ARCHIVES
OF
BIOCHEMISTRY
Cytochrome
AND
BIOPHYSICS
176, 103-112 (1976)
P-450 Heme and the Regulation Synthetase
D. MONTGOMERY Department
of Medicine
AND
University
Received
Acid
in the Liver’
BISSELL2
and the Liver Center,
of &Aminolevulinic
January
LYDIA
E. HAMMAKER
of California,
San Francisco,
California
94143
9, 1976
Hepatic &aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of 6-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates &aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of 6-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.
The activity of the heme synthetic pathway in the liver is determined by the rate of its initial step, the condensation of glytine and succinyl CoA to form &aminolevulinic acid (ALA).4 The mitochondrial enzyme, ALA synthetase, catalyzes this reaction and, thus, is the decisive controlling factor for the overall rate of heme synthesis in the liver (1). The activity of this enzyme appears to be regulated by the end-product of the pathway, heme, and for this reason a regulatory “pool” of hepatic heme has been postulated (2-6). While the precise mechanism of this regulation is controversial (7-g), evidence for its exist-
ence has come from numerous experimental studies. When heme is administered to rats (4, 10) or added to the incubation medium of cultured chick embryo liver cells (8, 11, 12), it blocks the response of ALA synthetase to inducing chemicals, such as phenobarbital or allylisopropylacetamide. While these data have implicated heme in the regulation of endogenous heme synthesis in the liver, the source (or sources) and metabolism of the postulated regulatory pool remained unclear, and certain inconsistencies have been noted. For example, the regulatory heme pool presumably is identical with the precursor pool for hepatic hemoprotein synthesis (2-6). Yet, exogenous heme, though it blocks induction5 of ALA synthetase, may not be available for incorporation into hemoprotein (13). Hence, it is difficult to understand how endogenous and exogenous heme con-
’ This work was aided by USPHS Grants GM21042, AM-11275, and (NLAMDD) P50 AM-18520, and by the Walter C. Pew Fund for Gastrointestinal Research. * Recipient of Research Career Development Award l-K04-GM-00149 from the National Institutes of Health, Bethesda, Md. ’ “Heme” refers to iron-protoporphyrin IX, without regard to its oxidation state, unless this is specified. 4 Abbreviations used: ALA, S-aminolevulinic acid; ETOX, endotoxin; AIA, allylisopropylacetamide.
’ Use of the term “induction” does not imply a specific mechanism for the increased activity of ALA synthetase that occurs after administration of allylthe terms, to isopropylacetamide. Similarly, “block,” “depress,” or “inhibit,” will be used interchangeably and do not imply a specific regulatory mechanism. 103
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
104
BISSELL
AND
stitute a single regulatory heme pool in the liver. The size of the regulatory pool will be affected by the various processes that either augment or deplete this heme fraction in the liver. The rate of endogenous heme synthesis undoubtedly is an important source of supply to the pool. Preformed heme, e.g., exogenously administered heme, may also add to the regulatory pool, as noted above. In the preceding paper, we studied degradation of cytochrome P-450 heme under various experimental conditions (14). A possible inference from those data was that the heme of cytochrome P-450 is not degraded directly on its apoprotein but rather dissociates feeding into a “free” heme pool prior to its degradation. In addition, it appeared that accelerated degradation of cytochrome P450 heme might be associated with impaired synthesis of endogenous hepatic heme. In rats that had been pulse-labeled with [5J4ClALA and subsequently treated with endotoxin (141, cytochrome P-450 was reduced, as a consequence of accelerated degradation of the hemoprotein. However, the specific activity of [14Chemelcytochrome P-450 was unaltered after administration of endotoxin, which suggested that compensatory synthesis of unlabeled cytochrome P-450 had failed to occur. These findings raised the possibility that endotoxin, in addition to stimulating the degradation of cytochrome P-450 heme, had blocked heme synthesis. In this paper, we have examined in detail the effect of endotoxin on heme synthesis, particularly with regard to the ability of this compound to perturb the metabolism of cytochrome P-450 heme. The experimental approach for these studies involved rats treated with allylisopropylacetamide, a porphyrinogenic compound (15, 16). A single injection of this chemical causes a striking and reproducible stimulation of hepatic ALA synthetase and, for this reason, has been widely used for studying the regulation of this enzyme in the liver (2,5,6,&g). A limitation of all such studies is that, despite the plausible hypotheses advanced (5, 61, the precise mechanism of action of allylisopropylacetamide remains unclear. In addition, it
HAMMAKER
is possible that the regulation of induced ALA synthetase differs from that of the basal or noninduced enzyme. The advantages of this approach are that ALA synthetase is stimulated to levels that are easily measurable by standard colorimetand decreased stimularic techniques, tion - as a result of experimental manipulation - is readily detectable. We have studied the effect of endotoxin, administered heme, or ALA on the induction of ALA synthetase by allylisopropylacetamide. Endotoxin appears to be a potent blocker of ALA synthetase induction, this effect being directly proportional to the ability of the compound to stimulate degradation of cytochrome P-450 heme. Administered heme also was found to block induction of ALA synthetase, as had been shown previously (2, 5, 10). As with endotoxin, the effect of heme correlates closely with its ability to perturb the metabolism of cytochrome P-450 heme. The data suggest that cytochrome P-450 heme, prior to its degradation, dissociates from its apoprotein and mixes with the regulatory heme pool in the liver (17). MATERL4LS
AND
METHODS
The animals, sources of materials, and preparation of endotoxin suspension were described in detail in the companion paper (14). Allylisopropylacetamide was provided by Hoffman-La Roche, Inc., Nutley, New Jersey. This compound was dissolved at a concentration of 15 mg/ml in isotonic saline and administered intraperitoneally. Animals were fasted for 24 h and then received 300 mg/kg as a single injection. In all studies involving administration of heme, the compound was injected as methemalbumin, prepared as described previously (141. The method of Marver et al. (181 was used for measurement of ALA synthetase activity. For each determination, livers from three animals were pooled. The amount of protoporphyrin in the liver also was measured by the method of Schwartz et al. (19), as an independent parameter of the porphyrinogenic effect of allylisopropylacetamide. Preliminary studies indicated that the inducing compound caused comparable stimulation of ALA synthetase, protoporphyrin content, and incorporation of labeled glycine into hepatic heme in uivo. In all the experiments to be reported, ALA synthetase was measured 16-18 h after injection of the inducer, allylisopropylacetamide, and was assumed to be maximal at that time. This assumption is based on previous investigations which have shown that after a single
REGULATION
OF HEPATIC
HEME
105
SYNTHESIS
toxin alone (without the inducer) caused no increase in ALA synthetase and in fact may have depressed the basal activity of the enzyme (data not shown). The latter possibility is under study with a radiometric method for ALA synthetase, since measurement of basal activity represents the lower limits of the sensitivity of the colorimetric assay. To rule out a trivial or nonspecific “toxic” effect of endotoxin on ALA synthetase, further studies were conducted, as RESULTS follows. Total hepatic protein synthesis Effect of Endotoxin on Induction of ALA was examined 5 h after administration of Synthetase by Allylisopropylacetamide endotoxin or saline to rats and was found The effect of endotoxin on induction of to be unaltered, as judged by the incorpoALA synthetase is shown in Table I. In ration of labeled leucine into protein in uiuo (see Materials and Methods). Drugthese studies, endotoxin was administered metabolizing activity was studied, since 2 h prior to injection of allylisopropylacetamide, because previous studies with ad- stimulation of this system by endotoxin ministered heme had indicated that heme might result in accelerated elimination of is maximally effective as a blocker of in- allylisopropylacetamide from the liver and duction when given 2 h prior to the inducer consequently a decreased effect of the inducer (27). However, hepatic drug-metabo(2). The dose of endotoxin was stimulatory for the degradation of cytochrome P-450 lizing activity tended to be diminished, heme (14). The results indicate that endo- rather than stimulated, after endotoxin toxin is a highly effective blocker of the (Table II). We explored the possibility that induction of ALA synthetase and of the endotoxin caused formation of an in vitro increase in hepatic protoporphyrin content inhibitor of ALA synthetase activity. This was investigated by incubating mixed that results from injection of allylisopropylacetamide. Administration of endo- liver homogenates from control or endointraperitoneal injection of the inducer, maximal stimulation of ALA synthetase is achieved by 12 h and maintained for at least 8 h thereafter (2, 20). Total hepatic protein synthesis was measured by the incorporation of a pulse of [Wleucine (301 Ci/ mol, New England Nuclear, Corp.), 7 &i/rat, into hepatic protein (21) over a 20-min period after injection of the label. Other methods were as follows: heme oxygenase (14), p-nitroanisole 0-demethylase (22), NADPH-cytochrome c reductase (23), aminopyrine N-demethylase (24), cytochrome P-450 (251, and protein (26).
TABLE INDUCTION OFALA
Treatment?
