Involvement of Trp-284, Val-296, and Val-297 of the human delta-opioid receptor in binding of delta-selective ligands.

Given the high homology in amino acid sequence between the delta-opioid receptor and the two other types (mu and kappa), distinct residues in this receptor may confer its selectivity to some ligands. In order to identify molecular determinants in the human delta receptor responsible for the selectivity of delta-selective ligands, two different delta/mu chimeras were constructed. In the first one, the delta sequence from the top of transmembrane 5 to the C terminus was replaced by the equivalent mu sequence, and in the second one, 13 consecutive residues in the third extracellular loop region of the delta receptor were replaced by the mu counterpart.

Neuropeptide Y receptor gene regulation in mouse adrenocortical Y-1 cells.

The mouse adrenocortical Y-1 cell line expresses a high level of neuropeptide Y1 receptor (NPY-Y1). Moreover the receptor density can be up-regulated by dexamethasone or down-regulated by cAMP. To determine whether such regulation occurs at the level of gene expression, Y1 receptor mRNA was measured using a reverse transcriptase-competitive PCR method.

Effects of phosphorothioated neuropeptide Y Y1-receptor antisense oligodeoxynucleotide in conscious rats and in human vessels.

1. Metabolically stabilized (phosphorothioate) human and rat NPY Y1 receptor oligodeoxynucleotides (ODNs) complimentary to the rat or human Y1 mRNA were synthesized; [sense (rY1-SODN, 5'-AATTCAACTCTGTTCTCC-3'), antisense (hY1-ASODN, 5'-CCTGGGAAAATAATGTTG-3' and rY1-ASODN, 5'-GGAGAACAGAGTTGAATT-3') and mismatches (hY1-MMODN, 5'-CCTGAGATAA-TAAGGTTG-3' and rY1-MM 5'-GTAGATCAGAGATGAAGT-3')] and used to modulate cardiovascular function in vitro in human vessels as well as in vivo in the rat. 2.

alpha-Trinositol: a functional (non-receptor) neuropeptide Y antagonist in vasculature.

Neuropeptide Y is a sympathetic co-neurotransmitter released with noradrenaline upon sympathetic nerve stimulation. This study describes the ability of a synthetic inositol phosphate, alpha-trinositol(D-myo-inositol 1,2,6-triphosphate; PP 56) to antagonize vasoconstrictor responses to neuropeptide Y in-vitro as well as in-vivo. In human and guinea-pig isolated arteries alpha-trinositol potently (10 nM to 1 microM extracellular concentration) suppressed the constriction evoked by neuropeptide Y alone, the potentiation by neuropeptide Y of noradrenaline-evoked constriction, and the neuropeptide Y-induced inhibition of relaxation.

Studies on neuropeptide Y receptors in a mouse adrenocortical cell line.

The mouse adrenocortical Y-1 cell line has been found to express high affinity binding sites for neuropeptide Y (NPY). Pharmacological studies have shown that these NPY binding sites are of the Y1 type. Reverse transcription-polymerase chain reaction using primers specific for the rat Y1 receptor revealed that the NPY Y1 receptor mRNA is present in Y-1 cells.

Effects of some novel D-myo-inositol-phosphate derivatives on binding and sympathetic transmission.

The vascular effects of myo-inositol and a series of D-myo-inositol phosphate derivatives: D-myo inositol-1-monophosphate (Ins[1]P1), D-myo-inositol-2-monophosphate (Ins[2]P1), D-myo-inositol-1, 2-biphosphate (Ins[1,2,6]P2), D-myo-inositol-1,2,6-trisphosphate (Ins[1,2,6]P3, alpha-trinositol; PP56), D-myo-inositol-1,2,5,6-tetraphosphate (Ins[1,2,5,6]P4), and D-myo-inositol-1,2,3,4,5,6-hexa-phosphate (InsP6, phytic acid) were studied in binding assays in rat heart membranes, in vitro in isolated guinea pig basilar artery, and in vivo in pithed rats. In binding assays in rat heart membranes, Ins[1,2,6]P3, Ins[1,2,5,6]P4, and InsP6 displaced the binding of [3H] alpha-trinositol [3H]Ins[1,2,6]P3). In the isolated guinea pig basilar artery, Ins[1,2]P2 and Ins[1,2,6]P3 inhibited the contractile effects of exogenous neuropeptide Y (NPY) in the concentration range of 10(-8)-10(-6) M.

Characterisation of an ATP receptor mediating mitogenesis in vascular smooth muscle cells.

Adenosine triphosphate (ATP), a co-transmitter in sympathetic nerves and released from platelets, has recently been shown to stimulate growth of vascular smooth muscle cells. It might therefore contribute to the development of vascular hypertrophy seen in hypertension and atherosclerosis. We aimed at characterising the receptor mediating this mitogenic effect in rat aorta smooth muscle cells.

