Nitric Oxide and Intracellular Heme1

Young-Myeong Kim,’ Hector A. Bergonia,’ Claudia Muller,i Bruce R. Pitt: W. David WatkinsJ and Jack R. Lancaster, Jr.8 Departments of * Surgery, t Anesthesiology and Critical Care Medicine, and Pharmacology University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania I526 I

*

Departments of Physiology and Medicine Louisiana State University Medical Center New Orleans, Louisiana 701 12

Nitric Oxide and Intrace1Iular Heme’

1. Introduction A primary intracellular target for the biological actions of nitric oxide (.NO) production is intracellular iron (Hibbs et al., 1990; Henry et a/., 1993).In activated macrophages and their tumor cell targets, a characteristic pattern of metabolic dysfunction is observed as a result of *NOsynthesis, which includes loss of nonheme iron-containing enzyme function, including aconitate hydratase, complexesf and I1 of the mitochondria1electron transfer chain (Hibbs et al., 1990) as well as the nonheme iron-containing enzyme ribonucleotide reductase (Lepoivre et al., 1991). Heme-containing proteins are also targets of .NO. Indeed, in vivo, quantitatively the major reaction of -NO is undoubtedly its reaction with oxyhemoglobin in the circulation to produce nitrate and methemoglobin This chapter is dedicated to Professor Lawrence H. Piette, who died on November 17,1992. Advances in Pharmacology, Volume 34 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.

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(Wennmalm et al., 1993). In addition, .NO binds to deoxyhemoglobin, yielding a complex that is observable by electron paramagnetic resonance (EPR) spectroscopy, for example, as detected in the whole blood of rats injected with endotoxin (Westenberger et al., 1990) or during rejection of heart allografts (Lancaster et al., 1992; Bastian et al., 1994a), during nitrovasodilator administration in humans (Cantilena et al., 1992), and during pregnancy in rats (Conrad et al., 1993). There is also evidence for interaction of endogenously produced .NO with intracellular heme-containing proteins. In its function as a messenger, it is heme-NO that stimulates guanylate cyclase (Craven and DeRubertis, 1983). .NO also stimulates [at low concentrations (Corbett et al., 1993; Salvemini et al., 1993)] and inhibits [at higher concentrations (Stadler et al., 1993b)] the heme-containing enzyme cyclooxygenase, although the mechanism of stimulation is independent of NO-heme interaction (Hajjar et al., 1995). Induction of * N Osynthesis in cultured vascular smooth muscle cells results in the appearance of EPR signals from both heme-NO and nonheme iron-NO complexes, with the heme-NO signals appearing, then declining, prior to maximal nonheme-NO complex formation (Geng et al., 1994). Heme-NO signals (in addition to nonheme iron-NO signals) are observed in tumor cells (Yim et al., 1993; Bastian etal., 1994b), and evidence has also been provided that heme is the first target of .NO (Bastian et al., 1994b). Both heme-NO and nonheme iron-NO signals are observed in the liver during conditions of inflammation with or without hepatotoxicant administration (Chamulitrat et al., 1994, 1995). Based on the spectral characteristics of the heme-NO species and the decrease in the signal from the low-spin oxidized heme spectrum, it was speculated that .NO interacts with cytochrome P-450 (CUP), the major drug-metabolizing system of the body. There is evidence that the effects of endogenous -NO production on CYPs may be important physiologically. It is well recognized that infection in patients is associated with altered drug disposition (Chang et al., 1978; Kraemer et al., 1982). Animal studies suggest that this may be a result of compromised metabolism by the CYP, as illustrated by the effects of injection of bacterial products and/or cytokines on hepatic CYP levels (Gorodischer et al., 1976; Stanley et al., 1988; Bertini et al., 1989; Morgan, 1989, 1993; Wright and Morgan, 1990; Raiford and Thigpen, 1994). In addition, it is known that cytokines modulate CYP expression (Ghezzi et al., 1986a-c; Bertini et al., 1988, 1989; Craig et al., 1990; Pous et al., 1990; Sujita et al., 1990; Williams et al., 1991; Stanley et al., 1991; Wright and Morgan, 1991; Ferrari et al., 1992, 1993b; Chen et al., 1992; Barker et al., 1992; Fukuda et al., 1992). Bissell and Hammaker (1976a,b) demonstrated that injection of endotoxin in rats results in increased hepatic heme oxygenase (HO) activity (which degrades free heme) and marked acceleration of the degradation of hepatic CYP heme, as well as decreased activity of S-aminolevulinate synthetase (ALAS), the rate-limiting enzyme in heme synthesis.

