The Mode of Secretion of the Clara Cell in Rat Bronchiole: A Freeze-Fracture Study

Kokichi Yoneda and Mary G. Birk

ABSTRACT: Freeze- fracture was used t o study the nonciliated epithelial (Clara) cell of rat bronchiole. En face views of the P face of the luminal cell membrane reveal particle-free bulgingandcircular depressions at the presumptive sites of the membrane fusion between the cell membrane and secre- tory granule membrane. Cross fracture views of the cytoplasm reveal the membranous continuity from the secretory granule to the luminal cell membrane with the decrease of the intramernbrane particles. These findings indicate that the mode of secretion of the Clara cell is a merocrine secre- tion by exocytosis.

INTRODUCTION The nonciliated epithelial cell of the bronchiole, the Clara cell, displays a characteristic morphology and is one of the cells unique to the lung [l). The presence of this type of cell is documented in various mammals [2 - 5). The lo- calization of the specialized cell in the bronchiole suggests an important func- tion in the peripheral airway. In spite of numerous morphological and cyto- chemical studies, the function of the Clara cell remains unresolved [6 - 12). The presence of abundant endoplasmic reticula, membrane-bound granules, well- developed Golgi apparatus, and high oxidative enzyme activity suggests a secre- tory function. The contents of the apparent secretory granules exhibit a crystal- line configuration with a certain fixation technique [lo), can be digested by pepsin "91, and can take up amino acids on autoradiography [l 31. Finally, we have shown cytochemically that these granules show a positive reaction for the phos- pholipase [14]. The function of the Clara cell, thus, appears to be production and secretion of lipid-splitting enzymes as well as other proteins ye t identified. In spite of these findings, some investigators maintain that Clara cells secrete pulmonary surfactant {15,16].

Another point of controversy is the secretory mechanism. Clara himself favored decapitation secretory mechanism and some investigators support this hypothesis [l2, 161. Kuhn and coworkers, on the other hand, proposed mero- crine secretion by exocytosis as the secretory mechanism [lo). We also support- ed this hypothesis based on a study of Clara cell secretion stimulated by pilo-

From the Veterans Administration Medical Center and Department of Pathology. University of Ken- tucky College ofMedicine. Lexington. Kentucky.

Correspondence should be addressed to K. Yoneda. M.D., Patbolou Service 11 I j C D , , Veterans Admin-

Receic~ed28 July 1980: accepted 2 March 1981. istration Medical Center. Lexington. K Y 405 I I .

Experimental Lung Research 2:177-185 (1981) @I Elsevier North Holland, Inc., 1981 52 Vanderbilr Ave., New York, N Y 10017

177 01 90-2 148jS 1 /030177 +9$02.50

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K. Yoneda and M. G. Birk 178

carpine [l 71. We undertook a freeze-fracture study of the Clara cell of the rat in order to answer these questions, specifically (a) the morphology of the secretory granules and (b) the membrane event on the luminar surface.

MATERIALS AND METHODS Six adult male Wistar- Furth rats, weighing approximately 150 g, were used for the experiment. Rats were exsanguinated under deep anesthesia with an intraper- itoneal injection of sodium pentobarbital (50 mg/lOO g body weight). In two rats, the thorax was opened immediately and the lungs were excised. Fresh tis- sue blocks containing bronchioles were disected under a stereoscopic micro- scope. The tissue blocks were frozen immediately in Freon 22 cooled in liquid nitrogen. The whole procedure for a rat took less than 10 min. In four rats, the trachea was canulated and the lungs were inflated with 2% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at a hydrostatic pressure of 25 cm. The lungs were excised en bloc and immersed in the same fixative for 2 hr at 4°C. After fixation, the lungs were sliced and the bronchioles were selected under a stereo- scopic microscope. The tissue blocks containing bronchioles were washed sever- al times with the cacodylate buffer, and then infiltrated with glycerol (with con- centrations increasing from 10 to 30%) in 0.1 M cacodylate buffer. The tissue blocks were frozen in Freon 22 cooled in liquid nitrogen. Freeze-fracture was performed in a Polaron E7900 freeze-fracture device (Polaron Co., Doylestown, PA) at a stage temperature of -1 15" C and in a vacuum of Torr. Carbon- platinum replicas were made of the fractured surface and the underlying tissue was dissolved with household bleach. The replicas were washed with distilled water and mounted on an uncoated copper grid and examined with a Philips EM300 electron microscope.

RESULTS The nomenclature of the freeze-fracture image is according to that of Branton et al. C17J.

No recognizable morphological difference between fresh or fixed tissues could be detected in the freeze-fractured Clara cells. We present electron micro- graphs of replicas of freeze-fractured Clara cells pretreated with fixation and cryoprotectant.

The ultrastructure of the Clara cell, as known from thin-section electron microscopic studies { 10,121, can be easily recognized in freeze-fracture replicas. The Clara cells are tall columnar cells with the cell apex projecting above the ciliated cells (Fig. 1). The center of the cell is occupied by an oval or more frequently a lobulated nucleus, which divides it into two zones. The basal zone contains rough endoplasmic reticulum and mitochondria The apical zone also contains mitochondria as well as secretory granules and smooth endoplasmic reticulum, A large Golgi apparatus is seen close to the nucleus in this figure.

