 and 51%, respectively, after the 40 and 400 μg · kg-1 · h-1 infusions. Finally, the ability of cAMP to increase protein kinase A activity was eliminated in ATII cells isolated from rats infused with Iso at 400 μg · kg-1 · h-1. These data demonstrate that prolonged β-AR agonist exposure can impair alveolar epithelial β-AR signaling downstream of the β-AR.