NO

and lntracellular Heme

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Wink et al. (1993b) demonstrated that -NO (andor reactive species formed from .NO autoxidation) inhibits CYP activity in both reversible and irreversible manners. Khatsenko et al. (1993) reported that the decrease in total microsomal CYP caused by endotoxin injection in rats is inhibited by coadministration of NG-nitro-L-argininemethyl ester (L-NAME),an inhibitor of NO synthase (NOS),indicating that the decrease in drug-metabolizing activity under inflammatory conditions is a result of endogenous *NOsynthesis. Stadler et al. (1994) demonstrated a decrease in CYP activity and protein expression in vitro in isolated hepatocytes as a result of -NO synthesis. We have investigated the mechanism of this inhibition of CYP in rat hepatocytes and have provided evidence for .NO-induced loss of proteinbound heme with consequent effects on cellular heme-metabolizing enzymatic activities (Kim et al., 1995). We present here an overview of our results and a review of possible mechanisms of this effect and its potential importance as a biological action of -NO production during conditions of infection and inflammation.

II. Effects of .NO on the Heme-Containing Enzyme Catalase We began our studies on the potential interactions of -NOwith intracelMar heme-containing enzymes with catalase, a heme-containing enzyme that provides protection against oxidative injury by dismutation of hydrogen peroxide to dioxygen and water. We utilized isolated rat hepatocytes for this study, which can be stimulated in vitro to produce prodigious amounts of -NO by induction of -NO synthase upon treatment with a mixture of inflammatory mediators [tumor necrosis factor a, interleukin-1P ( IL-lP), interferon-y, and endotoxin (“CME”)] (Curran et al., 1990; Stadler et al., 1993a) and in which catalase is an important endogenous defense against hydrogen peroxide (Starke and Farber, 1985). Table I presents the results of a study of the effects of endogenous .NO synthesis on catalase levels. Twelve hours after the addition of CME, there is a dramatic (74%) loss in catalase activity, coincident with appreciable .NO synthesis. Addition of the NOS inhibitor NG-monomethyl-L-arginine (NMMA) prevents .NO formation as well as the loss in catalase activity. These results indicate that increased -NO production may predispose cells and tissues to increased oxidative injury by compromised defense against H202.

111. Effects of .NO Synthesis Induced by Endotoxin or Corynebacterium parvum on Total CYP Heme and Total Microsomal Heme As also demonstrated by Khatsenko et al. (1993), Fig. 1 shows that endotoxin injection induces a decrease in total hepatic microsomal CYP

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TABLE I Effects of Endogenous Nitric Oxide Formation on Hepatocyte Catalase Activity’

Treatment

Catalase activity (Ulmg)

NO; formation ( p M )

Control CME CME + NMMA

177.8 5 4.8 46.6 5 4.6 161.1 ? 15.1

2.4 L 0.2 157.9 2 3.7 8.2 5 0.6

a

Hepatocytes were incubated in vitro without (control) or with (CME) a mixture of cytokines (tumor necrosis a,interleukin-16, and interferon-y) plus endotoxin for 12 hr, after which time catalase activity was measured. NMMA, Same experiment as CME except 0.5 mM P-monomethyl-Larginine (NMMA) was added at the same time as CME. Detailed methods were as described previously (Kim et al., 1995). NO;, Serum nitrite plus nitrite.

heme [measured spectroscopically by the reduced versus reduced plus CO absorption spectrum in isolated hepatic microsomes (Kim et al., 1995)], from 0.71 down to 0.29 nmol/mg. Similar to L-NAME (Khatsenko et al., Spectra of Heme-CO complex

NOx IpM in serum)

Cyt P450 (nmolfmg protein)

Heme (nmol/mg protein)

450

LPS

AG

A-

0.821 0.07

--

802.9 1 35.2

289.4i 30.3

0.29 f 0.06

0.37f 0.05

0.48 10.05

0.55i O . 0 8

0.5810.03

0.69 t 0.10

J I

400

.

