Studies on the uptake, metabolism, and release of endogenous and exogenous chemicals by use of the isolated perfused lung.

The isolated perfused lung is a valuable tool for studying many lung functions. The kinds of information one can obtain from the isolated perfused lung are illustrated by examples from our studies on the uptake, accumulation, and metabolism of endogenous and exogenous chemicals.

In recent years it has been recognized that the lung possesses a number of important functions other than serving as an organ for the exchange of gases. It has often been assumed that the lung plays only a passive role in the removal and metabolism of various chemicals from the circulation. Today it is recognized that the lung serves as a filter for the blood, removing and detoxifying various endogenous substances; for example, 5hydroxytryptamine (5-HT), norepinephrine (NE), and prostaglandin (PG) (1) and in other cases activating endogenous substances, e.g., angiotensin. Under normal conditions or under stress, the lung serves as an endocrine organ releasing vasoactive material into the circulation (1). In addition, the lung possesses the ability to metabolize exogenous chemicals (2). Since the lung is in direct contact with the environment, toxicity produced by environmental chemicals may be related to these nonrespiratory functions.
Many studies on the nonrespiratory lung functions have been made possible or aided by the isolated perfused lung preparation (IPL). We * Pharmacology Branch, National Institute of Environmental Health Sciences, Research Triangle Park, N. C. 27709. have used this isolated perfused rat and rabbit lung preparation to examine the uptake and metabolism of various chemicals. Figure 1 illustrates our IPL, which is a modification of the basic lung perfusion technique developed by Niemeyer and Bingham (3). Our modified perfusion apparatus can be used in three ways: (1) the perfusate or blood is recirculated, and the uptake and metabolism of a chemical by the lung is investigated by examination of the changes in the perfusate concentrations with time; (2) the perfusate is not recirculated (single pass), the labeled test compounds and labeled vascular maker dextran being injected as a bolus into the pulmonary artery and the effluent collected as a series of timed samples; (3) the perfusate is not recirculated, but lungs being perfused with "chemical-free" media are then perfused with perfusate containing the test compound. Effluent from the lung is collected as a series of timed samples during this time. In addition, the efflux of accumulated chemicals from the lung can then be studied by perfusing with "chemical-free" perfusate.
From each of these types of experiments one can obtain a variety of information concerning the uptake, accumulation, release and metabolism of  chemicals from the lung. We would like to illustrate this point using an example from our work. We have studied the accumulation and metabolism of a number of xenobiotics by the recirculating rabbit IPL (4-6). As illustrated in Figure  2, the basic amine, methadone, rapidly disappears from the circulation and accumulates in the lung. Metabolites of methadone slowly appear in the perfusate. By using this technique, we found that the rabbit IPL metabolized methadone, chlorcyclizine, pentobarbital, and parathion but did not  degrade imipramine (IMIP). However, all these chemicals, including imipramine, were degraded by lung homogenates or subcellular fractions, indicating important differences between metabolism in vitro and metabolism by IPL. Also, basic amines were found to accumulate in lung tissue with high tissue-to-blood ratios. By examination of the steady-state accumulation, we found that several mechanism(s) were responsible for the accumulation of basic amines in lung tissue (4,5). The single pass or nonrecirculating IPL is useful for examining the removal, metabolism, and release of chemicals by the lung. We have used these techniques to examine the accumulation of basic amines by the lung. The lung can be perfused with a constant concentration of a chemical and then the efflux of that accumulated chemical from the lung examined. Using this procedure we studied the accumulation of the basic amine (imipramine and methadone) by the lung. The rate of efflux of accumulated imipramine (Fig. 3)   solved into a series of exponentials indicating various pool or binding sites for the IMIP. Each of these pools, with its half-life, is in part responsible for the high accumulation of IMIP found in the lung. Further examination of these efflux rates indicated a pool with a half-life in excess of 5 hr and that the amount of imipramine