Elizabeth R. Walker

Role of phospholipase A inhibition in amiodarone pulmonary toxicity in rats

Amiodarone is effective in the treatment of ventricular and supraventricular arrhythmias. In man its clinical use is associated with the accumulation of phospholipid-rich multilamellar inclusions in various tissues including lung, liver and others. This report presents evidence showing that amiodarone is a potent inhibitor of lysosomal phospholipase A from rat alveolar macrophages, J-744 macrophages and rat liver. When compared with other cationic amphiphilic agents which are known to produce phospholipidosis, amiodarone is one of the most potent inhibitors yet discovered. The subcellular localization of amiodarone has been determined in lung and its distribution was consistent with a lysosomal localization. It is hypothesized that amiodarone causes cellular phospholipidosis by concentrating in lysosomes and inhibiting phospholipid catabolism.

Surface characteristics, morphology, and ultrastructure of human adherent lymphokine-activated killer cells.

Human adherent lymphokine‐activated killer (A‐LAK) cells represent a population of highly cytotoxic, interleukin‐2 (IL‐2)‐activated peripheral blood lymphocytes that have large granular lymphocyte (LGL) morphology and display natural killer (NK) cell‐associated surface markers (CD3‐CD56+). The ultrastructure of A‐LAK cells also is consistent with that of highly activated NK cells. After their initial isolation, continued culture of A‐LAK cells in the presence of IL‐2 resulted in cyclic shifts between adherent and nonadherent phases with about 90% of the cells floating and non‐adherent. All of these A‐LAK cells were spheroidal with numerous villi and pseudopodia. In the adherent phase, A‐LAK cells were motile, moving along the solid surface at a speed of about 1 μm per minute, and polar, with a ruffled leading edge at one end of the cell and a terminal tuft of villi at the opposite end. Transmission electron microscopy of these cells also demonstrated a high degree of Internal polarity, with the nucleus at the leading edge of the cell and richly granulated cytoplasm at the terminal end. Extensive cytoskeletal structures, multivesicular bodies, and an active Golgi complex characterized these cells. A‐LAK cells in the adherent phase were found to express numerous point contacts (podosomes) and larger adherence structures containing polymerized actin, which appear to be important for interactions of these cells with the substrate. Increased expression of adhesion molecules in association with surface structures mediating adherence is also responsible for effective binding of A‐LAK cells to solid surfaces.

Recovery from chlorphentermine-induced phospholipidosis in rat alveolar macrophages: Morphological features

Abstract Rats were treated for 4 weeks with the amphiphilic, anorexic drug chlorphentermine resulting in the induction of a phospholipid storage disorder in alveolar macrophages. When examined with the electron microscope, the cells were found to be filled with lamellarshaped and membranous inclusions often associated with amorphous granular and membranous material. These features presumably represent the accumulated phospholipid which occurs with this disorder. We have previously reported that following withdrawal of the rats from chlorphentermine treatment, the phospholipid content of the macrophages returned to the control level by 14 days of recovery (Reasor and Koshut, Toxicol. Appl. Pharmacol.55, 334–341, 1980). Morphological features of the cells were examined at various times after termination of drug treatment in order to monitor the recovery process from this perspective. After 7 days of recovery, the macrophages were generally free of the amorphous material, however, they contained prominent lamellar inclusions. At 14 days of recovery, most of the cells contained large pleomorphic bodies, which were probably autophagic vacuoles. These structures appeared to be involved in degrading the lamellar material and converting it into residual bodies. At 21 days of recovery, most of the macrophages appeared normal, although some contained structures similar to those present at 14 days of recovery. By 28 days of recovery, essentially all of the macrophages were normal in appearance. Therefore, while the phospholipid content of the cells returned to the control level by 14 days of recovery, the cells still had an altered morphological appearance. Thus, assessment of recovery from phospholipidosis should not be based on phospholipid content alone.

Changes in lysosomal properties of alveolar macrophages of rats treated with chlorphentermine.

Abstract Rats were treated with chlorphentermine (30 mg/kg) 5 days per week for 4 weeks. Alveolar macrophages washed from the lungs were greatly enlarged and filled with lamellated inclusions. Lysosomal acid hydrolase activity was increased in these cells when compared to macrophages from untreated rats. Chlorphentermine treatment appeared to have marked effects on lysosomal stability. The latency of acid phosphatase was increased. The effects on β- N -acetyl-glucosaminidase were somewhat different, however, with the latency of this enzyme characterized by an increased lysosomal fragility. These findings indicated that in addition to producing morphological changes within alveolar macrophages, chlorphentermine administration leads to lysosomal changes as well.

