Acetylated polyamines in lungs from rats with monocrotaline-induced pneumotoxicity

FUNDAMENTAL

AND

APPLIED

TOXICOLOGY

13,277-284 (1989)

Acetylated Polyamines in Lungs from Rats with Monocrotaline-Induced Pneumotoxicity’ URSZULA

ORLINSKA,

JACK W. OLSON,

SARAH ALLEN

GEBB, AND MARK

N. GILLESPIE’

Division ofPharmacology and Toxicology, University of Kentucky College of Pharmacy, Lexington, Kentucky 405360082

Received June 27,1988; accepted January 25, I989 Acetylated Polyamines in Lungs from Rats with Monocrotaline-Induced Pneumotoxicity. U., OLSON, J. W., ALLEN-GEBB, S., AND GILLESPIE, M. N. (1989). Fundam. Appl. Toxicol. 13,277-284. Multiple lines of evidence implicate the polyamines, putrescine, spermidine, and spermine in the lung injury and hypertensive pulmonary vascular disease produced in rats by the pyrrolizidine alkaloid monocrotaline. While increases in lung polyamine content evoked by monocrotaline can be attributed in part to induction of the two rate-limiting enzymes in de nova polyamine synthesis, omithine decarboxylase and S-adenosylmethionine decarboxylase, little attention has been paid to the role that catabolic interconversion processes might play in lung polyamine accumulation. Accordingly, the present study evaluated dose (lo-60 mg/ kg)- and time (O-2 1 days)-dependent effects of monocrotahne on lung contents of acetylated polyamines and on the activity of spermidine/spermine acetyltransferase (SAT), the enzyme affecting spermidine acetylation. A single subcutaneous injection of monocrotaline produced dose- and time-dependent increases in the lung contents of N’-acetylspermidine. Neither N’acetylspermine nor N’-acetylputrescine could be detected in lungs from control rats or from rats treated with monocrotahne. SAT activity also was increased in monocrotaline-treated rat lungs in a dose- and time-dependent manner that was closely related to increases in the lung burden of N’-acetylspermidine. As expected, monocrotaline also caused dose- and timedependent elevations in the lung contents of the primary polyamines, putrescine, spermidine, and spermine. Right ventricular hypertrophy, an index of sustained pulmonary hypertension, did not develop in animals treated with 10 or 20 mg/kg monocrotahne despite elevations in the lung contents ofputrescine and N’-acetylspermidine and increases in the activity ofSAT. In contrast, 30 and 60 mg/kg monocrotaline provoked right ventricular hypertrophy accompanied by elevations in the primary polyamines, N’-acetylspermidine, and SAT activity. These observations indicate that monocrotaline-induced pneumotoxicity, characterized by development of sustained pulmonary hypertension, is associated with increased activity of SAT and accumulation of N’-acetylspermidine as well as the primary polyamines. This association suggests that polyamine interconversion pathways may be important in development of monocrotaline-induced pneumotoxicity. 0 1989 society ofToxicology. ORLINSKA,

Administration of the pyrrolizidine alkaloid monocrotaline to rats provokes edematous lung injury followed by progressive pulmo’ This investigation was supported in part by grants from the National Institutes of Health (HG38495, HG 36404), the Kentucky Affiliate of the American Heart Association, and the Cystic Fibrosis Foundation. * To whom correspondence should be addressed.

nary vascular remodeling and chronic pulmonary hypertension. Because these abnormalities mimic various aspects of primary pulmonary hypertension in humans, monocrotaline-treated rats may provide clues regarding the pathogenesis of the human disorder. Pathologic studies implicate lung cell hyperplasia and hypertrophy in monocrotaline-induced hypertensive pulmonary vascu217

0272-0590189 $3.00 Copyright 0 1989 by the society ofToxic&gy. AI1 rights of reproduction in any form reserved.