remaining in the lung appeared to approach a constant amount (Fig. 4). The pool (or pools) did not result from irreversible binding and appeared to be associated with the phospholipid in the lung. We think that this pool is responsible for the persistence of imipramine and other basic amines in lung tissue and may be associated with drug-induced phospholipidosis (7). Single-pass IPL preparations are also useful in examining the mechanism responsible for the removal of chemicals from the circulation by the   lung. We have used these methods for examining the detoxification of prostaglandins by the lung. It has been established that the lung extensively degrades circulating prostaglandins (8). The enzymes responsible for this PG metabolism are found in the 100,000 g supernatant fraction of lung and thus are presumably found in the cytosol. Removal of PG was investigated by bolus injections of 3H-PGF2<, and the vascular marker '4C-dextran into the pulmonary artery of the isolated perfused lung. 3H-PGF2a metabolites appeared in the venous effluent at a later time than than the "4C-dextran (Fig. 5), which indicated a rapid uptake and release of PG by the lung. 3H-PGB1 or 3H-PGA1 was also injected as a bolus with the vascular marker 14C-dextran. These prostaglandins appeared with the '4C-dextran in the effluent and no metabolites were detected, indicating little or no removal of these PG by the lung. In contrast, PGE1, PGF2a, and PGA1 were degraded in vitro by the 100,00g supernatant. This selective degradation of PG by the lung in vivo indicates selective removal of the PG from the circulation. These data imply that PGEl and PGF2a are removed from the circulation by a transport system rather than by diffusion or binding.
To establish the existence of a pulmonary transport for PG, it is necessary to examine the unidirectional flux of the PG into the lung tissue. Rat lungs were perfused with either 3H-PGE1 or 3H-PGF20, and the vascular marker 14C-dextran at a final concentration and flow rate. By measuring the radioactivity in the effluent one can calculate the net rate of flux of radioactivity into the lung, as seen in Figure 6. After subtraction of the dextran dilution into the vascular space the rate of PG flux from the perfusate into the lung was estimated by extrapolation to zero time indicated by the arrow. At zero time, no PG has accumulated in the lung and no metabolism has occurred. The rate of PG flux was then investigated at various perfusate concentrations. The unidirectional flux of PGE1 saturated with respect to supply rate (Fig. 7). This hyperbolic relationship is consistent with the hypothesis that PGE1 is removed from the circulation by a carrier-mediated transport process.
Thus we have shown that PGE1, PGF2., but not PGA1 nor PGB1, were accumulated in the lung by the carrier-mediated process. The presence of a second unlabeled PG in the perfusate inhibited both the metabolism and uptake of a labeled PG (Fig. 7). The uptake of PGE1 in the presence of 5 nmole/ml of PGE2 was reduced. The Vmax for PGEI was unaltered by PGE2 while the Km of PGE1 increased. Thus, PGE2 was a competitive inhibitor of PGE,. This again is consistent with the hypothesis that PGs are accumulated in lung by a carrier-mediated transport process. Thus, the inactivation system for PG in rat lung consists of a transport system and subsequent enzymatic inactivation. The relative effectiveness of these systems is shown in Figure 8. At a low supply rate, approximately 80% of PGE1 was removed (into lung) and inactivated. As the supply rate increased, the percentage removal of PGEO slowly decreased to 20%. However, the percentage degradation of PGE1 decreased very rapidly with increasing supply rate, and this removed, but unmetabolized PGE1, returned to the circulation. Thus, at low concentrations the transport system is rate limiting while at higher concentrations the enzymatic inactivation becomes rate-limiting. In conclusion, we have developed and used an isolated perfused lung system that allows examination of the uptake, accumulation, metabolism and release of chemicals by the lung. Studies on some aspects of this problem would be difficult or impossible without the IPL preparation. We think that the system is highly suitable for the following: studies on the transport and metabolism of chemicals by lung; examination of accumulation of chemicals by lung; studies on metabolism of inhaled as well as circulating substances; investigation of the release of substances by the lung into the circulation or studies of the endocrine function of the lung. We believe that the IPL is a valuable tool in examination of these nonrespiratory lung functions.