.

440

.

,

480

.

,

.

520

,

560

Wavelength (nm)

FIGURE I In vivo effects of endotoxin [lipopolysaccharide (LPS)on hepatic microsomal total cytochrome P-450 (Cyt P450) heme and total extractable heme. Rats were injected with LPS (10 mgtkg intraportally) and 12 hr later serum nitrite plus nitrate and microsomal cytochrome P-450 heme and total extractable heme were determined as described previously (Kim et al., 1995). Where indicated, NG-monomethyl-L-arginine (NMMA) or aminoguanidine (AG) were injected intraportally beginning 4 hr after LPS injection and continuing at 3-hr intervals. CTRL, Control; NOx, nitric oxide.

NO and lntacellular Heme

28 I

1993), NMMA as well as aminoguanidine [AG, a NOS inhibitor reported to be more selective for the inducible versus constitutive isoform of NOS (Corbett et al., 1992)] inhibit *NO synthesis (as judged by measurements of circulating nitrite plus nitrate levels) and attenuate the loss in total hepatic microsomal CYP. This effect is not a result of conversion to the inactive CYP form of these enzymes, as judged by inspection of the small shoulders at 420 nm. AG is more effective at inhibiting .NO synthesis than NMMA, and is consequently more effective in preventing CYP heme loss than NMMA. In addition, total extractable microsomal heme [determined as pyridine hemochromogen (Kim et al., 199S)l is also decreased by an amount (46%) comparable to the decrease in CYP heme (52%), and this loss is attenuated by NMMA and AG by amounts comparable to that of total CYP heme. Administration of killed Corynebacterium parvum to rodents induces massive circulating NOx production as well as liver necrosis (Billiar et al., 1990; Geller etal., 1993), and also decreases hepatic CYP levels and attenuates CYP-dependent hepatotoxicant injury (Raiford and Thigpen, 1994). Table I1 shows that there is a substantial loss of total hepatic microsomal heme and total CYP heme in vivo under these inflammatory conditions. As demonstrated previously (Billiar et al., 1990), hepatocytes isolated from C. parvum-treated animals continue to produce .NO for at least 24 hr after

TABLE II Effects of Endogenous Nitric Oxide Synthesis on Total Cytochrome P450 Heme and Total Microsomal Heme in Microsomes from Freshly Isolated and Cultured Hepatocytes of Corynebocteriurn porvum-Treated Rats’ ~~~~~~

Treatment

In vivo (5 days) Control C. parvum After culture (24 hr) Contro1 C. parvum C. parvum plus NMMA

~

~

Cytochrome P-450 (nmolhg)

Heme (nmollmg)

4.1 t 0.4 818.3 2 57.3

0.81 2 0.06 0.34 2 0.05

0.88 +- 0.07 0.42 2 0.06

10.2 t 1.2 341.5 2 19.4

0.77 t 0.08 0.22 +- 0.04

0.84 t 0.10 0.29 t 0.09

7.3 t 0.6

0.64 t 0.10

0.71 t 0.12

NO;

*

Rats were injected with killed C. parvum (intraportally 9 mgkg). For the in vivo experiments the serum nitrite plus nitrate (NO;) level was determined 4.5 days after C. parvum injection, at which time hepatic microsomes were isolated and cytochrome P-450 heme and total heme content were determined as described previously (Kim et al., 1995). For the “after culture” experiments hepatocytes were isolated, also 4.5 days after C. parvum, and cultured for 24 hr, without or with 0.5 mM W-monomethyl-L-arginine (NMMA). Medium NO; was determined, and microsomal cytochrome P-450 heme and total heme were measured. bunits are in micromolar nitrite plus nitrate in serum (for the in vivo experiments) and in medium (for the in vitro experiments). a

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Young-Myeong Kim et a/.

being placed in culture. Under these conditions the amount of total microsoma1 and CYP heme remains low compared to control hepatocytes. However, inhibition of - N O synthesis during this 24-hr period results in a substantial recovery of total microsomal and CYP heme, demonstrating that cells recover from this heme loss once .NO production ceases.