ALA synthetase nmol ALA/g liver/30 (mean + SD)
Saline AIA AL4 + endotoxin a Animals sacrifice.
I
SYNTHETASE AND PROTOPORPHYRIN PRODUCTION BY ALLYLISOPROPYLACETAMIDE (AIA): EFFECT OF ADMINISTERED ENDOT~XIN
were injected
Hepatic
promporphyrin pg/liver (mean * SD)
min
17.2 -c 8.9 (n = 10) 73.9 k 21.3 (n = 10) 28.0 z? 8.3 (n = 5) intraperitoneally.
Endotoxin
TABLE
was injected
3.2 + 3.1 (n = 12) 13.9 ? 6.5 (n = 12) 2.9 ‘- 1.2 (n = 16) 18 h, and AIA or saline 16 h, prior to
II
EFFECT OF ENDOTOXIN ON THE DRUG-METABOLIZING SYSTEM IN RAT LIVER Aminop rine NA;h;Ilm;ytoTreatment Cytochrome P-450 p-Nitroanisole (nmol/mg protein) 0-demethylase N-demet x ylase c reductase (nmol of product formed/min/mg protein) Isotonic saline (n = 4) Endotoxin” (n = 4)
0.20 -+ 0.05 0.14 2 0.02
L Endotoxin, 1.5 mg/kg, was given 11 h prior to sacrifice : 20,OOOg supernatant of liver homogenate.
0.30 r 0.09 0.19 2 0.05 of the animals.
19.8 ” 3.5 18.2 ? 2.2 Assays were carried
1.5 2 0.4 1.2 * 0.2 out on the
106
BISSELL
AND
toxin-treated animals, and no evidence for an in vitro inhibitor was found (Table III). The low control value for ALA synthetase in this study (cf. Table I) reflects the level of enzyme in the liver sample rather than variation in the assay. Finally, inhibition of the induction process was found to depend on the time of injection of endotoxin, relative to the administration of allylisopropylacetamide. Endotoxin was most effective when given with, or just before, the inducer, although it exhibited some effect when injected 20-30 h prior to inducer. By contrast, administration of endotoxin 15 h ufier allylisopropylacetamide (1 h prior to sacrifice of the animals) failed to alter the induction seen in control animals (Fig. 1). These data suggest that endotoxin exerts a relatively specific effect on ALA synthetase induction in the liver. In seeking to relate the effect of endotoxin on the induction of ALA synthetase to its ability to stimulate degradation of cytochrome P-450 heme, we examined the dose range for these two responses to the compound. The results are shown in Fig. 2. Endotoxin was effective as a blocker of TABLE
Treatment
100” 50 -
AIA (%I
100” 50 50
FIG. 1. Effect of endotoxin on induction of ALA synthetase in relation to the time of endotoxin administration. Groups of three animals were treated with endotoxin (ETOX) (1.5 mg/kg) at the indicated times and/or with allylisopropylacetamide (AIA) as shown. The activity of ALA synthetase was determined 16 h after injection of the inducer in all groups of animals. The values from animals treated with endotoxin (horizontal bars) are expressed as a percentage of the activity in animals treated with allylisopropylacetamide alone.
III
ALA SYNTHETASE ACTIVITY IN MIXED EXTRACTS OF SALINE-, ALLYLISOPROPYLACETAMIDE (AIA)-, OR AIA + ENDOTOXIN-TREATED LIVER”
Saline (%I
HAMMAKER
AIA + endotoxin (%I 100” 50
ALA synthetase (nmol ALA/ g/30 min)
7 90 29 47 (48)’ 52 (59)”
n Three rats in each treatment group were fasted for 24 h and then injected intraperitoneally with isotonic saline, AIA, or AIA + endotoxin. The rats were sacrificed 16 h later and livers were removed and pooled in each treatment group. Extracts were prepared as described in Methods-and assayed for ALA synthetase, either alone or in mixtures as indicated in the table. * Percentage of liver extract in the assay mixture derived from animals treated as indicated. c Figure in parentheses indicates the expected activity.
DOSE OF ENDOTOXIN
hg
kg;
FIG. 2. Dose-response of endotoxin and inhibition of ALA synthetase activity (A) or stimulation of WO production from [‘4C-hemelcytochrome P-450 (B). Endotoxin in varying doses was administered to fasted rats, followed 2 h later by injection of allylisopropylacetamide. Hepatic ALA synthetase activity was determined 16 h after injection of the inducer. For measurement of the rate of ‘%O production from labeled cytochrome P-450 heme, pairs of animals were treated with a pulse of [5-‘ClALA, as described previously (141, and then injected either with endotoxin or isotonic saline according to previous protocol (14). The points and brackets (Fig. 2B) represent mean values * SD.
ALA synthetase induction at doses as low as 0.05 mg/kg and was maximally effective at approximately 0.15 mg/kg. By contrast, detectable stimulation of 14C0 production- under the protocol described in the preceding paper (14) -required doses
REGULATION
OF HEPATIC
greater than 0.50 mg/kg. These data appeared to indicate a lack of relationship between the rate of degradation of cytochrome P-450 heme and regulation of ALA synthetase induction. However, it remained possible that doses of endotoxin between 0.01 and 0.50 mg in fact had caused a shift of cytochrome P-450 heme to the postulated regulatory pool but failed to stimulate 14C0 production -either because the CO measurements were insensitive to small changes in the rate of heme degradation or because the labeled heme transferred to the regulatory pool had been reutilized in the synthesis of hemoprotein, in place of endogenously formed heme. To test these possibilities, a “chase” experiment was designed, in which unlabeled ALA was co-administered with endotoxin. The rationale was that unlabeled heme, formed from the injected ALA, would transiently flood the regulatory pool, chasing labeled heme to the degradative mechanism and/or preventing its re-uptake into hemoprotein. Effect of Administered ALA on Production of 14C0 and Induction of ALA Synthetase by Allylisopropylacetamide In preliminary experiments, the effect of ALA alone, 5 mg/kg, on 14C0 production was studied, according to the standard protocol (14). ALA was found to stimulate a small but reproducible increase in 14C0 excretion (Fig. 3). Next, ALA was given together with doses of endotoxin that, when injected alone, failed to stimulate 14C0 production (less than 0.5 mg/kg). In the experiment shown in Fig. 4, ALA was administered together with 0.15 mg/kg
--
0
4
6
8
HEME
SYNTHESIS
I
1 2
4
6
8
IO
12
HOURS
FIG. 4. Effect of co-administered ALA and endotoxin on production of WO from hepatic [Wlheme. The experimental procedure was similar to that described for Fig. 3.
endotoxin, and a substantial peak of 14C0 emerged which was of greater magnitude and somewhat delayed, by comparison with the increase that followed injection of ALA alone. The time-course of the peak from the combined endotoxin-ALA treated animals approximated that from animals given larger doses of endotoxin alone. Because of this finding, the dose-response range for stimulation of 14C0 production was examined between 0.01 and 0.50 mgl kg of endotoxin, each dose being administered together with 5 mg/kg of ALA. As demonstrated in Fig. 5, when examined in the presence of co-administered ALA, endotoxin stimulated 14C0 production over precisely the same dose range as that for inhibition of ALA synthetase (cf. Fig. 5 and Fig. 2A). The effect of endotoxin on induction of ALA synthetase was not altered by co-administration of ALA, as judged by the fact that injection of ALA alone had no significant effect on induction of ALA synthetase or on the increase in hepatic protoporphyrin caused by allylisopropylacetamide (Table IV). In the study shown, ALA was administered 2 h after injection of allylisopropylacetamide. Comparable results were obtained when ALA was injected with, or 2 h prior to, the inducer.
IO
HOURS
FIG. 3. Effect of administered ALA on production of WO from hepatic [Wlheme. Pairs of animals were studied according to the protocol presented previously (14). ALA was injected intraperitoneally; the control animal received an equal volume of isotonic saline by the same route.
