Molecular genetic analysis of a human neuropeptide Y receptor. The human homolog of the murine "Y5" receptor may be a pseudogene.

Neuropeptide Y is a 36-amino-acid peptide amide with numerous biological activities. These functions are mediated through several pharmacologically distinct receptors. To date five receptor subtypes have been cloned. Here we report the isolation, by low stringency homology cloning from a hypothalamic library, of a cDNA encoding the human homolog of the murine neuropeptide Y receptor subsequently reported (). Translation of the human Y1-like receptor clone suggested that it encoded a receptor which is truncated in the third extracellular loop. Comparison of the human Y1-like sequence to that of the human Y1 receptor suggested that the truncated receptor could have resulted from a frameshift due to a single nucleotide deletion in the sixth transmembrane domain. Southern blot analysis suggested that the gene is single copy in the human genome. The gene is located on chromosome 5q. To test the hypothesis that allelic variation of nucleic acid length within the sixth transmembrane domain of the Y1-like receptor may exist to produce a functional receptor, genomic DNA from 192 individuals of various ages, ethnic backgrounds, and degrees of obesity were analyzed electrophoretically and by direct sequencing. No variation was detected in any of the subjects, indicating that this receptor subtype may be a transcribed pseudogene in humans.

Extracellular activities of human granzyme A. Monocyte activation by granzyme A versus alpha-thrombin.

Granzymes, serine proteases located in the granules of cytotoxic T cel ls and NK cells, are essential for induction of target cell apoptosis. However, since cytotoxic cells constitutively secrete a portion of their synthesized granzymes, these proteases could mediate extracellular functions independent of their role in the lytic event. Thrombin, another serine protease, can induce cytokine production in a number of different cell types. In this study, we test the hypothesis that granzymes, like thrombin, can regulate cell-mediated immunity by inducing the production of different cytokines. We show that granzyme A (GA) stimulates IL-6, IL-8, and TNF-alpha production by human PBMC and purified monocytes. In contrast, monocytes exposed to thrombin had enhanced IL-8 production with no induction of IL-6 or TNF-alpha production. However, monocytes exposed to either GA or thrombin had enhanced phagocytic activity. The enzymatic activity of GA and thrombin was required for the induction of cytokine production and for the enhancement of phagocytic activity. The induction of different cytokine profiles by GA vs thrombin suggested that GA activates monocytes via a receptor that was different from the thrombin receptor. This conclusion was strengthened by the fact that GA was incapable of inducing Ca2+ mobilization in insect cells transfected with the thrombin receptor. These results suggest that enzymatically active GA mediates important immunoregulatory functions through signaling pathways that does not involve thrombin receptor activation.

Mutational analysis of the endothelin type A receptor (ETA): interactions and model of selective ETA antagonist BMS-182874 with putative ETA receptor binding cavity.

Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist-ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype-selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383-12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxlic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors an, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS-182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ETA receptor complex formation.

Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor.

Neuropeptide Y (NPY) is a 36-amino acid polypeptide that is widely distributed in the central nervous system and periphery. Pharmacological studies have suggested that there are at least three receptor subtypes, Y1, Y2, and Y3. Cloning of the Y1 subtype has been reported previously. Here we report the isolation by expression cloning of a cDNA encoding a human NPY receptor displaying a pharmacology typical of a Y2 receptor. COS-7 cells transfected with the cDNA express high affinity binding sites for NPY, peptide YY, and NPY13-36, whereas [Leu31,Pro34]NPY binds with lower affinity. The receptor is 381 amino acids in length and has seven putative transmembrane regions typical of G-protein-coupled receptors. Comparison of the amino acid sequence of this Y2 receptor to that of the human Y1 receptor indicates that the two receptors are 31% identical at the amino acid level. Northern blot analyses reveal a single 4-kilobase mRNA species and indicate that the messenger RNA is present in many areas of the central nervous system. NPY induced calcium mobilization and inhibited forskolin-stimulated cAMP accumulation in Chinese hamster ovary cells that stably express the Y2 receptor cDNA, indicating that the recombinant Y2 receptor is functionally coupled to second messenger systems.

Selective blockade of the endothelin subtype A receptor decreases early atherosclerosis in hamsters fed cholesterol.