IV. Cultured Hepatocytes: Effects of Endogenous and Exogenous .NO on Total CYP Heme, Total Microsomal Heme, and CYP Protein As shown in Table 111, in vitro induction of endogenous .NO synthesis in isolated hepatocytes (similar to the data on catalase in Table I ) results in a marked reduction in both total CYP heme (0.053 down to 0.024 nmollmg) and total extractable heme (0.062 down to 0.031 nmol/ mg). This decline is prevented by NMMA, demonstrating that it is a result of .NO synthesis. A similar effect is observed with the compound Snitrosoacetylpenicillamine (SNAP),which is a nitrosonium carrier and liberates - N O both nonenzymatically and as a result of cellular metabolism (Ignarro et af., 1981; Kowaluk and Fung, 1990). The loss of total extractable heme comparable to the loss of total CYP heme in the experiments above indicates that the decrease in spectrally detectable CYP heme is not due to prevention of the formation of the reduced carbon monoxy complex of CYP (Kahl et al., 1978; Khatsenko et al., 1993), but rather a loss of CYP heme and/or protein. To differentiate between these two possibilities, we attempted to reconstitute CYP heme in microsomes that had been depleted by endogenous or exogenous .NO, by dialyzing the microsomes versus heme. Heme reconstitution of rat hepatic apo-CYP has been demonstrated previously (Bonkovsky et a/.,1984; Bornheim et af., TABLE 111 Decrease in Total Cytochrome P-450 Heme and Total Microsomal Heme by Endogenous or Exogenous Nitric Oxide in Cultured Hepatocytes”

Treatment

Microsomal protein (mglmg cell protein)

CYP heme (nmolhg cell protein)

Total heme (nmol/mg cell protein)

Control CME CME + NMMA SNAP

0.072 2 0.003 0.066 t 0.008 0.067 ? 0.010 0.066 ? 0.003

0.053 ? 0.002 0.024 t 0.001 0.058 ? 0.010 0.022 ? 0.004

0.062 ? 0.006 0.031 ? 0.007 0.068 ? 0.006 0.031 ? 0.002

~~

a

~

Conditions were similar to those in Table I, except that microsomal total protein, cytochrome P-450 heme, and total heme were measured as described previously (Kim et al., 1995). For the S-nitrosoacetylpenicillamine(SNAP)experiments hepatocytes were incubated with 1 mM SNAP for 9 hr. NMMA, NG-monomethyl-L-arginine.

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1986). The results of a representative experiment are shown in Table IV (Kim et al., 1995). As expected, dialysis versus heme has little effect on the CYP heme content in microsomes from control hepatocytes (no .NO exposure) or in hepatocytes treated with CME in the presence of NMMA. However, this dialysis restored most of the total CYP heme that was lost due to treatment with either endogenous (CME)or exogenous (SNAP).NO. It is important to note that this restoration results in reappearance of virtually only the peak at 450 nm [no increase at 420 nm (Kim et al., 1995)l. This result demonstrates that the *NO exposure results in liberation of heme from CYP proteins, and that the apoproteins remain relatively intact after heme removal and available for heme reconstitution to the native heme coordination.

V. Modulation of Heme-Metabolizing Enzymes as a Result of Heme Liberation

Heme metabolism in hepatocytes is highly regulated (Bonkovsky, 1982). Free heme down-regulates its own synthesis (by decreased mRNA stability and mitochondria1 import of ALAS) and up-regulates its degradation (by transcriptional increase in HO). If heme is liberated from hepatocyte CYP [which is the major heme pool in this cell (Bonkovsky, 1982)], this could result in modulation of the activities of these enzymes. As shown in Table V, treatment of hepatocytes in vitro with CME indeed results in a decline (80%) in ALAS and an increase (3.9-fold) in H O activities. This effect is inhibited by NMMA, demonstrating it to be a result of -NO synthesis, and TABLE IV

Effect of Dialysis versus Heme on Total Cytochrome P-450 Heme Content in Isolated Microsomes’ Cytochrome P-450heme (nmol/mg)

Treatment

- Heme

+ Heme

Control CME CME + NMMA SNAP

0.89 0.40 0.80 0.35

0.87 0.78 0.88 0.76

Hepatocytes were treated as described in Table 111. The microsomal fraction was then dialyzed for 6 hr versus buffer without (“- Heme”) or with (“+ Heme”) 60 p M heme and cytochrome P-450 heme was determined (Kim et al., 1995).NMMA, NG-monomethyl-L-arginine; SNAP, S-nitrosoacetylpenicillamine.