Effect of Administered Heme on Induction of ALA Synthetase: Correlation with Stimulation of Hepatic Heme Oxygenuse Activity The studies with endotoxin indicated a close correlation between stimulation of
BISSELL AND HAMMAKER
108
z I
TABLE V
0
G 2l)o ZU 0 t=Z 40 It, gg
0
EFFECT OF ADMINISTERED HEME ON HEPATIC HEME OXYGENASE ACTIVITY AND ON INDUCTION OF ALA SYNTHETASE AND PROTOPORPHYRIN FORMATION BY THE LIVER AFTER ALLYLISOPROPYLACETAMIDE (AL4Y’
Treatment
ALA synthetase (rim01 ALA/g/ 30 min)
Heme oxygenase (rin$l+’ min/lO me)
Heme-free albumin; isotonic saline Heme-free albumin; AIA Heme: AL4
27
0.15
6.2
77
0.11
39.2
28
0.61
13.2
100
:2 ;; -9
0
l!!l//c
05
10
15
DOSE OF ENDOTOXIN Img/kg ADMINISTERED WITH 5mg/kg ALA
FIG. 5. Dose-response of co-administered endotoxin and ALA for stimulation of WO production. In a series of studies similar to that shown in Fig. 4, varying doses of endotoxin were administered together with a fixed amount of ALA (5 mg/kg) and the effect of peak 14C0 production was determined. TABLE IV EFFECT OF ADMINISTERED ALA ON INDUCTION OF ALA SYNTHETASE OR PROTOPORPHYRIN SYNTHESIS BY ALLYLISOPROPYLACETAMIDE (AIA)”
Treatment
ALA synthetase (nmol ALA/ g/30 min)
Protoporphyrin (cLg/liver)
Isotonic saline (two injections) Isotonic saline; ALA AIA; isotonic saline AIA; ALA
28 5 6
4.3 -+ 3.1
27 2 4 92 2 9 102 ?z 29
16.3 -e 5.8 19.2 k 9.6
3.9 2 1.5
a Nine rats were assigned to each treatment group and fasted for 24 h. According to the indicated protocol, rats were injected with saline or AIA and then, 2 h later, with saline or ALA (5 mg/kg). The animals were sacrificed 16 h after the final injection. Livers from groups of three rats were pooled, homogenized, and assayed as described in Methods. The results are expressed as mean t SE.
ProtoKo’: p yrm (Pd liver)
D Rats were fasted for 24 h and then treated in groups of three animals, receiving heme (25 pmol/ kg) or heme-free albumin 18 h prior to sacrifice; 2 h later, AIA was administered. Both injections were given intraperitoneally. Liver from the three animals of each group was pooled and assayed as described in Methods.
ALA synthetase and the increase in hepatic protoporphyrin content. To assess the relationship of this effect to changes in the degradation of cytochrome P-450 heme, exogenous heme was administered in a range of doses 2 h prior to administration of allylisopropylacetamide. Rats were sacrificed 16 h after injection of the inducer, for determination of heme oxygenase or ALA synthetase activity, respectively. The results revealed a close reciprocal relationship between stimulation of heme oxygenase activity and inhibition of ALA synthetase for a given dose of administered heme (Fig. 6). DISCUSSION
14C0 production by the compound and inhibition of ALA synthetase induction. This approach was extended to studies of administered heme, since heme was shown in the companion paper (14) to stimulate 14C0 production and is known to block induction of ALA synthetase (2-6). The latter finding was confirmed, as is demonstrated by the data in Table V. Administration of 25 pmol/kg of heme, 2 h prior to injection of allylisopropylacetamide, blocked almost completely stimulation of
These studies provide evidence that cytochrome P-450-associated heme has metabolically important functions in addition to being a constituent of cytochrome P-450. The findings of the preceding paper linked the degradation of cytochrome P-450 heme to stimulation of hepatic heme oxygenase activity. The present data suggest that a similar or identical heme fraction participates in the regulation of hepatic ALA synthetase. A hypothesis that encompasses both of these effects of cytochrome
REGULATION
3 4 DOSE OF HEME j,, molerj
OF HEPATIC
5
FIG. 6. Dose-response of heme for stimulation of hepatic heme oxygenase and inhibition of ALA synthetase induction. Groups of three rats were injected intraperitoneally with varying doses of heme in a total volume of 2 ml. Two hours later, allylisopropylacetamide was administered intraperitoneally, and the animals were sacrificed 16 h after injection of the inducer. The results are expressed as a percentage of the activity in animals treated with allylisopropylacetamide alone.
P-450 heme is presented in Fig. 7.‘j According to this scheme, the regulatory heme pool is fed both by endogenous synthesis and by dissociation of heme from cytochrome P-450. The suggestion that heme dissociates from the apocytochrome appears to be the most plausible and parsimonious explanation for the effect of endotoxin or administered heme on the induction of ALA synthetase by allylisopropylacetamide. The lack of direct toxic effect of endotoxin and the correlation with stimulation of 14C0 production and heme oxygenase activity all suggest that heme detached from cytochrome P-450 mixes, prior to its degradation, with the regulatory heme pool for ALA synthetase activity. Since it is assumed that the regulatory pool is the precursor heme pool for hemoprotein formation (2-6), these data lead to the conclusion that heme moves bidirectionally between the regulatory pool and apocytochrome P-450. Moreover, the 6 The mechanism of regulation of ALA synthetase by heme is still an open question (7-91, and the “feedback” loop in Fig. 7 does not imply a specific mode of regulation for the enzyme.
HEME SYNTHESIS
109
“chase” effect of administered ALA - presumably involving the same regulatory heme pool -suggests that a bidirectional flux of heme exists in the steady state, since administered ALA, without co-administered endotoxin, provokes an increase in 14C0 excretion. A two-way flux of heme from the regulatory pool to apocytochrome P-450 is attractive, teleologically, since such a system would account for the exquisite sensitivity -demonstrated by the studies with low doses of endotoxin - of the heme-synthesizing mechanism to changes in the metabolism of cytochrome P-450. The possibility that heme reversibly dissociates from apocytochrome P-450 in rat liver has received little attention, although it has been suggested that the heme of cytochrome b, may exchange with precursor pool in the liver (28). Dissociation of cytochrome P-450 in vitro has not been observed, except when the hemoprotein was converted first to a denatured form (“cytochrome P-420”) (29). Thus, heme and protein usually are assumed to be tightly bound, in this cytochrome. On the other hand, the in vitro approach, involving isolated microsomes in aqueous salt solution, may not be the optimal approach to this problem, since it fails to reproduce the milieu of the heme moiety in the intact cell. Heme is synthesized in mitochondria, passing to the endoplasmic reticulum to combine with apocytochrome. The latter translocation may occur at points of close apposition of mitochondria and endoplasmic reticulum (301, in which case association or dissociation of heme presumably occurs lal. Jy in a lipid environment. Moreover, recent studies in intact hepatocytes suggest that, in the absence of administered substrate for the microsomal oxidase system, a major fraction of cytochrome P-450 exists in the oxidized (ferri-heme) state (31). This would favor dissociation of heme and apocytochrome, if this hemoprotein behaves in analogous fashion to hemoglobin; for globin, the affinity of oxidized (ferri-1 heme is much less than that of reduced (ferro-> heme (32). The hypothesis of Fig. 7 suggests two routes for disposal of heme from the regu-
110
BISSELL
FIG. 7. Proposed scheme for the regulation
AND
co of ALA synthetase
latory pool: formation of hemoprotein or oxidation of heme to bile pigment and carbon monoxide. These pathways can be inferred logically from the present and previous findings. However, important related questions can be treated only speculatively. A long-standing problem concerns the regulation of hepatic hemoprotein synthesis. With administration of phenobarbital to rats, increased activity of ALA synthetase precedes changes in the level of cytochrome P-450 (2). On this basis, it originally had been proposed that the primary effect of phenobarbital is on the rate of heme synthesis in the liver and that an increased heme pool secondarily triggered new synthesis of apocytochrome. According to the model presented here (Fig. 7), enlargement of the heme pool should stimulate heme oxygenase activity. However, treatment of rats with phenobarbital fails to produce this result. Indeed, the drug may decrease hepatic heme oxygenase activity (33).? Moreover, addition of “free” heme to microsomes by injection of ALA to rats fails to increase the amount of cytochrome P-450 in the liver (5, 34). In view of these data, phenobarbital treatment may stimulate primarily the synthesis of apocytochrome P-450, which draws heme from the regulatory pool, secondarily “derepressing” the heme synthetic pathway. In fact, recent studies, involving direct measurement of apocytochrome P-450, offer a similar conclusion (35, 36). A second route for discharge of heme from the regulatory pool is by way of its degradation. The degradative process ap7 Bissell, D. M., and Hammaker, lished data.
