Intermediate filament-mediated stretch-induced changes in chromatin: a hypothesis for growth initiation in cardiac myocytes.

Excessive stretching of the myocardium leads to hypertrophy, but how the stretch message is communicated to hypertrophy-initiating genes is unknown. Candidates hypothesized as couplers of physical stretch to growth initiation include neural and hormonal factors, stretch-activated and stretch-inactivated ion channels, microtubules, microfilaments, and contractile activity. Upon investigation, however, all were ruled out. We provide evidence here that it is the intermediate filaments in the mechanically stressed myocyte that transmit the stretch message to the chromatin. Using rat myocytes and an immunogold desmin-lamin-labeling technique, we found that when cardiac myocytes are stretched there are changes in the spatial arrangement of both the desmin-lamin intermediate filament network and the nuclear-envelope-associated chromatin. We hypothesize that (a) by physically linking the sarcomere to chromatin, the desmin-lamin intermediate filament network couples sarcomere length to chromatin distribution, and (b) stretch-induced changes in chromatin (mediated by the intermediate filament network) activate hypertrophy-associated genes. Further investigation is needed to test this hypothesis.

Does level of ligation influence results in a murine biliary obstruction model?

BACKGROUND: Despite advances in perioperative management, patients with extrahepatic biliary obstruction still experience a high rate of complications and death after surgery. The rat is commonly used as an experimental animal for research in obstructive jaundice. Ligation of the rat bile duct high in the liver hilum is assumed to produce a more severe model of biliary obstruction than low ligation. The differences are attributed to the ability of the rat bile duct to dilate. Differences in level of ligation may, thus, explain some discrepancies between studies. MATERIALS AND METHODS: To test this hypothesis, female Lewis rats underwent high ligation (HL), low ligation (LL), and sham celiotomy. Colloidal carbon clearance, bilirubin, total serum bile acids, and hematocrit were measured 12 days later. Liver and spleen weight, presence or absence of ascites, infection, and adequacy of ligation were noted and the liver was processed for routine histology and electron microscopy. RESULTS: Although bilirubin levels were higher after HL than after LL, liver and spleen weight, total serum bile salts, and phagocytic constants K and alpha were not different between these two groups. Gross, histologic, and ultrastructural appearance did not differ between HL and LL groups. CONCLUSION: High ligation causes greater hyperbilirubinemia than low ligation, but does not alter other parameters including phagocytic constants. The present study does not confirm the hypothesis that HL creates a more severe model than LL; therefore, it is unlikely that differences in level of ligation explain variability in results between studies.

Trans-cellular desmin-lamin B intermediate filament network in cardiac myocytes.

Excessive stretch of heart muscle is thought to be a determinant of myocardial hypertrophy. Because cell shape and nuclear shape are closely coupled in cardiac myocytes, we hypothesize that excessive stretch causes physical deformation of the nucleus which might be responsible for some molecular events leading to hypertrophy. Cell shape and nuclear shape are most likely to be coupled by cytoskeletal elements. With this in mind, we have used immunogold labeling to examine the topological associations of desmin cytoskeletal and lamin B nucleoskeletal intermediate filaments with various intracellular structures in mammalian cardiac myocytes. We found that desmin filaments form a sarcoplasmic network radiating from the sarcolemma to the nuclear surface. Perpendicular to the long axis of the cell, strands of desmin filaments traverse the interfibrillary space in a co-linear arrangement with Z-discs. The desmin filament strands extend between peripheral regions of adjacent Z-discs. Desmin filaments traversing the interfibrillary space closely associate with the surface of mitochondria. At the cell surface, desmin filaments extend from Z-discs to terminate immediately beneath the sarcolemma. Close to the nucleus, desmin filaments extend from Z-discs towards nuclear pores. At the same time, lamin B filaments, which co-localize with heterochromatin immediately beneath the inner nuclear membrane, encircle the inner aspect of each nuclear pore. We hypothesize that desmin and lamin B are functionally anchored to each other at the nuclear pore, either directly or through anchorage proteins within the pore complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural immunolabeling in the evaluation, diagnosis, and characterization of neuroendocrine neoplasms.