Morphological and functional changes in mouse splenic lymphocytes following in vivo and in vitro exposure to chlorphentermine

With repeated administration to animals, the cationic, amphiphilic drug, chlorphentermine (CP), has been shown by others to induce a phospholipidosis in lymphocytes. In the present study mouse splenic lymphocytes, exposed to CP, either in vivo or in vitro, developed morphological changes consistant with the induction of phospholipidosis. In addition, CP induced functional changes in lymphocytes. Mice, treated with CP in vivo, demonstrated a significantly depressed ability to generate a delayed hypersensitivity response or to produce antibody-secreting cells against de novo antigens. Mouse splenic lymphocytes, exposed to 10(-7) M CP for 3 days in vitro, demonstrated a significantly depressed blastogenic response to the mitogens phytohemagglutinin, concanavalin A and lipopolysaccharide. CP inhibited an event that occurred early during lymphocyte activation, but was subsequent to mitogen/receptor coupling. In addition, CP significantly depressed the increased uptake of choline that occurs in lymphocytes following cellular activation. Since the presence of phospholipidosis is indicative of an impairment in phospholipid metabolism, these results taken together provide evidence for a relationship between this phenomenon and altered immune function.

Neonatal toxicity in rats following in utero exposure to chlorphentermine or phentermine

The administration of chlorphentermine (30 mg/kg) to pregnant rats during the last 5 days of gestation resulted in the development of a phospholipidosis in the lungs of the dams. The disorder developed in utero, as the phospholipidosis was evident in the lungs of the neonates when examined at 12 h postpartum. In contrast, a phospholipidosis was not observed in the lungs of the dams or neonates following phentermine treatment (30 mg/kg). Concurrently, neonates of the chlorphentermine-treated dams displayed a significant decrease in body weight in comparison to controls. Between 16 h and 24 h postpartum, 83% of the neonates of chlorphentermine-treated dams died. Cross-fostering and starvation experiments revealed that the lethality was not due to aberrant maternal behavior by the chlorphentermine-treated dams or malnutrition of the neonates. Histological examinations revealed endothelial and septal alterations in the lungs of neonates from chlorphentermine-treated dams. No signs of toxicity, as evidenced by the maintenance of body weight, or lethality were observed in the neonates of the phentermine-treated dams.

Chlorphentermine-induced neonatal and maternal pulmonary phospholipidosis in rats

Pregnant rats were treated on Days 16-20 of pregnancy with 30 mg/kg chlorphentermine hydrochloride (CP) or an equal volume of 0.9% NaCl. Females delivered on Day 21 and neonates were examined 12 hr postpartum. Electron micrographs showed the CP neonates to have lamellar inclusions, characteristic of drug-induced phospholipidosis in Type I and Type II cells, macrophages, and nonciliated bronchiolar epithelial cells of the lung. These inclusions were either unicentric, multicentric, or consisted of whorls of membranous material. The content of total phospholipids was increased in neonatal lung as a result of CP treatment. A decrease in the percentage of total phospholipid present as phosphatidylinositol plus phosphatidylserine and an increase in the content of disaturated phosphatidylcholine were found in neonatal lung as a result of maternal chlorphentermine treatment. In maternal lung, increases were seen in the content of total phospholipids, total phosphatidylcholine, disaturated phosphatidylcholine, and phosphatidylinositol plus phosphatidylserine. A decrease in the percentage of sphingomyelin was also found. The results of this study indicate that following prepartum maternal administration of CP morphological changes are accompanied by biochemical alterations which are consistent with the induction of phospholipidosis as observed in neonatal lung. These biochemical changes were different than those associated with the induction of this disorder in the maternal lung.

Lysosomal stabilizing effect of chlorphentermine in the rabbit alveolar macrophage

Abstract 1. Rabbits were injected (i.p.) daily with chlorphentermine (30 mg/kg), 5 days a week for 7 weeks. 2. Alveolar macrophages were collected from the lungs of control and treated rabbits by pulmonary lavage. 3. When compared to controls, the latencies of the lysosomal acid hydrolases, acid phosphatase and β-N-acetyl glucosaminidase were increased in lysosome-containing fractions from alveolar macrophages of treated rabbits. The same results were obtained when chlorphentermine was incubated in vitro with a lysosomal fraction from macrophages of normal rabbits. 4. The increased latency of lysosomal enzymes following chlorphentermine treatment can be explained by the drugs ability to stabilize lysosomal membranes.