Gi-Protein alpha-subunit mRNA antisense oligonucleotide inhibition of Gi-coupled receptor contractile activity in the epididymis of the guinea-pig.

We have used a reversible permeabilization method to facilitate the entry of Gialpha1, 2 and 3 G-protein subunit mRNA antisense or mismatch oligonucleotides into intact tissue, to investigate the G-protein alpha-subunit coupling of alpha2-adrenoceptors, neuropeptide Y (NPY) Y1, and A1 adenosine receptors in preparations of the epididymis of the guinea-pig. The alpha2-adrenoceptor agonist, xylazine, elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 6.52+/-0.39, maximum response 236+/-41 mg force). Compared to respective mismatch controls the incubation of preparations with Gialpha2, but not with Gialpha1 or Gialpha3 mRNA antisense oligonucleotides (30 microM) reduced the maximal xylazine-potentiation of phenylephrine (3 microM)-stimulated contractility (to 51+/-12% of Gialpha2 mismatch control). The oligonucleotide incubations had no effect upon the pEC50 values of xylazine. The A1 adenosine receptor agonist, cyclopentyladenosine (CPA) elicited concentration dependent contractions from preparations of phenylephrine (3 microM)-stimulated epididymis (pEC50 value 7.66+/-0.57, maximum response 208+/-54 mg force). Incubation of preparations of epididymis with Gialpha1, but neither Gialpha2 nor Gialpha3 antisense oligonucleotides reduced the maximal CPA-potentiation of phenylephrine (3 microM)-stimulated contractions (to 55+/-17% of Gialpha1 mismatch control), pEC50 values were not affected. The incubation of preparations with Gialpha2 antisense mRNA oligonucleotides reduced the maximal NPY-potentiation of phenylephrine (3 microM)-stimulated contractions (to 62+/-15% of Gialpha mismatch control). Compared with Gialpha2 mismatch controls, the incubation of preparations with Gialpha1 and Gialpha3 oligonucleotides also reduced the NPY-potentiation of phenylephrine (3 microM)-stimulated contractions. These studies indicate that, in the guinea-pig epididymis, alpha2-adrenoceptors and A1 adenosine receptors preferentially couple to effectors through Gialpha2 and Gialpha1 subunits respectively. In contrast NPY receptors may elicit effects through either Gialpha1, 2 or 3 subunits.

Neuropeptide Y Y1 and Y2 receptor-mediated stimulation of mitogen-activated protein kinase activity.

Neuropeptide Y (NPY) regulates cardiovascular function, smooth muscle contraction and smooth muscle cell proliferation. Stimulation of NPY Y1 and Y2 receptor subtypes has been shown to result in increases in second messengers, such as cytosolic calcium concentrations, which precede physiological events such as cell contraction. To assess whether NPY receptors also stimulate second messengers which may precede mitogenic effects, we measured the mitogen-activated protein kinase (MAPK) activity in NPY receptor-expressing cell lines in response to NPY. CHO K1 cells stably expressing either NPY Y1 or Y2 receptors were shown to specifically bind radiolabelled Peptide YY (PYY), and MAPK activity in these cells was assessed using a peptide kinase assay. NPY stimulated dose-dependent increases in MAPK activity in both NPY Y1 and Y2 receptor-expressing cell lines. The NPY-stimulated MAPK activity was sensitive to pretreatment with pertussis toxin, the MAPK specific inhibitor PD098059 or wortmannin, an inhibitor of phosphatidylinositol-3-kinase (PI-3-K). These results indicate that both NPY Y1 and Y2 receptors stimulate wortmannin-sensitive increases in MAPK activity via Gi proteins and suggest a role for NPY Y1 and Y2 receptors in the regulation of smooth muscle cell growth involved in hypertrophy.

G protein-coupled-receptor cross-talk: the fine-tuning of multiple receptor-signalling pathways.

