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Purpose A fundamental feature of asthma is an enhanced contractile response to stimuli. Given the physiological interplay between airway epithelia and smooth muscle cells, cell culture model systems are of limited utility in defining functionally relevant mechanisms impacting airway contraction and relaxation. We have established a lung explant culture system that retains contractile characteristics of intact airways. Methods Tracheal rings (˜ 4 mm) were isolated from Sprague-Dawley rats. Viability of explants was confirmed by microscopic observation of ciliary beating prior to treatment and by contractile response to 90 mM KCl after treatment. Explants were contracted using 2.5 μM methacholine (MCh) followed by relaxation using 100 μM isoproterenol (Iso) or 1 mM 8-Br-cAMP. Airway responsiveness was assessed by measuring the cross sectional area of the lumen using time-lapse video microscopy. Results Explants from 6 animals were analyzed and the cross sectional area expressed as percent reduction in the area at baseline. Mean (SD) sustained contractility of all explants following MCh was 21(11)% (n = 47). Subsequent addition of Iso and 8-Br-cAMP significantly reversed contraction by 14% (n = 21) and 52% (n = 15), respectively. 8-Br-cAMP induced relaxation was significantly better than that by Iso. Repetitive treatment with Iso at 30-minute intervals following Mch-induced contractility demonstrated tolerance to the relaxing effect of Iso. Conclusion Ex vivo cultures of rat airways can be utilized as a model to study mechanisms for relaxing hyperresponsive airways. This model provides an intact airway system that can be used to study interaction between airway smooth muscle and epithelial cells to better understand airway disease.

In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

We have previously shown that the hamster sperm acrosome reaction, an event essential for fertilization, is stimulated by beta-adrenergic agonists and inhibited by beta-adrenergic antagonists. In the present report, we describe attempts to use (+/-)-9-aminoacridylpropranolol (9-AAP), a fluorescent derivative of a potent beta-adrenergic antagonist, to microscopically detect beta-adrenergic receptors on cauda epididymal hamster spermatozoa in vitro. 9-AAP binding to washed hamster spermatozoa was localized primarily in the acrosomal region, but we were unable to consistently displace the 9-AAP with the biologically active (-)-stereoisomers of several beta-adrenertic agonists and antagonists. Such displacement is necessary in order to separate binding to receptors from "nonspecific binding." Thus we did not detect sperm beta-adrenertic receptors by this method. Failure to detect the receptors with 9-AAP may be due to their presence in numbers too low for detection by this compound or to the masking of the receptors in these uncapacitated sperm. However, we could displace 9-AAP with either 5.0 mM NH4Cl or 1.2 microgram/ml nigericin, compounds capable of discharging pH gradients across cell membranes. These compounds have also been previously reported to displace the fluorescent portion of 9-AAP, 9-aminoacridine from the acrosome by such mechanism. The present results suggest that 9-AAP fluorescence does not always represent binding to beta-adrenergic receptors or "nonspecific binding," but may also represent the concentration of 9-AAP in acidic compartments within a cell.