Alpha-1 Globulin Trypsin Inhibitor in Canine Surfactant Protein

Abstract

The observation in man of a relationship between an inherited deficiency of serum alpha-1-antitrypsin and the occurrence of pulmonary emphysema suggests that alpha-1-antitrypsin may play an important role in the normal and pathologic physiology of the lung ( 1 ). Alpha-1-antitrypsin inhibits the activity of trypsin, collagenase, elastase, and leukocytic proteases (2, 3 ) , and therefore, may protect the lung from proteolytic damage resulting from inhalation of a variety of toxicants.The distribution of alpha-1-antitrypsin in normal lung tissue is not known. In order to determine if alpha-1-antitrypsin may be a part of or found in situ with the surfactant lipid-protein complex, we have evaluated the trypsin inhibitory capacity of canine surfactant protein.Methods. Surfactant was obtained by bronchopulmonary lavage of healthy adult Beagle dog lungs by the method of Kylstra (4) as modified by Boecker et al. (5). Unilateral lavage with normal saline was performed in vivo on nine animals under light halothane anesthesia. In order to reduce the possibility of contamination of the surfactant protein with whole blood protein, we also obtained surfactant by lavage of isolated, saline-perfused lungs. For this purpose, four animals were anesthetized with sodium pentobarbital and sacrificed by exsanguination. The heart and lungs were re-1 This research was conducted under AEC Contract AT(29-2) 1013, and in animal care facilities fully accredited by the American Association for Accreditation of Laboratory Animal Care. moved en bloc and irrigated with saline. The lungs were ventilated with a Harvard constant volume pump at the rate of 12 cycles per minute and the pulmonary vasculature was perfused with normal saline. When the lungs became white, bilateral lavage with normal saline was performed.The lavage fluid was treated identically whether it was obtained from live anesthetized animals or from the isolated, perfused lung preparations. Cells were removed from the whole lavage fluid by slow speed centrifugation (3,000 g-min) . Precipitation of the surfactant was then accomplished by high speed centrifugation (2 X lo6 g-min) at 4°C. The fluffy pellet obtained by high speed centrifugation has been shown by previous studies in this laboratory (6) to contain 94-96% of the pulmonary washing phospholipid and to be highly surface-active. Following dialysis against distilled water for 48 hr, the surfactant pellet was lyophilized. Lipids were extracted from the freeze-dried material with 4: 1 v/v hexane-ethanol. The hexane-ethanol insoluble fraction, hereafter referred to as surfactant protein, was suspended in normal saline.Immunoelectrophoresis of the surf actant protein from each animal was conducted as follows. Electrophoresis was performed at 4°C in 2% agarose in barbital buffer (pH 8.6, ionic strength 0.05) poured on 1 X 3 in. microscope slides. Current was regulated at 5 mA/slide. Following electrophoresis, troughs were filled with either canine antiserum globulin (rabbit origin, Colorado Serum Co.), antidog whole serum antiser...

Cigarette Smoke Inhalation Decreases α 1 -Antitrypsin Activity in Rat Lung

“…A widely accepted explanation for this destruction holds that elastolytic proteases from lung leukocytes and macrophages attack alveolar connective tissue components (1). Normally, these components are protected against elastase-mediated attack by a1-antitrypsin, a circulating antiprotease that is present in alveolar epithelial fluid (2) and that constitutes the predominant endogenous regulator of leukocyte elastase activity (3). Indeed, inherited deficiency of this antiprotease predisposes an individual to development of pulmonary emphysema (4).…”

Use of bronchoalveolar lavage to detect lung damage.

“…The balance between these two types of activities is crucial to the maintenance of the structural integrity of lung tissue (30,31). The presence of a1-antitrypsin has been demonstrated in lung washings from dogs (32) and patients with alveolar proteinosis (33). The t2-macroglobulin was also found in lavage fluid (34,35), has been detected in lavage fluid (33).…”