Two distinct receptor subtypes for mammalian bombesin-like peptides

The mammalian bombesin-like peptides, gastrinreleasing peptide (GRP) and neuromedin B (NMB), are structurally related neuropeptides that elicit a wide spectrum of biological activities including regulation of smooth muscle contraction, stimulation of secretion, modulation of neural activity, and growth regulation. Earlier studies have shown that GRP and NMB are expressed in different regions of both the CNS and peripheral organs. Recent ligand-binding and molecular-cloning studies have revealed two pharmacologically distinct G- protein-coupled receptor subtypes for mammalian bombesin-like peptides that have different relative affinities for GRP, NMB and bombesin receptor antagonists. Similar to the peptide ligands, the two receptor subtypes are expressed in a distinct but overlapping set of CNS regions, some of which have been identified in functional studies as sites where bombesin peptides elicit defined biological responses. Delineation of these peptide ligands and receptor subtypes will be important in future studies that explore the molecular basis for the heterogeneous nature of the responses to bombesin observed in mammalian systems.     

Distribution of VIP mRNA and two distinct VIP binding sites in the developing rat brain: Relation to ontogenic events

The peptide neurotransmitter vasoactive intestinal peptide (VIP) has neurotrophic properties and influences neurobehavioral development. To assess the role of VIP during neural ontogeny, the present work traces the development of VIP mRNA with in situ hybridization and VIP receptors with in vitro autoradiography in rat central nervous system (CNS) from embryonic day 14 (E14) to the adult. VIP mRNA was not evident in the CNS until birth. Postnatally, it was expressed in several distinct brain regions, but its distribution bore little relation to that of VIP receptors. VIP receptors were present and expressed changing patterns of distribution throughout CNS development. The changing patterns were the result of (1) the transient appearance of GTP-insensitive VIP receptors in several regions undergoing mitosis or glial fasciculation and (2) the transient appearance of GTP-sensitive VIP receptors homogeneously distributed throughout the CNS during the first 2 postnatal weeks, the period of the brain growth spurt. At E14-16 VIP binding was dense throughout the brainstem and spinal cord, but limited in the rest of the brain. From E19 to postnatal day 14 (P14), while VIP binding was higher in germinal zones, it tended to be uniformly dense throughout the remainder of the brain. By P21 the adult pattern began to emerge; VIP binding was unevenly distributed and was related to specific cytoarchitectural sites. Since the expression of VIP in the CNS is limited to postnatal development but VIP receptors are abundant prenatally, we suggest that extraembryonic VIP may act upon prenatal VIP receptors to regulate ontogenic events in the brain. © 1994 Wiley-Liss, Inc.     

Expression of the orphan receptor TR4 during brain development of the rat

The orphan receptor TR4, member of the nuclear hormone receptor family, is related to the orphan receptors TR2, COUP-TFI and ARP-1, and was originally cloned from the adult rat brain. The latter two orphan receptors have been implicated in central nervous system (CNS) development. To investigate a possible role for TR4 in brain development, expression of TR4 was studied in rat embryos. At embryonic days 14.5 and 19.5, high expression of TR4 was found in the CNS, while low expression was detected throughout the embryo. In postnatal rats, TR4 was mainly expressed in the hippocampus and cerebellum, resembling the expression pattern found in adult brain. These data show that like COUP-TFI and ARP-1, expression of TR4 becomes restricted to distinct areas. In adult brain, TR4 is predominantly expressed in granule cells of both hippocampus and cerebellum. The data suggest a possible role for TR4 during proliferation and maturation of brain structures.     

Family 1 G protein-coupled receptor function in the CNS: Insights from gene knockout mice

Family 1 G protein-coupled receptors (GPCRs) are activated by a large number of ligands including photons, odorants, neurotransmitters and hormones and are involved in a wide variety of central and peripheral functions. Due to their wide distribution in the central nervous system (CNS), family 1 GPCRs play a major role in the regulation of neuronal activity and behaviour. In general, the lack of selective ligands for each member of the GPCR subfamilies has made it difficult to assign specific central functions to each receptor subtype. Advances in gene targeting techniques have allowed the inactivation of receptor genes in the mouse through homologous recombination leading to the generation of mouse ‘knockout’ models lacking one or more GPCRs. In this review, we have listed the family 1 GPCR knockout models produced in the past decade and we have summarized the findings obtained from studies on these mice with respect to CNS function.     