HAMMAKER
L. E., unpub-
and heme oxygenase
by hepatic
heme.
pears to be mediated by heme oxygenase, as judged by the close correlation between rates of 14C0 production from [14Chemelcytochrome P-450 and stimulation of heme oxygenase activity (14). A question that arises is whether heme oxygenase can be stimulated as a primary event, secondarily causing depletion of heme from the regulatory pool or from cytochrome P-450 (37). While this sequence of events is a theoretical possibility, the present data argue against it. It was shown in the preceding paper (14) that increased 14C0 production clearly precedes stimulation of heme oxygenase. Thus, stimulation of hepatic heme oxygenase appears to be a mechanism for discharge of “excess” heme from the regulatory pool. The inhibitory effect of administered heme on induction of ALA synthetase correlates well with the ability of this compound to perturb the metabolism of cytochrome P-450 heme - assessedeither as an increase in heme oxygenase activity or as stimulation of 14C0 production from labeled cytochrome P-450 heme. These data suggest that the effect of heme on ALA synthetase is largely-if not entirely- indirect. This hypothesis is consistent with the proposed scheme (Fig. 7), if it is assumed that exogenously administered heme taken up by the liver, cannot mix with the regulatory pool, i.e., it does not serve as a precursor for cytochrome P-450 (13). However, the latter point is controversial. Indeed, some of the data in the preceding paper suggest indirectly that administered heme may be incorporated into cytochrome P-450. It was shown that after injection of unlabeled heme to rats containing [14C-heme]cytochrome P-450, the specific activity of the hemoprotein was
REGULATION
OF HEPATIC
reduced. Since endogenous heme synthesis presumably is blocked under these conditions, the administered heme may be the precursor for the unlabeled hemoprotein that was formed. If exogenous heme or heme from administered ALA enter the regulatory pool, they presumably affect the regulation of ALA synthetase. Indeed, Hayashi et al. have shown that administered ALA blocks the induction of ALA synthetase (9). They used a large dose of ALA (100 mg/kg) and measured enzyme activity very early (2 h) after injection of allylisopropylacetamide. In the present studies, ALA had little effect on induction of ALA synthetase, probably because a relatively small dose was given (5 mg/kg) and enzyme activity was examined 16-18 h after injection of inducer. The dose of ALA was selected on the basis that it provided a maximal “chase” effect for 14C0 production, in the type of experiment shown in Fig. 4. Moreover, the period of 16-18 h may allow time for elimination of heme formed from ALA and, therefore, removal of its regulatory effect on induction of the enzyme. Heme formed from ALA appears to be degraded largely within l-2 h, as reflected by the size of the “early peak” of bilirubin or CO after administration of ALA (14, 38). By contrast, the half-life of allylisopropylacetamide in rat liver is 6-7 h (28). Thus, the inducer (if the native compound is the inducing species) persists much longer than does heme generated from administered ALA. Similarly, exogenous heme is converted relatively rapidly to bile pigment, exhibiting a half-life of about 2.5 h (39). It appears likely, therefore, that the modest effect of administered ALA on induction of ALA synthetase in the present studies is due to the rapid elimination of endogenous heme from the liver. By similar reasoning, a direct effect of administered heme- if it reaches the regulatory pool - on the induction process would be expected to be transient. By contrast, the effect of endotoxin or exogenous heme on the dissociation of heme from cytochrome P-450 is protracted, providing for a sustained change in the regulatory heme pool and, hence, a
HEME
SYNTHESIS
111
marked effect on induction of ALA synthetase by allylisopropylacetamide. This model (Fig. 7) has implications for studies of cytochrome turnover. Because methods for measuring apocytochrome P450 have not been available until very recently, turnover has been estimated from the rate at which pulse-labeled heme disappears from the hemoprotein (20, 40). The implicit assumption in this approach is that the heme moiety does not cycle or exchange to any extent with other heme fractions in the liver. The present findings challenge this assumption and suggest that use of a heme label may lead to an overestimation of the half-life of holocytochrome P-450. Reinvestigation of this question, with direct measurement of the turnover of apocytochrome P-450, ,appears to be needed and should be feasible with the recent preparation of highly purified cytochrome P-450. Also, it has been noted that loss of [14C!]hemefrom cytochrome P450 is bi-exponential, a finding that is consistent with two separate pools of heme in association with cytochrome P-450, with half-lives of 8 and 2440 h (20, 40). This finding has been interpreted as evidence for two types of cytochrome P-450 with differing half-lives in the liver (40). While it is now established that the liver contains multiple forms of cytochrome P-450 (41), the turnover rates of these different forms have not been analyzed individually. It seems equally probable that the two components of heme disappearance represent the rate of elimination of labeled heme from the regulatory pool and from cytochrome P-450, respectively. Finally, the model suggests that hepatic ALA synthetase and heme oxygenase are interrelated, with respect to their regulation by heme. It further predicts that these individual enzyme activities vary reciprocally-a possibility that has received recent experimental support (42). Thus, depletion of the regulatory pool would be expected to stimulate ALA synthetase but depress heme oxygenase, while repletion of the pool should affect each enzyme activity in the opposite direction. With regard to the size and regulatory effect of the
112
BISSELL
AND
postulated heme pool, synthesis and degradation of cytochrome P-450 appear to play major roles. ACKNOWLEDGMENTS We are grateful to Dr. Rudi Schmid for support and for a critical review of the manuscript, and to Dr. Philip Guzelian for stimulating discussions. Mr. Jose Aronce provided very capable technical assistance. REFERENCES 1. MARVER, H. S., AND SCHMID, R. (1972) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.), 3rd ed., p. 1087, McGrawHill, New York. 2. MARVER, H. S. (1969) in Microsomes and Drug Oxidations (Gillette, J. R., Conney, A. H., Cosmides, G. J., Estabrook, R. W., Fouts, J. R., and Mannering, G. J., eds.), p. 495, Academic Press, New York. 3. MEYER, U. A., AND SCHMID, R. (1973) Fed. Proc. 32, 1649-1655. 4. WATSON, C. J. (1975) New Engl. J. Med. 239, 605-607. 5. DE MATTEIS, F. (1971) Biochem. J. 124, 767-777. 6. TSCHUDY, D. P., AND BONKOWSKY, H. L. (1972) Fed. Proc. 31, 147-159. 7. SCHOLNICK, P. L., HAMMAKER, L. E., AND MARVER, H. S. (1969) Proc. Nut. Acad. Sci. USA 63, 65-70. 8. SASSA, S., AND GRANICK, S. (1970) Proc. Nut. Acad. Sci. USA 67, 517-522. 9. HAYASHI, N., KURASHIMA, Y., AND KIKUCHI, G. (1972) Arch. Biochem. Biophys. 148, 10-21. 10. MARVER, H. S., SCHMID, R., AND SCH~TZEL, H. (1968) Biochem. Biophys. Res. Commun. 33, 969-974. 11. GRANICK, S. (1966) J. Biol. Chem. 241, 13591375. 12. STRAND, L. J., MANNING, J., AND MARVER, H. S. (1972) J. Biol. Chem. 247, 2820-2827. 13. SCHMID, R. (1973) Drug Metab. Dispos. 1, 256258. 14. BISSELL, D. M., AND HAMMAKER, L. E. (1976) Arch. Biochem. Biophys. 176, 91-102. 15. SCHMID, R., AND SCHWARTZ, S. (1952) Proc. Sot. Exp. Biol. Med. 81, 685-689. 16. GOLDBERG, A., AND RIMINGTON, C. (1955) Proc. Roy. Sot., London, Ser. B 143, 257-280. 17. BISSELL, D. M., AND HAMMAKER, L. E. (1975) Clin. Res. 23, 384A. 18. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A. (1966)J. Biol. Chem. 241, 2803-2809. 19. SCHWARTZ, S., BERG, M. H., BOSSENMAIER, I.,
HAMMAKER
20. 21. 22. 23.
24.
25. 26.
27. 28. 29. 30.
31.
32. 33. 34. 35. 36.
37. 38. 39. 40. 41. 42.
AND DINSMORE, H. (1960) Methods Biochem. Anal. 8, 221-293. MEYER, U. A., AND MARVER, H. S. (1971) Science 171, 64-66. FARBER, J. L., AND FARMAR, R. (1973) Biochem. Biophys. Res. Commun. 51, 626-630. NETTER, K. J., AND SEIDEL, G. (196415. Pharmacol. Exp. Therap. 146, 61-65. MASTERS, B. S. S., WILLIAMS, C. H., JR., AND KAMIN, H. (1967) in Methods in Enzymology, Vol. X (Estabrook, R. W., and Pullman, M. E., eds.), p. 565, Academic Press, New York. ESTABROOK, R. W., FRANKLIN, M. R., COHEN, B., SHIGAMATZU, A., AND HILDEBRANDT, A. G. (1971) Metabolism 20, 187-199. OMURA, T., AND SATO, R. (1964) J. Biol. Chem. 239, 2370-2378. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 265-275. KAUFMAN, L., SWANSON, A. L., AND MARVER, H. S. (1970) Science 1’70, 320-322. BOCK, K. W., AND SIEKEVITZ, P. (1970) Biochem. Biophys. Res. Commun. 41, 374-380. MAINES, M. D., AND ANDERS, M. W. (1973) Mol. Pharmacol. 9, 219-228. JONES, A. L., AND EMANS, J. B. (1969) in Metabolic Effects of Gonadal Hormones and Contraceptive Steroids (Salhanick, H. A., Kipnis, D. M., and Vande Wiele, R. L., eds.), p. 68, Plenum Press, New York. MOLDBUS, P., GRUNDIN, R., VON BAHR, C., AND ORRENIUS, S. (1973) Biochem. Biophys. Res. Commun. 55, 937-944. BUNN, H. F., AND JANDL, J. H. (1968) J. Biol. Chem. 243, 465-475. ROTHWELL, J. D., LACROIX, S., AND SWEENEY, G. D. (1973) Biochim. Biophys. Actu 304,871-874. DRUYAN, R., AND KELLY, A. (1972) Biochem. J. 129, 1095-1099. CORREIA, M. A., AND MEYER, U. A. (1975) Proc. Nat. Acad. Sci. USA 72, 400-404. RAJAMANICKAM, C., RAO, M. R. S., AND PADMANABAN, G. (1975) J. Biol. Chem. 250, 23052310. MAINES, M. D., AND KAPPAS, A. (1975) J. Biol. Chem. 250, 4171-4177. ROBINSON, S. H. (1968) New Engl. J. Med. 279, 143-149. OSTROW, J. D., JANDL, J. H., AND SCHMID, R. (1962) J. Clin. Znuest. 41, 1628-1637. LEVIN, W., AND KUNTZMAN, R. (1969) J. Biol. Chem. 244, 3671-3676. GILLETTE, J. R., DAVIS, D. C., AND SASAME, H. A. (1972) Ann. Rev. Pharmacol. 12, 57-84. SCHACTER, B. A. (1975) in Jaundice (Goresky, C. A., and Fisher, M. M., eds.), p. 85, Plenum Press, New York.