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ORLINSKA

lar disease (Meyrick and Reid, 1979, 1982). Based on the recognition that the polyamines are essential for normal cell growth and differentiation (Pegg and McCann, 1982; Pegg, 1986), we have proposed that augmented polyamine contents in key resident lung cells may play a central role in monocrotaline-induced hypertensive pulmonary vascular remodeling. In support of this contention, we have shown that a single subcutaneous injection of monocrotaline increases the lung activities of ornithine decarboxylase (EC 4.1.1.17) and S-adenosylmethionine decarboxylase (EC 4.1.1 SO) (Olson et al., 1984a,b), the rate-limiting enzymes in polyamine biosynthesis, and augments the lung contents of the three “primary” polyamines, putrescine, spermidine, and spermine (Olson et al., 1984b). These increases are temporally related to development of pulmonary hypertension and right ventricular hypertrophy. More importantly, blockade of ornithine decarboxylase with the highly selective “suicide” inhibitor, cu-difluoromethylornithine, attenuates the monocrotaline-induced accumulation of lung polyamines and prevents or forestalls development of hypertensive pulmonary vascular disease, pulmonary hypertension, and right ventricular hypertrophy (Olson et al., 1984b, 1985; Gillespie et al., 1985). In addition to de nova synthesis of polyamines, their cellular content can be regulated by catabolic interconversion pathways involving the acetylated intermediates, N’acetylspermidine and N’-acetylspermine (Pegg and McCann, 1982; Pegg, 1986). These intermediates are formed by the action of cytosolic sperminelspermidine-W-acetyltransferase (SAT: EC number not yet assigned) on their respective “primary” polyamine precursors. The acetylated intermediates are natural substrates for polyamine oxidase, which degrades N’-acetylspermidine and N’-acetylspermine to form putrescine and spermidine, respectively. It is not known whether these interconversion pathways can be induced in the lung by injurious stimuli and, as such, it is

ET AL.

uncertain whether they contribute to the increased lung burden of polyamines believed to be important to the development of monocrotaline-induced pneumotoxicity. Accordingly, the objectives of the studies reported herein were to determine whether monocrotaline administration to rats promoted doseand time-dependent increases in the lung burden of acetylated polyamines and in the activity of SAT and to determine whether these changes were temporally related to accumulation of the primary polyamines and to development of right ventricular hypertrophy indicative of pulmonary hypertension. METHODS Animal model. Adult male Sprague-Dawley rats weighing 225-275 g were given a single subcutaneous injection of monocrotaline in doses ranging from 10 to 60 mg/kg. Additional animals received equivalent volumes of the vehicle for monocrotaline, saline. Monocrotaline and its vehicle were prepared as described previously (Hayashi and Lalich, 1967). Two types of studies were performed: To evaluate dose-dependent effectsof monocrotaline on lung polyamine biosynthesis, animals were treated with a range of monocrotaline doses and sacrificed 2 1 days thereafter. To assesstime-dependent effects of monocrotahne on lung polyamine synthesis, animals received an injection of 60 mg/kg monocrotaline and were studied at Days 1,7, and 2 1 post-treatment. In both cases, vehicle control animals were treated identically and were studied at the same time points. Additional experiments were conducted to determine if exogenous spermidine could be converted in vivo to putrescine via the acetylated intermediate. In this instance, spermidine, 0.8 mmol/kg, was injected intraperitoneally and animals were killed 6 hr thereafter for determination of lung polyamine contents, including N’-acetylspermidine, and SAT activity. To detect the presence of chronic pulmonary hypertension, the extent of right ventricular hypertrophy was evaluated as described previously (Meyrick and Reid, 1979; Olson et aZ., 1984a,b, 1985). Immediately after the animals were killed, the heart was excised, the atria were removed, and the right ventricular free wall (RV) was dissected from the left ventricle plus septum (LV + S). The RV and LV + S were then weighed separately to the nearest milligram and the ratio RV (LV + S) was used as an index of the extent of right ventricular hypertrophy. Polyamine and cytosolic SAT analysis. Lungs removed from control and monocrotaline-treated rats were cleared of excess tissue, blotted, weighed, frozen,

