H. Ishihara

Tachykinins induce a [Ca2+]i rise in the acinar cells of feline tracheal submucosal gland

The intracellular Ca2+ concentration ([Ca2+]i) of acinar cells of isolated submucosal glands from trachea was measured using a fluorescent dye, Fura-2. Neurokinin A (NK-A) produced a sustained rise in [Ca2+]i in a dose-dependent manner, reaching a response of 500 to 600% of the prior baseline value at 10(-6) or 10(-5) M, and the NK-A evoked [Ca2+]i was significantly higher than that by substance P (SP) at similar concentrations. NK-B did not induce significant increases in [Ca2+]i. In a Ca(2+)-free solution, NK-A produced a transient rise in [Ca2+]i, which returned to the baseline within 3 min. Mucus glycoprotein (MGP) secretion, estimated by measuring trichloroacetic-acid (TCA) precipitable glycoconjugates, was stimulated by NK-A or SP. These findings indicate that tachykinins produce a rise in [Ca2+]i by both entry from the extracellular solution and release from intracellular storage, probably by NK-2 receptor stimulation, and stimulate MGP secretion from airway submucosal glands.

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.

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.

Effect of surfactant on bioelectric properties of canine tracheal epithelium

The functional significance of pulmonary surfactant in the airways is not well known and the effects of surfactant on bioelectrical properties of airway epithelium have not been investigated. In the present study, we examined the effect of synthetic surfactant (TR-14) or calf lung surfactant extract (surfactant TA) on transepithelial potential difference (PD) and short circuit current (SCC) in canine trachea. The conductance (G) was calculated as the ratio of SCC per open-circuited PD. The posterior membrane without muscular layer from canine trachea was mounted in an Ussing-type chamber, bathed with Krebs-Ringer buffer solution at 37 degrees C and gassed with 95% O2-5% CO2, pH 7.4. Treatment with mucosal surfactant produced an increase in both PD and SCC in a dose-dependent fashion. PD and SCC reached 132% and 124% of before control at 0.25 mg/ml after the addition of each surfactant respectively, whereas G remained unchanged. Both ouabain and furosemide abolished surfactant-evoked increases in PD and SCC, whereas amiloride did not alter the surfactant-evoked increases. No significant differences in the effect on bioelectric parameters were observed between TR-14 and surfactant TA. These findings suggest that lung surfactant affects bioelectrical properties and changes ion transport (Cl- secretion) across airway epithelium, probably through the activity of ion pumps in the cellular membrane.

Direct write-out of particle size distribution of inhaled aerosol deposition using a laser scattering method.

We developed a device to measure inhaled aerosol deposition in the lung from the difference in particle size distribution of the aerosol during inspiration and expiration. To measure quantity and size distribution of nebulized aerosol particles directly at the subjects mouth we used a He-Ne gas laser scattering technique. To test the reliability of the instrument, we measured particle size distributions of saline aerosols generated by four kinds of nebulizer and compared the results with those from a Malvern 2600D particle sizer as a standard of comparison. Measured mass median diameters (MMDs) were almost identical by both methods. Aerosol deposition in five normal subjects showed that the deposition fraction of the particles increased the larger the particle sizes inhaled. The data suggest that the newly developed aerosol spectrometer can directly analyze aerosol particle size distribution at the mouth during tidal breathing, and that the instrument is useful for quantitative analysis of aerosol inhaled and of inhaled aerosol deposition.