Cloning of human cDNA encoding a novel heptahelix receptor expressed in Burkitt's lymphoma and widely distributed in brain and peripheral tissues.

Using PCR with degenerate primers and screening of a human B-cell lymphoblast cDNA library, a full-length cDNA encoding a 375-amino-acid protein was isolated. It contains seven regions of hydrophobic amino acids probably representing membrane-spanning domains of a novel heptahelix receptor, tentatively named CMKRL2. It shows nearly 30% overall identity with the high-affinity IL8 receptor and similar degree of homology with other chemoattractant receptors, including the "fusin" coreceptors for HIV1. Measurements of various transduction pathways following application of a panel of chemokines to transfected cells failed to evoke any reproducible response. Although the natural ligand for CMKRL2 could, thus, not be identified, receptor expression in spleen and lymph nodes as well as in Burkitt's lymphoma (irrespective of EBV status) supports a functional role in activated B-cells. Receptor message was ubiquitously distributed in normal peripheral tissues and CNS, suggesting that CMKRL2 is expressed in widespread cell populations, such as macrophages and neuroglia.

Cloning of cDNA encoding a putative chemoattractant receptor.

Based on polymerase chain reaction (PCR) utilizing degenerate primers directed to the second and sixth transmembrane domains of several G-protein-coupled neurotransmitter receptors and screening of a human B-lymphoblast cDNA library, we isolated a cDNA whose predicted amino acid sequence shows considerable homology with human chemoattractant receptors, e.g., 30% overall identity with the C5a anaphylatoxin receptors. The coding region consists of 1056 bp corresponding to 352 amino acid residues and giving an approximate molecular weight of 43 kDa. Northern blot analysis showed hybridizing transcripts in spleen, thymus, and lymph nodes, as well as in bone marrow and peripheral blood leukocytes. Message was also found in lymphoid tumor cell lines. Chromosome mapping with FISH/DAPI technique showed the corresponding gene to reside on human chromosome 14q11.2-q12. In accordance with the Genome Database Nomenclature the receptor was designated CMKRL1 ("chemoattractant receptor-like 1"). Stably transfected mammalian cells (CHO cells and LVIP2.0Zc reporter cells) expressing high levels of corresponding receptor RNA were analyzed for changes in cAMP concentration and cellular calcium fluxes. Chemokines tested to date (GRO-a, MCP-1, MCP-3, MIP-1a, MIP-1b, C5a, RANTES, and LTB4) have failed to elicit any reproducible response. Although the ligand for CMKRL1 could thus not be identified among chemotactic peptides, the high expression in lymphoid cells and tissues suggests that the receptor may function in the regulation of the inflammatory system.

Extrapituitary expression of the rat V1b vasopressin receptor gene.

[Arg8]vasopressin (AVP) stimulates adrenocorticotropic hormone release from the anterior pituitary by acting on the V1b AVP receptor. This receptor can be distinguished from the vascular/hepatic V1a and renal V2 AVP receptors by its differential binding affinities for structural analogous of AVP. Recent studies have shown that the cloned V1a and V2 receptors are structurally related. We have isolated a clone encoding the V1b receptor from a rat pituitary cDNA library using polymerase chain reaction (PCR)-based methodology. The rat V1b receptor is a protein of 421 amino acids that has 37-50% identity with the V1a and V2 receptors. Homology is particularly high in the seven putative membrane-spanning domains of these guanine nucleotide-binding protein-coupled receptors. Expression of the recombinant receptor in mammalian cells shows the same binding specificity for AVP agonists and antagonists as the rat pituitary V1b receptor. AVP-stimulated phosphotidylinositol hydrolysis and intracellular Ca2+ mobilization in Chinese hamster ovary or COS-7 cells expressing the cloned receptor suggest second messenger signaling through phospholipase C. RNA blot analysis, reverse transcription PCR, and in situ hybridization studies reveal that V1b receptor mRNA is expressed in the majority of pituitary corticotropes as well as in multiple brain regions and a number of peripheral tissues, including kidney, thymus, heart, lung, spleen, uterus, and breast. Thus, the V1b receptor must mediate some of the diverse biological effects of AVP in the pituitary as well as other organs.

Cellular localization of vasopressin V1a receptor messenger ribonucleic acid in adult male rat brain, pineal, and brain vasculature.

Vasopressin V1a receptor (V1aR) transcripts were localized in brain, pineal, and superficial brain vascular tissues of adult male rats using hybridization histochemistry and an [35S]riboprobe complementary to the messenger ribonucleic acid (mRNA) encoding the fifth to the midseventh transmembrane regions of the receptor. V1aR mRNA was extensively distributed throughout brain and was expressed in 1) superficial cells of the granule cell layers of the main olfactory bulb, hippocampal dentate gyrus, and cerebellum; 2) numerous anatomically distinct brain nuclei; 3) isolated cells dispersed throughout the central nervous system; 4) cells of the choroid plexus, occasional blood vessels in the olfactory bulb and interpeduncular nucleus, and extraparenchymal intracranial vasculature; and 5) some white matter structures. Numerous cells expressing V1aR transcripts were found in forebrain structures, including primary olfactory (piriform) cortex, the anterior and posterior olfactory nuclei; dorsal, intermediate, and ventral lateral septal nuclei; the septo-fimbrial nucleus and accumbens nucleus; and numerous hypothalamic regions with the most intense hypothalamic labeling in the arcuate, stigmoid, suprachiasmatic, and periventricular nuclei and the lateral hypothalamic area. Cells expressing V1aR transcripts were ubiquitous throughout the midbrain, pontine, and medullary regions. A lower intensity signal was found in cells of the parvocellular paraventricular and anteroventral nucleus of the thalamus, circumventricular organs including the pineal, and the subfornical organ. V1aR transcripts were not generally detected in parenchymal vasculature, but could be found over large blood vessels in the interpeduncular nucleus and medial olfactory bulb; transcripts were commonly detected in perivascular brain cells. V1aR mRNA was abundantly expressed by choroid plexus, endothelial cells of midline blood vessels between the main olfactory bulbs, and superficial vascular tissue on all brain surfaces. These data confirm the presence of the vascular/hepatic-type V1aR gene in brain tissue and document an extensive expression. The distribution of V1aR mRNA suggests that there are at least two types of vasopressin-responsive cells in brain: one type exemplified by lateral septal ara neurons innervated by classical axodendritic/somatic synaptic vasopressinergic terminals and a second, perivascular/vascular type that would facilitate humoral vasopressinergic signaling in the brain.