POLYAMINES

IN LUNG

and stored at -8o’C until subsequent determination of polyamine contents and SAT activity. Frozen lungs were homogenized in 4 vol of 0.2 N HClO, using an UltraTurrax homogenizer (full speed, three bursts for 20 set each). Homogenates were then centrifuged at 40,OOOgfor 20 min and polyamine contents in the resulting supematants were determined according to methods described by Stefanelli et al. (1986) and Kabra and co-workers ( 1986). In brief, polyamines in the resulting supematants were derivatized with dansyl chloride and transferred to a Bond-Elut Cls column, and dansylated polyamines were eluted with 1500 ~1 methanol. Twenty-five-microliter aliquots of the methanol elutrate were applied to a 5-pm reversed-phase Beckman Ultrasphere ODS HPLC column (4.6 mm X 25 cm). The dansylated polyamines were separated by gradient elution at a tlow rate of 1.0 ml/min using acetonitrile:methanol:water (5:3:2) as solvent A and acetonitrile:methanol(3:2) as solvent B. The gradient consisted of linear increases in solvent B from 10 to 95% within 29 min followed by a decrease from 95 to 10% solvent B from 37-39 min and then a column reequilibration period of 7 min. Polyamines were quantitated using a Beckman Model 157 fluorescence detector with a limit of detection for the dansyl derivatives of less than 1 pmol. Lung polyamine contents are expressed in terms of the amount present per total wet lung. The activity of cytosolic SAT was determined according to methods described by Erwin et al. (1984). SAT activity was assayed in a 100~~1reaction volume containing approximately 100 pg supematant protein (derived from a 100,OOOgcentrifugation), 300 nmol spermidine, 0.8 nmol(40 nCi) of [I-?Z]acetyl coenzyme A, and 10 rmol of Tris-HCl (pH 7.8). After 10 min at 3o’C, the reaction was terminated by addition of 20 ~1 of 1 M NH,OH-HCl. Protein was then precipitated by boiling the samples for 3 min and removed by centrifugation. Fifty microliters of the supematant was applied to cellulose phosphate paper and then washed five times with water and five times with ethanol. Subsequently, the cellulose phosphate paper was placed in vials containing 5 ml scintillation fluid and radioactivity was determined using a Beckman liquid scintillation spectrometer. SAT activity was expressed as transfer of radiolabeled acetylCoA to spermidine per 10 min incubation time per total lung weight. As noted above, lung contents of the polyamines and SAT activity are expressed in terms of amount or activity, respectively, per total lung. As reported previously (Olson et al., 1984a), the parameter used to normalize these values, i.e., wet or dry lung weight, protein content, etc., influences the magnitudes of differences between monocrotahne-treated and control lungs. This is expected based on the ability of monocrotaline to promote changes in lung weights (both wet and dry) and DNA and protein contents (Meyrick and Reid, 1979, 1982). Nevertheless, the qualitative effects of monocrotaline on lung polyamine content or enzyme activities are appar-

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Dose

MCT

(mglkg)

FIG. 1. Dose-dependent effects of monocrotaline (MCT) on rat lung contents of N’-acetylspermidine, Control animals received the vehicle for monocrotaline. Contents of IV’-acetylspermidine were assessed 2 1 days after monocrotaline treatment. Each point is the mean + SE for six animals. *Significantly different from control at p < 0.05.

ent regardless of the manner in which the data are. normalized. Because it is difficult to defend any of the above normalization procedures, we elected to express polyamine contents and SAT activity in terms of the total lung. Statistics. Data are presented as means plus or minus the standard errors. Depending on the specific experimental protocol, either one- or two-way analyses of variance combined with Dunnett’s test when appropriate were employed to evaluate time- and/or monocrotahne dose-dependent differences. A p value equal to or less than 0.05 was considered to denote statistical significance.

RESULTS Dose-dependent effects of monocrotaline on lung contents of IV’-acetylspermidine are shown in Fig. 1. Monocrotaline treatment increased the lung content of Nr-acetylspermidine at all doses examined, including the lowest dose of 10 mg/kg. Time-dependent effects of 60 mg/kg monocrotaline on lung IV’-acetylspermidine content are shown in Fig. 2. Although there was a tendency for N’acetylspermidine to be increased as early as Day 7 after monocrotaline, a significant elevation was not observed until Day 21. Neither IV’-acetylputrescine nor IV’-acetylsper-

ORLINSKA

ET AL.

01 0 Time

(days)

FIG. 2. Timedependent effects of 60 mgjkg monocrotaline on rat lung content of N’-acetylspermidine (0). Control animals (u) received the vehicle for monocrotaline. Each point is the mean + SE for six animals. *Significantly different from time-matched control at p c 0.05.

mine could be detected at any dose of monocrotaline or at any time after treatment with 60 mg/kg of the alkaloid (data not shown). The increases in lung N’-acetylspermidine content provoked by monocrotaline were associated with dose- and time-dependent increases in lung SAT activity. As shown in Fig. 3, SAT activity tended to be elevated by the 10 mg/kg dose of monocrotaline and was significantly and progressively elevated with in-

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Time

I

15

20

25

(days)

