Molecular cloning and expression of inducible nitric oxide synthase ...

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3491-3495, April 1993 Biochemistry

Molecular cloning and expression of inducible nitric oxide synthase from human hepatocytes (lipopolysaccharide/endotoxin/interferon y/interleukin/tumor necrosis factor)

DAVID A. GELLER*t, CHARLES J. LOWENSTEINt, RICHARD A. SHAPIRO*, ANDREAS K. NUSSLER* MAURIcIo DI SILVIO*, STEWART C. WANG*, DON K. NAKAYAMA§, RICHARD L. SIMMONS*, SOLOMON H. SNYDER¶, AND TIMOTHY R. BILLIAR* *Department of Surgery and Division of §Pediatric Surgery, University of Pittsburgh, Pittsburgh, PA 15261; and lDepartments of Neuroscience, Pharmacology and Molecular Sciences and Psychiatry, tDivision of Cardiology, Johns Hopkins University School of Medicine, Baltimore, MD 21205

Contributed by Solomon H. Snyder, January 4, 1993

ABSTRACT Nitric oxide is a short-lived biologic mediator for diverse cell types. Synthesis of an inducible nitric oxide synthase (NOS) in murine macrophages is stimulated by lipopolysaccharide (LPS) and interferon y. In human hepatocytes, NOS activity is induced by treatment with a combination of tumor necrosis factor, interleukin 1, interferon y, and LPS. We now report the molecular cloning and expression of an inducible human hepatocyte NOS (hep-NOS) cDNA. hep-NOS has 80% amino acid sequence homology to macrophage NOS (mac-NOS). Like other NOS isoforms, recognition sites for FMN, FAD, and NADPH are present, as well as a consensus calmodulin binding site. NOS activity in human 293 kidney cells transfected with hep-NOS cDNA is diminished by Ca2+ chelation and a calmodulin antagonist, reflecting a Ca2+ dependence not evident for mac-NOS. Northern blot analysis with hep-NOS cDNA reveals a 4.5-kb mRNA in both human hepatocytes and aortic smooth muscle cells following stimulation with LPS and cytokines. Human genomic Southern blots probed with human hep-NOS and human endothelial NOS cDNA clones display different genomic restriction enzyme fragments, suggesting distinct gene products for these NOS isoforms. hep-NOS appears to be an inducible form of NOS that is distinct from mac-NOS as well as brain and endothelial NOS isozymes.

same combination of LPS and cytokines as rat hepatocytes, providing evidence that a specific human cell expresses inducible NOS (30). We now report the cloning and functional expression of a distinct form of inducible NOS from human hepatocytes. 11

MATERIALS AND METHODS Isolation of Human Hepatocytes. Human hepatocytes were isolated from histologically normal operative wedge resections (in accordance with institutional approval) by using a modification of an in situ collagenase procedure (type IV; Sigma) (30). Briefly, hepatocytes were separated from nonparenchymal cells by differential centrifugation four times at 50 x g. The hepatocytes were then further purified over a 30% Percoll gradient at a concentration of 106 hepatocytes per ml of Percoll to obtain a highly purified cell population (31). Hepatocyte purity by microscopy was >98%, and viability consistently exceeded 95% by trypan blue exclusion. Cell Culture. Hepatocytes (5 x 106) were plated onto 100-mm gelatin-coated Petri dishes (Coming) in 6 ml of culture medium. Medium consisted of Williams media E (GIBCO) with L-arginine (0.50 mM), insulin (1 uM), Hepes (15 mM), L-glutamine, penicillin, streptomycin, and 10%o (vol/vol) low endotoxin calf serum (HyClone). After a 24-hr incubation, the medium was changed to a cytokine/LPS mixture (CM) of recombinant human IL-1l8 (Cistron, Pine Brook, NJ) at 5 units/ml, recombinant human TNF-a (Genzyme) at 500 units/ml, recombinant human IFN-y(Amgen) at 100 units/ml, and LPS (Escherichia coli 0111:B4; Sigma) at 10 ,ug/ml. Human aortic smooth muscle cells were explanted from operative specimens, cultured as described (8), and stimulated between passages three and five with CM. Molecular Biology. Total RNA was extracted from cultured hepatocytes 8 hr after LPS and cytokine stimulation using the RNAzol B (Biotecx Laboratories, Houston) modified method of Chomczynski and Sacchi (32) and poly(A) mRNA isolated by using oligo(dT)-cellulose (Collaborative Research). An induced human hepatocyte cDNA library was constructed in the phage vector AZap II (Stratagene), by oligo(dT) and random priming from 20 ,ug of poly(A) mRNA. A 2.7-kb fragment of murine mac-NOS cDNA (19) was radiolabeled and used to screen the human cDNA library by plaque hybridization. Positive phages were isolated, and

