ULTRASTRUCTURE OF THE EPITHELIUM OF TERMINAL BRONCHIOLES IN RABBITS AFTER THE ADMINISTRATION OF ACETYLCHOLINE

Uhlík J . , V. Konrádová, L. Vajner , J . Zocová: Ultrastructure of the Epithelium of Terminal Bronchioles in Rabbits After the Administration of Acetylcholine. Acta Vet. Brno 1999, 68: 179–184. In this experiment, the ultrastructure of the epithelium of terminal bronchioles was studied 5 and 20 minutes after intravenous administration of two different doses of acetylcholine. The administration of 0.5 mg acetylcholine resulted in pathological alteration of the cytoplasm of epithelial cells recorded 5 and 20 minutes post exposure. In the first phase, the secretion of Clara cells was inhibited. Twenty minutes after the acetylcholine administration, a mild stimulation of these cells to discharge the content of their secretory granules was observed. After the administration of 0.1 mg acetylcholine, the signs of pathological alteration of the epithelial cell cytoplasm were mild. Twenty minutes post exposure, the findings did almost not differ from those in the epithelium of healthy control rabbits. After an initial inhibition, the secretory activity of Clara cells was resumed and did not differ from that found in controls. No increased proliferation of epithelial cells was recorded in any experimental group. Airways, ciliated cells, Clara cells, cholinergic stimulation, electron microscopy Recently, the mechanisms controlling the functions of respiratory organs have become one of the main topics of authors dealing with the structure and function of the respiratory system. One of the most important mechanisms controlling the functions of airways, including the function of secretory elements and ciliary border, is the parasympathetic innervation with acetylcholine serving as a neurotransmitter (Fung et al. 1992; Kami jo et al. 1993; Ramnar ine et al. 1996; S tee l and Hanrahan 1997; Sa la the et al. 1997; Wess le r et al. 1998). The stimulating effect of the acetylcholine administration on the secretory cells of the lining epithelium of airways and digestive tube was repeatedly demonstrated in experiments in vitro or in vivo (Spec ian and Neut ra 1980; Ph i l l ips and Wilson 1993; Tokuyama et al. 1990; Konrádová et al. 1996ab). The authors described the effect of the acetylcholine administration on the mucus-producing goblet cells. However, the reaction of the distal airways, where the secretory elements are represented by Clara cells, has not been studied in detail and therefore we investigated the effect of the intravenous administration of two different doses of acetylcholine on the ultrastructure of the epithelium of the terminal bronchioles in rabbits. Materials and Methods Fifteen clinically healthy rabbits (males, body weight 1,520 g 3,800 g) were used. Three untreated rabbits served as controls and 12 animals were divided in two experimental groups (n = 6). They were given 0.1 mg or 0.5 mg of acetylcholine chloride (Acetylcholinum ophthalmicum Dispersa, Ciba, Niederwangen, Belgium) i.v., respectively. The material was always collected from 3 rabbits 5 and 20 minutes after i.v. administration of each dose of the drug, respectively. In the animals, the thorax was opened under general anaesthesia (ketamine 35 mg/kg and xylazine 5 mg/kg i.m.), the lungs were removed and immediately perfused by 5% glutaraldehyde in 0.1 M cacodylate buffer ACTA VET. BRNO 1999, 68: 179–184 Address for correspondence: MUDr. Jifií Uhlík Institute of Histology and Embryology 2nd Medical Faculty, Charles University V úvalu 84, CZ-150 06 Praha 5, Czech Republic Phone: ++420 2 2443 5982 Fax: ++420 2 2443 5820 E-mail: jiri.uhlik@lfmotol.cuni.cz http://www.vfu.cz/acta-vet/actavet.htm (pH 7.2). From one pulmonary lobe, tiny pieces of the tissue were collected, fixed for 90 minutes in the same fixative and then for 60 minutes in 2% OsO4 in 0.1 M cacodylate buffer (pH 7.4). The material was dehydrated in graded series of alcohol and embedded in a Durcupan-Epon mixture. Terminal bronchioles were localized in semithin sections stained with toluidine blue. Ultrathin sections were prepared on Ultrotome Nova (LKB, Broma, Sweden), contrasted with uranyl acetate and lead citrate and examined in JEM 100 C electron microscope (Jeol, Tokyo, Japan). For quantitative evaluation, the total number of ciliated and Clara cells, and the functional state of Clara cells were recorded in the electron microscope using the method described in our previous paper (Uhlík 1996). The actual values are given in Table 1. To compare the results in all experimental groups, the χ2 test of homogeneity in frequency tables was used. To specify categories causing deflections from the hypothesis of homogeneity, adjusted standardized deviations were used.