Signalling via the large family of G protein-coupled receptors (GPCRs) can lead to many cellular responses, ranging from regulation of intracellular levels of cAMP to stimulation of gene transcription. Members of this receptor family have been grouped into different categories dependent on the particular G protein subtypes that they predominantly interact with. Thus, receptors that couple to GS proteins will stimulate adenylate cyclase in many cells, while Gq/11-coupled receptors can mobilize intracellular Ca2+ via activation of phospholipase C. There is accumulating evidence, however, that activation of one particular signalling pathway by a GPCR can amplify intracellular signalling within a parallel but separate pathway. In this article Lisa Selbie and Stephen Hill review some of the evidence for these synergistic interactions and suggest that they may have an important role in finetuning signals from multiple receptor signalling pathways.

Responses to neuropeptide Y in adult hamster suprachiasmatic nucleus neurones in vitro.

We investigated the effects of neuropeptide Y and related analogues on the extracellularly recorded spontaneous firing rate activity of adult Syrian hamster suprachiasmatic nucleus neurones in vitro. Sixty-seven neurones were tested with neuropeptide Y: 45% were suppressed, 4% were activated, and the remaining 51% were unresponsive. These responses were not blocked by the GABA receptor antagonist bicuculline, indicating that neuropeptide Y-evoked responses did not appear to be dependent on GABA(A) receptor activation. We tested the effects of the neuropeptide Y Y1 receptor agonist [Leu31, Pro34]neuropeptide Y and the neuropeptide Y Y2 receptor agonist neuropeptide Y-(13-36) on nine cells suppressed by neuropeptide Y in order to determine the receptor subtype(s) mediating the effects of neuropeptide Y. Four of nine cells were suppressed by [Leu31, Pro34]neuropeptide Y only, one of nine was suppressed by neuropeptide Y-(13-36) only, two of nine were suppressed by both compounds, while the remaining two cells did not respond to either compound. These data suggest that neuropeptide Y can modulate suprachiasmatic nucleus function directly, without recruitment of GABA(A) interneurones. Further, our results indicate that neuropeptide Y may act on more than one receptor subtype within the adult hamster suprachiasmatic nucleus.

Role of G-protein beta gamma subunits in the augmentation of P2Y2 (P2U)receptor-stimulated responses by neuropeptide Y Y1 Gi/o-coupled receptors.

Neuropeptide Y (NPY) significantly potentiates the constrictor actions of noradrenaline and ATP on blood vessels via a pertussis toxin (PTX)-sensitive mechanism involving Gi/o (alpha beta gamma) protein subunits (Gi/o, GTP-binding proteins sensitive to PTX). In Chinese hamster ovary K1 (CHO K1) cells expressing specific receptors for these neurotransmitters, stimulation of Gi/o protein-coupled receptors for NPY and other neurotransmitters can augment the Gq/11-coupled (Gq/11, GTP-binding proteins insensitive to PTX) alpha 1B adrenoceptor- or ATP receptor-induced arachidonic acid (AA) release and inositol phosphate (IP) production (early events which may precede vasoconstriction). In this study, we have assessed the role of G beta gamma subunits in the synergistic interaction between Gi/o- (NPY Y1, 5-hydroxytryptamine 5-HT1B, adenosine A1) and Gq/11- [ATP P2Y2 (P2U)]-coupled receptors on AA release by using the specific abilities of regions of the beta-adrenergic receptor kinase (beta ARK1 residues 495-689) and the transducin alpha subunit to associate with G-protein beta gamma subunit dimers and to act as G beta gamma subunit scavengers. Transient expression of beta ARK1(495-689) in CHO K1 cells heterologously expressing NPY Y1 receptors had no significant effect on the PTX-insensitive ability of ATP to stimulate AA release. Stimulation of NPY Y1 receptors (as well as the endogenous 5-hydroxytryptamine 5-HT1B receptor and the transiently expressed human adenosine A1 receptor) resulted in a PTX-sensitive augmentation of ATP-stimulated AA release, which was inhibited by expression of both G beta gamma subunit scavengers. Expression of beta ARK1(495-689) similarly inhibited NPY Y1 receptor augmentation of ATP-stimulated IP production (a measure of phospholipase C activity), a step thought to precede the NPY Y1 receptor-augmented protein kinase C-dependent AA release previously observed in these cells. These experiments demonstrate that G beta gamma subunits, as inhibited by two different G beta gamma scavengers, significantly contribute to the synergistic interaction between NPY Y1 Gi/o- and Gq/11-coupled receptor activity, and are required for the augmentation of IP production and AA release observed in this model cell system.