FIG. 4. Time-dependent effects of 60 mgjkg monocrotaline on activity of rat lung spermidine/spermine N’acetyltransferase (SAT: 0). Control animals (m) received the vehicle for monocrotaline. Each point is the mean f SE for six animals. *Significantly different from timematched control at p c 0.05.

creasing doses of the alkaloid. Time-dependent changes in SAT activity induced by 60 mg/kg monocrotaline are depicted in Fig. 4. SAT activity was augmented as early as 7 days after monocrotaline administration and remained so until the 2 1-day time point. As expected, monocrotaline produced dose-dependent increases in the lung contents of the primary polyamines, putrescine, spermidine, and spermine. As shown in Fig. 5, putrescine was increased at all monocrotaline doses examined while spermidine and spermine were elevated at the 30 and 60 mg/ kg doses. Time courses for increased lung contents of the primary polyamines after administration of 60 mg/kg monocrotaline are illustrated in Fig. 6. Lung putrescine contents were augmented as early as Day 7 post-treatment and remained so throughout the 2 1-day observation period. In contrast, the contents of spermidine and spermine were not inCON 10 20 30 60 creased above controls until 21 days postDOSO MCT OWkg) treatment. As shown in Table 1, administration of exFIG. 3. Dose-dependent effects of monocrotaline (MCT) on activity of rat lung spermidine/spermine N’ogenous spermidine (0.8 mmol/kg) was assoacetyltransferase (SAT). Control animals received the ve- ciated with marked increases in the lung conhicle for monocrotaline. SAT activity was assessed 21 tents of spermidine, N’-acetylspermidine, days after monocrotaline treatment. Each point is the and, importantly, putrescine. Contents of mean f SE for six animals. *Significantly different from spermine did not change significantly. The control at p 6 0.05.

POLYAMINBS

281

IN LUNG INJURY

1982; Persson et al., 1985; Pegs and Erwin, 1985). The principal observations of the present study are that monocrotaline causes doseand time-dependent induction of lung SAT activity and associated increases in the lung content of N’-acetylspermidine. These changes occur in concert with accumulation of the

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20 MCT

30

60

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FIG. 5. Dosedependent effects of monocrotaline (MCT) on rat lung contents of the “primary” polyamines, putrescine, spermidine, and spermine. Control animals received the vehicle for monocrotaline. Contents of the primary polyamines were assessed 21 days after monocrotaline treatment. Each point is the mean f SE for six animals. *Significantly different from control at p < 0.05.

activity of SAT also was significantly augmented by exogenous spermidine. Dose- and timedependent effects of monocrotaline on the RV/(LV + S) ratio indicative of right ventricular hypertrophy are shown in Figs. 7A and B, respectively. Whereas 20 mg/kg monocrotaline tended to produce a modest degree of right ventricular hypertrophy, a significant increase in the RV(LV + S) ratio was not observed until 30 mg/kg. At 8 days post-treatment with 60 mg/ kg, there was no evidence of right ventricular hypertrophy. However, the ratio had increased by approximately 75% at 21 days post-treatment.

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DISCUSSION In a number of organs other than the lung, a wide variety of pathological and physiological stimuli have been shown to induce SAT activity and promote accumulation of N1acetylspermidine (Seiler, 1987). In the lung, the only stimuli previously demonstrated to enhance SAT activity and promote accumulation of ZV’-acetylspermidine are exogenous spermidine and spermine (Matsui et al.,

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Tlma (daya) FIG. 6. Time-dependent effects of 60 mgjkg monocrotaline on rat lung contents of the “primary” polyamines (0): (A) putrescine, (B) spermidine, (C) spermine. Control animals (a) received the vehicle for monocrotaline. Each point is the mean f SE error for six animals *Sip nificantly different from time-matched control at p c 0.05.

282

ORLINSKA TABLE 1 EFFECT

OF EXOGENOUS

SPERMIDINE

POLYAMINECONTENTSANDONLUNG Control

ON LUNG

SATAcr~vrrv Spermidine”

Pntrescine(nmol/total lung) 41.5? 3.9 292.4 k 44.4* Spermidine(nmol/total lung) 587.7 +_19.6 1250.5 + 287.8* Spennine (nmol/total lung) 327.5 zk 11.4 335.9 ? 32.5 N’-Acetylspermidine (nmol/total lung) 4.4* 1.0 102.8 f 21.6* SAT activity 127.2 + 10.4 717.2 +_ 34.7’ (pmol/total lune/ 10 min) n Spermidine was administered as an intraperitoneal injection of 0.8 mmol/kg. Animals were killed and the lungs excised for analysis of polyamines and SAT activity 6 hr aRer spermidine administration. Control animals were treated identically except they received intraperitoneal injections of isotonic saline. * Significantly different from control at p < 0.05.