Neuroendocrine neoplasia represents a heterogenous entity with variable morphologic light microscopic expressions. In many cases a definite diagnosis is easily made by light microscopic examination, but in some cases this does not suffice. In the latter instances, immunocytochemistry, ultrastructural examination, or both are required to diagnose a neuroendocrine neoplasm. However, basing a diagnosis of neuroendocrine neoplasia exclusively on the results obtained from immunocytochemical or ultrastructural evaluation of these tumors may not be entirely accurate in some instances. Ultrastructural immunolabeling plays a key role in accurately defining localization of immunoreactive substances in well-characterized neuroendocrine neoplasms, can assess colocalization of antigenic epitopes, helps define specificity and significance of immunocytochemistry results obtained at the light microscopic level, and is more sensitive than light microscopic immunocyto-chemistry. Some evolving diagnostic entities can be further characterized by utilization of ultrastructural labeling techniques. Controversies concerning the neuroendocrine nature of electron-dense structures identifiable at the ultrastructural level can be readily and accurately resolved. By providing a way to evaluate combined immunomorphologic parameters, ultrastructural immunogold labeling can settle important questions pertaining to neuroendocrine neoplasia. The present article illustrates a series of cases where the above-mentioned applications were tested.

A simple technique for postembedding ultrastructural immunogold labeling.

A simple and reproducible method for postembedding ultrastructural immunogold labeling is described. Tissues are fixed in Carson-Millonig phosphate-buffered 4% formaldehyde, and then are embedded in LR White resin. Immunogold labeling of ultrathin sections is accomplished by means of an indirect method that employs a nonconjugated primary antibody and gold particles conjugated to antibody to immunoglobulin G. Technical aspects of fixation, embedding, and immunolabeling that influence the success of this protocol are discussed.

Morphologic features and nuclide composition of infarction-associated cardiac myocyte mineralization in humans.

Low dietary Mg results in Ca loading of cardiac myocytes, which increases the likelihood of myocyte calcification in the event of acute myocardial infarction (AMI), and possibly increases myocyte vulnerability to necrosis. Bloom and Peric-Golia1 previously reported an autopsy study of cases from the Washington, D.C. area (a region with low levels of Mg in the drinking water), demonstrating AMI-associated mineralization in myocytes with histologically normal nuclei and cross striations, as well as in obviously necrotic myocytes. The authors have re-examined mineralized myocytes from the same autopsy material, using electron probe microanalysis, light microscopy, and transmission electron microscopy. Microprobe analysis identified Ca and P as the nuclides composing the inorganic phase of the mineral deposits. Ultrastructurally, all Ca deposits, regardless of size or intracellular location, were composed of aggregates of needlelike hydroxyapatite crystals. The mildest form of intracellular Ca deposition was observed as small Ca deposits limited to some mitochondria of myocytes, which demonstrated intact nuclei and regular sarcomere pattern. More advanced stages of intracellular calcification, in the form of Ca deposits associated with mitochondria, Z-band regions and nuclei, were observed in other myocytes that also retained intact nuclei and sarcomeres. Massive Ca deposits were associated with myocytes which showed morphologic features of advanced necrosis, including loss of nuclei, disruption of sarcomere structure and masses of cellular debris. These observations support the theory originally proposed by Bloom and Peric-Golia1 suggesting that Ca loading of myocytes, possibly related to Mg deficiency in humans, increased vulnerability of the myocytes to subsequent AMI-associated necrosis and dystrophic calcification. In addition, the light microscopic impression of calcification of otherwise normal myocytes is contradicted by the electron microscopic identification of hydroxyapatite crystals free in the sarcoplasm, a condition unlikely to be compatible with viability. Lastly, the fact that all Ca deposits were in the form of hydroxyapatite supports the view that they were formed in a Mg-poor environment, which favors conversion of the more common amorphous form of Ca phosphate into the needlelike crystals of hydroxyapatite.

Soft tissue myofibroblastomas.

Five well-circumscribed solitary soft tissue tumors composed of myofibroblasts are described and termed myofibroblastomas. By light microscopy these lesions are characterized by short, intersecting, or crisscrossing fascicles of spindle cells, sometimes associated with foci of necrosis and/or mitotic activity with less than three mitoses per 10 high power fields. Myofibroblastomas show well-defined myofibroblastic differentiation ultrastructurally with peripheral myofilaments and vimentin, actin, and desmin immunocytochemistry positivity. The five tumors described occurred in patients of various age groups, including one congenital, and in a variety of soft tissue locations. It is important to recognize this benign soft tissue neoplasm to avoid confusion with other soft tissue tumors and to separate this lesion from other myofibromatosis. This study elucidates the spectrum of light microscopic, ultrastructural, and immunocytochemistry findings of soft tissue myofibroblastomas and establishes this soft tissue tumor as a specific clinico-pathologic entity.