Neuropeptide Y (NPY) and peptide YY (PYY) effects in the epididymis of the guinea-pig: evidence of a pre-junctional PYY-selective receptor.

1. The effects of peptide YY (PYY), neuropeptide Y (NPY) and structurally related peptides upon field stimulation-induced and phenylephrine-mediated contractile responses in the cauda epididymis of the guinea-pig were investigated. 2. Preparations of cauda epididymis responded to field stimulation with contractions which were completely attenuated by both the neurotoxin, tetrodotoxin (500 nM), and also by the alpha-adrenoceptor antagonist, phentolamine (3 microM). PYY (n=7) and the truncated peptide analogue PYY(3-36) (n=5) inhibited field stimulation-induced contractions (pIC50+s.e.mean: 8.9+/-0.2 and 9.4+/-0.2, respectively). Pancreatic polypeptide (PP, up to 1 microM, n=6), NPY (up to 100 nM, n=6) and the NPY analogues [Leu31,Pro34]NPY (n=6) and NPY(13-36) (both up to 1 microM, n=5) had no significant effect. 3. The NPY Y1 receptor antagonist BIBP3226 ((R)-N2-(diphenylacetyl)-N[(4-hydroxyphenyl)-methyl]-argininami de) at 750 nM (n=6) and 7.5 microM (n=6) did not affect the PYY-mediated inhibition of field stimulation-induced contractions (pIC50 8.9+/-0.3 and 9.0+/-0.3, respectively). In the presence of BIBP3226 (7.5 microM), NPY (n=6) inhibited field stimulation-induced contractions (pIC50 8.0+/-0.2). 4. NPY, PYY and PYY(3-36) inhibited [3H]-noradrenaline release from preparations of epididymis (pIC50 values 7.9+/-0.7, 9.6+/-0.8 and 10.0+/-0.9, respectively, all n=6). The agonists PP and [Leu31,Pro34]PYY (both up to 100 nM) were without significant effect (both n=6). 5. In preparations of cauda epididymis, stimulated with threshold concentrations of the alpha1-adrenoceptor agonist, phenylephrine (1 microM), both NPY (n=6) and PYY (n=7) elicited concentration-dependent increases in contractile force (with pEC50 values of 8.9+/-0.2 and 8.6+/-0.1, respectively). The effects of both NPY (n=6) and PYY (n=6) were antagonized by preincubation with BIBP3226 (75 nM; apparent pK(B)+/-s.e. values 8.3+/-1.0 and 8.2+/-0.6, respectively). The peptide analogues NPY(13-36) (n=5), PYY(3-36) (n=7) and [Leu31,Pro34]NPY (n=5) did not significantly augment responses to threshold concentrations of phenylephrine. 6. These results are consistent with the proposal that distinct NPY receptors mediate the (prejunctional) inhibition of field stimulation-induced contractions and the (postjunctional) potentiation of responses to phenylephrine in the cauda epididymis of the guinea-pig. The rank order of agonist potency (NPY > or = PYY >> NPY(13-36), [Leu31,Pro34]NPY and PYY(3-36) and the high potency of BIBP3226 indicate that the postjunctional receptor may be Y1-like. The rank orders of agonist potency in inhibiting field stimulation-induced contractile responses and [3H]-noradrenaline release (PYY(3-36) > or = PYY > NPY >> PP, NPY(13-36), [Leu31,Pro34]NPY and PYY(3-36) > or = PYY > NPY >> PP, [Leu31,Pro34]PYY, respectively) are consistent with the action of these peptides at a PYY-preferring receptor subtype, which may be distinct from the presently characterized NPY receptor subtypes.