GPCR drug discovery: novel ligands for CNS receptors

G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. They convey extracellular signals into the cell interior by activating intracellular processes such as heterotrimeric G protein-dependent signaling pathways. They are widely distributed in the nervous system, and mediate key physiological processes including cognition, mood, appetite, pain and synaptic transmission. With at least 30% of marketed drugs being GPCR modulators, they are a major therapeutic target in the pharmaceutical industry's drug discovery programs. This review will survey recently patented ligands for GPCRs implicated in CNS disorders, in particular the metabotropic glutamate, adenosine and cannabinoid receptors. Metabotropic glutamate receptors regulate signaling by glutamate, the major excitatory brain neurotransmitter, while adenosine is a ubiquitous neuromodulater mediating diverse physiological effects. Recent patents for ligands of these receptors include mGluR5 antagonists and adenosine A(1) receptor agonists. Cannabinoid receptors remain one of the most important GPCR drug discovery target due to the intense interest in CB(1) receptor antagonists for treating obesity and metabolic syndrome. Such small molecule ligands are the outcome of the continuing focus of many pharmaceutical companies to identify novel GPCR agonist, antagonist or allosteric modulators useful for CNS disorders, for which more effective drugs are eagerly awaited.     

Purinergic-receptor oligomerization: Implications for neural functions in the central nervous system

It is becoming clear that the functions of G protein-coupled receptors (GPCRs), the largest family of plasma membrane-localized receptors, are regulated by direct oligomeric formation between GPCRs, as either homo-or hetero-oligomers. This review article explores the mechanistic implications of GPCR dimerization, especially among purinergic receptors, adenosine receptors and P2 receptors, which play critical roles in the regulation of neuro-transmission in the central nervous system. Briefly, adenosine receptors are able to form a heteromeric complex with P2 receptors that generates an adenosine receptor with P2 receptor-like agonistic pharmacology. This mechanism may be used to fine-tune purinergic inhibition locally at sites where there is a particular oligomerization structure between purinergic receptors, and to explain the undefined adenosine-like purinergic functions of adenine nucleotides. Purinergic receptors also form oligomers with GPCRs of other families present in the brain, such as dopamine receptors and metabotropic glutamate receptors, to alter the functional properties. The effect of GPCR oligomerization on receptor functions is thus considered as an important system in the central nervous system.     

Cloning of a novel TGF-beta related cytokine, the vgr, from rat brain: cloning of and comparison to homologous human cytokines.

Here we describe cloning of a TGF-beta related cytokine from a rat brain cDNA library. This novel cytokine, the vgr (vegetal related), is homologous to the vegetal (Vg1) gene of Xenopus (DL Weeks and DA Melton, Cell, 51:861-867, 1987). In rat brain mRNA a single 3.5 kb RNA could be detected by Northern blot analysis. Thus, this new cytokine is constitutively expressed in the central nervous system. A monoclonal antibody reactive with a synthetic peptide of vgr revealed a faint vgr-like immunoreactivity throughout the CNS, with more pronounced staining of hippocampal neurons, ependymal cells, cells of the choroid plexus, and hypophysis. Using the rat cDNA, two homologous human cytokine cDNAs encoding the human vgr and op-1 were cloned.     