Recent studies suggest that endothelin and its receptors may be involved in atherogenesis. To test this hypothesis, cholesterol-fed hamsters were treated with a selective endothelin subtype A (ETA) receptor antagonist BMS-182874. Characterization of hamster atherosclerotic plaques indicated that they contained a fibrous cap of smooth muscle cells, large macrophage-foam cells, and epitopes of oxidized low density lipoprotein. Messenger RNA for both ETA and ETB receptors was detected in aortic endothelial cells, in medial smooth muscle cells, and in macrophage-foam cells and smooth muscle cells of the fibro-fatty plaques. BMS-182874 inhibited the endothelin-1-induced pressor response whereas the depressor effect was unaltered, suggesting that vascular ETA receptors were selectively blocked in vivo. In hyperlipidemic hamsters, BMS-182874 decreased the area of the fatty streak by reducing the number and size of macrophage-foam cells. The results indicated that ETA receptors and thus endothelin promoted the early inflammatory phase of atherosclerosis.

Aspartate mutation distinguishes ETA but not ETB receptor subtype-selective ligand binding while abolishing phospholipase C activation in both receptors.

The endothelin receptors, ETA and ETB, are G protein-coupled receptors (GPCR) that show distinctively different binding profiles for the endothelin peptides and other ligands. We recently reported that Tyr129 in the second transmembrane region (TM2) of the ETA receptor was critical for subtype-specific ligand binding [Krystek, S.R. et al. (1994) J. Biol. Chem. 269, 12383-12386]. Receptor models indicated that aspartic acids located one helical turn above (Asp133) and below (Asp126) Tyr129 in ETA had their side chains directed toward the putative binding cavity. Similarly in ETB, Asp147 and Asp154 are located one turn below and above His150, the residue that corresponds to Tyr129. Asp126 in ETA and Asp147 in ETB correspond to the highly conserved aspartate present in TM2 of many GPCR that has frequently been shown to be crucial for agonist efficacy. Mutagenesis of Asp126 of the human ETA receptor to alanine resulted in an unaltered affinity for ET-1, a 160-fold increase in ET-3 affinity and a decrease in affinity for the ETA selective naphthalenesulfonamide, BMS-182874. ET-1 activation of phospholipase C was abolished. In addition, despite the gain in binding affinity, ET-3 failed to activate phospholipase C, suggesting that Asp126 is required for signal transduction. Mutagenesis of Asp133 to alanine indicated that it was critical only for the binding of BMS-182874. In the ETB receptor, mutation of His150 to alanine or tyrosine indicated that it plays a minor role in ETB subtype-selective ligand binding; mutation of the aspartates in TM2 of ETB did not alter ligand binding. As in the Asp126 Ala ETA variant, ET-1 and ET-3 failed to increase intracellular levels of inositol phosphates in the Asp147Ala ETB mutant. Taken together, these data support the hypothesis that Asp126 and Asp133 flanking Tyr129 in TM2 of the ETA receptor play a role in defining ETA subtype-selective ligand binding but Asp147 and Asp154 that flank the His150 in TM2 of the ETB receptor do not. Furthermore, these data indicate that Asp126 in ETA and Asp147 in ETB are important for transmembrane signaling via phospholipase C.

BMS-182874 is a selective, nonpeptide endothelin ETA receptor antagonist.

BMS-182874 [5-(dimethylamino)-N-(3,4-dimethyl-5-isoxazolyl)-1-naphthalene sulfonamide] is a recently discovered, low molecular weight, nonpeptide endothelin (ET) receptor antagonist. BMS-182874 competitively inhibited the binding of [125I]ET-1 to ETA receptors in rat vascular smooth muscle A10 (VSM-A10) cell membranes (Ki = 61 nM) and in CHO cells stably expressing the human ETA receptor (Ki = 48 nM), but was a weak inhibitor at ETB receptors (Ki > 50 microM) and non-ET receptors. BMS-182874 inhibited ET-1-stimulated inositol phosphate accumulation (KB = 75 nM) and calcium mobilization (KB = 140 nM) without suppressing the maximal responses in VSM-A10 cells. BMS-182874 was a competitive antagonist of force development elicited by stimulation of ETA, but not other, receptors in isolated blood vessels such as the rabbit carotid artery (KB = 520 nM). The apparent discrepancy between efficacy in cell and tissue models was likely related to the high degree of protein binding exhibited by BMS-182874. When administered either orally (ED50 = 30 mumol/kg) or intravenously (ED50 = 24 mumol/kg) to conscious, normotensive rats, BMS-182874 blunted the pressor response to exogenous ET-1. These data demonstrate that BMS-182874 is a competitive, selective and orally active ETA receptor antagonist that will be useful in understanding the role of ET in normal and disease states.