Nitric oxide (NO) is a recently recognized messenger molecule mediating diverse functions including vasodilation, neurotransmission, and antimicrobial and antitumor activities. Different cells such as macrophages (1, 2), endothelial cells (3, 4), neurons (5, 6), smooth muscle cells (7, 8), and cardiac myocytes (9) produce NO from L-arginine. Constitutive and inducible isoforms of NO synthase (NOS) differ in structure and regulation (reviewed in refs. 10 and 11). Constitutive NOS has been cloned from rat cerebellum (12) and bovine (13, 14) and human (15, 16) endothelial cells, whereas inducible murine macrophage NOS (mac-NOS) has been cloned from RAW 264.7 cells (17-19). Rat hepatocytes make NO in vivo during chronic hepatic inflammation (20, 21) and in vitro in response to conditioned Kupffer cell supernatant (22) or to lipopolysaccharide (LPS) and the cytokines tumor necrosis factor (TNF), interleukin 1 (IL-1), and interferon y (IFN-y) (23-25). Since rats treated with LPS manifest inducible NOS in numerous tissues with few macrophages (26), it is possible that more than one isoform of inducible NOS exists. Evidence for inducible human NOS activity has been shown in patients receiving IL-2 cancer therapy (27, 28) and during sepsis (29). Recently, human hepatocytes were stimulated to produce NO by the

Abbreviations: LPS, lipopolysaccharide; CM, cytokine/LPS mixture; IFN-y, interferon y; NOS, NO synthase; hep-NOS, hepatocyte NOS; mac-NOS, macrophage NOS; IL-1, interleukin-1; TNF-a, tumor necrosis factor a. tTo whom reprint requests should be addressed. IThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L09210).

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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plasmids containing the inserts were rescued by helper phage superinfection according to the manufacturer's instructions. Plasmid inserts were sequenced by the dideoxynucleotide chain-termination method with a Genesis 2000 system (DuPont). Sequence analysis was done with software from the Genetics Computer Group. For Northern blot analysis, hepatocyte and smooth muscle cell RNA were isolated, and hybridization was performed with 20-,ug aliquots as described (25) by using the hepatic NOS (hep-NOS) cDNA as probe. To compare hep-NOS mRNA levels with NOS activity, the culture supernatants were assayed for the stable end products of NO oxidation, NO- plus NO-, using an automated procedure based on the Griess reaction (33). For Southern blot analysis, human genomic DNA was purchased from Promega. After restriction enzyme digestion, duplicate DNA samples were electrophoresed on a 0.7% agarose gel, transferred to GenescreenPlus (NEN) under alkaline conditions, and hybridized (34). The hep-NOS cDNA probe was a 0.24-kb EcoRI/BamHI fragment, and the human endothelial NOS cDNA was a 0.21-kb EcoRI/Sac I fragment (15).

hep-NOS cDNA Expression. The hep-NOS cDNA was inserted into the pCIS expression vector (Genentech). Human embryonic kidney 293 cells (American Type Culture Collection) were transfected by the transient Ca2+ phosphate method (35). NOS activity was assayed by conversion of [3H]arginine to [3H]citrulline as described (6), under the following conditions: 10 mM Tris, 2 mM NADPH, and, when added, 2 mM EDTA, 2 mM EGTA, and 2 mM Ca2+.

RESULTS Cloning Strategy. A 3' terminal 2.7-kb fragment of macNOS cDNA was used to screen a cDNA library constructed from human hepatocytes after an 8-hr stimulation with LPS and the cytokines TNF-a, IL-1, and IFN-y. Ten positive clones were partially sequenced. The longest two (clones 3 and 7) were fully sequenced on both the sense and antisense strands by using overlapping internal primers. Clone 3 was 4013 bp in length and contained a start codon beginning at nucleotide 207 and a stop codon ending at nucleotide 3668,