primary polyamines and development of right ventricular hypertrophy. Further, in accord with previous observations (Seiler, 1987) administration of exogenous spermidine augments SAT activity and increases the contents of N’-acetylspermidine and putrestine, thereby suggesting that induction of the interconversion pathway can lead to the formation of putrescine without participation of the initial enzyme in polyamine synthesis, ornithine decarboxylase. These observations are the first to demonstrate that a pneumotoxic stimulus, monocrotaline, induces an important pathway regulating cellular polyamine contents. The lung thus appears to respond like other organs, notably the liver and kidneys, in terms of the polyamine biosynthetic response to injury. In addition, insofar as polyamine synthesis blockade affords significant protection against monocrotaline-induced lung injury and pulmonary hypertension (Olson et al., 1984a,b, 1985; Gillespie et al., 1985), these observations suggest that N’acetylspermidine along with the primary polyamines may play a central pathogenic role in this animal model of pulmonary vascular disease. The mechanisms by which polyamines in general and acetylated polyamines in particu-

ET AL.

lar are linked to hypertensive pulmonary vascular disease are somewhat speculative. Given the essential role that polyamines subserve in regulation of cell growth and differentiation (Pegg and McCann, 1982) their involvement in hyperplastic and hypertrophic responses of lung cells affecting the hypertensive vascular remodeling is likely. The cellular function of acetylated polyamines is less well understood. They could contribute to the remodeling process by directly influencing proliferation and/or differentiation of lung cells. However, as long-term elevations of acetylated intermediates in brain and other tissues do not produce overt physiological or pathological abnormalities (Blankenship and

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RG. 7. Dose (A)- and time (B)-dependent effects of monocrotaline on the RV/LV + S ratio, an index of right ventricular hypertrophy and chronic pulmonary hypertension. In the dose-response studies, animals were killed 2 I days after monocrotaline administration. Animals received 60 mg/kg monocrotaline to assessthe time response. Bach point is the mean + SE for six animals. *Significantly different from control at p C 0.05.

POLYAMINES

IN LUNG

Al Shabanak, 1983; Bolkenius et al., 1985; Bolkenius and Seiler, 1987), this possibility seems remote. Perhaps more likely is the hypothesis that acetylated polyamines contribute to hypertensive pulmonary vascular remodeling through regulating cellular contents of the primary polyamines. In this context, it has been proposed that the interconversion pathway permits resynthesis of putrescine which, in turn, promotes formation of the other polyamines without involvement of omithine decarboxylase (Seiler, 1987). In support of this contention, both the present study and earlier reports (Matsui et al., 1982; Persson et al., 1985; Pegg and Erwin, 1985) demonstrate that exogenous spermidine enhances lung SAT activity and augments the lung content of N’-acetylspermidine with the attendant accumulation of putrescine. Other postulated functions of acetylated intermediates are to promote detachment of primary polyamines from anionic binding sites (on DNA and elsewhere) and to affect rapid reduction in cellular polyamine contents (Seiler, 1987). The latter proposal is critically dependent on there being an appropriate export system for the acetylated intermediates, which has yet to be demonstrated. Clearly, understanding the precise mechanism by which the interconversion pathway interacts with the primary biosynthetic route to regulate lung polyamine content and to mediate development of monocrotaline-induced pulmonary hypertension will require additional studies. Our previous experiments on the role of polyamines in monocrotaline-induced lung injury and pulmonary hypertension have focused on the temporal relation between monocrotaline administration and changes in lung polyamine synthesis and on salutary effects of polyamine synthesis blockade (Olson et al., 1984a,b, 1985; Gillespie et al., 1985). While these observations provide compelling support for a pathogenic role for polyamines in this animal model of pulmonary hypertension, they do not establish the dose-response relation between monocrota-