Synergistic interaction of Y1-neuropeptide Y and alpha 1b-adrenergic receptors in the regulation of phospholipase C, protein kinase C, and arachidonic acid production.

Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

Neuropeptide Y analog with selective antagonism of effects mediated by postjunctional Y1 receptors.

Neuropeptide, a 36 amino acid peptide, is one of the most ubiquitous neuropeptides in the nervous system. It is released during stimulation of sympathetic nerves and is implicated as an important neurotransmitter regulating cardiovascular activity. Administration of neuropeptide Y results in vasoconstriction and inhibition of neurotransmitter release. However, the absence of any effective inhibitors of neuropeptide Y action have precluded the examination of its possible role in hypertension. Here we describe a synthetic hexapeptide (BRC 672), corresponding to residues 22-27 of neuropeptide Y. Following the administration of BRC 672 (6.7 mumol/kg), neuropeptide Y-induced pressor responses were reduced by 32-48% in a dose-dependent fashion. The inhibition was specific for neuropeptide Y, as the pressor response to phenylephrine, an alpha-adrenoceptor agonist, was unchanged. It was selective for the postsynaptic (neuropeptide Y Y1 receptor-mediated) vasoconstrictor activity, because the presynaptic (neuropeptide Y Y2 receptor-mediated) cardiac vagal inhibition evoked by injection of neuropeptide Y to rats was not affected. The hexapeptide inhibited the neuropeptide Y-induced increase in cytosolic free Ca2+ in mammalian cells expressing the cloned human neuropeptide Y Y1 receptor. Injections of BRC 672 significantly reduced blood pressure in anaesthetised rats and in conscious spontaneously hypertensive rats. Resting arterial blood pressure decreased from 136 +/- 4 mm Hg to 122 +/- 3 mm Hg and remained depressed 2 h after the administration of the hexapeptide in anaesthetised rats. In spontaneously hypertensive rats blood pressure was decreased for up to 4 h.

Neuropeptide Y and regulation of the cardiovascular system.

CONTROL OF CARDIOVASCULAR SYSTEM: Neuropeptide Y has three major activities which are important in the modulation of blood pressure homeostasis. When released from sympathetic neurons innervating the cardiovascular system, this peptide causes direct long-lasting vasoconstriction, inhibits the release of noradrenaline and other neurotransmitters and potentiates the action of noradrenaline and other pressor agents. RECEPTOR SUBTYPE DIVERSITY: At least two major subtypes of neuropeptide Y receptor have been defined by pharmacological criteria, and the major subtype involved in the control of blood pressure (Y1) has been isolated by molecular cloning. Analysis of the cloned DNA sequence has confirmed that the receptor is a member of the G protein-coupled receptor superfamily and when expressed in various cell lines can couple to both the inhibition of adenylate cyclase and the elevation of intracellular calcium. NEUROPEPTIDE Y ANTAGONISTS: A specific neuropeptide Y antagonist has been developed which significantly lowers the dose-dependent neuropeptide Y-induced pressor response in normal rats. The inhibition is specific for the peptide and also selective for the postsynaptic Y1 receptor-mediated vasoconstrictor activity. Administration of this specific and selective inhibitor significantly reduces resting arterial blood pressure, which remains depressed for up to 4 h in normal and spontaneously hypertensive rats. CONCLUSIONS: Inhibition of endogenous neuropeptide Y activity demonstrates that this peptide makes a significant contribution to the control of blood pressure and indicates the therapeutic potential of neuropeptide Y inhibitors as a new class of antihypertensive agent. The molecular cloning of the neuropeptide Y receptor subtype responsible for both the direct vasoconstrictor activity of the peptide and the potentiation of the action of other pressor agents represents an important advance in our understanding of the molecular basis of neuropeptide Y action and will help in the development of selective neuropeptide Y antagonists.