OF
BIOCHEMISTRY
Cytochrome
AND
BIOPHYSICS
176, 103-112 (1976)
P-450 Heme and the Regulation Synthetase
D. MONTGOMERY Department
of Medicine
AND
University
Received
Acid
in the Liver’
BISSELL2
and the Liver Center,
of &Aminolevulinic
January
LYDIA
E. HAMMAKER
of California,
San Francisco,
California
94143
9, 1976
Hepatic &aminolevulinic acid synthetase was induced in rats injected with allylisopropylacetamide. The induction process was studied in relation to experimental perturbation of cytochrome P-450 in the liver. Animals were treated with either administered endotoxin or exogenous heme, both of which accelerate degradation of cytochrome P-450 heme. These manipulations were effective in blocking induction of 6-aminolevulinic acid synthetase, and the effect of each compound was proportional to its ability to stimulate degradation of cytochrome P-450 heme. The findings suggest that the heme moiety of cytochrome P-450 dissociates reversibly from its apoprotein and, prior to its degradation, mixes with endogenously synthesized heme to form a pool that regulates &aminolevulinic acid synthetase activity. A similar or identical heme fraction appears to mediate stimulation of heme oxygenase, which suggests that the regulation of 6-aminolevulinic acid synthetase and of heme oxygenase in the liver are closely interrelated.
The activity of the heme synthetic pathway in the liver is determined by the rate of its initial step, the condensation of glytine and succinyl CoA to form &aminolevulinic acid (ALA).4 The mitochondrial enzyme, ALA synthetase, catalyzes this reaction and, thus, is the decisive controlling factor for the overall rate of heme synthesis in the liver (1). The activity of this enzyme appears to be regulated by the end-product of the pathway, heme, and for this reason a regulatory “pool” of hepatic heme has been postulated (2-6). While the precise mechanism of this regulation is controversial (7-g), evidence for its exist-
ence has come from numerous experimental studies. When heme is administered to rats (4, 10) or added to the incubation medium of cultured chick embryo liver cells (8, 11, 12), it blocks the response of ALA synthetase to inducing chemicals, such as phenobarbital or allylisopropylacetamide. While these data have implicated heme in the regulation of endogenous heme synthesis in the liver, the source (or sources) and metabolism of the postulated regulatory pool remained unclear, and certain inconsistencies have been noted. For example, the regulatory heme pool presumably is identical with the precursor pool for hepatic hemoprotein synthesis (2-6). Yet, exogenous heme, though it blocks induction5 of ALA synthetase, may not be available for incorporation into hemoprotein (13). Hence, it is difficult to understand how endogenous and exogenous heme con-
’ This work was aided by USPHS Grants GM21042, AM-11275, and (NLAMDD) P50 AM-18520, and by the Walter C. Pew Fund for Gastrointestinal Research. * Recipient of Research Career Development Award l-K04-GM-00149 from the National Institutes of Health, Bethesda, Md. ’ “Heme” refers to iron-protoporphyrin IX, without regard to its oxidation state, unless this is specified. 4 Abbreviations used: ALA, S-aminolevulinic acid; ETOX, endotoxin; AIA, allylisopropylacetamide.
’ Use of the term “induction” does not imply a specific mechanism for the increased activity of ALA synthetase that occurs after administration of allylthe terms, to isopropylacetamide. Similarly, “block,” “depress,” or “inhibit,” will be used interchangeably and do not imply a specific regulatory mechanism. 103
Copyright All rights
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
104
BISSELL
AND
stitute a single regulatory heme pool in the liver. The size of the regulatory pool will be affected by the various processes that either augment or deplete this heme fraction in the liver. The rate of endogenous heme synthesis undoubtedly is an important source of supply to the pool. Preformed heme, e.g., exogenously administered heme, may also add to the regulatory pool, as noted above. In the preceding paper, we studied degradation of cytochrome P-450 heme under various experimental conditions (14). A possible inference from those data was that the heme of cytochrome P-450 is not degraded directly on its apoprotein but rather dissociates feeding into a “free” heme pool prior to its degradation. In addition, it appeared that accelerated degradation of cytochrome P450 heme might be associated with impaired synthesis of endogenous hepatic heme. In rats that had been pulse-labeled with [5J4ClALA and subsequently treated with endotoxin (141, cytochrome P-450 was reduced, as a consequence of accelerated degradation of the hemoprotein. However, the specific activity of [14Chemelcytochrome P-450 was unaltered after administration of endotoxin, which suggested that compensatory synthesis of unlabeled cytochrome P-450 had failed to occur. These findings raised the possibility that endotoxin, in addition to stimulating the degradation of cytochrome P-450 heme, had blocked heme synthesis. In this paper, we have examined in detail the effect of endotoxin on heme synthesis, particularly with regard to the ability of this compound to perturb the metabolism of cytochrome P-450 heme. The experimental approach for these studies involved rats treated with allylisopropylacetamide, a porphyrinogenic compound (15, 16). A single injection of this chemical causes a striking and reproducible stimulation of hepatic ALA synthetase and, for this reason, has been widely used for studying the regulation of this enzyme in the liver (2,5,6,&g). A limitation of all such studies is that, despite the plausible hypotheses advanced (5, 61, the precise mechanism of action of allylisopropylacetamide remains unclear. In addition, it
HAMMAKER
is possible that the regulation of induced ALA synthetase differs from that of the basal or noninduced enzyme. The advantages of this approach are that ALA synthetase is stimulated to levels that are easily measurable by standard colorimetand decreased stimularic techniques, tion - as a result of experimental manipulation - is readily detectable. We have studied the effect of endotoxin, administered heme, or ALA on the induction of ALA synthetase by allylisopropylacetamide. Endotoxin appears to be a potent blocker of ALA synthetase induction, this effect being directly proportional to the ability of the compound to stimulate degradation of cytochrome P-450 heme. Administered heme also was found to block induction of ALA synthetase, as had been shown previously (2, 5, 10). As with endotoxin, the effect of heme correlates closely with its ability to perturb the metabolism of cytochrome P-450 heme. The data suggest that cytochrome P-450 heme, prior to its degradation, dissociates from its apoprotein and mixes with the regulatory heme pool in the liver (17). MATERL4LS
AND
METHODS
The animals, sources of materials, and preparation of endotoxin suspension were described in detail in the companion paper (14). Allylisopropylacetamide was provided by Hoffman-La Roche, Inc., Nutley, New Jersey. This compound was dissolved at a concentration of 15 mg/ml in isotonic saline and administered intraperitoneally. Animals were fasted for 24 h and then received 300 mg/kg as a single injection. In all studies involving administration of heme, the compound was injected as methemalbumin, prepared as described previously (141. The method of Marver et al. (181 was used for measurement of ALA synthetase activity. For each determination, livers from three animals were pooled. The amount of protoporphyrin in the liver also was measured by the method of Schwartz et al. (19), as an independent parameter of the porphyrinogenic effect of allylisopropylacetamide. Preliminary studies indicated that the inducing compound caused comparable stimulation of ALA synthetase, protoporphyrin content, and incorporation of labeled glycine into hepatic heme in uivo. In all the experiments to be reported, ALA synthetase was measured 16-18 h after injection of the inducer, allylisopropylacetamide, and was assumed to be maximal at that time. This assumption is based on previous investigations which have shown that after a single
REGULATION
OF HEPATIC
HEME
105
SYNTHESIS
toxin alone (without the inducer) caused no increase in ALA synthetase and in fact may have depressed the basal activity of the enzyme (data not shown). The latter possibility is under study with a radiometric method for ALA synthetase, since measurement of basal activity represents the lower limits of the sensitivity of the colorimetric assay. To rule out a trivial or nonspecific “toxic” effect of endotoxin on ALA synthetase, further studies were conducted, as RESULTS follows. Total hepatic protein synthesis Effect of Endotoxin on Induction of ALA was examined 5 h after administration of Synthetase by Allylisopropylacetamide endotoxin or saline to rats and was found The effect of endotoxin on induction of to be unaltered, as judged by the incorpoALA synthetase is shown in Table I. In ration of labeled leucine into protein in uiuo (see Materials and Methods). Drugthese studies, endotoxin was administered metabolizing activity was studied, since 2 h prior to injection of allylisopropylacetamide, because previous studies with ad- stimulation of this system by endotoxin ministered heme had indicated that heme might result in accelerated elimination of is maximally effective as a blocker of in- allylisopropylacetamide from the liver and duction when given 2 h prior to the inducer consequently a decreased effect of the inducer (27). However, hepatic drug-metabo(2). The dose of endotoxin was stimulatory for the degradation of cytochrome P-450 lizing activity tended to be diminished, heme (14). The results indicate that endo- rather than stimulated, after endotoxin toxin is a highly effective blocker of the (Table II). We explored the possibility that induction of ALA synthetase and of the endotoxin caused formation of an in vitro increase in hepatic protoporphyrin content inhibitor of ALA synthetase activity. This was investigated by incubating mixed that results from injection of allylisopropylacetamide. Administration of endo- liver homogenates from control or endointraperitoneal injection of the inducer, maximal stimulation of ALA synthetase is achieved by 12 h and maintained for at least 8 h thereafter (2, 20). Total hepatic protein synthesis was measured by the incorporation of a pulse of [Wleucine (301 Ci/ mol, New England Nuclear, Corp.), 7 &i/rat, into hepatic protein (21) over a 20-min period after injection of the label. Other methods were as follows: heme oxygenase (14), p-nitroanisole 0-demethylase (22), NADPH-cytochrome c reductase (23), aminopyrine N-demethylase (24), cytochrome P-450 (251, and protein (26).