The cloned vasopressin V1a receptor stimulates phospholipase A2, phospholipase C, and phospholipase D through activation of receptor-operated calcium channels.

Arginine vasopressin mediates its effects through vasopressin receptor activation and second messenger production. Recent cloning of the V1a receptor provided the opportunity to investigate the possible signal transduction pathways associated with this single vasopressin receptor subtype. When stably expressed in CHO cells, vasopressin stimulated several signal transduction pathways simultaneously including calcium influx, phospholipase A2, phospholipase C, and phospholipase D. Vasopressin-stimulated release of arachidonic acid, IP3 formation, and phosphatidylethanol formation (in the presence of 1% ethanol) were used as indexes of phospholipase A2, phospholipase C, and phospholipase D activation, respectively. V1a receptor-activation stimulated a peak followed by a sustained plateau phase of intracellular calcium. The plateau phase was dependent on extracellular calcium, insensitive to blockers of voltage sensitive calcium channels, blocked by heavy metals, and quenched when MnCl2 was present in the extracellular media. Removal of extracellular calcium blunted the release of IP3, and blocked the release of arachidonic acid and phosphatidylethanol indicating that these responses were at least in part regulated by receptor-operated calcium influx. Vasopressin-stimulated release of arachidonic acid and phosphatidylethanol were augmented with the phorbol ester PMA, and this augmentation was blocked by inhibitors of protein kinase C and absent with long-term PMA treatment. Vasopressin-stimulated IP3 release was inhibited with PMA and the inhibition reversed with protein kinase C inhibitors.

Expression of the gene for arginine vasopressin in Brattleboro rats.

There is now overwhelming evidence--radio-immunoassay, immunofluorescence, Northern blot analysis with highly specific cDNA probes--for the production of vasopressin and its congeners in the rat ovary as well as the hypothalamus. Ontogeny studies and studies over the oestrous cycle suggest that such production is controlled by different mechanisms in the two tissues. Studies in Brattleboro rats suggest that the production of vasopressin and its congeners involves different post-transcriptional mechanisms in these tissues. In Brattleboro rats hypothalamic mRNA is either not translated, or yields a very short-lived product, whereas ovarian levels of immunoreactive vasopressin are indistinguishable from those in control animals. The mechanism of this tissue specificity, and its implications in terms of potential paracrine roles for vasopressin and, or its congeners awaits explanation.

Immunoreactive arginine-vasopressin in Brattleboro rat ovary.

Homozygous (di/di) Brattleboro rats have normal hypothalamic levels of oxytocin and neurophysin I, but undetectable levels of neurophysin II and arginine-vasopressin (AVP). This defect has been presumed to be at the genomic or transcriptional level, as AVP messenger is reported to be drastically reduced, if not absent, from the hypothalamus of Brattleboro rats. Recent studies suggest de novo production of various neuropeptides in the mammalian gonad, including AVP. We report here the detection and localization of immunoreactive (ir) AVP in the luteal cells of adult homozygous Brattleboro rats, and the modulation of this ovarian ir-AVP by gonadotropins. These findings are thus consistent with a tissue-specific defect of AVP expression in the magnocellular neurones of the Brattleboro rat, and suggest that a comparable defect does not occur in the ovaries of such animals.

Cells in neonatal rat hypothalamus primary culture--an immunofluorescence study.

Hypothalami from 1 day neonatal rats were dissociated and cultured for 4-16 days. Using immunofluorescence and antisera against neurofilament (NF) peptides, glial fibrillary acidic protein (GFAP), galactocerebroside and fibronectin we have distinguished neurons, astrocytes, oligodendrocytes and fibroblast-like cells in culture. Astrocytes initially grew as islets of 15-30 cells which dispersed as the culture aged. These cells, together with fibronectin-reactive flat cells, formed a monolayer upon which ovoid and process-bearing cells grew after 4 days in culture. Neurofilament-positive neurons constituted 5-10% of the total cell population. In maturing cultures the number of neurons decreased and fibroblasts increased. Oligodendrocytes represented less than 1% of total cell population. These studies emphasize the necessity of using the complementary techniques of morphology and immunocytochemistry for the characterization of hypothalamic neural cells in vitro.

Neonatal rat hypothalamus cell culture: neuron subpopulations secrete immunoreactive beta-endorphin but not immunoreactive ACTH.

Primary cultures of dissociated hypothalamic cells were prepared from 1-day-old rat neonates. Studies with cell-specific antisera revealed the presence of neurons, glial cells, oligodendrocytes and fibroblast-like cells. By immunohistochemistry, two morphologically distinct cells in culture were positive for immunoreactive beta-endorphin (IR-beta-EP). The medium derived from these cultures contained radioimmunoassayable IR-beta-EP, but not IR-ACTH. These data suggest that, in rat neonatal hypothalamic cultures, two subpopulations of cells exist which store and secrete IR-beta-EP, but not IR-ACTH.