M H

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GSGE I L HDT L HHKAT SDF T C KSKSCL GS I M N P K SL T RGPRD KPT PL E E L LPHAIE F I N QYYGSFKE AKIE E HL AR L E AVT KE IE TTGT YQ GSG MT FQDT L HHKA KG I L T CR SK SCL GS I M T P K SL T R GPRD KPT PP DE L L PQ AIE FV N QYYGSFKE AKI E E HL ARVE AVT KE IE TT GT YQ

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LT L DE L F A T KMAW R NAPRC I GR IQW SN L QVFDAR N C ST AQE MFQHI CRH I L YAT NNGN I R SAIT VFPQRSDGKHDFR L W N S QL I R Y AGY LT GDE L I F A T KQ AW R NAPRC I GR I QW SN L QV FDAR S C ST ARE MFE HI CRHVR YS T NNGNI R SAIT VFPQR SDGKHDFRVW N AQL I R YAGY

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YR ARGGCPADW I W L VPPV SGSITPVFHQE ML NYVL SPFYYYQI EPW KT Hi W QNEKL RP RRRE IRF RVLVK VV F FASMLMRKVMASRVRAT YRSRGGCPADW W L VPPMSGSITPVFHQE ML NYVL SPFYYYQVEAW KTHv QDEKRRP K RREIPL KVLVK AVL FACMLMRKT MASRVRVT

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VL FATE T G K SE AL A R DL AT L FSY AFNTKV VC MDQY KAST L E EE QL L L VVT ST FGNGDCPSNGQT L KKSL FML RE L NHT FR YAVFGL,GS,,SM

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M 805 H 811

SYWVKDKRLPPCSLSQALTYFLDITTPPTQLQLHKLARFATDETDRQRLEALCQPSEYNDWKFSNNPTFLEVLEEFPSLHVPAAFLLSQL

YPRFCAFAHDIDQKLSHLGASQLTPMGEGDELSGQEDAFRSWAVQTFKAACETFDVRGKQHIQIPKLYTSNVTWDPHHYRLVQDSQPLDL

SKALSSMHAKNVFTMRLKSRQNLQSPTSSRATILVELSCEDGQGLN YL-PG-GVCPGNQPALVQGILERVVDGPTPHQTVRLEDLDESG

SYWVSDKRLPPCSLSQALTYSPDITTPPTQLLLQKLAQVATEEPERQRLEALCQPSEYSKWKFTNSPTFLEVLEEFPSLRVSAGFLLSQL

M 895

PIL KPRyYSISSaQDHTPSEVHLTVAVVTYRTRDGQGPLHHGVCSTWIRNLKPQDPVPCFVRSVSGFQLPEDPSQPCILIGPGTGIAPFR

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PILKPRF YS1SS SRDHTPTEIHLTVAVVTYHTGDGQGPLHHGVCSTWLNSLKPQDPVPCFVRNASAFHLPEDPSHPCILIGPGTGIVPFR

M 985 H 9 91

SFWQQRLHDSQHKGVRGGRMTLVFGCRRPDEDHI YQEE4MLEMAQKGVLHAVHTAYSRLPGKPKVYVQDILRQQLASEVLRVLHKEPGHLY

M 1075 H 1081

SFWQQRLHDSQHKGLKGGRMSLVFGCRHPEEDHLYQEEMQEMVRIKRVLFQVHTGYSRLPGKPKVYVQDILQKQLANEVLSVLHGEQGHLY I CGDVRMARDVATTLKKLVATKLNLSEEQVEDYFFQLKSQKRYHEDIFGAVFSYGAKKGSALEEPKATRL VCGDVRMARDVAHTLKQLVAAKLKLNEEQVEDYFFQLKSQKRYHEDIFGAVFPYEAKKDRVAVQPSSLEMSAL

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FIG. 1. Comparison of deduced amino acid sequence of inducible human hep-NOS and murine mac-NOS proteins. (Upper) Single-letter amino acid sequence of mac-NOS (M) and hep-NOS (H). Gaps in mac-NOS are indicated by dots ( ). Putative binding sites are indicated as follows: calmodulin, boldface type; FMN, dashed underline; FAD, single underline; NADPH, double underline. (Lower) Schematic alignment of NOS proteins with consensus binding sites labeled. CAL, calmodulin. Compared to mac-NOS, hep-NOS contains 6 additional amino acids in the amino-terminal portion and 3 additional amino acids at the carboxyl terminus. The consensus nucleotide cDNA sequence has been deposited in the GenBank data base.