Expression patterns of epidermal growth factor receptor and fibroblast growth factor receptor 1 mRNA in fetal human brain

Factors that interact with the epidermal growth factor and fibroblast growth factor receptors have numerous effects in the central nervous system (CNS), inducing the proliferation of CNS stem cells and astrocytes and the survival and differentiation of neurons. Both receptors are expressed in the embryonic rodent brain in proliferative and nonproliferative regions, suggesting roles in numerous developmental processes. However, the roles of these factors in human brain development are not known. In the current study, we examined the expression of human epidermal growth factor receptor (HEGFR) and human fibroblast growth factor receptor 1 (HFGFR1) mRNAs in the human fetal brain. The expression of both receptors is strikingly conserved relative to previously reported patterns in the rodent. In the germinal zones, the sites of cellular proliferation, HFGFR1 was expressed primarily in the ventricular zone, whereas HEGFR was expressed in the subventricular zone, suggesting different roles in CNS progenitor proliferation. Differential expression was also observed in other brain areas examined, including the hippocampus and the cerebellum. The current study suggests that HEGFR and HFGFR1 are likely to play different roles during human brain development, but that these roles will be similar to those observed in the rodent brain.     

Isolation of cDNA clones encoding rat glial fibrillary acidic protein: expression in astrocytes and in Schwann cells.

Glial fibrillary acidic protein (GFAP) expressed by astrocytes in the central nervous system (CNS) has been extensively characterized but the molecular identity of related molecules in the peripheral nervous system (PNS) remains unclear. To examine possible structural differences between CNS and PNS GFAP, we have isolated cDNA clones for rat GFAP from both cultured astrocyte and Schwann cell libraries. Nucleotide sequence analysis indicated that the PNS and CNS GFAP clones contained identical coding regions, with a predicted protein product of 430 amino acids. However, the 5'-untranslated region of clone rGFA15, isolated from the Schwann cell library, was longer than that predicted for brain-derived GFAP mRNA. Primer extension analysis of RNA isolated from the RT4-D6 Schwann cell line indicated that the start site for PNS GFAP mRNA lies 169 bases upstream from that used in the CNS. In addition, tryptic peptide mapping of GFAP prepared from cultured astrocytes and Schwann cells revealed one major peptide fragment present in CNS GFAP but absent from PNS GFAP. These results suggest structural differences between GFAP in these two cell types, at both the nucleic acid and protein level, and are consistent with previous observations of immunochemical differences existing between CNS and PNS GFAP.     

Developmental regulation of pituitary adenylate cyclase-activating polypeptide and PAC(1) receptor mRNA expression in the rat central nervous system

As the brain develops, a homogeneous population of mitotically active progenitors generates the molecularly heterogeneous post-mitotic cells of the mature brain. The balance between cell division, growth arrest and differentiation of these progenitors undoubtedly requires the activation of a vast array of genes. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. Within the nervous system, PACAP has been shown to stimulate neurite outgrowth, regulate neurotransmitter production and neuronal survival. These diverse biological actions are mediated through interaction with two types of receptors, a PACAP-selective receptor (PAC(1)-R) and receptors which interact almost equally with both VIP and PACAP. Since several lines of evidence suggest that PACAP acts as a neurotrophic factor, we sought to characterize PACAP and PAC(1)-R expression in the developing rat nervous system. The PAC(1)-R is expressed at very high levels in ventricular zones throughout the neuraxis. In addition to the embryonic enrichment in proliferative zones, PAC(1)-R expression is maintained in areas of neurogenesis in the adult central nervous system (CNS), namely, the subventricular zone of the olfactory bulb and hippocampal dentate gyrus. In contrast, PACAP is expressed primarily in the post-mitotic parenchyma. This temporal regulation and cellular distribution suggests that PACAP, through its interaction with the PAC(1)-R, may play a role in mammalian neurogenesis.     

The natriuretic peptide system in the brain: implications in the central control of cardiovascular and neuroendocrine functions.