284

Young-Myeong Kim eta/.

TABLE V

Effects of Endogenous and Exogenous Nitric Oxide on Activity of Heme-Metabolizing Enzymesa

Treatment Control CME CME + NMMA SNAP a

ALAS (nmol ALAlmgh) 2.0 5 0.1 0.4 t 0.1

1.5 2 0.2 0.5 0.1

*

Ferrochelatase (nmol hemelmgl h r)

Heme oxygenase (nmol bilirubin1 mgh)

49.5 7.5 31.2 16.5

1.38 2 0.12 5.32 t 0.32 2.68 2 0.24 5.62 t 0.41

? 6.1

t 0.9 ? 4.4 t 1.2

Hepatocytes were treated as described in Table 111, and activities of 6-arninolevulinate synthetase (ALAS), ferrochelatase, and heme oxygenase were determined as described previously (Kim et al., 1995). NMMA, W-monomethyl-L-arginine; SNAP, S-nitrosoacetylpenacillamine.

also occurs with exogenous *NO(SNAP). We have shown previously (Kim et al., 1995) that parallel changes occur in the expression of HO mRNA. We note that HO activity is still substantially increased (although to a lesser extent) in hepatocytes treated with CME plus NMMA. This could be due to -NO-independent up-regulation of this protein by cytokine stimulation (Helqvist et al., 1991; Mitani et al., 1992; Fukuda and Sassa, 1993). Finally, as shown in Table V, activity of ferrochelatase is also decreased by .NO. This mitochondria1 enzyme catalyzes the final step in heme synthesis, the insertion of ferrous iron into porphyrin, and has been shown to contain an Fe2S2nonheme iron-sulfur cluster which is required for activity (Dailey et al., 1994). This result suggests that loss of activity may occur by nitrogen oxide-mediated [i.e., .NO in the presence of dioxygen (Wink et al,, 1993a)l destruction of its iron-sulfur cluster. Consistent with this result, while inhibition of ALAS and increase in HO induced by SNAP requires a period of 4-8 hr (consistent with the established effects of heme on these enzymes, as described above), the inhibition of ferrochelatase is virtually complete within 1 hr (Kim et al., 1995).

VI. Discussion As early as the late 1960s, it was demonstrated that, like virtually all hemoproteins, the CYPs bind .NO (Miyake et al., 1968), and this has been used in numerous studies as a spectroscopic probe to examine the heme ligand environment using EPR spectroscopy (Miyake et a/., 1968; Saprin et al., 1977; O’Keeffe et al., 1978). It has been shown that supraphysiological amounts of nitrogen oxides inhibit CYP activity, by both reversible and irreversible mechanisms (Wink et al., 1993b; Khatsenko et al., 1993; Stadler et al., 1994). It is thus possible that endogenous .NO production inhibits activity via this mechanism. It is also possible that .NO could inhibit spectral