Hep-iNOS

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Ca EDTA EDTA+Ca EGTA

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End-cNOS 2 3 4 5

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control mac-NOS hepatic NOS TRANSFECTED CLONES FIG. 2. Expression of human hep-NOS cDNA clones in transfected 293 kidney cells. Human embryonic 293 kidney cells contain NOS catalytic activity after transfection with human hep-NOS cDNA clone 3 [but not after transfection with clone 7, which contains an intron (data not shown)]. The cDNA clones were inserted into the pCIS expression vector and transiently transfected into 293 kidney cells, which were assayed for NOS activity after 48 hr.

which corresponded to the start and stop codons in mac-NOS cDNA. Clone 7 was 120-bp longer than clone 3 at the 3' untranslated region and also contained a 129-bp intron, which contained an in-frame stop codon. Otherwise, clones 3 and 7 were identical. hep-NOS Sequence Compared to mac-NOS. The 4145-bp human hep-NOS cDNA consensus sequence contains a 3459-bp open reading frame, which encodes a polypeptide of 1153 aa with a calculated molecular mass of 131 kDa (Fig. 1). The ATG start site at nucleotide 207 conforms to the Kozak consensus sequence TAGAGATGG (36). (Interestingly, a second ATG upstream at nucleotide 29 does not fulfill the o

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Hep-

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FIG. 3. Northern blot hybridization of cultured human hepatocytes and smooth muscle cells using the human hep-NOS cDNA as

probe. (Upper) Northern blot analysis of inducible human hep-NOS mRNA after a 2- to 48-hr stimulation in vitro with a CM of TNF-a (500 units/ml), IL-1Xp (5 units/ml), IFN-y (100 units/ml), and LPS (10 ,g/ml). (Lower) Northern blot analysis of inducible human aorta smooth muscle cell NOS (SM-NOS) mRNA after a 4- and 24-hr stimulation in vitro with CM.

FIG. 4. Southern blot analysis of human genomic DNA with the inducible human hep-NOS (Hep-iNOS) cDNA or constitutive human endothelial NOS (End-cNOS) cDNA as probes. For each lane, 5 ,ug of human genomic DNA was digested with the restriction enzymes BamHI (lane 1), EcoRI (lane 2), Hindlll (lane 3), HindII (lane 4), or Kpn I (lane 5). After electrophoresis and blot transfer, membranes were individually hybridized with either a 0.24-kb fragment of inducible human hep-NOS cDNA (Left) or a 0.21-kb fragment of constitutive endothelial NOS cDNA (Right) and washed under high stringency.

Kozak consensus sequence requirements for initiation of translation and is also followed by an in-frame stop codon, which precedes the next ATG at position 207.) The hep-NOS sequence is longer than mac-NOS by 6 aa in the aminoterminal portion of the protein and 3 aa at the carboxyl terminus. hep-NOS, like other NOS isoforms, possesses consensus recognition sites for the cofactors FMN, FAD, and NADPH in the carboxyl half of the protein. A consensus calmodulin recognition site is also present. Overall, hep-NOS has 80% sequence identity to mac-NOS at both the nucleotide and amino acid levels. The sites for binding of the oxidative cofactors FMN, FAD, and NADPH display even greater similarity. hep-NOS has 51% and 53% amino acid sequence identity, respectively, to human endothelial NOS (16) and rat neuronal NOS (12). A consensus phosphorylation site has been identified in the amino-terminal portion of rat neuronal (12) and human endothelial (16) NOS. Consensus sites for a cAMP-dependent protein kinase (37) are identified at residues 232, 576, and 890 in hep-NOS. A functional role for these potential phosphorylation sites remains to be determined. hep-NOS, like mac-NOS, contains 400-500 bp in the 3' untranslated region of the cDNA. No polyadenylylation signal is identified in either cDNA sequence. However, several ATTTA sequences are present, which have been implicated in the posttranscriptional regulation of gene expression in many cytokines and protooncogenes by conferring instability to the mRNA (38). In addition, hep-NOS cDNA contains a TTATTTAT sequence at nucleotide 4028, which has been identified as a destabilizing signal in the mRNA for TNF-a (39). Such 3' untranslated regions in hep-NOS might account for the relatively rapid degradation of the mRNA following induction (see below). Transfection Establishes Functional Activity of the hep-NOS cDNA Clones. The hep-NOS cDNA clone was ligated into the expression vector pCIS and transfected into 293 embryonic kidney cells, which were then assayed for NOS catalytic activity alongside cells transfected with mac-NOS (Fig. 2). Control cells had negligible levels of NOS activity. Cells transfected with the hep-NOS cDNA containing an intron did not have NOS activity (data not shown). In contrast, cells transfected with hep-NOS clones displayed substantial activity in the presence of NADPH. The chelating agents EDTA and EGTA lowered hep-NOS activity 70% but failed

