Control of blood vessel tone is central to vascular homeostasis. Here, we show that metabolism of tryptophan to kynurenine by indoleamine 2,3-dioxygenase (IDO) expressed in endothelial cells contributes to arterial vessel relaxation and the control of blood pressure. Infection of mice with malarial parasites (Plasmodium berghei), and experimental induction of endotoxemia, caused endothelial expression of IDO, resulting in decreased plasma tryptophan, increased kynurenine, and hypotension. Pharmacological inhibition of IDO increased blood pressure in systemically inflamed mice, but not in mice deficient for IDO or interferon-γ, which is required for IDO induction. Tryptophan dilated pre-constricted porcine coronary arteries only if active IDO and an intact endothelium were both present. Kynurenine dose-dependently decreased blood pressure in spontaneously hypertensive rats, inhibited contraction of arteries, and relaxed pre-constricted rings endothelium-independently. Arterial relaxation by kynurenine was mediated by activation of the adenylate and soluble guanylate cyclase pathways.

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Neuropeptide Y (NPY) is one of the most abundant neuropeptides in the mammalian nervous system and exhibits a diverse range of important physiological activities, including effects on psychomotor activity, food intake, regulation of central endocrine secretion, and potent vasoactive effects on the cardiovascular system. Two major subtypes of NPY receptor (Y1 and Y2) have been defined by pharmaclgical criteria. We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor and the corrected sequence for a rat homologue. Analysis ofthis sequence confirms that the receptor is a member of the G protein-coupled receptor superfamily. When expressed in Chinese hamster ovary (CHO) or human embryonic kidney (293) cells, the receptor exhibits the characteristic ligand specificity of a Y1 type of NPY receptor. In the 293 cell line, the receptor is coupled to a pertussis toxinsensitive G protein that mediates the inhibition of cyclic AMP accumulation. In the CHO cell line, the receptor is coupled not to the inhibition of adenylate cyclase but rather to the elevation of intraceflular calcium. These results demonstrate that second messenger coupling of the NPY-Y1 receptor is cell type specific, depending on the specific repertoire of G proteins and effector systems present in any cell type.Neuropeptide Y (NPY), a 36-amino acid peptide, is an important regulator in both the central and peripheral nervous systems (1). NPY is highly conserved in primary structure between species, as the sequences of human, rat, rabbit, and guinea pig are identical and differ from the porcine sequence by only a single amino acid (2). NPY also shares close sequence homology and a common tertiary structure with a family of peptides which include peptide YY (PYY) and pancreatic polypeptide (PP) This G protein complex in turn activates a variety of second messenger systems, including a decrease in cyclic AMP and an increase in intracellular calcium (10). However, there are reports of NPY receptors coupled to phosphoinositol metabolism, suggesting the existence ofadditional receptor subtypes and/or multiple functions for the Y1 and Y2 subtypes (6, 11).We report here the molecular cloning of a cDNA sequence encoding a human NPY receptor,* which exhibits the characteristic ligand specificity of a Y1 receptor. When expressed in different cell lines, the receptor couples via pertussis toxin-sensitive G proteins to different second messenger systems.MATERIAL AND METHODS Nucleotide Sequence Determination. Total RNA (3 pug) from rat brain was used as a template to synthesize random primed single-stranded cDNA. The cDNA was used in a polymerase chain reaction (PCR) together with the oligonucleotide primers, which correspond to positions 672-584 and 48-78 in the rat cDNA clone FC5 (12). PCR conditions: 30 cycles at 950C for 1 min, 630C for 2 min, and 720C for 1 min. The reaction product was digested with EcoRI and Pst I, gel purified, and subcloned for sequencing in the Bluescript vector (Stratagene) for confirmation of the seq...

Chromosomal translocation t(11;17)(q23;21) is associated with a retinoic acid-resistant form of acute promyelocytic leukemia. The translocation fuses the RAR␣ gene to the PLZF gene, resulting in the formation of reciprocal fusion proteins, hypothesized to play prominent roles in leukemogenesis. Promyelocytic leukemia zinc finger (PLZF) encodes a transcription factor with nine Krü ppel-like zinc fingers, seven of which are retained in the t(11;17) fusion protein RAR␣-PLZF. We identified a specific DNA-binding site for the PLZF protein and showed that PLZF binds to this site through its most carboxyl seven zinc fingers. In co-transfection experiments, PLZF repressed transcription through its cognate binding site. This repression function of PLZF was mapped to two regions on the protein, including the evolutionarily conserved POZ domain. In contrast, the RAR␣-PLZF protein activated transcription of a promoter containing a PLZF response element. These results suggest that RAR␣-PLZF, generated in acute promyelocytic leukemia, is an aberrant transcription factor that can deregulate the expression of PLZF target genes and contribute to leukemogenesis.

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The promyelocytic leukemia zinc finger (PLZF) protein is a transcription factor disrupted in patients with t(11;17)(q23;q21)-associated acute promyelocytic leukemia. PLZF contains an N-terminal BTB/POZ domain which is required for dimerization, transcriptional repression, formation of high-molecular-weight DNAprotein complexes, nuclear sublocalization, and growth suppression. X-ray crystallographic data show that the PLZF BTB/POZ domain forms an obligate homodimer via an extensive interface. In addition, the dimer possesses several highly conserved features, including a charged pocket, a hydrophobic monomer core, an exposed hydrophobic surface on the floor of the dimer, and two negatively charged surface patches. To determine the role of these structures, mutational analysis of the BTB/POZ domain was performed. We found that point mutations in conserved residues that disrupt the dimer interface or the monomer core result in a misfolded nonfunctional protein. Mutation of key residues from the exposed hydrophobic surface suggests that these are also important for the stability of PLZF complexes. The integrity of the charged-pocket region was crucial for proper folding of the BTB/POZ domain. In addition, the pocket was critical for the ability of the BTB/POZ domain to repress transcription. Alteration of charged-pocket residue arginine 49 to a glutamine (mutant R49Q) yields a domain that can still dimerize but activates rather than represses transcription. In the context of full-length PLZF, a properly folded BTB/POZ domain was required for all PLZF functions. However, PLZF with the single pocket mutation R49Q repressed transcription, while the double mutant D35N/R49Q could not, despite its ability to dimerize. These results indicate that PLZF requires the BTB/POZ domain for dimerization and the charged pocket for transcriptional repression.

The PLZF gene was identi®ed by its fusion with the RARa locus in a therapy resistant form of acute promyelocytic leukemia (APL) associated with the t(11;17)(q23;q21) translocation. Here we describe PLZF as a negative regulator of cell cycle progression ultimately leading to growth suppression. PLZF can bind and repress the cyclin A2 promoter while expression of cyclin A2 reverts the growth suppressed phenotype of myeloid cells expressing PLZF. In contrast RARa-PLZF, a fusion protein generated in t(11;17)(q23;q21)-APL activates cyclin A2 transcription and allows expression of cyclin A in anchorage-deprived NIH3T3 cells. Therefore, cyclin A2 is a candidate target gene for PLZF and inhibition of cyclin A expression may contribute to the growth suppressive properties of PLZF. Deregulation of cyclin A2 by RARa-PLZF may represent an oncogenic mechanism of this chimeric protein and contribute to the aggressive clinical phenotype of t(11;17)(q23;q21)-associated APL.

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