Unusual pigmented vesical lesion in a middle-aged woman.

Excess lipofuscin deposition in tissues is a phenomenon observed in normal aging. The amount of lipofuscin in myocardium, liver, skeletal muscle, and adnexal skin structures varies considerably with age and increases in older individuals. In addition, lipofuscin deposition occurs in association with specific, non-age-related processes; these include melanosis coli, the so-called brown bowel syndrome, and the black thyroid. Localized lipofuscinosis has also been described in the gallbladder, esophagus, and fallopian tubes. The present article adds lipofuscinosis vesicalis to the list of entities characterized by focal lipofuscin deposition. All cases have a characteristic gross appearance that is somewhat variable from entity to entity and in some cases may suggest clinically an ominous pathologic process.

In vivo toxicity and pulmonary effects of promazine and chlorpromazine in rats.

Cationic amphiphilic drugs induce a phospholipid storage disorder known as phospholipidosis. Halogenated analogs of the drugs are more potent inducers of phospholipidosis when compared to nonhalogenated analogs. Two such antipsychotic drugs, promazine and chlorpromazine, are effectively taken up by the lungs and induce lamellar inclusions in vitro. We compared the in vivo toxicity and efficacy of promazine and chlorpromazine to induce phospholipidosis in the lung and in pulmonary alveolar macrophages. Male Sprague-Dawley rats were given promazine or chlorpromazine (25 mg/kg/day, P.O., in water) for 5 weeks. Food intake was decreased in promazine- and chlorpromazine-treated rats, chlorpromazine rats being affected more than promazine rats. To minimize experimental error due to starvation, control rats were pair-fed. The body weight gain was decreased in chlorpromazine rats in comparison to pair-fed controls. Chlorpromazine-treated rats, but not promazine-treated rats, showed increased mortality over the 5-week treatment period. Histopathologic examination of lung revealed loss of alveolar macrophages with no other gross abnormalities in chlorpromazine-treated rats. Quantitative analysis of lung lavage also showed significant reduction in the number of macrophages. This finding is in contrast to other cationic amphiphilic drugs, which induce phospholipidosis as well as accumulation of alveolar macrophages. Phospholipid level increased in alveolar macrophages but not in lavaged lung following chlorpromazine treatment. Acid phosphatase activity in lavaged lung homogenate and macrophages of promazine- and chlorpromazine-treated rats, taken as an index of toxicity to cells, did not differ significantly from control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlordecone (Kepone)-potentiated carbon tetrachloride hepatotoxicity in partially hepatectomized rats--a histomorphometric study.

Our previous studies indicated the involvement of some unidentified mechanisms, apart from the bioactivation phenomenon, in chlordecone (CD)-potentiated CCl4 hepatotoxicity and lethality. Recent studies revealed that hepatocellular regeneration is suppressed in CD + CCl4 toxicity. The present work is a continuation of our earlier work employing a partial hepatectomy model for stimulating hepatocellular division, in normal (N) or CD-treated (10 ppm for 15 days) rats. Male Sprague-Dawley rats maintained on an appropriate dietary protocol and undergoing sham (SH) or partial hepatectomies (PH) were employed. Hepatocellular regeneration was assessed by measuring the percentage mitotic figures and by autoradiography of liver sections from rats given 3H-thymidine in vivo. Hepatotoxicity was assessed by examining liver sections for necrotic cells, swollen cells and cells having lipid droplets. CCl4 (100 microliters kg-1)-induced histopathological alterations in CD-pretreated rats were significantly decreased in rats 2 days post-PH (PH2) as compared to SH rats or rats 7 days post-PH (PH7), indicating that amplification of CCl4 toxicity is significantly reduced when there is a greater regenerative activity. The percentage of mitoses as well as the percentage of labelled cells were significantly elevated at 2-6 h after CCl4 administration in N rats but remained suppressed in CD rats. In CD-pretreated PH2 rats where the percentage of mitoses and the percentage of labelled cells were many-fold greater when compared to SH or PH7 rats, a portion of the stimulated hepatocellular division decreased significantly at 2-6 h after CCl4 administration, but remained significantly greater when compared to basal level of regeneration.(ABSTRACT TRUNCATED AT 250 WORDS)

The time course of liver injury and [3H]thymidine incorporation in chlordecone-potentiated CHCl3 hepatotoxicity.