Exclusion of close linkage of bipolar disorder to the Gs-alpha subunit gene in nine Australian pedigrees.

Growing evidence suggests that guanine nucleotide binding proteins (G proteins) may be involved in both the pathogenesis and treatment of bipolar affective disorder. Both overactive G proteins and increased levels of the alpha subunit of the stimulatory form (Gs-alpha) have been demonstrated in peripheral leucocytes of manic patients while an increase of Gs-alpha subunit levels has also been found in a postmortem study of bipolar disorder. The function of Gs and Gi alpha subunits has now been shown to be affected by lithium. The present study aimed to determine whether bipolar affective disorder was linked to the Gs-alpha subunit gene which has been mapped to chromosomal region 20q13.2. Linkage analysis utilized the PCR amplification of a portion of the Gs-alpha gene that contains a dinucleotide repeat (CA repeat) polymorphism. Linkage of bipolar disorder and recurrent depression to the Gs-alpha subunit gene was tested using a series of autosomal dominant and recessive models with varying penetrance levels. Additionally, linkage was examined using a series of levels of definitions of affective illness. Close linkage to the Gs-alpha subunit gene was strongly excluded using each model and definition. Thus, our study indicates that a genetic defect in the Gs-alpha subunit gene is unlikely to be the cause of bipolar disorder.

Stabilized structure of the presynaptic (Y2) receptor-specific neuropeptide Y analog N-acetyl[Leu-28,Leu-31]NPY(24-36).

Neuropeptide Y analog N-acetyl[Leu-28,Leu-31]NPY(24-36)-amide binds specifically to prejunctional or Y2 receptors acting to inhibit neurotransmitter release. The structure of this biologically active mutant was studied by two-dimensional proton nuclear magnetic resonance spectroscopy. Assignments of all backbone and side chain hydrogens were made by using totally correlated spectroscopy (TOCSY) experiments providing through-bond 1H-1H connectivities, and nuclear Overhauser effect spectroscopy (NOESY), which provided through-space and sequential backbone connectivities. Structure analysis of the peptide was performed using distance geometry and dynamic simulated annealing revealing the presence of a helical structure exhibiting an amphiphilic character and slight constriction in the segment 24-29.

A novel neuropeptide Y analog, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36), with functional specificity for the presynaptic (Y2) receptor.

We have carried out functional and in vitro studies on a novel analog of neuropeptide Y which shows selectivity for the prejunctional or neuropeptide Y Y2 receptor. In anaesthetised rats N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) attenuates cardiac vagal action (a prejunctional or neuropeptide Y Y2 action) and has no significant pressor effects (postjunctional or neuropeptide Y Y1 action). In the human neuroblastoma cell line (SMS-KAN) which expresses and endogenous Y2-like neuropeptide Y receptor, N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) competes with peptide YY for binding sites with an IC50 of 0.5 +/- 0.1 nM. In contrast in a fibroblast Chinese hamster ovary cell line which expresses the cloned human neuropeptide Y Y1 receptor and is used to study changes in cytosolic calcium evoked by (a neuropeptide Y Y1 effect), N-acetyl [Leu28,Leu31]neuropeptide Y-(24-36) showed no activity even at high concentrations. The steric structure for this novel compound has been determined using proton nuclear magnetic resonance (NMR) spectroscopy and it is consistent with the C-terminal end of published structures of neuropeptide Y. We suggest acetylation and amino acid substitutions stabilise the molecule and allow it to bind only to the neuropeptide Y Y2 receptor.

Molecular cloning and characterization of PKC iota, an atypical isoform of protein kinase C derived from insulin-secreting cells.