TABLE INDUCTION OFALA
Treatment?
ALA synthetase nmol ALA/g liver/30 (mean + SD)
Saline AIA AL4 + endotoxin a Animals sacrifice.
I
SYNTHETASE AND PROTOPORPHYRIN PRODUCTION BY ALLYLISOPROPYLACETAMIDE (AIA): EFFECT OF ADMINISTERED ENDOT~XIN
were injected
Hepatic
promporphyrin pg/liver (mean * SD)
min
17.2 -c 8.9 (n = 10) 73.9 k 21.3 (n = 10) 28.0 z? 8.3 (n = 5) intraperitoneally.
Endotoxin
TABLE
was injected
3.2 + 3.1 (n = 12) 13.9 ? 6.5 (n = 12) 2.9 ‘- 1.2 (n = 16) 18 h, and AIA or saline 16 h, prior to
II
EFFECT OF ENDOTOXIN ON THE DRUG-METABOLIZING SYSTEM IN RAT LIVER Aminop rine NA;h;Ilm;ytoTreatment Cytochrome P-450 p-Nitroanisole (nmol/mg protein) 0-demethylase N-demet x ylase c reductase (nmol of product formed/min/mg protein) Isotonic saline (n = 4) Endotoxin” (n = 4)
0.20 -+ 0.05 0.14 2 0.02
L Endotoxin, 1.5 mg/kg, was given 11 h prior to sacrifice : 20,OOOg supernatant of liver homogenate.
0.30 r 0.09 0.19 2 0.05 of the animals.
19.8 ” 3.5 18.2 ? 2.2 Assays were carried
1.5 2 0.4 1.2 * 0.2 out on the
106
BISSELL
AND
toxin-treated animals, and no evidence for an in vitro inhibitor was found (Table III). The low control value for ALA synthetase in this study (cf. Table I) reflects the level of enzyme in the liver sample rather than variation in the assay. Finally, inhibition of the induction process was found to depend on the time of injection of endotoxin, relative to the administration of allylisopropylacetamide. Endotoxin was most effective when given with, or just before, the inducer, although it exhibited some effect when injected 20-30 h prior to inducer. By contrast, administration of endotoxin 15 h ufier allylisopropylacetamide (1 h prior to sacrifice of the animals) failed to alter the induction seen in control animals (Fig. 1). These data suggest that endotoxin exerts a relatively specific effect on ALA synthetase induction in the liver. In seeking to relate the effect of endotoxin on the induction of ALA synthetase to its ability to stimulate degradation of cytochrome P-450 heme, we examined the dose range for these two responses to the compound. The results are shown in Fig. 2. Endotoxin was effective as a blocker of TABLE
Treatment
100” 50 -
AIA (%I
100” 50 50
FIG. 1. Effect of endotoxin on induction of ALA synthetase in relation to the time of endotoxin administration. Groups of three animals were treated with endotoxin (ETOX) (1.5 mg/kg) at the indicated times and/or with allylisopropylacetamide (AIA) as shown. The activity of ALA synthetase was determined 16 h after injection of the inducer in all groups of animals. The values from animals treated with endotoxin (horizontal bars) are expressed as a percentage of the activity in animals treated with allylisopropylacetamide alone.
III
ALA SYNTHETASE ACTIVITY IN MIXED EXTRACTS OF SALINE-, ALLYLISOPROPYLACETAMIDE (AIA)-, OR AIA + ENDOTOXIN-TREATED LIVER”
Saline (%I
HAMMAKER
AIA + endotoxin (%I 100” 50
ALA synthetase (nmol ALA/ g/30 min)
7 90 29 47 (48)’ 52 (59)”
n Three rats in each treatment group were fasted for 24 h and then injected intraperitoneally with isotonic saline, AIA, or AIA + endotoxin. The rats were sacrificed 16 h later and livers were removed and pooled in each treatment group. Extracts were prepared as described in Methods-and assayed for ALA synthetase, either alone or in mixtures as indicated in the table. * Percentage of liver extract in the assay mixture derived from animals treated as indicated. c Figure in parentheses indicates the expected activity.
DOSE OF ENDOTOXIN
hg
kg;
FIG. 2. Dose-response of endotoxin and inhibition of ALA synthetase activity (A) or stimulation of WO production from [‘4C-hemelcytochrome P-450 (B). Endotoxin in varying doses was administered to fasted rats, followed 2 h later by injection of allylisopropylacetamide. Hepatic ALA synthetase activity was determined 16 h after injection of the inducer. For measurement of the rate of ‘%O production from labeled cytochrome P-450 heme, pairs of animals were treated with a pulse of [5-‘ClALA, as described previously (141, and then injected either with endotoxin or isotonic saline according to previous protocol (14). The points and brackets (Fig. 2B) represent mean values * SD.
ALA synthetase induction at doses as low as 0.05 mg/kg and was maximally effective at approximately 0.15 mg/kg. By contrast, detectable stimulation of 14C0 production- under the protocol described in the preceding paper (14) -required doses
REGULATION
OF HEPATIC
greater than 0.50 mg/kg. These data appeared to indicate a lack of relationship between the rate of degradation of cytochrome P-450 heme and regulation of ALA synthetase induction. However, it remained possible that doses of endotoxin between 0.01 and 0.50 mg in fact had caused a shift of cytochrome P-450 heme to the postulated regulatory pool but failed to stimulate 14C0 production -either because the CO measurements were insensitive to small changes in the rate of heme degradation or because the labeled heme transferred to the regulatory pool had been reutilized in the synthesis of hemoprotein, in place of endogenously formed heme. To test these possibilities, a “chase” experiment was designed, in which unlabeled ALA was co-administered with endotoxin. The rationale was that unlabeled heme, formed from the injected ALA, would transiently flood the regulatory pool, chasing labeled heme to the degradative mechanism and/or preventing its re-uptake into hemoprotein. Effect of Administered ALA on Production of 14C0 and Induction of ALA Synthetase by Allylisopropylacetamide In preliminary experiments, the effect of ALA alone, 5 mg/kg, on 14C0 production was studied, according to the standard protocol (14). ALA was found to stimulate a small but reproducible increase in 14C0 excretion (Fig. 3). Next, ALA was given together with doses of endotoxin that, when injected alone, failed to stimulate 14C0 production (less than 0.5 mg/kg). In the experiment shown in Fig. 4, ALA was administered together with 0.15 mg/kg
--
0
4
6
8
HEME
SYNTHESIS
I
1 2
4
6
8
IO
12
HOURS
FIG. 4. Effect of co-administered ALA and endotoxin on production of WO from hepatic [Wlheme. The experimental procedure was similar to that described for Fig. 3.
endotoxin, and a substantial peak of 14C0 emerged which was of greater magnitude and somewhat delayed, by comparison with the increase that followed injection of ALA alone. The time-course of the peak from the combined endotoxin-ALA treated animals approximated that from animals given larger doses of endotoxin alone. Because of this finding, the dose-response range for stimulation of 14C0 production was examined between 0.01 and 0.50 mgl kg of endotoxin, each dose being administered together with 5 mg/kg of ALA. As demonstrated in Fig. 5, when examined in the presence of co-administered ALA, endotoxin stimulated 14C0 production over precisely the same dose range as that for inhibition of ALA synthetase (cf. Fig. 5 and Fig. 2A). The effect of endotoxin on induction of ALA synthetase was not altered by co-administration of ALA, as judged by the fact that injection of ALA alone had no significant effect on induction of ALA synthetase or on the increase in hepatic protoporphyrin caused by allylisopropylacetamide (Table IV). In the study shown, ALA was administered 2 h after injection of allylisopropylacetamide. Comparable results were obtained when ALA was injected with, or 2 h prior to, the inducer.
