Microsomal fractions were prepared from canine and bovine airway smooth muscle (ASM) by differential and gradient centrifugations. Surface membrane vesicles were characterized by binding assays and incorporated into planar lipid bilayers. Single-channel activities were recorded in symmetric or asymmetric K+ buffer systems and studied under voltage and Ca2+ clamp conditions. A large-conductance K(+)-selective channel (greater than 220 pS in 150 mM K+) displaying a high Ca2+, low Ba2+, and charybdotoxin (CTX) sensitivity was identified. Time analysis of single-channel recordings revealed a complex kinetic behavior compatible with the previous schemes proposed for Ca(2+)-activated K+ channels in a variety of biological surface membranes. We now report that the open probability of the channel at low Ca2+ concentration is enhanced on in vitro phosphorylation, which is mediated via an adenosine 3',5'-cyclic monophosphate-dependent protein kinase. In addition to this characterization at the molecular level, a second series of pharmacological experiments were designed to assess the putative role of this channel in ASM strips. Our results show that 50 nM CTX, a specific inhibitor of the large conducting Ca(2+)-dependent K+ channel, prevents norepinephrine transient relaxation on carbamylcholine-precontracted ASM strips. It was also shown that CTX reversed the steady-state relaxation induced by vasoactive intestinal peptide and partially antagonized further relaxation induced by cumulative doses of this potent bronchodilatator. Thus it is proposed that the Ca(2+)-activated K+ channels have a physiological role because they are indirectly activated on stimulation of various membrane receptors via intracellular mechanisms.

1 The effects of rat and human x-calcitonin gene-related peptide (a-CGRP) were investigated in isolated smooth muscle preparations obtained from three levels of the rat respiratory tract.2 Neither peptide (10--10-16M) had any effect on resting tension or on carbamylcholine (10-6M)-induced tone of trachea or main bronchus. In contrast, CGRP sometimes reduced spontaneous or carbamylcholine-induced tone of lung parenchymal strips. 3 CGRP produced a significant rightward shift of the log concentration-response curves to carbamylcholine and 5-hydroxytryptamine (5-HT) in the main bronchus. A rightward shift was also seen in trachea and parenchymal strips but this did not achieve the level of significance. The maximal response to 5-HT was reduced in the main bronchus and lung parenchyma whereas the maximal contraction to carbamylcholine was decreased in parenchymal strip only. 4 In all three airway preparations, CGRP caused concentration-dependent inhibition of responses elicited by challenges with 10-7M carbamylcholine or 5 x 10-7M 5-HT. The inhibitory effect of the peptide was inversely related to the size of the airways: the smaller the calibre, the greater the inhibition. 5 The inhibitory action of CGRP was not modified by pretreatment with tetrodotoxin (10-6M), propranolol (10-6M) or indomethacin (10-6 M). 6 The results strongly suggest that (a) CGRP has a nonspecific inhibitory action on airway smooth muscle cells, (b) CGRP may act as a potent inhibitor of responses elicited by bronchoconstrictor substances and (c) its inhibitory activity may be most powerfully expressed in peripheral regions of the respiratory tract.

Calcitonin gene-related peptide (CGRP), a neuropeptide released from sensory nerves during axonal reflexes, has strong bronchoprotector properties in rat isolated airways. In this study, we examined this ability of CGRP to prevent agonist-induced contraction in guinea pig and human airways and determined whether inflammatory reaction affects its function. CGRP administered intravenously (0.38 to 114 microgram/kg) in anesthesized guinea pig had no effect per se on airway resistance but caused a dose-related inhibition of substance P (SP; 13.5 microgram/kg)-induced bronchoconstriction (60% at 114 microgram/kg). Similarly, CGRP (10(-)9 to 10(-)6 M) prevented in a concentration-dependent manner the contraction elicited by SP (5 x 10(-)8 M) in guinea pig isolated main bronchi and parenchymal strips, the inhibition caused by CGRP being more pronounced in distal than in proximal airways (47 and 20%, respectively, at 10(-)6 M). The breaking effect of CGRP on SP-induced constriction was however significantly reduced (p < 0.05) in guinea pig actively sensitized to ovalbumin (OA) and the loss in its potency was of similar magnitude (> 40%) whether it was administered in vivo or in vitro. A same phenomenon was observed in human isolated peripheral bronchi. CGRP (10(-)6 M) reduced by more than 75% the extent of the contraction evoked by 10(-)6 M of carbamylcholine and its protector effect was totally abolished in bronchi showing clear morphological manifestation of inflammatory reaction. It is concluded that CGRP acts as a potent bronchoprotector agent on both guinea pig and human airways but its ability to limit the extent of airway responsiveness is strongly impaired in inflammatory conditions.

The effects of an intravenous injection of Sephadex beads on lung eosinophil infiltration and eosinophil peroxydase activity and its relationship to bronchial hyperresponsiveness was examined in guinea pigs. This Sephadex beads injection led to blood, lung and airway eosinophilia in association with bronchial hyperresponsiveness. Histologic examination of the lower bronchus indicated that the eosinophil number increased markedly in the mucosa and submucosa. In addition, the eosinophils surrounding the bronchioles 1 day after the Sephadex injection migrated further in airway submucosa and mucosa 7 and 14 days after. Moreover, the bronchial hyperresponsiveness is observed without histologic evidence of airway epithelium damage. Therefore, the bronchial hyperresponsiveness seems to be more related to the eosinophil infiltration in the airway epithelium and possibly eosinophil activation rather than to the eosinophil number recovered in the BAL fluid. We conclude that the maintenance of hyperresponsiveness state could be associated with the persistence of blood and airway eosinophilia.