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to affect mac-NOS. Ca2+ did not enhance NOS activity in EDTA- and EGTA-treated samples. The calmodulin antagonist trifluoperazine (40-60 ,uM) reduced hep-NOS activity 50% (data not shown). NOS mRNA is Inducible in Human Hepatocytes and Smooth Muscle Cells. To assess the kinetics of hep-NOS induction, the hep-NOS cDNA was used to probe RNA from cultured human hepatocytes stimulated with LPS, TNF-a, IL-1, and IFN-'y for 2-48 hr (Fig. 3 Upper). A single mRNA band at 4.5-kb was first identified 4 hr after stimulation, peaked at 8 hr, and was barely detectable by 48 hr. In control or unstimulated hepatocytes, no mRNA signal was detectable. Equal RNA loading was verified by subsequent probing for 18S ribosomal RNA (data not shown). NOS activity closely follows hep-NOS mRNA levels. NO2 plus NO- levels were measured in the culture supernatants collected at the time of RNA harvesting. hep-NOS activity increased from a basal NO2 plus NO- level of 3 ,uM to 71 ,uM at 24 hr and 109 ,M at 48 hr. To determine if NOS was induced in other human cell types, hep-NOS cDNA was used to probe cultured human aortic smooth muscle cells stimulated with the same cytokines and LPS (Fig. 3 Lower). A single 4.5-kb mRNA band was seen on Northern blot at both 4 and 24 hr after stimulation and was not detected in control smooth muscle cells. This is consistent with a recent report demonstrating inducible NOS activity in human aortic smooth muscle cells following IL-183 stimulation (40). Southern Blot Establishes hep-NOS and Endothelial NOS as Distinct Gene Products. To determine if different human NOS isoforms are the products of different genes, Southern blot hybridization was performed on identical human genomic DNA restriction enzyme fragments using human hep-NOS or human endothelial NOS cDNA probes (Fig. 4). In each lane, hep-NOS and endothelial NOS probes identified individual, unique bands, suggesting that these NOS isoforms are the products of distinct genes.

DISCUSSION To our knowledge, hep-NOS is the first inducible NOS cloned from humans. Despite extensive efforts, it has previously been difficult to demonstrate induction of NO formation in humans, especially in macrophages, though recently inducible NO formation has been observed in humans (2729). The cloning of human hep-NOS establishes definitively the ability of human tissues to form NO in an inducible fashion. Several lines of evidence indicate that hep-NOS is a distinct form of NOS. Its amino acid sequence is -50% identical to rat neuronal and human endothelial NOS and 80% identical to murine mac-NOS. Whether hep-NOS is a different isoform from human mac-NOS is unknown. Hybridization has been demonstrated for the murine mac-NOS cDNA to inducible rat smooth muscle NOS mRNA and for the human hep-NOS cDNA to inducible human smooth muscle cell NOS mRNA (8). NOS sequences are extremely well conserved between species [e.g., human and bovine endothelial NOS display >93% amino acid sequence identity (13-16)]. Yet human hep-NOS and murine mac-NOS show only 80% sequence identity. Also, the apparent Ca2+ dependence of hep-NOS differs strikingly from the Ca2+ independence of mac-NOS. The number of unique inducible NOS isoforms will be resolved by cloning and sequence analysis from different cell lines. Neuronal NOS is absolutely dependent upon calcium and calmodulin; enzyme activity is abolished by calcium chelating agents and enhanced by low concentrations of added calcium and calmodulin (6). hep-NOS activity is markedly decreased by EDTA, but about 25-30% of the enzyme activity remains in the presence of EDTA or the calmodulin