Chondromalacia patellae: fat-suppressed MR imaging.

PURPOSE: To evaluate the accuracy of fat-suppressed magnetic resonance (MR) imaging in diagnosing chondromalacia patellae. MATERIALS AND METHODS: Seventy-one patients underwent fat-suppressed MR imaging and arthroscopy of the patellofemoral compartment. Findings were classified as early or advanced chondromalacia or as normal and were correlated with arthroscopic findings. RESULTS: Early and advanced stages of chondromalacia patellae were reliably detected, with positive predictive values of 85% and 92%, respectively. Specificity in early stages was 94% and in late stages was 98%. However, the overall accuracies did not differ substantially from those reported in studies that did not use fat-suppressed imaging. CONCLUSION: Axial, fat-suppressed MR imaging accurately depicts changes caused by chondromalacia patellae. Early stages can be seen as intrasubstance changes of increased signal intensity. Results of this study suggest a high degree of specificity in excluding both early and advanced changes.

Endothelins stimulate mitogen-activated protein kinase cascade through either ETA or ETB.

Endothelin (ET) has been shown to activate mitogen-activated protein kinase (MAPK). However, it has been unclear which of the ET receptors is coupled to MAPK activation. In the present study, we conducted experiments to determine which ET receptor is linked to MAPK activation. We found that both human ETA and ETB were coupled to the MAPK cascade in ETA or ETB cDNA-transfected Chinese hamster ovary cells. ET-1 was more potent than ET-3 in the activation of p42 MAPK, induction of MAPK kinase (MAPKK) gel retardation and uptake of [3H]thymidine in ETA-transfected cells, whereas sarafotoxin (S6c) showed no stimulatory effect on the kinases and [3H]thymidine uptake. ET-1, ET-3, and S6c had approximately the same potency to activate p42 MAPK, MAPKK gel retardation, and [3H]thymidine uptake in ETB-transfected cells. These data suggest that 1) ET isopeptides, through either ETA or ETB receptors, induce the MAPK cascade as well as cell proliferation; and 2) the different potencies of ET isopeptides for activation of the MAPK cascade and induction of cell growth are mainly due to their different affinities toward ETA and ETB.

MR imaging of the patellofemoral compartment.

This article reviews the applications of MR imaging of the patellofemoral compartment. Axial plane images are the most informative for abnormalities of this compartment. The role of MR imaging in the evaluation of the medial synovial plica and in the detection of chondromalacia is discussed. MR imaging can reliably detect and delineate the complex of injuries associated with patellar dislocations and valgus hyperextension.

Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.

The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.

Endothelin-1 stimulates cytosolic phospholipase A2 in Chinese hamster ovary cells stably expressing the human ETA or ETB receptor subtype.

The ability of endothelin-1 (ET-1) to activate cytosolic phospholipase A2 (cPLA2) has been studied in Chinese Hamster ovary (CHO) cells stably expressing either the human ETA (CHO/ETA) or the human ETB (CHO/ETB) receptor subtype. ET-1 dose-dependently increased a dithiothreitol-insensitive cPLA2 activity in both cell types. In CHO/ETA cells, BQ-123, an ETA-selective antagonist, completely blocked ET-1-induced cPLA2 stimulation. In CHO/ETB cells, the ET-1 response was mimicked by 4AlaET-1 which could be blocked partially by PD 145065, a nonselective antagonist of ETA and ETB. As expected, ET-1 stimulated PGE2 synthesis in CHO cells transfected with ET receptors. We conclude that ET-1 can stimulate cPLA2 via both the human ETA and ETB receptor subtype.

Expression of endothelin receptor subtypes in rabbit saphenous vein.

