T. Takishima

Endothelin-1 Stimulates Chloride Secretion across Canine Tracheal Epithelium

Although much attention has been paid to the effect of endothelin on the bronchopulmonary system, there are few reports concerning the effect of endothelin on Cl- secretion across airway epithelium. We examined the effects of endothelin-1, -2 and -3 on bioelectric parameters of canine tracheal epithelium, a tissue in which Cl- is actively secreted and Na+ is absorbed. Potential difference (PD) and short-circuit current (SCC) were measured using an Ussing chamber with 0.5 cm2 of exposed area, and conductance (G) was calculated by dividing SCC by PD. Luminal endothelin-1 produced transient increases in PD and SCC, returning to baseline values within 5 min after stimulation in a dose-dependent fashion and reached mean responses of 123 and 126% of baseline PD and SCC, respectively, at 10(-6) M of endothelin-1, whereas submucosal endothelin-1 did not alter PD and SCC. G remained unchanged when stimulated by endothelin-1. Endothelin-2 and -3 did not produce any significant alterations in PD and SCC. Endothelin-1 evoked increases in PD and SCC, which were not altered by pretreatment with luminal amiloride (10(-4) M), or by treatment with submucosal propranolol (10(-5) M). Ionic substitution of Cl- with nontransported anions, iodide and gluconate, inhibited endothelin-1-induced increases in PD and SCC. Pretreatment with 10(-5) M indomethacin partially inhibited Endothelin-1-evoked increases in PD and SCC. These findings indicate that endothelin-1 stimulates Cl- secretion partially through the generation of cyclooxygenase products of arachidonic acid.

Surfactant apoprotein A secretion by human tracheobronchial epithelial cells

To determine whether surfactant apoproteins are produced locally by the airway walls, we used a two-site simultaneous immunoassay with monoclonal antibodies and measured the surfactant apoprotein-A (SP-A) content in media cultured with human airway explants or cultured epithelial cells. Tracheobronchial explants were cultured for 2 successive periods (periods I and II). Significant SP-A concentrations were detected in both periods. Methacholine (MCh) or isoproterenol (ISP), added to the medium at the beginning of period II, reduced the ratio of SP-A concentration in period II to that in period I, compared to samples without treatment. The SP-A concentration in the medium at the confluent period of cultured epithelial cells was significantly higher than at the mid-period, indicating that SP-A secretion is dependent on the cell number. The supernatant from explants stimulated by MCh was capable of reducing SP-A secretion from cultured epithelial cells. Immunohistochemical study of SP-A using monoclonal antibody demonstrated positive immunoperoxidase staining in the cytoplasm of epithelial cells. Further, Western blots of electrophoresed proteins from epithelial cells showed the characteristic properties of SP-A. These findings indicate that tracheobronchial epithelium can secrete SP-A.

A stimulatory role of protein kinase C in feline tracheal submucosal gland secretion.

To determine the role of protein kinase C (PKC) in airway submucosal gland secretion, we examined the effect of a selective PKC stimulant, phorbol 12-myristate 13-acetate (PMA), on mucus glycoprotein (MGP) secretion, fluid secretion and intracellular Ca2+ concentration ([Ca2+]i) in isolated feline submucosal glands. MGP and fluid secretions were estimated by measuring trichloroacetic acid (TCA)-precipitable glycoconjugates and 22Na-efflux, respectively, from isolated glands. [Ca2+]i was measured using a Ca(2+)-sensitive fluorescent dye, Fura 2. PMA itself produced a significant increase in MGP secretion in a dose-dependent fashion (173% of control at 10(-5) M). PMA also produced a significant increase in 22Na-efflux (151% of baseline rate constant at 10(-5) M). Indomethacin failed to alter the increase in MGP secretion or in 22Na-efflux in response to PMA. Two PKC inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) and sphingosine, inhibited both MGP secretion and 22Na-efflux stimulated by PMA; there was only a partial inhibition after stimulation by methacholine (MCh). PMA did not significantly alter [Ca2+]i and H-7 did not alter the MCh-induced [Ca2+]i rise. These findings indicate that PKC has a direct stimulatory role in stimulus-secretion coupling of airway submucosal gland secretion.

HMT regulates histamine-induced Cl- secretion across the canine tracheal epithelium.

Although histamine N-methyltransferase (HMT), the primary enzyme responsible for the inactivation of histamine, has been shown to exist in the airway epithelium, it is still unknown whether this enzyme regulates ion transport across the airway epithelium. Using an Ussing chamber, we examined the effect of a HMT inhibitor, SKF 91488, on potential difference (PD) and short circuit current (SCC) in epithelial membranes from the posterior portion of canine trachea. SKF 91488 itself did not significantly alter PD or SCC values. Pretreatment with SKF 91488 significantly augmented PD and SCC induced by histamine. Amiloride did not significantly alter the augmentation by SKF 91488 in histamine-induced PD and SCC rises. These findings indicate that HMT regulates Cl- secretion across airway epithelium.

Magnesium regulates ion transport across canine tracheal epithelium

We examined the effect of Mg2+ on potential difference (PD) and short circuit current (SCC) of the posterior epithelial membrane of canine trachea using an Ussing chamber. After the exchange to a Mg(2+)-free solution, PD and SCC rapidly increased, reaching maximal values within 3 min, followed by a gradual return towards the baseline over 60 min. In a Ca(2+)-free solution, Mg2+ removal did not alter PD and SCC values. Increased Mg2+ in the solution produced significant gradual decreases in PD and SCC. The decreases in PD and SCC were reversed by the addition of excessive Ca2+ to the solution. Mg2+ removal did not alter significantly isoproterenol-induced increases in PD and SCC values, while increased Mg2+ significantly reduced the increases. These findings indicate that extracellular Mg2+ is an important determinant in ion transport across the airway epithelium, probably through antagonistic actions of Mg2+ and Ca2+.