(pH 7.2).From one pulmonary lobe, tiny pieces of the tissue were collected, fixed for 90 minutes in the same fixative and then for 60 minutes in 2% OsO 4 in 0.1 M cacodylate buffer (pH 7.4).The material was dehydrated in graded series of alcohol and embedded in a Durcupan-Epon mixture.Terminal bronchioles were localized in semithin sections stained with toluidine blue.Ultrathin sections were prepared on Ultrotome Nova (LKB, Broma, Sweden), contrasted with uranyl acetate and lead citrate and examined in JEM 100 C electron microscope (Jeol, Tokyo, Japan).
For quantitative evaluation, the total number of ciliated and Clara cells, and the functional state of Clara cells were recorded in the electron microscope using the method described in our previous paper (Uhlík 1996).The actual values are given in Table 1.To compare the results in all experimental groups, the χ 2 test of homogeneity in frequency tables was used.To specify categories causing deflections from the hypothesis of homogeneity, adjusted standardized deviations were used.

The ultrastructure of the epithelium of terminal bronchioles in control rabbits
Terminal bronchioles of healthy control rabbits were lined by a simple epithelium where low columnar or cuboidal ciliated cells and high columnar Clara cells alternated almost regularly.In our previous paper, the arrangement and ultrastructure of both types of epithelial cells were described in detail (Uhlík 1996).
In the epithelium of the terminal bronchioles of healthy control rabbits, the Clara cells and the ciliated ones represented 52.7 ± 3.6% and 47.3 ± 3.6% of epithelial cells, respectively (Fig. 1).In the majority of Clara cells (73.5 ± 9.4%), secretory granules were discovered.The granules were not revealed only in 26.5 ± 9.4% of them (Fig. 2).2. The ultrastructure of the epithelium of terminal bronchioles in rabbits 5 minutes after i.v.administration of 0.5 mg acetylcholine Five minutes after administration of 0.5 mg acetylcholine, the terminal bronchioles were lined by an altered simple epithelium consisting of ciliated and Clara cells.Intercellular spaces were narrow, apical junctional complexes remained intact (Plate V., Fig 3).
The cytoplasm of ciliated cells contained voluminous lysosomes and many smaller vacuoles.Mitochondria revealed an electron-lucent matrix and altered cristae.Cisternae of the Golgi complex were dilated.On the apical surfaces of the ciliated cells, the formation of cytoplasmic protrusions sometimes containing axonemes of degenerating cilia was observed (Fig. 4).Less altered, pathological cilia were often encountered, too.
In the cytoplasm of Clara cells, a distinct dilatation of tubules of the smooth endoplasmic reticulum filled with an electron-lucent content was revealed.The alteration of the smooth endoplasmic reticulum was unequally pronounced in individual cells.In several of them, signs of the vacuolar degeneration (Fig. 5) and impairment of the apical plasma membrane were revealed.Remnants of their degenerated cytoplasm were then found above the epithelium in the lumen of terminal bronchioles (Fig. 6).In the less altered Clara cells, mitochondria with an electron-lucent matrix and dilated spaces of the rough endoplasmic reticulum were encountered.Secretory granules were usually present in the cytoplasm of Clara cells, but the exocytosis of these granules was noticed only exceptionally (Plate VI., Fig. 7).
3. The ultrastructure of the epithelium of terminal bronchioles in rabbits 20 minutes after i.v.administration of 0.5 mg acetylcholine Twenty minutes after administration of 0.5 mg acetylcholine, the epithelium of terminal bronchioles was still altered.Intercellular spaces remained narrow and apical junctional complexes were intact (Fig. 8).Ciliated cells contained slightly altered mitochondria, dilated Golgi complex, tiny vacuoles, numerous lysosomes and exceptionally isolated ciliated vacuoles in their cytoplasm.In the small apical cytoplasmic blebs, disintegrating axonemes of cilia were often contained.Differentiating ciliated cells were observed more frequently than in the previous experimental group.
In the cytoplasm of Clara cells, tubules of smooth endoplasmic reticulum were dilated, but the degree of their swelling was less pronounced compared with the previous experimental group (Fig. 8).Mitochondria revealed mild signs of pathological alteration and tiny lysosomes were occasionally observed.Apical cytoplasmic protrusions containing secretory granules were often found on the Clara cells.
The epithelium was composed of 49.5 ± 13.5% and 50.5 ± 13.5% of ciliated and Clara cells, respectively (Fig. 1).Secretory granules were found in only 63.4 ± 24.1% of Clara cells (Fig. 2).This value differed significantly (α = 0.01) from those revealed 5 and 20 minutes after administration of the lower dose and 5 minutes after the administration of the same dose of acetylcholine.
4. The ultrastructure of the epithelium of terminal bronchioles in rabbits 5 minutes after i.v.administration of 0.1 mg acetylcholine Five minutes after administration of 0.1 mg acetylcholine, the degree of alteration of the epithelium of rabbits' terminal bronchioles was mild.
In the cytoplasm of ciliated cells, only a small increase in number of lysosomes and tiny vacuoles was observed.Slightly dilated tubules of smooth endoplasmic reticulum represented the only signs of pathological alteration of the cytoplasm of Clara cells.
5. The ultrastructure of the epithelium of terminal bronchioles in rabbits 20 minutes after i.v.administration of 0.1 mg acetylcholine Twenty minutes after administration of 0.1 mg acetylcholine, the epithelium of terminal bronchioles revealed almost no signs of pathological alteration.
In the cytoplasm of ciliated cells, only individual lysosomes with heterogeneous contents were seen (Fig. 9).On their apical surfaces, isolated small cytoplasmic protrusions were developed.The Clara cells contained an intact cytoplasm and the contents of their secretory granules were discharged only occasionally (Fig. 10).
In the epithelium, 40.4 ± 8.1% of ciliated cells and 59.6 ± 8.1% of the Clara cells were found (Fig. 1); 78.2 ± 10.4% of Clara cells contained secretory granules in their cytoplasm (Fig. 2).This value differed significantly (α = 0.01) from the lowest value found 20 minutes after administration of the higher dose of acetylcholine.