The mechanism by which chlordecone (CD) potentiates CHCl3 hepatotoxicity and lethality remains unknown. We examined the time course of the hepatotoxicity by following serum enzymes, liver histopathology, hepatocellular regeneration, and tissue repair by morphometric analysis and [3H]thymidine (3H-T) incorporation into nuclear DNA. Male mice fed control, or CD (10 ppm), mirex (Mx. 10 ppm), or phenobarbital (PB. 225 ppm) diets for 15 days and receiving a single ip dose of 0.1 ml CHCl3/kg in corn oil vehicle were used. Liver damage was assessed by plasma alanine and aspartate transaminases and by histopathology at 4, 12, 24, 36, 48, 72, and 96 hr after CHCl3 administration. None of the dietary pretreatments caused plasma transaminase elevations, any liver necrosis, or any increase in 3H-T incorporation in nuclear DNA at any time. CHCl3 alone caused only limited hepatocellular necrosis without any increase in plasma transaminases. The same dose of CHCl3 given to CD-pretreated mice resulted in greatly increased liver injury. Plasma transaminases were elevated starting at 4 hr, reaching a maximum value at 12 hr and a decline starting at 48 hr. Centrilobular and midzonal necroses were evident at 12 hr onward. PB pretreatment caused some increase in CHCl3-induced necrosis and a moderate rise in transaminases at 24 hr, but Mx pretreatment caused neither effect. 3H-T incorporation was increased at 72 and 96 hr after CHCl3 alone. The same dose of CHCl3 caused only a modest increase in PB and Mx and a significant and maximal biphasic increase at 36 and 72 hr CD-pretreated mice. Morphometry of liver sections indicated that hepatocellular regeneration is stimulated at 72 hr after CHCl3 alone. The same dose of CHCl3 results in a greater stimulation of hepatocellular regeneration in CD-pretreated mice, and this event is pushed forward at 48 hr, continuing through 96 hr to compensate for greater hepatocellular necrosis associated with this treatment. Lesser stimulation of hepatocellular regeneration was observed in PB + CHCl3 and Mx + CHCl3 groups of mice consonance with much lesser hepatotoxicity. These results suggest that the critical decisive event in the recovery from limited hepatocellular injury is the hepatocellular regeneration and tissue repair, which appear to be stimulated in proportion to the injury.

Fluorescence studies on binding of amphiphilic drugs to isolated lamellar bodies: relevance to phospholipidosis.

Lysosomal phospholipid storage disorder in lung tissue was observed during chronic treatment with amphiphilic amine drugs. The prevailing and widely accepted mechanism of phospholipidosis is that amphiphilic drugs bind to phospholipids and make the phospholipids unsuitable substrates for the action of phospholipases. We investigated hydrophobic and hydrophilic binding of fifteen drugs to the phospholipid storage organelle, lung lamellar bodies, isolated from male Sprague-Dawley rats. Hydrophobic interactions were studied using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe and hydrophilic binding was studied using 1-anilino-8-naphthalene sulfonate as a fluorescent probe. The binding parameters were calculated using Scatchard equations. Of the fifteen drugs used, nine drugs bound to the hydrophobic moiety of lamellar bodies. The order of binding capacities was promethazine greater than chloramphenicol greater than amiodarone = desethylamiodarone greater than promazine greater than chlorpromazine greater than trimipramine greater than propranolol greater than imipramine much greater than chlorphentermine, phentermine, chloroquine, chlorimipramine, cyclizine and chlorcyclizine. Two binding affinities were calculated for all the bound drugs. Binding affinities to hydrophilic sites of lamellar bodies were calculated in terms of emission coefficients for 1-anilino-8-naphthalene sulfonate in the presence of drugs. Hydrophilic binding was in the order chlorpromazine greater than chlorimipramine greater than promazine greater than trimipramine greater than imipramine greater than chlorcyclizine greater than propranolol greater than promethazine greater than chlorphentermine greater than cyclizine greater than phentermine greater than chloroquine much greater than chloramphenicol, amiodarone and desethylamiodarone. The binding affinities of chlorinated analogs were stronger to hydrophilic sites when compared to the parent compound. Amiodarone, which is known to induce pulmonary phospholipidosis and its major non-polar metabolite, desethylamiodarone, bound strongly to lamellar bodies. These two drugs also inhibit phospholipases in vitro. The drugs with weak phospholipidosis-inducing capacity and extensive in vivo metabolism, namely, imipramine, chlorpromazine and promazine, also bound strongly to lamellar bodies with hydrophilic as well as hydrophobic interactions. On the other hand, chloroquine, which is known to induce phospholipidosis and to inhibit phospholipases, did not bind to lamellar bodies. Two major conclusions could be drawn from this study: one is that the drug interactions with isolated lamellar bodies could be studied using membrane fluorescence probes, 1,6-diphenyl-1,3,5-hexatriene and 1-anilino-8-naphthalene sulfonate; second is that the amphiphilic drugs bind to lamellar bodies, as reported for phospholipid vesicles, and the binding of drugs to lamellar bodies could be correlated with their phospholipidosis-inducing capacity only if