Secretory Granules On thin-section electron microscopy, the secretory granules in rat vary consider- ably in size and shape [lo]. Two shapes predominate, round or oval granules up to 0.5 p m in diameter and granules which appear rod-shaped, which vary up to 3 pm in length and 0.1 -0.1 p m in thickness. On freeze fracture, the round granules appear as hemispherical, sometimes cuboidal, cavities (P face) or eleva-

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Mode of Secretion of Clara Cell 179

Figure 1 Overview of a freeze-fractured Clara cell. Fracture has exposed an outer leaf- let of the plasma membrane, namely, E face. The cytoplasm was cross-fractured, exposing mostly E faces of the secretory granules and the nucleus ( x 16,300).

tions (E face) [Fig. 2). When the fracture plane passes through the center of the granule, the content appears to be finely granular. The P face of the secretory granule contains numerous intramembrane particles. The E face of the secretory granule, on the other hand, shows only a few intramembrane particles. Occa- sional cells show secretory granules at the apex of the cell (Fig. 2). At the apex, the membrane of the secretory granule is continuous with the plasma membrane. This figure indicates the discharge of the contents into the bronchiolar lumen.

On a cross-fractured plane, the contents of both rod-shaped and spherical secretory granules appear finely granular. The E face of the rod-shaped granule is smooth with few intramembrane particles, like that of the spherical secretory

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180 K. Yoneda and M. G. Birk

Figure 2 High magnification view of the secretory granules. Cross-fracture of the content shows finely granular appearance. E face of the membrane shows some intra- membrane particles. The surface of the content is smooth (arrow). One of the secretory granules is in the process of luminal discharge (double arrow). S; secretory granule, L: bronchiolar lumen ( x 82,000).

Figure 3 and smooth endoplasmic reticulum (SER) (x 42,000).

Rod-shaped secretory granules IS]. Notice the similarity of secretory granules

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Mode of Secretion of Clara Cell 181

Figure 4 (arrow) corresponding to the attachment of a secretory granule [ 5 ] ( x 82,000).

High magnification of the luminal plasma membrane. Notice a depression

granule. (Fig. 3) . The P face of the rod-shaped granule, on the other hand, shows also few intramembrane particles compared to that of the spherical secretory granule. The “rod-shaped” granule appears cylindroid.

Plasma Membrane Occasional cells are fractured on the plane which follows the plasma membrane and the cross section of the cytoplasmic interior (Fig. 4). This view is essential for the interpretation of secretory activity, since it allows for the examination of the event on the plasma membrane and the identification of the cell character at the same time. In Clara cells fractured in this fashion, the plasma membrane in contact with a secretory granule showed an indentation (Fig. 4). En face view of these cells showed these figures (Fig. 5). One is a small circular elevation free of intramembrane particles. In the cytoplasm underlying such an elevation, a secretory granule was found in close proximity to the plasma membrane. An- other was depression which corresponds to the image shown in Fig. 4. And the third was an oval “crater” with granular material in the center. This corresponds with a secretory granule in the process of discharge shown in Fig. 2. When a fortuitous fracture revealed the continuity of the membrane from the secretory granule to the plasma membrane (Fig. 6) , the portion of the membrane connect- ing to the luminal plasma membrane contained few, if any, intramembrane parti- cles.

DISCUSSION The Clara cell remains an enigma in spite of extensive studies. Niden proposed that it is the source of pulmonary surfactant [15]. This view has been challenged by many investigators [lo, 11 1. We proposed that the Clara cell secretes enzymes which catabolize pulmonary surfactant [14}.

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182 K. Yoneda and M. G. Birk

Figure 5 En face view of the luminal plasma membrane, three phases of membrane changes are seen; particle-free elevation (arrow), depression (double arrow) and crater (triple arrow). Fractured microvilli (MV) are clearly differentiated by their morphology ( X 82,000).

Also controversial is the mode of secretion of the Clara cell. Clara himself favored an apocrine secretion El). This view was supported by some investiga- tors {12,16]. Kuhn and coworkers proposed a merocrine secretion by exocytosis based on electron microscopic study 11 01. We supported this hypothesis based on a study of stimulated Clara cell secretion by pilocarpine administration 11 71. The present study was conducted to clarify the morphology of the secretory granule of the Clara cell and the membrane event on the luminar surface.

Freeze fracture has been used extensively for the study of membranes. The study of exocytosis in protozoa yielded a distinct membrane morphology, termed a “rosette”, at the site of membrane fusion E19-21). So far no counter- part of this protozoan rosette has been convincingly shown in mammalian secre- tory cells. On the other hand, a clearance of intramembrane particles occurs at the point of exocytotic membrane fusion. This has been documented in endo- crine t227, exocrine 1231, and neurosecretory cells [24]. Our present study, al- though limited to random Clara cells in the normal state, shows a similar mem- brane phenomenon of exocytosis.

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Mode of Secretion of Clara Cell 183

Figure 6 Higher magnification of the plasma membrane. Notice the difference in the density of intramembrane particles between the luminal membrane (top) and the lateral membrane (right lower edge). One of the secretory granules (S) is in the process of dis- charge (arrow). Notice the difference in the particle density between the granule mem- brane proper and stoma ( x 82,000).

The morphology of the lamellar body of type I1 pneumocyyte and the intra- alveolar substance has been studied by several investigators {25 - 27). They agree that the lamellar body and the intraalveolar substance consist of staks of membrane free of intramembrane particles. The contents of the secretory gran- ule of the Clara cell, on the other hand, showed granular appearance without any membranous structure. This morphology is more characteristic of protein secre- tion [23].

Another point of interest in this study is the structure of rod-shaped secretory

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184 K. Yoneda and M. G. Birk

granules. The cylindrical appearance of the membrane strongly suggests a simi- larity to the smooth endoplasmic reticulum [28,29]. It is tempting to speculate that this is a specialized storage site for the secretory product before it is trans- ported to the secretory granule. Autoradiography and cytochemistry have dem- onstrated the similarity of the contents in the spherical and rod-shaped granules C9,13,141.

Based on these findings, we conclude that the mode of secretion of the Clara cell in the rat bronchiole is merocrine secretion by exocytosis.

This work is supported by the Medical Research Service of the Veterans Administration.

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