Discussion
The influence of cholinergic stimulation on the structure and function of the secretory epithelial cells was described by several authors.Nevertheless, all these papers dealt with mucus-producing elements, usually the goblet cells in the intestinal or airway epithelium.Both in vitro and in vivo, S p e c i a n and N e u t r a (1980) and later also P h i l l i p s and W i l s o n (1993) demonstrated that the administration of acetylcholine on the intestinal mucous membrane of rats and rabbits resulted not only in very rapid evacuation of goblet cells, but changed also their secretory mechanism.R o u m a g n a c and L a b o i s s e (1987) described an increase in mucus secretion in the organ culture of human intestinal carcinoma after the addition of acetylcholine to the culture medium.The effect of cholinergic stimulation on the secretion of goblet cells was studied by T o k u y a m a and his co-workers in the airways of guinea pigs ( T o k u y a m a et al. 1990).Using a light microscope, they observed a decrease in the mucus content in the goblet cells of trachea and large bronchi after an electric stimulation of cervical vagal nerves.Kamijo with coworkers studied the exocytosis of the secretion from the goblet cells and glands in the rat nasal mucous membrane due to the administration of various neurotransmitters ( K a m i j o et al. 1993).The authors again demonstrated a pronounced stimulating effect of the acetylcholine administration.
The effect of acetylcholine administration on the ultrastructure of the rabbit tracheal epithelium was studied in our laboratory several years ago (Konrádová et al. 1996ab).The acetylcholine administration affected mainly goblet cells.In highly stimulated goblet cells, the mechanism of secretion was accelerated.In some secretory elements, the apocrine secretion and also chain exocytosis were recorded.The completely exhausted goblet cells usually did not take part in the next secretory cycles, but degenerated and were gradually expelled from the epithelium.After the administration of both doses of acetylcholine, more than 90% of tracheal goblet cells were stimulated.After the administration of 0.5 mg acetylcholine, the peak of the reaction was reached within 5 minutes.After the administration of 0.1 mg acetylcholine, the reaction was more prolonged.Twenty minutes after the administration of 0.5 mg acetylcholine, a marked increase in the number of differentiating secretory elements accompanied with changes in their distribution resulting in the formation of intraepithelial mucous glands was recorded.
The reaction of the epithelium of terminal bronchioles differed from that of the tracheal epithelium.The influence of acetylcholine was also concentrated on the secretory cells.Similar to the effects of administration of various toxicants (naphthalene, 4-ipomeanol, trichloroethylene) (Plopper et al. 1992ab, 1994aVan Winkle et al. 1995;Lakritz et al. 1996;Giovanetti et al. 1998), inhalation of ozone (Harkema et al. 1993;Plopper et al. 1994b), or tracheobronchography (Uhlík and TÛma 1998), response of the Clara cells to the administration of acetylcholine resulted in pathological alteration of their cytoplasm.In the first phase post exposure, the secretion of the Clara cells was inhibited and the number of cells containing secretory granules slightly increased.Twenty minutes after administration of the higher dose of acetylcholine, the Clara cells were mildly stimulated to discharge their secretion.However, the quantitative evaluation did not show any significant changes in the distribution of the cells in any of the experimental groups compared to control groups.
In contrast to the stimulation, degeneration and consecutive differentiation of tracheal goblet cells, the reaction of the secretory elements of terminal bronchioles on the acetylcholine administration was less pronounced.Nevertheless, significant changes in the secretory activity of Clara cells were recorded.