antagonist trifluoperazine. Moreover, Ca2+ does not increase hep-NOS activity in EDTA- or EGTA-treated samples. Perhaps hep-NOS cannot rebind calmodulin after its removal by calcium-chelating agents. This partial dependency on calcium resembles the behavior of a form of liver NOS purified from rats treated with LPS and Propionibacterium acnes, whose dependency on calmodulin varies with differing treatments of the enzyme preparation (41). In rats treated with LPS, NOS activity is enhanced in a wide range of tissues, indicating the ubiquitous distribution of inducible NOS, which may reflect hep-NOS (26). The apparent resistance to chelating agents of the induced NOS in these studies (26) may relate to the need to purify forms of inducible NOS to demonstrate calmodulin dependence (41, 42). The presence of an inducible form of NOS in virtually every tissue of the body has been suggested to reflect a primitive type of immune effector system. Such a system would be relatively nonspecific, attacking any type of invading organism. Perhaps hep-NOS represents the molecular substrate of this system. We thank Dr. Ken Bloch of Massachusetts General Hospital for the kind gift of the human endothelial NOS cDNA and Drs. Sidney M. Morris and David J. Tweardy of the University of Pittsburgh for helpful discussions and suggestions. This work was supported by National Institutes of Health Grants GM-44100 (T.R.B.), GM-37753 (R.L.S.), Kll HL02451 (C.J.L.), MH-18501 (S.H.S.), and DA-00266 (S.H.S.) and contract DA-271-90-7408 (S.H.S.), Research Scientist Award DA-00074 (S.H.S.), the W. M. Keck Foundation, and Pittsburgh Supercomputing Center Grant CLROLJP (T.R.B.). D.A.G. is the recipient of the Society of University Surgeons/Ethicon Fellowship award. 1. Stuehr, D. J. & Marletta, M. A. (1985) Proc. Natl. Acad. Sci. USA 82, 7738-7742. 2. Hibbs, J. B., Jr., Taintor, R. R. & Vavrin, Z. (1987) Science 235, 473-476. 3. Palmer, R. M. J., Ferrige, A. G. & Moncada, S. (1987) Nature (London) 327, 524-526. 4. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E. & Chaudhuri, G. (1987) Proc. Natl. Acad. Sci. USA 84, 92659269. 5. Garthwaite, J., Charles, S. L. & Chess-Williams, R. (1988) Nature (London) 336, 385-388. 6. Bredt, D. S. & Snyder, S. H. (1990) Proc. Natl. Acad. Sci. USA 87, 682-685. 7. Busse, R. & Mulsch, A. (1990) FEBS Lett. 265, 133-136. 8. Nakayama, D. K., Geller, D. A., Lowenstein, C. J., Davies, P., Pitt, B. R., Simmons, R. L. & Billiar, T. R. (1992) Am. J. Respir. Cell Mol. Biol. 7, 471-476. 9. Finkel, M. S., Oddis, C. V., Jacob, T. D., Watkins, S. C., Hattler, B. G. & Simmons, R. L. (1992) Science 257, 387-389. 10. Moncada, S., Palmer, R. M. & Higgs, E. A. (1991) Pharmacol. Rev. 43, 109-142. 11. Nathan, C. (1992) FASEB J. 6, 3051-3064. 12. Bredt, D. S., Hwang, P. M., Glatt, C. E., Lowenstein, C., Reed, R. R. & Snyder, S. H. (1991) Nature (London) 351, 714-718. 13. Lamas, S., Marsden, P. A., Li, G. K., Tempst, P. & Michel, T. (1992) Proc. Natl. Acad. Sci. USA 89, 6348-6352. 14. Nishida, K., Harrison, D. G., Navas, J. P., Fisher, A. A., Dockery, S. P., Uematsu, M., Nerem, R. M., Alexander, R. W. & Murphy, T. J. (1992) J. Clin. Invest. 90, 2092-2096. 15. Janssens, S. P., Shimouchi, A., Quertermous, T., Bloch, D. B. & Bloch, K. D. (1992) J. Biol. Chem. 267, 14519-14522. 16. Marsden, P. A., Schappert, K. T., Chen, H. S., Flowers, M., Sundell, C. L., Wilcox, J. N., Lamas, S. & Michel, T. (1992) FEBS Lett. 307, 287-293. 17. Lyons, C. R., Orloff, G. J. & Cunningham, J. M. (1992) J. Biol. Chem. 267, 6370-6374. 18. Xie, Q. W., Cho, H. J., Calaycay, J., Mumford, R. A., Swiderek, K. M., Lee, T. D., Ding, A., Troso, T. & Nathan, C. (1992) Science 256, 225-228. 19. Lowenstein, C. J., Glatt, C. S., Bredt, D. S. & Snyder, S. H. (1992) Proc. Natl. Acad. Sci. USA 89, 6711-6715.

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