Hypothalamic neuropeptide Y, its gene expression and receptor activity: relation to circulating corticosterone in adrenalectomized rats.

Previous evidence has suggested a possible relationship between the adrenal steroid, corticosterone (CORT) and neuropeptide Y (NPY) in the brain. To provide a more systematic analysis of this interaction, the present study employed a variety of techniques, including in situ hybridization to measure NPY gene expression, radioimmunoassay to examine peptide levels and radioligand [125I]peptide YY (PYY) binding for analysis of peptide receptors. The results show that adrenalectomy (ADX), which caused a decline in CORT to levels < 0.

Cellular uptake of intracerebroventricularly administered biotin- or digoxigenin-labeled antisense oligodeoxynucleotides in the rat.

1. Antisense oligodeoxynucleotides (ODNs) internally labeled with biotin or digoxigenin were injected into the lateral ventricle of rats and the distribution of the labeled ODNs was examined at several timepoints following the intracerebroventricular (icv) injections. The stability of these injected antisense ODNs, which had no backbone modifications, was also studied by performing recovery experiments.

Molecular cloning of a potential proteinase activated receptor.

A DNA sequence encoding a G-protein-coupled receptor was isolated from a mouse genomic library. The predicted protein is similar in structure to the thrombin receptor and has a similar activation mechanism. When expressed in Xenopus laevis oocytes, the receptor was activated by low concentrations of trypsin (EC 3.

Human endothelin ETA receptor antisense oligodeoxynucleotides inhibit endothelin-1 evoked vasoconstriction.

Antisense oligodeoxynucleotides to endothelin ETA receptor mRNA were used to characterize vascular smooth muscle receptors. The concentration-response curve showed a significant attenuation of endothelin-1-induced contraction in circular segments of the human superficial temporal artery. Endothelin ETB receptor antisense or mismatch oligodeoxynucleotides showed no alteration of the endothelin-1-induced contraction.

Characterization of specific binding sites for alpha-trinositol (D-myo-inositol 1,2,6-trisphosphate) in rat tissues.

Alfa-trinositol (or D-myo-inositol 1,2,6-trisphosphate) was recently found to, e.g., inhibit agonist-induced vasoconstriction and display antiinflammatory properties.

Selective loss of delta opioid analgesia and binding by antisense oligodeoxynucleotides to a delta opioid receptor.

Antisense oligodeoxynucleotides (18-20 bases) to a cloned delta opioid receptor (DOR-1) lower delta binding in NG108-15 cells by 40%-50%. Changing 4 bases to generate a mismatch antisense oligodeoxynucleotide or mixing the corresponding sense and antisense oligodeoxynucleotides prior to treatment of the cells eliminates the inhibition of binding, confirming the specificity of the response. In vivo, an antisense oligodeoxynucleotide to DOR-1 given intrathecally lowers delta, but not mu or kappa 1 spinal analgesia.

Specific inhibition of endogenous neuropeptide Y synthesis in arcuate nucleus by antisense oligonucleotides suppresses feeding behavior and insulin secretion.

Neuropeptide Y (NPY), which is synthesized in neurons of the arcuate nucleus (ARC) that project to different hypothalamic nuclei, is known to have potent effects on eating behavior and hormone secretion after hypothalamic administration. To test the hypothesis that endogenous NPY is essential for the normal expression of these responses, the present study used to unmodified antisense oligodeoxynucleotides (ODNs) to disrupt the synthesis of NPY in the ARC and to examine the impact of this disturbance on nutrient intake, as well as on circulating levels of insulin and the adrenal steroids, corticosterone and aldosterone. Brain-cannulated rats maintained on macronutrient diets were given daily, bilateral injections, over a 4-day period, of NPY antisense ODNs, sense ODNs or saline into the ARC.

Modulation of vascular function by neuropeptide Y during development of hypertension in spontaneously hypertensive rats.

Neuropeptide Y (NPY) is a sympathetic cotransmitter and a platelet-derived factor which causes vasoconstriction, potentiation of norepinephrine (NE) action, and vascular mitogenic effects. Reciprocally, NE markedly enhances the actions of NPY. We studied vasopressor effects of NPY and sources of peptide release during the development of hypertension in spontaneously hypertensive rats (SHR).

Mitogenic effects of ATP on vascular smooth muscle cells vs. other growth factors and sympathetic cotransmitters.

The sympathetic nervous system has been shown to exert a trophic influence on vascular smooth muscle cells (VSMC). Therefore, we studied the growth-regulating effects of the sympathetic cotransmitters ATP, neuropeptide Y (NPY), and norepinephrine (NE). ATP in concentrations of 1-100 microM greatly increased the incorporation of [3H]thymidine in VSMC from rat aorta and vena cava.