283

INJURY

line, increased lung polyamine contents, and development of right ventricular hypertrophy indicative of pulmonary hypertension. In the present report, we demonstrate that monocrotaline does indeed cause dose-dependent accumulation of lung polyamines, including iV’-acetylspermidine. Only the two highest doses of monocrotaline, 30 and 60 mg/kg, caused significant accumulation of all three primary polyamines and acetylated spermidine coincident with right ventricular hypertrophy. Lower doses of monocrotaline increased both putrescine and N’-acetylspermidine contents without increases in spermidine or spermine and without development of right ventricular hypertrophy. These relations may indicate that accumulation of spermidine and spermine, but not putrescine and N’-acetylspermidine, is necessary for the expression of monocrotaline-induced pneumotoxicity. It is perhaps more likely that right ventricular hypertrophy is a rather insensitive index of the actions of monocrotaline on the lung and that the increases in putrescine and N’-acetylspermidine reflect an undetected toxic manifestation. In summary, the present study provides the first demonstration that polyamine interconversion pathways in the lung involving acetylated intermediates are induced by a pneumotoxic stimulus, monocrotaline. Insofar as the monocrotaline-induced increase in SAT activity and accumulation of Nl-acetylspermidine is dose and time dependent and appears to be temporally related to accumulation of the primary polyamines, it is reasonable to propose that the acetylated derivative is involved with the integrated polyamine biosynthetic response in this animal model of lung injury and pulmonary hypertension. REFERENCES BLANKENSHIP, J., AND AL SHABANAK, 0. A. (1983). Toxicology of N’-acetylspermidine and Ns-acetylspermidine in mice. Fed. Proc. 42,7625 (abstr.).

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inhibition

of polyamine

oxidase

in

Spevivo

is a

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GILLESPIE, M. N., DYER, K. K., OLSON, J. W., O’CONNOR, W. N., AND ALTIERE, R. J. (1985). Alpha difluoromethylornithine, an inhibitor of polyamine synthesis, attenuates monocrotaline-induced pulmonary vascular hyperresponsiveness in isolated perfused rat lungs. Res. Commun. Chem. Pathol. Pharmacol. SO, 365-378.

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KABRA, P. M., LEE, H. K., LUBICH, W. P., AND MARTON, L. J. (1986). Solid-phase extraction and determination of dansyl derivatives of unconjugated and acetylated polyamines by reversed-phase liquid chromatography: Improved separation systems for polyamines in cerebrospinal fluid, urine and tissue. J. Chromatog. 380, 19-32. MATSUI, I., Poso, H., AND PEGG, A. E. (1982). Conversion of exogenous spermidine into putrescine after administration to rats. B&hem. Biophys. Acta 719,199207.

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ET AL. midine by the cells of the pulmonary circulation and alveolar walls. Amer. J. Pathol. 106,84-94. OLSON, J. W., ALTIERE, R. J., AND GILLESPIE, M. N. (1984a). Prolonged elevation in rat lung omithine decarboxylase in monocrotaline-induced pulmonary hypertension. Biochem. Pharmacol. 33,3633-3637. OLSON, J. W., ATKINSON, J. W., HACKER, A. D., AL TIERE, R. J., AND GILLESPIE, M. N. (1985). Suppression of polyamine synthesis prevents monocrotalineinduced pulmonary edema and arterial medial thickening. Toxicol. Appl. Pharmacol. 81,91-99. OLSON, J. W., HACKER, A. D., ALTIERE, R. J., AND GILLESPIE, M. N. (1984b). Polyamines and the develop ment of monocrotaline-induced pulmonary hypertension. Amer. J. Physiol. 247, H682-H685. PEGG, A. E. (1986). Recent advances in the biochemistry of polyamines in eukaryotes. Biochem. J. 234, 249262.

PEGG, A. E., AND ERWIN, B. G. (1985). Induction of spermidine/spermine N’-acetyltransferase in rat tissues by polyamines. Biochem. J. 231,285-289. PEGG, A. E., AND MCCANN, P. P. (1982). Polyamine metabolism and function. Amer. J. Physiol. 243, C2 12c221. PERSSON, L., ERWIN, B. G., AND PEGG, A. E. (1985). Spermidine/spermine Nr-acetyltransferase: studies using a specific antiserum. In Recent Progress in Polyamine Research (L. Selmeci, M. Brosnan, and N. Seiler, Eds.), pp. 287-292. Akademiai Kiado, Budapest. SEILER, N. (1987). Functions of polyamine acetylation. Canad. J. Physiol. Pharmacol. 65,2024-2035. STEFANELLI, C., CARATI, D., AND ROSSONI, C. (1986). Separation of N’- and N8-acetylspermidine isomers by reversed-phase column liquid chromatography after derivitization with dansyl chloride. J. Chromatog. 375, 49-55.