Fig. 1 .
Fig. 1.Quantitative evaluation of the epithelium of terminal bronchioles in rabbits 5 and 20 minutes after the i. v. administration of 0.1 and 0.5 mg of acetylcholine.

Fig. 2 .
Fig. 2. Clara cells containing secretory granules in the epithelium of terminal bronchioles in rabbits 5 and 20 minutes after the i. v. administration of 0.1 and 0.5 mg of acetylcholine.
Fig. 3: A portion of the altered epithelium of a terminal bronchiole.The ciliated (1) and Clara (2) cells reveal signs of the pathological alteration of their cytoplasm.Five minutes after the administration of 0.5 mg acetylcholine Fig. 4: A small cytoplasmic protrusion (p) containing an axoneme of a cilium and vacuoles on the apical surface of the ciliated cell.Five minutes after the administration of 0.5 mg acetylcholine.

Fig. 5 :
Fig. 5: An apical portion of the Clara cell containing moderately altered mitochondria (m) and extremely dilated tubules of the smooth endoplasmic reticulum (e) in its cytoplasm.Five minutes after the administration of 0.5 mg acetylcholine.

Fig. 6 :
Fig. 6: An impairment of the apical plasma membrane of the altered Clara cell resulting in the liberation of remnants of its degenerated cytoplasm into the lumen (arrowhead).Five minutes after the administration of 0.5 mg acetylcholine.

Fig. 8 :
Fig. 8: An intact apical junctional complex (arrowhead) between two Clara cells revealing different signs of the pathological alteration of their cytoplasm.Twenty minutes after the administration of 0.5 mg acetylcholine.

Fig. 9 :
Fig. 9: Lysosomes (l) and a multivesicular body (m) in the cytoplasm of the ciliated cell.Twenty minutes after the administration of 0.1 mg acetylcholine.

Fig. 10 :
Fig. 10: A secretory granule (g) situated in the close vicinity to the apical surface of the Clara cell with the intact cytoplasm.Twenty minutes after the administration of 0.1 mg acetylcholine.

Table 1
Quantitative evaluation of the epithelium of terminal bronchioles in rabbits 5 and 20 minutes after i.v.administration of 0.1 and 0.5 mg acetylcholine (actual values)