IO
HOURS
FIG. 3. Effect of administered ALA on production of WO from hepatic [Wlheme. Pairs of animals were studied according to the protocol presented previously (14). ALA was injected intraperitoneally; the control animal received an equal volume of isotonic saline by the same route.
Effect of Administered Heme on Induction of ALA Synthetase: Correlation with Stimulation of Hepatic Heme Oxygenuse Activity The studies with endotoxin indicated a close correlation between stimulation of
BISSELL AND HAMMAKER
108
z I
TABLE V
0
G 2l)o ZU 0 t=Z 40 It, gg
0
EFFECT OF ADMINISTERED HEME ON HEPATIC HEME OXYGENASE ACTIVITY AND ON INDUCTION OF ALA SYNTHETASE AND PROTOPORPHYRIN FORMATION BY THE LIVER AFTER ALLYLISOPROPYLACETAMIDE (AL4Y’
Treatment
ALA synthetase (rim01 ALA/g/ 30 min)
Heme oxygenase (rin$l+’ min/lO me)
Heme-free albumin; isotonic saline Heme-free albumin; AIA Heme: AL4
27
0.15
6.2
77
0.11
39.2
28
0.61
13.2
100
:2 ;; -9
0
l!!l//c
05
10
15
DOSE OF ENDOTOXIN Img/kg ADMINISTERED WITH 5mg/kg ALA
FIG. 5. Dose-response of co-administered endotoxin and ALA for stimulation of WO production. In a series of studies similar to that shown in Fig. 4, varying doses of endotoxin were administered together with a fixed amount of ALA (5 mg/kg) and the effect of peak 14C0 production was determined. TABLE IV EFFECT OF ADMINISTERED ALA ON INDUCTION OF ALA SYNTHETASE OR PROTOPORPHYRIN SYNTHESIS BY ALLYLISOPROPYLACETAMIDE (AIA)”
Treatment
ALA synthetase (nmol ALA/ g/30 min)
Protoporphyrin (cLg/liver)
Isotonic saline (two injections) Isotonic saline; ALA AIA; isotonic saline AIA; ALA
28 5 6
4.3 -+ 3.1
27 2 4 92 2 9 102 ?z 29
16.3 -e 5.8 19.2 k 9.6
3.9 2 1.5
a Nine rats were assigned to each treatment group and fasted for 24 h. According to the indicated protocol, rats were injected with saline or AIA and then, 2 h later, with saline or ALA (5 mg/kg). The animals were sacrificed 16 h after the final injection. Livers from groups of three rats were pooled, homogenized, and assayed as described in Methods. The results are expressed as mean t SE.
ProtoKo’: p yrm (Pd liver)
D Rats were fasted for 24 h and then treated in groups of three animals, receiving heme (25 pmol/ kg) or heme-free albumin 18 h prior to sacrifice; 2 h later, AIA was administered. Both injections were given intraperitoneally. Liver from the three animals of each group was pooled and assayed as described in Methods.
ALA synthetase and the increase in hepatic protoporphyrin content. To assess the relationship of this effect to changes in the degradation of cytochrome P-450 heme, exogenous heme was administered in a range of doses 2 h prior to administration of allylisopropylacetamide. Rats were sacrificed 16 h after injection of the inducer, for determination of heme oxygenase or ALA synthetase activity, respectively. The results revealed a close reciprocal relationship between stimulation of heme oxygenase activity and inhibition of ALA synthetase for a given dose of administered heme (Fig. 6). DISCUSSION
14C0 production by the compound and inhibition of ALA synthetase induction. This approach was extended to studies of administered heme, since heme was shown in the companion paper (14) to stimulate 14C0 production and is known to block induction of ALA synthetase (2-6). The latter finding was confirmed, as is demonstrated by the data in Table V. Administration of 25 pmol/kg of heme, 2 h prior to injection of allylisopropylacetamide, blocked almost completely stimulation of
These studies provide evidence that cytochrome P-450-associated heme has metabolically important functions in addition to being a constituent of cytochrome P-450. The findings of the preceding paper linked the degradation of cytochrome P-450 heme to stimulation of hepatic heme oxygenase activity. The present data suggest that a similar or identical heme fraction participates in the regulation of hepatic ALA synthetase. A hypothesis that encompasses both of these effects of cytochrome
REGULATION
3 4 DOSE OF HEME j,, molerj
OF HEPATIC
5
FIG. 6. Dose-response of heme for stimulation of hepatic heme oxygenase and inhibition of ALA synthetase induction. Groups of three rats were injected intraperitoneally with varying doses of heme in a total volume of 2 ml. Two hours later, allylisopropylacetamide was administered intraperitoneally, and the animals were sacrificed 16 h after injection of the inducer. The results are expressed as a percentage of the activity in animals treated with allylisopropylacetamide alone.
P-450 heme is presented in Fig. 7.‘j According to this scheme, the regulatory heme pool is fed both by endogenous synthesis and by dissociation of heme from cytochrome P-450. The suggestion that heme dissociates from the apocytochrome appears to be the most plausible and parsimonious explanation for the effect of endotoxin or administered heme on the induction of ALA synthetase by allylisopropylacetamide. The lack of direct toxic effect of endotoxin and the correlation with stimulation of 14C0 production and heme oxygenase activity all suggest that heme detached from cytochrome P-450 mixes, prior to its degradation, with the regulatory heme pool for ALA synthetase activity. Since it is assumed that the regulatory pool is the precursor heme pool for hemoprotein formation (2-6), these data lead to the conclusion that heme moves bidirectionally between the regulatory pool and apocytochrome P-450. Moreover, the 6 The mechanism of regulation of ALA synthetase by heme is still an open question (7-91, and the “feedback” loop in Fig. 7 does not imply a specific mode of regulation for the enzyme.
HEME SYNTHESIS
109
“chase” effect of administered ALA - presumably involving the same regulatory heme pool -suggests that a bidirectional flux of heme exists in the steady state, since administered ALA, without co-administered endotoxin, provokes an increase in 14C0 excretion. A two-way flux of heme from the regulatory pool to apocytochrome P-450 is attractive, teleologically, since such a system would account for the exquisite sensitivity -demonstrated by the studies with low doses of endotoxin - of the heme-synthesizing mechanism to changes in the metabolism of cytochrome P-450. The possibility that heme reversibly dissociates from apocytochrome P-450 in rat liver has received little attention, although it has been suggested that the heme of cytochrome b, may exchange with precursor pool in the liver (28). Dissociation of cytochrome P-450 in vitro has not been observed, except when the hemoprotein was converted first to a denatured form (“cytochrome P-420”) (29). Thus, heme and protein usually are assumed to be tightly bound, in this cytochrome. On the other hand, the in vitro approach, involving isolated microsomes in aqueous salt solution, may not be the optimal approach to this problem, since it fails to reproduce the milieu of the heme moiety in the intact cell. Heme is synthesized in mitochondria, passing to the endoplasmic reticulum to combine with apocytochrome. The latter translocation may occur at points of close apposition of mitochondria and endoplasmic reticulum (301, in which case association or dissociation of heme presumably occurs lal. Jy in a lipid environment. Moreover, recent studies in intact hepatocytes suggest that, in the absence of administered substrate for the microsomal oxidase system, a major fraction of cytochrome P-450 exists in the oxidized (ferri-heme) state (31). This would favor dissociation of heme and apocytochrome, if this hemoprotein behaves in analogous fashion to hemoglobin; for globin, the affinity of oxidized (ferri-1 heme is much less than that of reduced (ferro-> heme (32). The hypothesis of Fig. 7 suggests two routes for disposal of heme from the regu-
110
BISSELL
FIG. 7. Proposed scheme for the regulation
AND
co of ALA synthetase
latory pool: formation of hemoprotein or oxidation of heme to bile pigment and carbon monoxide. These pathways can be inferred logically from the present and previous findings. However, important related questions can be treated only speculatively. A long-standing problem concerns the regulation of hepatic hemoprotein synthesis. With administration of phenobarbital to rats, increased activity of ALA synthetase precedes changes in the level of cytochrome P-450 (2). On this basis, it originally had been proposed that the primary effect of phenobarbital is on the rate of heme synthesis in the liver and that an increased heme pool secondarily triggered new synthesis of apocytochrome. According to the model presented here (Fig. 7), enlargement of the heme pool should stimulate heme oxygenase activity. However, treatment of rats with phenobarbital fails to produce this result. Indeed, the drug may decrease hepatic heme oxygenase activity (33).? Moreover, addition of “free” heme to microsomes by injection of ALA to rats fails to increase the amount of cytochrome P-450 in the liver (5, 34). In view of these data, phenobarbital treatment may stimulate primarily the synthesis of apocytochrome P-450, which draws heme from the regulatory pool, secondarily “derepressing” the heme synthetic pathway. In fact, recent studies, involving direct measurement of apocytochrome P-450, offer a similar conclusion (35, 36). A second route for discharge of heme from the regulatory pool is by way of its degradation. The degradative process ap7 Bissell, D. M., and Hammaker, lished data.