Effects of drug-induced pulmonary phospholipidosis on lung mechanics in rats.

Chronic administration of amphiphilic drugs to rats induces pulmonary phospholipidosis (P), a disease characterized by accumulation of phospholipids and large foamy macrophages in alveolar spaces. We investigated whether P induced by chlorphentermine (CPH) causes changes in lung volumes and mechanics in this species. Groups of rats were fed CPH (50 mg.kg-1.day-1) for 1, 2, 3, 5, 9, and 14 wk. After each treatment period, lung volumes and mechanics were studied in the anesthetized, paralyzed, supine rat. Partial pressure-volume (PV) curves were developed at 3 and 6 ml above functional residual capacity (FRC; PV3, PV6), followed by maximal [up to total lung capacity (TLC)] PV curves. FRC was determined by saline displacement. Lungs were then fixed for histopathological examination. A subgroup of animals was allowed a recovery period of 6 wk, after the 9 wk of CPH administration. Pair-fed rats served as controls (CTR) at each time point. Lung weight increased in CPH-treated (CPH-T) rats from 1.5 +/- 0.2 (SD) g at week 1 to 5.8 +/- 1.4 g at week 14, reflecting the development of P. TLC, FRC, transpulmonary pressure at FRC, the shape of maximal PV curves, and static expiratory lung compliance computed from maximal PV data points did not change in CPH-T rats. However, partial PV curves of CPH-T lungs (particularly PV3) were shifted downward and to the right of those of CTR at 2, 3, 5, and 9 wk, indicating increased recoil pressure in phospholipidotic lungs at these time points.(ABSTRACT TRUNCATED AT 250 WORDS)

Protection of chlordecone-potentiated carbon tetrachloride hepatotoxicity and lethality by partial hepatectomy.

Chlordecone (CD) pretreatment is known to markedly potentiate CCl4 hepatotoxicity. Previous studies have shown that prior exposure to CD obtunds the increased hepatocellular regeneration and repair observed in non-treated rats challenged with a single, low dose of CCl4. These observations allowed us to hypothesize that suppression of hepatic regeneration and tissue repair by CD + CCl4 combination treatment might be involved in this interaction. To test this hypothesis, CCl4 hepatotoxicity was evaluated in actively regenerating livers using CD-treated (10 ppm in the diet for 15 days), surgically partially hepatectomized (PH) male Sprague-Dawley rats. Rats undergoing no surgical manipulation (CTRL) and sham operation (SH) were included as appropriate controls. Surgical manipulations were conducted on day 15 of the dietary protocol. Based on liver-to-body weight ratios (LW/BW), mitotic indices, hepatic cytochrome P-450 content, and hepatic glutathione (GSH and GSSG) levels, PH-induced hepatocellular regeneration was not affected by pretreatment with CD. Thus, the PH model was considered valid for assessing the effects of CD + CCl4 combination treatment. CCl4 (100 microliter/kg; i.p.) was administered 1, 2, 4 or 7 days after the surgical manipulations. Hepatotoxicity was assessed 24 h later by measuring LW/BW and serum enzymes (SGPT, SGOT and ICD) in all four groups. Hepatic histopathological, histomorphometric and lethal effects were assessed in animals receiving CCl4 1 or 7 days after the surgical manipulations. CCl4-induced increases in LW/BW were observed in CD + PH rats receiving CCl4 4 or 7 days post-PH, but not in the 1 or 2 day post-PH groups in which the hepatocellular regeneration was maximal. CCl4-induced serum enzyme elevations were significantly less in the CD + PH rats as compared to CD + SH. This decrease in the serum enzyme elevations was most prominent in the 1 day post-PH group, where the hepatocellular mitotic activity was most pronounced. CCl4 lethality, assessed in the 1 day post-surgical manipulation group, was also decreased in the CD + PH rats in comparison to CD + SH rats. Such a protection was not observed in rats receiving CCl4 7 days post-PH. These data are consistent with and are supportive of the hypothesis that a suppression of otherwise normally stimulated hepatocellular regeneration following low-dose CCl4 administration is involved in the marked amplification of CCl4 toxicity by CD.

