A novel function of thyrotropin as a potentiator of electrolyte secretion from the tracheal gland.

Accumulating evidence suggests that thyrotropin (thyroid-stimulating hormone [TSH]) plays some roles in immunoregulation by an extrathyroidal action. Because airway submucosal glands are responsible for nonspecific and specific airway defense, we tested the effect of TSH on feline tracheal submucosal gland using a whole-cell patch-clamp technique, immunohistochemistry, and reverse transcription/polymerase chain reaction (RT-PCR). TSH potentiated neurotransmitter-induced ionic currents significantly in a dose-dependent manner. Acetylcholine (10(-)(8) M)- and norepinephrine (10(-)(7) M)-induced inward current (I(i)), which we previously showed to be a Cl(-) current, were increased to about 3-fold the pre-TSH control responses, respectively, by 2.0 ng/ml TSH; and to 6- and 23-fold the control values by 20.0 ng/ml TSH, respectively. TSH alone was without effect up to 20.0 ng/ml. Follicular stimulating hormone only slightly affected the I(i) (1. 5-fold the control). Analyses with immunohistochemistry and RT-PCR failed to identify TSH receptors on the glandular tissue. Maneuvers to raise the cellular adenosine 3',5'-cyclic monophosphate also failed to mimic the TSH-mediated potentiation. The TSH effect appeared to be mediated by a signaling pathway involving tyrosine kinase because its inhibitors (genistein and herbimycin A) abolished the augmentation completely, and interferon-gamma, a tyrosine kinase activator, imitated the TSH action on submucosal gland. Thus, TSH may be an important regulator of airway fluid secretion.

Cholinomimetic action of macrolide antibiotics on airway gland electrolyte secretion.

We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl- currents were reduced to 54 and 83% and the outward K+ currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+ channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl- secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.

Effects of histamine and endothelin-1 on membrane potentials and ion currents in bovine tracheal smooth-muscle cells.

We tested the effects of tetraethylammonium (TEA), acetylcholine (ACh), histamine, and endothelin-1 on single airway smooth-muscle cells from bovine trachea, using the patch-clamp technique. Resting membrane potential was -48 +/- 1 mV (n = 47). Both TEA and ACh significantly depolarized the membrane, by +28 +/- 4 mV (P < 0.001, n = 12) and +21 +/- 2 mV (P < 0.01, n = 7), respectively, in the whole-cell configuration. In contrast, both histamine and endothelin-1 hyperpolarized the membrane, by -21 +/- 6 mV (P < 0.01, n = 8) and -15 +/- 2 mV (P < 0.01, n = 8), respectively. Calcium-dependent large-conductance K+-channels (127 pS) and small-conductance K+ channels (21 pS) were identified in excised patches. The small-conductance K+ channel was inhibited by 4-aminopyridine and activated by both histamine and endothelin-1. Furthermore, TEA did not alter the membrane hyperpolarization by these agonists, suggesting that the small-conductance K+ channel or delayed-rectifier K+ channel was involved in the membrane hyperpolarization. Membrane hyperpolarization by histamine and endothelin-1 suggests that activation of voltage-dependent calcium channels (VDCCs) or of calcium influx does not contribute substantially to the contractile response of airway smooth-muscle contraction to these agonists.

HMT regulates histamine-induced glycoconjugate secretion from human airways in vitro.

To determine whether histamine N-methyltransferase (HMT) regulates mucus glycoprotein (MGP) secretion from airways, we examined the effect of an HMT inhibitor, SKF 91488, on MGP secretion from human airways in vitro. MGP secretion from human airway explants (with epithelium) and isolated submucosal glands was estimated by measuring trichloroacetic acid (TCA) precipitable glycoconjugates using secretory indices. Histamine induced significant MGP secretion from both explants and isolated glands. Pretreatment with SKF 91488 significantly inhibited histamine-induced secretion from explants, while it did not alter significantly the secretion from isolated glands. H1-blocker significantly reversed the inhibition by SKF 91488 of the secretion from explants, while H2-blocker abolished histamine-induced secretion from both explants and isolated glands. Prostaglandin E2 (PGE2) significantly inhibited histamine-induced secretion from isolated glands. The inhibitory action of SKF 91488 in airway explants was blocked by indomethacin and was significantly reduced by a prostanoid EP4 receptor antagonist (AH23848B). These findings suggest that HMT regulates MGP secretion from human airway submucosal glands through an interaction with epithelial cells which involves the release of PGE2.