A proposed bovine neuropeptide Y (NPY) receptor cDNA clone, or its human homologue, confers neither NPY binding sites nor NPY responsiveness on transfected cells.

Receptors with seven transmembrane domains (7TM) constitute a large family of structurally and functionally related proteins which respond to various types of ligands. We describe here the cloning and expression of a human 7TM receptor, denoted hFB22 (human Fetal Brain 22), which is the homologue (92% amino acid identity) of a bovine receptor (LCR1) reported by others to bind neuropeptide Y (NPY) with a pharmacological profile of the Y3 receptor subtype. However, upon expression in COS1 (confirmed by Northern analysis), COS7 or CHO-K1 cells, the hFB22 receptor did not confer specific 125I-Bolton-Hunter-NPY, 3H-propionyl-NPY or 125I-peptide YY (PYY) binding sites, in either intact cells or in membrane preparations.

Human neuropeptide Y Y1 receptor antisense oligodeoxynucleotide specifically inhibits neuropeptide Y-evoked vasoconstriction.

This paper describes a new approach for the development of an inhibitor of the contractile responses of neuropeptide Y in human blood vessels by the use of an antisense oligodeoxynucleotide complementary to human neuropeptide Y Y1 receptor mRNA. One micromolar of an antisense 18-base oligodeoxynucleotide (hY1-AS), corresponding to the human Y1 receptor NH2-terminus, was incubated with segments of human subcutaneous arteries and veins for 48 h at 37 degrees C. Control vessels were incubated with the corresponding sense oligodeoxynucleotide (hY1-S) or a 3-base mismatched antisense oligodeoxynucleotide (hY1-MM) or no oligodeoxynucleotide.

Quantitation of NMDA receptor (NMDAR1) mRNA levels in the adult and developing rat CNS.

A rapid and sensitive solution hybridization assay was used to quantitate N-methyl-D-aspartate (NMDA) receptor mRNA levels in the central nervous system (CNS) of rat, mouse and human. A riboprobe labelled with 32P was prepared from a plasmid containing a 1413 base sequence from the cDNA for the functional rat NMDA receptor subunit, NMDAR1. Using a full length sense transcript as the calibration standard, the assay reliably measures 8 pg of NMDAR1 mRNA.

Antisense oligodeoxynucleotides to NMDA-R1 receptor channel protect cortical neurons from excitotoxicity and reduce focal ischaemic infarctions.

The excitatory amino acid, L-glutamate, acting through its N-methyl-D-aspartate (NMDA) receptor, may contribute to neuronal death following cerebral vascular occlusion. In support of this hypothesis, NMDA receptor antagonists reduce the volume of infarction produced by occlusion of the middle cerebral artery in vivo and attenuate Ca2+ influx and neuronal death elicited by L-glutamate or NMDA in vitro. A complementary DNA coding for a major component of the NMDA receptor channel complex, a single protein of M(r) 105.

Mitogenic effect of neuropeptide Y in rat vascular smooth muscle cells.

Neuropeptide Y (NPY) is a vasoconstrictor released with norepinephrine from perivascular sympathetic nerves. Since sympathetic nerves appear to play a role in vascular smooth muscle cell (SMC) hypertrophy, we studied the effects of NPY on proliferation of cultured rat aorta- and vena cava-derived SMC. Both cell types displayed high-affinity NPY binding sites with displacement characteristics of [Pro34]NPY > NPY(13-36) > NPY(18-36) in aorta and [Pro34]NPY = NPY(13-36) = NPY(18-36) in the vena cava.

Neuropeptide Y suppresses the neurogenic inflammatory response in the rabbit eye; mode of action.

Ocular injury in the rabbit causes miosis and breakdown of the blood aqueous barrier (aqueous flare response, AFR), reflecting a sensory nerve-mediated inflammatory response, elicited by the release of tachykinins and calcitonin gene-related peptide (CGRP) from C-fibers. Neuropeptide Y (NPY) occurs in sympathetic fibers in the eye. The study was designed to examine whether NPY and related peptides interfere with the inflammatory response to ocular injury in the rabbit in vivo.

Modulation of anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides.

The function of neuropeptide Y, one of the most abundant peptide transmitters of the mammalian brain, remains unclear because of a lack of specific receptor antagonists. An antisense oligodeoxynucleotide corresponding to the NH2-terminus of the rat Y1 receptor was constructed and added to cultures of rat cortical neurons. This treatment resulted in a reduced density of Y1 (but not Y2) receptors and diminished the decrease in adenosine 3',5'-monophosphate (cAMP) usually seen after Y1 receptor activation.