Amplification of chloroform hepatotoxicity and lethality by dietary chlordecone (kepone) in mice.

Male Swiss Webster mice (25-30 g) maintained on powdered control diet, or on diets containing chlordecone (CD, 10 ppm), mirex (M, 10 ppm), or phenobarbital (PB, 225 ppm) were used in this study. At these low levels, chlorinated hydrocarbon pesticides are not toxic, they neither affect food or water consumption, nor the body weight of mice. After a 15-day dietary protocol, a single challenge dose of CHCl3 (0.1 ml/kg) was administered intraperitoneally in corn oil vehicle. Liver damage was assessed 24 hours later using serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, histopathology, and lethality. For comparison, serum enzymes were measured in a separate group of mice receiving a high dose of CHCl3 (1.0 ml/kg) alone. None of the dietary treatments alone affected any of the serum transaminases. The serum enzymes were remarkably elevated in the mice treated with CD and CHCl3. A high dose of CHCl3 (1.0 ml/kg) elevated the serum enzymes more than 10-fold over those in the mice fed normal diet receiving only the corn oil vehicle. The histopathology of the liver indicated midzonal necrosis typical of liver injury from CHCl3 and depletion of PAS positive glycogen deposits. These effects were not evident in mice treated with 0.1 ml/kg CHCl3 alone. Additional histological alterations in the livers of the CD + CHCl3 group include the degenerated cells, loss of basophilic staining characteristics, and an increased degree of cytoplasmic vacuolation. The amplification of CHCl3 hepatotoxicity by CD was also reflected by a 4.2-fold increase in lethality determined by 48-hour LD50.(ABSTRACT TRUNCATED AT 250 WORDS)

Pentachlorophenol intoxication: report of a fatal case, with comments on the clinical course and pathologic anatomy.

A case of a 33-yr-old man who died following occupational exposure to pentachlorophenol is presented. Postmortem examination revealed cerebral edema and fatty degeneration of the viscera. Review of the literature indicates that the clinical syndrome of poisoning with the compound results from mitochondrial toxicity with derangement of aerobic metabolism.

Ultrastructural changes in Blastomyces dermatitidis after in vitro exposure to lidocaine.

Electron microscopic examination of yeasts of Blastomyces dermatitidis, exposed in vitro to concentrations of lidocaine that occur when the drug is used for topical anesthesia, showed that lidocaine rapidly damaged intracellular structures. The extent of damage was dependent on the concentration of drug and length of exposure. The observed ultrastructural changes were very similar to those reported for other drugs that directly damage membranes. This relationship suggests that the antifungal effect of lidocaine is the result of direct membrane damage.

Effect of chlorphentermine pretreatment on the distribution of chlorphentermine in isolated perfused rat lungs.

Previous studies have shown that a 7-day treatment of rats with chlorphentermine (CP) enhances total pulmonary phospholipids and significantly enhances the accumulation of CP in perfused lungs. This study was conducted to determine if the accumulation of CP was associated with a particular fraction of the lung, and if the enhanced uptake correlated with the increased phospholipids. Rats were treated daily for 7 days with 50 mg/kg CP dissolved in saline and pair-fed controls received the vehicle only. On Day 8, the rats were sacrificed, and the lungs were removed, ventilated, and perfused. [14C]Chlorphentermine (5 mumol) was added to the perfusate of control and CP-pretreated lungs. After perfusion, the lung was homogenized and subjected to standard fractionation procedures. Some lungs were examined by light and electron microscopy. After 60 min of perfusion, CP uptake by lungs obtained from CP-treated rats was enhanced in comparison to uptake by lungs from control rats. However, CP was distributed uniformly among subcellular fractions, and CP pretreatment did not alter this pattern. Pulmonary macrophages obtained from CP-treated animals contained 8 times more CP than controls. Increased CP uptake following pretreatment can be accounted for by increased CP in the macrophages of treated rats. Macrophages in the lung tissue and in the lavage fluid from rats treated with CP were more numerous and larger than in controls lungs. This suggests that a close association may be found between accumulated CP and macrophage uptake.