The protein kinase C (PKC) family of serine-threonine kinases comprises at least eight members. These are differentially expressed, show varying affinities for activators such as Ca2+ and lipid species, and are therefore thought to play wide-ranging roles in the regulation of such cellular processes as differentiation, growth, and secretion. The aim of this study was to identify new PKC isoforms in the insulin-secreting cell line RINm5F that might be activated by the alterations in lipid metabolism that accompany nutrient-stimulated insulin release. Fragments of cDNA, derived from RINm5F cell mRNA, were amplified by the polymerase chain reaction using degenerate oligonucleotide primers corresponding to highly conserved regions in the catalytic domains of all known PKCs. A novel sequence generated by this approach was subsequently used to screen cDNA libraries. The entire 587-amino acid coding region of a new PKC isoform, PKC iota, was deduced from two overlapping clones isolated from a human kidney cDNA library. The amino acid sequence of PKC iota showed greatest homology to PKC zeta, with 72% identity overall rising to 84% in the catalytic domain. In contrast, the homology of PKC iota to the other isoforms was less pronounced, with < 53% identity even in the highly conserved catalytic region. Further similarities between PKC zeta and PKC iota included a highly conserved pseudosubstrate sequence, the absence of an apparent Ca(2+)-binding region, and the presence of only one cysteine-rich, zinc finger-like domain. Northern blot analysis, using the full-length PKC iota clone as a probe, revealed a single 4.6-kilobase transcript present predominantly in lung and brain, but also expressed at lower levels in many tissues including pancreatic islets. In CHO-K1 cells stably expressing the PKC iota cDNA under the human beta-actin promoter, the protein was detected as a 65-kDa band by Western blotting using an antibody to the COOH terminus of PKC zeta (conserved in PKC iota). Extracts of transfected CHO-K1 cells also displayed a significantly increased kinase activity using myelin basic protein as a substrate. The results suggest that PKC iota should be included in the atypical subgroup of PKCs whose definitive member is PKC zeta. As such, PKC iota is unlikely to be activated by the diacylglycerol that is derived from phosphoinositide hydrolysis, but might be a target for novel lipid activators that are elevated during nutrient-stimulated insulin secretion.

Neuropeptide-Y Y1 receptor gene polymorphism: cross-sectional analyses in essential hypertension and obesity.

Neuropeptide Y increases blood pressure and appetite, disorders of which have a genetic component. The present study examined the neuropeptide-Y Y1 receptor gene (NPYY1R) for involvement in essential hypertension (HT) and obesity. Frequency of alleles of the only known variant, involving a point mutation in intron 1, was determined by PCR and PstI digestion. Minor allele frequency was 0.37 in 75 HT offspring of two HT parents, compared with 0.35 in 86 normotensives (NTs) ( chi 2 = 0.11; P = 0.73). In obese and lean HTs frequency was 0.40 and 0.35 (chi 2 = 0.51; P = 0.46); and was 0.38 and 0.34 in obese and lean NTs (chi 2 = 0.16; P = 0.69). In conclusion, variant(s) in linkage disequilibrium with the NPYY1R RFLP are not involved in HT or obesity.

Molecular cloning, characterization, and localization of the human homolog to the reported bovine NPY Y3 receptor: lack of NPY binding and activation.

A cDNA clone encoding the human homolog of the bovine cDNA clone LCR1 was isolated from a human lung cDNA library. The 1,670-bp-long nucleotide sequence predicts a single open reading frame of 352 amino acids, with a 92% amino acid identity to a bovine sequence reported to represent the neuropeptide Y (NPY) Y3 receptor. The amino acid sequence shares features common to many other G-protein-coupled receptors, including the seven transmembrane regions and putative glycosylation and phosphorylation sites. Polymerase chain reaction (PCR) analysis of human-hamster hybrid cell DNA reveals that the corresponding gene is located on human chromosome 2. Although the ligand for the bovine receptor has previously been identified as NPY in binding studies, extensive analysis with the human homolog transfected in several different cell lines failed to confirm this classification. Furthermore, the receptor shows 36% identity to both the human interleukin-8 (IL-8) and angiotensin II receptors but only 21% identity to the human NPY Y1 receptor. In addition, NPY and a number of other ligands fail to induce any change in cytosolic calcium levels in transfected cells, suggesting that this clone represents a novel neuropeptide receptor.