Signature current of SO2-induced bronchitis in rabbit.

To investigate abnormalities of airway epithelial ion transport underlying chronic inflammatory airway diseases, we performed electrophysiological, histological, and molecular biological experiments using rabbits exposed to SO2 as a model of bronchitis. By comparison with control, the SO2-exposed trachea exhibited decreased short circuit current (Isc) and conductance associated with increased potential difference. In normal trachea, apical ATP induced a transient Isc activation followed by a suppression, whereas the bronchitis model exhibited a prolonged activation without suppression. This pathological ATP response was abolished by diphenylamine 2-carboxylate or Cl--free bath solution. A significant increase in net Cl- flux toward the lumen was observed after ATP in our bronchitis model. Isoproterenol or adenosine evoked a sustained Isc increase in SO2-exposed, but not in normal, tracheas. The Northern blot analysis showed a strong expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in SO2-exposed epithelium. The immunohistochemical study revealed a positive label of CFTR on cells located luminally only in SO2-exposed rabbits. We concluded that the prolonged ATP response in our bronchitis model was of a superimposed normal and adenosine-activated current. The latter current was also activated by isoproterenol and appeared as a signature current for the bronchitis model airway. This was likely mediated by CFTR expressed in the course of chronic inflammation.

Bradykinin regulation of airway submucosal gland secretion: role of bradykinin receptor subtype.

To clarify the role of bradykinin receptor subtypes, we examined the effect of bradykinin on feline tracheal and human airway submucosal gland secretion using an isolated gland preparation. Bradykinin induced a significant increase in [3H]glycoconjugate secretion in a dose-dependent manner from isolated glands, which was significantly inhibited by D-Arg-(Hyp3, Thi5,8, D-Phe7)-bradykinin (the B2-receptor antagonist), whereas Des-Arg9-(Leu8)-bradykinin (B1-receptor antagonist) or indomethacin did not significantly alter it. Nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) caused a significant inhibition of bradykinin-induced glycoconjugate secretion, which was reversed by the addition of L-arginine. Bradykinin evoked bidirectional current responses, and an initial inward current (Cl- current) was followed by an outward current (K+ current) of the acinar cells in a whole cell configuration by patch-clamp technique. Bradykinin induced an immediate increase in intracellular calcium concentration ([Ca2+]i) of the acinar cells followed by a prolonged plateau, and Ca2+ removal resulted in an initial increase alone. [Ca2+]i rise was significantly inhibited by the B2-receptor antagonist, whereas the B1-receptor antagonist did not significantly alter it. These findings suggest that B2-receptor stimulation and the resultant [Ca2+]i rise induced both mucus glycoprotein and electrolyte secretions, involving NO formation in airway submucosal gland cells.

Nitric oxide regulation of glycoconjugate secretion from feline and human airways in vitro.

To determine whether nitric oxide (NO) regulates mucus secretion from airway submucosal glands which are the main source of human airway secretion, we examined the effects of NO synthase inhibitors (L-NAME and L-NMMA) on mucus glycoprotein (MGP) secretion from feline and human airway explants (with epithelium) and isolated submucosal glands. MGP secretion was estimated by measuring trichloroacetic-acid (TCA) precipitable [H3]-glycoconjugates using secretory indices. NO synthase inhibitors alone did not alter significantly MGP secretion from explants or isolated glands. Pretreatment with NO synthase inhibitors significantly inhibited both methacholine (MCh) and bradykinin (BK)-induced secretion from isolated glands, but not significantly inhibit the secretion from explants. The inhibition by L-NAME was reversed by the addition of L-arginine in both MCh- and BK-induced secretions from isolated glands. Further, a NO generator isosorbide dinitrate induced a significant increase in the secretion. These findings suggest that endogenous NO has a stimulatory action in airway submucosal gland secretion and directly regulates the secretion from submucosal glands independently of superficial epithelial cells.