Recent investigations have revealed the presence of vasoconstrictory endothelin (ET)-B receptors in several tissues, including the rabbit saphenous vein (RSV). To determine the nature of the ET binding sites in RSV, radioligand-receptor binding studies with selective ligands and Northern analyses with probes from the ET-A and ET-B receptor cDNAs were conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner, with an inhibition constant (Ki) of 0.08 +/- 0.02 nM and a slope factor of 0.9 +/- 0.1. ET-3 inhibition of 125I-ET-1 binding was biphasic, with 68% of the 125I-ET-1 binding sites being displaceable with a Ki value of 31 +/- 4 nM. The remaining 32% of the sites displayed high affinity for ET-3 (Ki = 0.2 +/- 0.1 nM). The ET-A-selective peptide BQ-123 inhibited 125I-ET-1 binding in a biphasic manner, with Ki values of 10.4 +/- 1.9 nM and 3.2 +/- 0.9 microM. The high affinity BQ-123 site comprised 70% of the binding sites, whereas the low affinity site comprised 30%. The correspondence of high affinity binding sites for BQ-123 and low affinity binding sites for ET-3 is consistent with the suggestion that 70% of the 125I-ET-1 binding sites in this tissue are ET-A receptors. To further investigate the nature of the ET-B binding sites in RSV, 125I-ET-3 competition binding experiments were conducted. ET-1 and BQ-123 inhibited 125I-ET-3 binding in RSV with Ki values of 40 +/- 7 pM and 7.2 microM, respectively, whereas inhibition curves for ET-3 and the ET-B receptor-selective agonist sarafotoxin S6c (S6c) were best fit to two-site models. Resultant Ki values for ET-3 and S6c were 50 pM (71%)/4 pM (29%) and 0.7 nM (71%)/95 nM (29%), respectively. Binding in RSV differed from that in rat cerebellum, where ET-3 and S6c inhibition of 125I-ET-3 binding was monophasic (Ki values of 70 pM and 1.1 nM for ET-3 and S6c, respectively). The presence of the nonhydrolyzable guanine nucleotide analog guanosine-5'-O-(3-thio)triphosphate (200 microM) did not affect 125I-ET-3 binding. Low stringency Northern analysis of RSV RNA with [alpha-32P]dCTP-labeled fragments from the ET-A or ET-B receptor cDNAs revealed similar hybridization patterns with both probes, with two resolved RNA species migrating at 4.7 and 1.8 kilobases.(ABSTRACT TRUNCATED AT 400 WORDS)

Transvaginal ultrasonographic findings in surgically verified ectopic pregnancy.

OBJECTIVE: Our purpose was to evaluate transvaginal ultrasonographic findings in ectopic pregnancies for positive ultrasonographic sign(s). STUDY DESIGN: Eighty-nine patients admitted with an ectopic pregnancy from September 1987 through September 1989 were retrospectively reviewed. Sixty-nine had undergone transvaginal ultrasonography within 10 days before surgery. The ultrasonographic examinations were reviewed by four radiologists. RESULTS: Ultrasonography revealed adnexal masses in 54 patients (78%). Thirty-six masses had an appearance consistent with an adnexal ring. Twenty-four adnexal rings demonstrated a thin sonolucent area surrounding the ring, a "halo sign" (67%). A control group of 116 intrauterine pregnancies were evaluated by ultrasonography. Forty-one women had adnexal cysts. Twenty-seven of these had an adnexal ring; only two of these had halos. CONCLUSION: The halo sign is presumptive evidence of a living ectopic pregnancy and, when identified, may allow earlier diagnosis and intervention.

Endothelin receptor B expression in the rat and rabbit lung as determined by in situ hybridization using nonisotopic probes.

Endothelins are a family of potent vasoactive peptides. Full-length cDNA clones to human endothelin receptor B (ETB) mRNA were random prime-labeled with nucleotides conjugated to digoxigenin for in situ hybridization. The labeled cDNA was used to probe frozen sections of rat and rabbit lung. Detection of the digoxigenin-labeled probe was accomplished by an antibody-enzyme conjugate, anti-digoxigenin alkaline phosphatase. The location of the antibody-antigen complex was visualized as an enzyme-linked color reaction. The hybridization, washings, and detection steps were performed under stringent conditions. The following cell types of the rat and rabbit lung had abundant positive reaction product to the ETB probe: bronchiolar and bronchial epithelium, endothelium of smooth-muscle--walled vessels, and bronchial and bronchiolar-associated lymphoid tissue. Abundant positive reaction product was also observed in cell populations in the lung parenchyma. Additional studies are being performed to identify those populations. The results of this study suggest that in addition to vasoactivity, endothelins play other important roles in the lung.

