Differential effect of epidermal growth factor on serous and mucous cells in porcine airway submucosal gland

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Abstract

Using a patch-clamp technique, we found that the fresh porcine submucosal gland acinar cells contained two functionally distinct cell populations, i.e. physiologically relevant concentration of acetylcholine (ACh, 30 nM) induced two distinct patterns of electric response in tracheal gland acinar cells. One was characterized by an outstanding oscillatory Cl-current activity, and the other was with poor Cl-current response but with a comparable K+-current. We examined the effect of epidermal growth factor (EGF) on the ACh-induced electric responses in these cells. EGF affected only the latter (K+-prominent) cell type to potentiate significantly the ACh-induced K+-current. An immunohistochemistry revealed that the receptor for EGF was identified preferentially on the mucous, but not serous, cells. Genistein, one of the tyrosine-kinase inhibitors, abolished the augmentation effect of EGF on the ACh-induced current. Thus, we identified the serous cell with a Cl-rich current in response to ACh and the mucous cell with a K+-dominant response. Moreover, EGF affected the mucous cells alone to potentiate the ACh-induced electric response. EGF may contribute to the pathophysiological alterations in chronic inflammatory airways both in morphological (mucous cell hypertrophy/hyperplasia) and functional (thick viscous hypersecretion) ways.

Introduction

The airway submucosal gland secretes mucins, various enzyme proteins, electrolytes and water (Basbaum et al., 1990) to washout inhaled particles and/or to buffer noxious stimuli. In addition, it transports immunoglobulins from submucosal side toward the airway lumen to neutralize and/or eliminate inhaled microbes or foreign substances (Loman et al., 1997, Godding et al., 1998). Thus, the submucosal gland plays a pivotal role in maintaining the integrity of airway defense mechanisms. In chronic inflammatory airways, however, the submucosal glands subject to morphological changes with hypertrophy and hyperplasia (Reid, 1960) resulting in a thick viscous secretion, which causes an airway obstruction that is predisposed to infectious diseases. Epidermal growth factor (EGF), a 6 kDa polypeptide originally identified in mouse submaxillary glands, is known by its mitogenic activity on a variety of epidermal cells including fibroblasts, glial cells, and mammary epithelial cells. Importantly, EGF and its receptor have revealed to be upregulated in airway epithelial cells and submucosal glands in bronchial asthma (Amishima et al., 1998), one of the chronic inflammatory airway diseases. We assumed that EGF might take some parts not only in the morphological changes but also in the functional alterations in the submucosal gland of airways.

Airway submucosal gland is a mixed mucous- and serous gland, and the differences between the two cell population had been investigated by morphological as well as functional studies. The cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., 1989) is a Cl channel with a pathognomonic correlation with chronic bronchitis (Iwase et al., 1997) as well as cystic fibrosis (CF). Immunocytochemical studies revealed that the highest concentration of CFTR in human airways is in the gland serous cells (Engelhardt et al., 1992). On the other hand, in a morphological study, the mucous cells have been found to greatly increase in number relative to serous cells in the airways with chronic bronchitis (Reid, 1960). From a functional point of view, the submucosal gland secretion of mucins and water seems to be controlled separately (Basbaum et al., 1981, Finkbeiner et al., 1986, Finkbeiner et al., 1994), that is, the mucus secretion can be regulated largely by β-adrenergic stimuli via adenosine 3′,5′-cyclic monophosphate (cAMP) (Finkbeiner et al., 1986) whereas the fluid is mainly by α-adrenergic, and the cholinergic stimuli such as acetylcholine (ACh) energize both the mucus and fluid secretions (Basbaum et al., 1981, Yang et al., 1988). The ACh-induced fluid secretion has been shown to be uncoupled from the mucus secretion by a pharmacological manipulation in porcine bronchi (Inglis et al., 1997). These findings indicate that the respective cell type is under different regulation in normal as well as in diseased airways, and also suggest different cellular mechanisms operating in the respective phenotype. Hitherto, however, there seems to be little information about the physiological hallmark for discriminating the cell type using fresh acinar cells.

In the present investigation, we found that the receptor for EGF was expressed predominantly on the mucous phenotype of the gland acinar cells in normal porcine trachea. Therefore, it is of interest to examine whether EGF acts on the acinar cells in a discriminative way dependent upon the cell type. We reported previously that human and feline tracheal gland acinar cells generated ionic currents in response to agonists, which were activated by the cellular Ca2+ (Sasaki et al., 1994, Irokawa et al., 1999, Tamada et al., 2000). In the present study, we used the ionic currents as a marker for the acinar cell activation, and examined the effects of ACh and/or EGF on the freshly isolated porcine tracheal gland acinar cells with a whole cell patch clamp technique. Our present report carries observations of twofold. First, we found two types of the electric response to ACh (30 nM), one was characterized by an outstanding oscillatory Cl-current activity, and the other was with poor Cl-current response but with a comparable K+-current. Second, EGF affected only to the latter K+-current-rich phenotype to potentiate the ACh-induced response, which was mediated by a pathway involving tyrosine-kinase activation. Combined with the results obtained from the present immunohistochemistry, we concluded that the cells with Cl-current-rich response corresponded to the serous phenotype and the K+-current dominant type to the mucous cells

Section snippets

Cell preparation

Porcine tracheas were obtained at a local slaughterhouse immediately after the animals had been sacrificed, which were transported to the laboratory in an ice-cold extracellular solution (see Section 2.3, Electrical recordings). The external surface of the trachea was cleaned of fat and connective tissues, cut into rings 3–4 cm long, and the posterior (membranous) strip of the tracheal wall was excised longitudinally attached both sides with about 1 cm width of cartilaginous portion and was

Distribution of EGF-receptor (EGF-R) on the porcine airway

It has been shown that epidermal growth factor (EGF) and its receptor are upregulated in airway epithelial cells and submucosal glands in bronchial asthma (Amishima et al., 1998). As a preliminary investigation, an immunohistochemical experiment was performed to localize EGF-R on the fresh porcine tracheal wall. As shown in Fig. 2a, dense positive label of the monoclonal antibody against EGF-R was identified on both the superficial epithelium and submucosal glands. In the glandular tissue, the

Discussion

In attempts to investigate the physiological properties of airway submucosal glands, we have made use of the isolated gland preparations (Shimura et al., 1986, Sasaki et al., 1989, Sasaki et al., 1990, Sasaki et al., 1994, Irokawa et al., 1999, Tamada et al., 2000). The freshly isolated gland preparation has some advantages in studying the mechanisms of airway secretion. The in vivo preparation such as counting the hillock formation on tracheal mucosa (Nadel and Davis, 1980) may be affected by

Acknowledgements

We gratefully acknowledge Brent K. Bell for reading the manuscript. This work was supported by Grant-in-Aid for Scientific Research from The Ministry of Education, Science, Sports and Culture, Japan to TS (no.09670593).

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