HAMMAKER
L. E., unpub-
and heme oxygenase
by hepatic
heme.
pears to be mediated by heme oxygenase, as judged by the close correlation between rates of 14C0 production from [14Chemelcytochrome P-450 and stimulation of heme oxygenase activity (14). A question that arises is whether heme oxygenase can be stimulated as a primary event, secondarily causing depletion of heme from the regulatory pool or from cytochrome P-450 (37). While this sequence of events is a theoretical possibility, the present data argue against it. It was shown in the preceding paper (14) that increased 14C0 production clearly precedes stimulation of heme oxygenase. Thus, stimulation of hepatic heme oxygenase appears to be a mechanism for discharge of “excess” heme from the regulatory pool. The inhibitory effect of administered heme on induction of ALA synthetase correlates well with the ability of this compound to perturb the metabolism of cytochrome P-450 heme - assessedeither as an increase in heme oxygenase activity or as stimulation of 14C0 production from labeled cytochrome P-450 heme. These data suggest that the effect of heme on ALA synthetase is largely-if not entirely- indirect. This hypothesis is consistent with the proposed scheme (Fig. 7), if it is assumed that exogenously administered heme taken up by the liver, cannot mix with the regulatory pool, i.e., it does not serve as a precursor for cytochrome P-450 (13). However, the latter point is controversial. Indeed, some of the data in the preceding paper suggest indirectly that administered heme may be incorporated into cytochrome P-450. It was shown that after injection of unlabeled heme to rats containing [14C-heme]cytochrome P-450, the specific activity of the hemoprotein was
REGULATION
OF HEPATIC
reduced. Since endogenous heme synthesis presumably is blocked under these conditions, the administered heme may be the precursor for the unlabeled hemoprotein that was formed. If exogenous heme or heme from administered ALA enter the regulatory pool, they presumably affect the regulation of ALA synthetase. Indeed, Hayashi et al. have shown that administered ALA blocks the induction of ALA synthetase (9). They used a large dose of ALA (100 mg/kg) and measured enzyme activity very early (2 h) after injection of allylisopropylacetamide. In the present studies, ALA had little effect on induction of ALA synthetase, probably because a relatively small dose was given (5 mg/kg) and enzyme activity was examined 16-18 h after injection of inducer. The dose of ALA was selected on the basis that it provided a maximal “chase” effect for 14C0 production, in the type of experiment shown in Fig. 4. Moreover, the period of 16-18 h may allow time for elimination of heme formed from ALA and, therefore, removal of its regulatory effect on induction of the enzyme. Heme formed from ALA appears to be degraded largely within l-2 h, as reflected by the size of the “early peak” of bilirubin or CO after administration of ALA (14, 38). By contrast, the half-life of allylisopropylacetamide in rat liver is 6-7 h (28). Thus, the inducer (if the native compound is the inducing species) persists much longer than does heme generated from administered ALA. Similarly, exogenous heme is converted relatively rapidly to bile pigment, exhibiting a half-life of about 2.5 h (39). It appears likely, therefore, that the modest effect of administered ALA on induction of ALA synthetase in the present studies is due to the rapid elimination of endogenous heme from the liver. By similar reasoning, a direct effect of administered heme- if it reaches the regulatory pool - on the induction process would be expected to be transient. By contrast, the effect of endotoxin or exogenous heme on the dissociation of heme from cytochrome P-450 is protracted, providing for a sustained change in the regulatory heme pool and, hence, a
HEME
SYNTHESIS
111
marked effect on induction of ALA synthetase by allylisopropylacetamide. This model (Fig. 7) has implications for studies of cytochrome turnover. Because methods for measuring apocytochrome P450 have not been available until very recently, turnover has been estimated from the rate at which pulse-labeled heme disappears from the hemoprotein (20, 40). The implicit assumption in this approach is that the heme moiety does not cycle or exchange to any extent with other heme fractions in the liver. The present findings challenge this assumption and suggest that use of a heme label may lead to an overestimation of the half-life of holocytochrome P-450. Reinvestigation of this question, with direct measurement of the turnover of apocytochrome P-450, ,appears to be needed and should be feasible with the recent preparation of highly purified cytochrome P-450. Also, it has been noted that loss of [14C!]hemefrom cytochrome P450 is bi-exponential, a finding that is consistent with two separate pools of heme in association with cytochrome P-450, with half-lives of 8 and 2440 h (20, 40). This finding has been interpreted as evidence for two types of cytochrome P-450 with differing half-lives in the liver (40). While it is now established that the liver contains multiple forms of cytochrome P-450 (41), the turnover rates of these different forms have not been analyzed individually. It seems equally probable that the two components of heme disappearance represent the rate of elimination of labeled heme from the regulatory pool and from cytochrome P-450, respectively. Finally, the model suggests that hepatic ALA synthetase and heme oxygenase are interrelated, with respect to their regulation by heme. It further predicts that these individual enzyme activities vary reciprocally-a possibility that has received recent experimental support (42). Thus, depletion of the regulatory pool would be expected to stimulate ALA synthetase but depress heme oxygenase, while repletion of the pool should affect each enzyme activity in the opposite direction. With regard to the size and regulatory effect of the
112
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postulated heme pool, synthesis and degradation of cytochrome P-450 appear to play major roles. ACKNOWLEDGMENTS We are grateful to Dr. Rudi Schmid for support and for a critical review of the manuscript, and to Dr. Philip Guzelian for stimulating discussions. Mr. Jose Aronce provided very capable technical assistance. REFERENCES 1. MARVER, H. S., AND SCHMID, R. (1972) in The Metabolic Basis of Inherited Disease (Stanbury, J. B., Wyngaarden, J. B., and Fredrickson, D. S., eds.), 3rd ed., p. 1087, McGrawHill, New York. 2. MARVER, H. S. (1969) in Microsomes and Drug Oxidations (Gillette, J. R., Conney, A. H., Cosmides, G. J., Estabrook, R. W., Fouts, J. R., and Mannering, G. J., eds.), p. 495, Academic Press, New York. 3. MEYER, U. A., AND SCHMID, R. (1973) Fed. Proc. 32, 1649-1655. 4. WATSON, C. J. (1975) New Engl. J. Med. 239, 605-607. 5. DE MATTEIS, F. (1971) Biochem. J. 124, 767-777. 6. TSCHUDY, D. P., AND BONKOWSKY, H. L. (1972) Fed. Proc. 31, 147-159. 7. SCHOLNICK, P. L., HAMMAKER, L. E., AND MARVER, H. S. (1969) Proc. Nut. Acad. Sci. USA 63, 65-70. 8. SASSA, S., AND GRANICK, S. (1970) Proc. Nut. Acad. Sci. USA 67, 517-522. 9. HAYASHI, N., KURASHIMA, Y., AND KIKUCHI, G. (1972) Arch. Biochem. Biophys. 148, 10-21. 10. MARVER, H. S., SCHMID, R., AND SCH~TZEL, H. (1968) Biochem. Biophys. Res. Commun. 33, 969-974. 11. GRANICK, S. (1966) J. Biol. Chem. 241, 13591375. 12. STRAND, L. J., MANNING, J., AND MARVER, H. S. (1972) J. Biol. Chem. 247, 2820-2827. 13. SCHMID, R. (1973) Drug Metab. Dispos. 1, 256258. 14. BISSELL, D. M., AND HAMMAKER, L. E. (1976) Arch. Biochem. Biophys. 176, 91-102. 15. SCHMID, R., AND SCHWARTZ, S. (1952) Proc. Sot. Exp. Biol. Med. 81, 685-689. 16. GOLDBERG, A., AND RIMINGTON, C. (1955) Proc. Roy. Sot., London, Ser. B 143, 257-280. 17. BISSELL, D. M., AND HAMMAKER, L. E. (1975) Clin. Res. 23, 384A. 18. MARVER, H. S., TSCHUDY, D. P., PERLROTH, M. G., AND COLLINS, A. (1966)J. Biol. Chem. 241, 2803-2809. 19. SCHWARTZ, S., BERG, M. H., BOSSENMAIER, I.,
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