Orthogonal Arrays in Normal and Respiratory Airway Epithelium Injured

Orthogonal arrays are found on plasma membranes of glial cells, in the central nervous system, on muscle plasma membranes at neuromuscular junctions, and on a variety of epithelial cells. These structures have been correlated with ion flux. With the aid of freeze fracture technique, orthogonal particle arrays were found on plasma membranes on airway epithelial cells of rats and hamsters. They have been found in abundance at the base of secretory cells throughout normal airway epithelium. These structures were found to increase in number during regeneration in response to injury and they were found in great numbers on plasma membranes of all airway cells in response to acute and chronic NO2 exposure. The lateral and basal plasma membranes of the respiratory epithelium are a new source for studying orthogonal arrays. The normal number and distribution of these arrays can be perturbed in response to mechanical and chemical injury. Orthogonal particle assemblies (rectangular arrays) are closely packed 6-nm intramembrane particles arranged in a square lattice. These assemblies have been found on the P-face of freeze-fractured plasma membranes of a variety of cells (111). Mammalian astrocytes, in particular, can be identified in freeze fractures by the abundance of these structures on their plasma membrane (12). The biochemical nature of these particles and their function in the membrane have not been determined. It has been speculated that these structures are associated with ion flux, at least in astrocytes (13). In this report we describe a rich source for these orthogonal structures in respiratory epithelium of rats and hamsters. We also describe conditions that increase their abundance, thereby perhaps providing a mechanism whereby these structures could be biochemically characterized and their function determined. MATERIALS AND METHODS 20 male Syrian Golden hamsters weighing 6 0 g for the chronic study and 125 g for the acute study were divided into two groups. One group of eight animals was exposed to NO2:2 for 6 h, 2 for 24 h, 2 for 48 h, and the last 2 remained as controls breathing room air. The second group of 12 animals was exposed to NO2:2 for 1 mo, 2 for 5 mo, 2 for 9 mo, and the remaining six animals were used as unexposed age controls, two animals corresponding to each exposure period (1, 5, and 9 mo). Rationale for NOz Exposure: NO2 is a noxious gas that has been known to cause emphysematous lesions in rats (14) and hamsters (15). We have been using continuous NO2 exposure in hamsters as an animal model for the study of the development and progression of lesions caused by this agent in bronchioles and alveolar epithelium. During the studies in which we examined barrier function disruption of respiratory epithelium we made the observations reported in this manuscript. N O 2 Exposure Techniques: The exposure groups were treated with NO2 at concentrations of 30 ppm for 22 h per day, 7 d per wk. 30 ppm was chosen because it was low enough not to cause death and yet high enough to cause a consistent lesion where lower levels do not. The NO2 exposed hamsters were housed separately in stainless steel cages according to the method of Kleinerman et al. (16). The NO2 was generated by passing dry N2 through icecooled NO2. The airflow in the chambers was controlled by an exhaust pump that generated a negative pressure of 2 cm H20. NO2 concentration was monitored by an in line Columbia Scientific Industries CSI 1600 NO2 (Columbia Scientific Industries, Corp., Austin, TX) analyzer and continuously recorded on a Brown-Honeywell recorder (Honeywell, Inc., Minneapolis, MN). The NO2 analyzer was calibrated and the chambers intermittently checked by the method of Saltzman (17). Tissue Preparation: At the prescribed times of sacrifice, the animals were anesthetized lightly with an intraperitoneal injection of 0.75 ml of a 1:1 dilution of sodium pentobarbital and exsanguinated via the abdominal aorta. The chest cavity was opened exposing the lungs and trachea. The lungs, trachea, and esophagus were carefully dissected from the chest cavity to avoid pleural perforation. The trachea was freed by an incision just above the larynx. The lungs were inflation-fixed via tracheal cannulation at 20 cm H~O pressure for 3 h with a solution containing 3% glutaraldehyde and 0.2 M Na cacodylate, buffered at pH 7.4. The bronchioles and surrounding alveoli were dissected out and prepared for freeze fracture. Freeze Fracture: Tissue cubes containing cross-sectionalareas of bronchi and/or bronchioles were obtained as described above. These blocks were transferred from cold cacodylate buffer to a 25% glycerine/cacodylate buffer at 4"C. Glycerination proceeded for 90 min with gentle agitation every 15 min. Tissue blocks were then transferred to nickel-gold specimen holders and rapidly frozen between Balzers double-replica supports by immersion in Freon 22, cooled with liquid nitrogen. Frozen specimens were transferred to a precooled stage of the Balzers 301 freeze-fracture plant (Hudson, NH). Vacuum was increased to 10 4 torr. Lung tissue samples were fractured at a stage temperature of -110°C. Within 1 s following fracture, the specimens were shadowed with platinum-carbon at 45"C and carbon at 90"C. Freeze fracture replicas were THE JOURNAL OF CELL BIOLOGY . VOLUME 100 FEBRUARY 1985 648-651 648 © The Rockefeller University Press . 0021-952518510210648104 $1.00 on July 1, 2017 jcb.rress.org D ow nladed fom removed, lung tissue was digested from the replicas by successive washings in increasing concentrations of sodium hypochlorite, rinsed in three changes of triple-distilled filtered water, and mounted on 300-mesh copper grids. All replicas were examined and photographed in a JEM 100CX electron microscope equipped with a goniometer stage. The lungs from each animal were coded by technician to prevent bias by the investigator and only after all statistical analyses were calculated was the data decoded. Each lung was evaluated independently and a minimum of five bronchioles from each animal were freeze fractured. The plasma membranes of at least 20 epithelial cells from each fracture were randomly photographed at 20,000 diameters magnification. A total of at least 100 micrographs at a final magnification of 60,000 diameters were morphometrically evaluated by counting the number of orthogonal arrays in each micrograph. The plasma membrane P face area was determined with the aid of a Numonics graphic calculator (Numonics Corp., Lansdale, PA). The number of arrays and surface area for each bronchiole was totaled and expressed as orthogonal arrays per square micrometer. The mean for each lung was calculated and compared with the other lung in each group and with its respective control by the Student's t test analysis,