Genomic organization, localization, and allelic differences in the gene for the human neuropeptide Y Y1 receptor.

A 14-kilobase pair (kb) region of genomic DNA encoding the human neuropeptide Y Y1-receptor gene including 3'- and 5'-flanking sequences has been cloned and the human gene localized to chromosome 4q(31.3-32). In contrast to the contiguous structure of most G protein-coupled receptor genes, the NPY Y1 receptor gene is divided into three exons. A small 5'-exon of the mRNA untranslated region is separated by a 6-kb intron from the second exon. The coding region of the receptor is interrupted by a small intron, containing an in-frame stop codon, shortly after the proposed fifth transmembrane domain. In the 5'-flanking region a potential cAMP-response element and an AP-2 site, in addition to a TATA-like sequence and a typical CAAT, box are present. A single point mutation within the 6-kb intron generates a PstI polymorphic site with a highly informative allele frequency of 54:46% in the population.

Expression of the human neuropeptide tyrosine Y1 receptor.

Neuropeptide tyrosine (NPY) is the predominant peptide in the innervation of many human tissues and is considered to play a role in the regulation of blood flow, gastrointestinal secretion and motility, and renal function. Three NPY receptors have been identified (Y1, Y2, and Y3) and the cDNAs encoding the human Y1 and bovine Y3 receptors have recently been cloned. We have demonstrated the expression of the Y1 receptor subtype in several fetal and adult human tissues, including the colon, kidney, adrenal gland, heart, and placenta. A single transcript was identified (approximately 2.2 kb) and localized in tissue sections by in situ hybridization. In the colon the receptor is expressed in the mucosa and basal glands, as well as the myenteric and submucous plexuses. Y1 receptor mRNA was detected in renal collecting ducts, loop of Henle, and juxtaglomerular apparatus and in the syncytiotrophoblast layer of placental villi. Fetal aorta and adult intramyocardial, colonic, and renal blood vessels also exhibited receptor expression, localized to the intima as well as the media. The distribution of Y1 receptor expression correlates with that of NPY-immunoreactive nerves and the apparent actions of NPY in the intestine, kidney, and heart. Although the placenta is devoid of nerves, an NPY-like transcript was detected in the villous trophoblast layer. The results indicate a tissue-specific regulation of NPY Y1 receptor expression.

Molecular characterization of a human brain adenosine A2 receptor.

A cDNA encoding a G protein-coupled receptor of unknown ligand specificity was isolated from a human hippocampal cDNA library by virtue of the high degree of structural homology between members of this receptor family. The cloned receptor DNA was transfected into human embryonic kidney 293 cells. Stably transfected cell lines bound a variety of adenosine agonists and antagonists with affinities characteristic of a brain adenosine A2a receptor. The A2a specific agonist CGS21680 stimulated cAMP production but did not alter intracellular calcium concentrations in transfected 293 cells.

Molecular cloning and expression of an adenosine A2b receptor from human brain.

A novel receptor cDNA was isolated from a human hippocampal cDNA library. The encoded polypeptide contains structural features consistent with its classification as a G protein-coupled receptor and shares 45% homology with the human A1 and A2a adenosine receptors. Chinese hamster ovary K1 cells expressing this receptor showed marked stimulation of adenylate cyclase when treated with 1mM adenosine. There was no response to ligands selective for A1 and A2a receptors but the general adenosine agonist N-ethylcarboxyamidoadenosine (NECA) caused a 10 fold increase in cyclic AMP accumulation with an EC50 of approximately 0.9 microM. This effect was inhibited by the adenosine receptor antagonist theophylline. Specific binding of A1 and A2a selective agonists and NECA was not detected. It is proposed that the novel receptor is a human brain adenosine A2b receptor subtype.

Cloned human neuropeptide Y receptor couples to two different second messenger systems.

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 pharmacological 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 of this 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 toxin-sensitive 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 intracellular 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.