Binding of 125I-endothelin-1 and 125I-endothelin-3 in rabbit saphenous vein: evidence for an atypical ET binding component.

Recent investigations have confirmed the presence of vasoconstrictory endothelinB (ETB) receptors in several tissues, including the rabbit saphenous vein (RSV). To determine the molecular nature of the ET receptor subtypes in RSV, radioligand-receptor binding with selective ligands was conducted. ET-1 inhibited 125I-ET-1 binding to RSV in a monophasic manner with an inhibition constant (Ki) of 0.08 +/- 0.03 nM. Inhibition of 125I-ET-1 binding by ET-3 or the ETA-selective peptide BQ-123 resulted in markedly biphasic inhibition curves with Ki values of 0.4 +/- 0.1 nM (36% of total sites)/37 +/- 10 nM (64% of total sites) for ET-3 and 10.4 +/- 1.9 nM (70%)/3.2 +/- 0.9 microM (30%) for BQ-123. The correspondence of high-affinity binding sites for BQ-123 with low-affinity binding sites for ET-3 agrees with the suggestion that 70% of the 125I-ET-1 binding sites in this tissue are ETA receptors. To further investigate the nature of the ET-B (non-ET-A) binding sites in RSV, 125I-ET-3 competition binding was conducted. ET-1 and BQ-123 inhibited 125I-ET-3 binding in RSV with Ki values of 40 +/- 7 pM and 7.2 microM, respectively, while ET-3 and the ETB receptor-selective agonist sarafotoxin S6c (S6c) inhibition curves were best fit to two-site models. Resultant Ki values for ET-3 and S6c were 50 pM (71%)/4 pM (29%) and 0.3 nM (76%)/115 nM (24%).(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of a human endothelin receptor: subtype A.

The polymerase chain reaction, employing degenerate primers specific for the intramembrane domains III and VI of G-coupled receptors, was used to generate partial clones encoding those receptors carried by cultured rat aorta smooth muscle cells. One clone, spanning the intramembrane domains IV-VI of a receptor specific for endothelin-1 (ET-R[A]), was used as a probe to screen a human placental cDNA library. The clone pL4-3, encoding a selective type of human endothelin receptor (ET-R[A]), has an open reading frame encoding a protein 427 amino acids in length, with a relative molecular weight of 48,625 daltons. The sequence analysis suggests the presence of a signal peptide, two potential sites for glycosylation in the N terminal extracellular domain, the seven transmembrane domains typical of G-protein receptors, and several potential sites for phosphorylation in the C terminal cytoplasmic domain. At the amino acid level, the human ET-R(A) shows 91% and 94% identity with the rat and bovine ET-R(A)s, respectively, and 59% similarity with the human ET-R(B). Xenopus laevis oocytes injected with the cloned cDNA express binding sites specific for endothelin-1. Expression of the message in COS 7 cells gave a membrane-bound product to which binding of the [125I]-ET-1 was inhibited by peptide analogues specific for ET-R(A).

Mental images can be ambiguous: reconstruals and reference-frame reversals.

Philosophers and psychologists have debated whether or not mental images of ambiguous figures are reversible as pictures of such figures are. Previously, empirical evidence both pro (Finke, Pinker, & Farah, 1989) and con (Chambers & Reisberg, 1985) has been obtained. In a series of four experiments, we identify the conditions under which images of classic ambiguous figures like the duck/rabbit and the snail/elephant are reversible. We distinguish between two types of reversal: those that entail a change in reference-frame specification as well as a reconstrual of image components (reference-frame realignments) and those that entail reconstruals only (reconstruals). We show that reference-frame realignments can occur in imagery, particularly if observers are given an explicit or an implicit suggestion; and that reconstruals of images occur commonly, regardless of experimental conditions. In addition, we show that images constructed from good parts are more likely to reverse than images constructed from poor parts. On the basis of these results, we propose a functional organization of shape memory that is consistent with shape recognition findings as well as with our reversal findings.