NO and lntracellular Heme

285

detection of total CYP heme by preventing formation of the carbon monoxy complex (Kahl et al., 1978; Khatsenko et al., 1993); however, we demonstrate here a loss of total extractable microsomal heme that parallels the decrease in CYP heme, and that CYP apoproteins are still present and available for heme reconstitution. EPR examination of liver tissue and isolated hepatocytes exposed to endogenous or exogenous nitrogen oxides reveals the disappearance of the low-spin absorption from CYP and the presence of a signal characteristic for heme-NO complexes (Chamulitrat et al., 1994,1995). The appearance of triplet hyperfine structure in the signal reveals that it is from the so-called pentacoordinate species, which can be formed from a variety of different -NO complexes of hemoproteins. However, the broad, relatively featureiess absorption on the low-field side of the spectrum is distinctly different from the well-resolved absorptions of native hemoprotein-NO complexes (Yonetani et al., 1972) and resemble those from denatured proteins or ironporphyrin complexes in nitrogenous solvents (Kon, 1975).This may indicate that this signal originates, at least in part, from relatively “free” heme, liberated from proteins as a result of exposure to *NO. Modulation of total heme synthesis and degradation is almost certainly not the entire explanation for the observation that inflammation decreases CYP-catalyzed reactions. Numerous studies describe the regulation of specific CYP isoforms by inflammatory mediators (Stanley et d., 1988, 1991; Wright and Morgan, 1990; Craig et al., 1990; Chen et al., 1992; Barker et al., 1992; Ferrari et al., 1992, 1993a,b; Morgan, 1993), and these findings argue against a generalized decrease in heme-containing enzymes as the sole cause of CYP loss. However, it is possible that specific CYPs exhibit different susceptibilitiesto heme depletion and degradation. Indeed, as demonstrated in Tables 11-IV, there appears to be a significant fraction of total CYP that is resistant to -NO-induced heme loss. More convincing evidence of a selective mechanism of CYP decrease comes from the numerous demonstrations that inflammatory mediators differentially modulate specific CYP proteins andlor mRNA levels (Stanley etal., 1988,1991; Morgan, 1989,1993; Wright and Morgan, 1990, 1991; Craig et al., 1990; Williams et al., 1991; Fukuda et al., 1992; Chen et al., 1992; Barker et al., 1992; Ferrari et al., 1993a,b). Interestingly, Stadler et al. (1994) demonstrated down-regulation of translation of at least one CYP, indicating that there may be selective effects of nitrogen oxides on CYP protein synthesis. Further studies are required to determine whether the differential (versus total) modulation of CYP and other hemoproteins is due to .NO production. It is also important to point out that HO, which catalyzes the breakdown of heme to biliverdin, is also induced during a variety of stress responses, including heat shock (Mitani et al., 1989; Dwyer et al., 1992), heavy metals (Keyse and Tyrrell, 1989; Taketani et al., 1989), oxidant stress (Keyse and Tyrrell, 1989, 1990; Taketani et al., 1990; Saunders et al., 1991), arsenite

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Young-Myeong Kim et a/.

Repair? Resynthesis?

Biliverdin

Protection? FIGURE 2 Schematic representation of the effects of nitric oxide (.NO) on hepatocyte cytochrome P-450, heme, and heme-metabolizing enzymes. ALAS, 6-arninolevulinate synthetase; FC, ferrochelatase; HO, heme oxygenase.

(Keyse and Tyrrell, 1989; Taketani et al., 1989, 1990), and ultraviolet A irradiation (Keyse and Tyrrell, 1989, 1990; Vile and Tyrrell, 1993). This raises the possibility that the *NO-induced heme loss may result in the upregulation of a protective enzyme (HO). HO is also induced by cytokines, including IL-6 (Mitani etal., 1992; Fukuda and Sassa, 1993),IL-11 (Fukuda and Sassa, 1993), and IFN (Ghezzi et al., 198613). We also demonstrate that inhibition of .NO synthesis in vitro results in substantial recovery of total CYP and microsomal heme. This result shows that, as described previously for hepatocyte mitochondria1 electron transfer (Stadler et al., 1991) and intracellular iron-nitrosyl complexes (Nussler et al., 2993), hepatocytes [as well as other cells (Corbett and McDaniel, 1994)] are capable of recovery or repair of the decrease in iron-containing enzyme function caused by .NO.

VII. Summary Figure 2 depicts a working hypothesis for these results. Activation of .NO synthesis results in nitrogen oxide-induced loss of protein-bound heme from CYP proteins, which remain relatively intact. This heme liberation results in a decrease in heme synthesis (decreased ALAS) and an increase in heme degradation (increased HO). In addition, - N O synthesis results in direct inhibition of ferrochelatase, which further contributes to inhibition of heme synthesis. There also appears to be a mechanism to repair or resynthesize CYP after .NO synthesis is inhibited. Finally, a result of this effect may be protection against cellular injury, since increased HO is an important response against cellular injury from a variety of insults. Acknowledgments This study was supported by research grants BE-128 from the American Cancer Society (to J.R.L.), DK-46935 from the National Institute of Diabetes and Digestive and Kidney Diseases

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(to J.R.L.), and NIHHL 32154 from the National Heart, Lung and Blood Institute (to B.R.P.), and funds from the Max Kade Foundation, New York (to W.D.W.). We thank Karla Wasserloos and William M. Konitsky for excellent technical assistance and are grateful to Dr. Shibeki Shibahara, Tohuku University, Japan, for the gift of plasmid pHHO1, containing cDNA to the human heme oxygenase 1 gene.

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