The natriuretic peptide system consists of three endogenous ligands, i.e., atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), and at least three subtypes of receptors. All of the peptides and receptors exist in the central nervous system (CNS). ANPs in the brain are N-terminally truncated forms: ANP (4-28) and ANP (5-28). The primary structure of BNP varies considerably among species, whereas that of CNP is highly conserved. ANP, BNP, and CNP are distributed in discrete brain regions, although the distribution varies in different species. Few immunohistochemical studies have so far been performed on BNP and CNP. There are three subtypes of receptors: ANP-A and ANP-B, which are bioactive, and the C receptor, which does not seem to be directly related to bioactivity. In the rat, the major subtype of ANP receptor in the CNS is the ANP-B receptor, based on the results of Northern blotting. Since the ligand for ANP-B receptor is CNP, the CNP-ANP-B receptor system may be most important, at least in rat brain. It is still unknown whether or not a specific receptor for BNP exists in central or peripheral tissues. Further studies should clarify the exact localization of ANP, BNP, and CNP and the three receptor subtypes in the CNS. Although natriuretic peptides and their receptors are distributed widely in the CNS, the AV3V regions, basal medial hypothalamus, brainstem, and circumventricular organs are the most prominent sites. This suggests an important physiological role of the natriuretic peptide system in the central control of cardiovascular homeostasis. The natriuretic peptide system seems to be involved in the regulation of water and salt intake, blood pressure, and secretion of vasopressin in the direction of reducing body fluid and lowering blood pressure. Such actions of natriuretic peptides are antagonistic to the central actions of angiotensin II (AII). In fact, the distribution of ANP and AII and their receptors in the CNS overlaps considerably. It is highly likely, therefore, that the central natriuretic peptide system and the renin-angiotensin system play important roles in the central control of cardiovascular and body fluid homeostasis in opposite directions. The natriuretic peptide system may also be involved in neuroendocrine control and some other CNS functions, although the physiological significance of these actions is less clear at the present time. It is now clear that there is considerable plasticity in the regulation of natriuretic peptides and their receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Expression patterns of Notch1, Notch2, and Notch3 suggest multiple functional roles for the Notch-DSL signaling system during brain development.

The Notch-DSL signaling system consists of multiple receptors and ligands, and plays many roles in development. The function of Notch receptors and ligands in mammalian brain, however, is poorly understood. In the current study, we examined the expression patterns for three receptors of this system, Notch1, 2, and 3, in late embryonic and postnatal rat brain by in situ hybridization. The three receptors have overlapping but different patterns of expression. Messenger RNA for all three proteins is found in postnatal central nervous system (CNS) germinal zones and, in early postnatal life, within numerous cells throughout the CNS. Within zones of cellular proliferation of the postnatal brain, Notch1 mRNA is found in both the subventricular and the ventricular germinal zones, whereas Notch2 and Notch3 mRNAs are more highly localized to the ventricular zones. Both Notch1 and Notch3 mRNAs are expressed along the inner aspect of the dentate gyrus, a site of adult neurogenesis. Notch2 mRNA is expressed in the external granule cell layer of the developing cerebellum. In several brain areas, Notch1 and Notch2 mRNAs are relatively concentrated in white matter, whereas Notch3 mRNA is not. Neurosphere cultures (which contain CNS stem cells), purified astrocyte cultures, and striatal neuron-enriched cultures express Notch1 mRNA. However, in these latter cultures, Notch1 mRNA is produced by nestin-containing cells, rather than by postmitotic neurons. Taken together, these results support multiple roles for Notch1, 2, and 3 receptor activation during CNS development, particularly during gliogenesis.     

Expression of the EGF receptor family members ErbB2, ErbB3, and ErbB4 in germinal zones of the developing brain and in neurosphere cultures containing CNS stem cells

The epidermal growth factor receptor family consists of four related tyrosine kinases: the epidermal growth factor receptor (EGF-R or ErbB), ErbB2, ErbB3, and ErbB4. These receptors are capable of extensive cross-activation upon the binding of their ligands - the EGF family of peptides for EGF-R and the neuregulins for ErbB3 and ErbB4. Since EGF-R is expressed by proliferating cells in the central nervous system (CNS), including multipotent CNS stem cells, we examined the expression of ErbB2, ErbB3 and ErbB4 in the germinal epithelia of the developing rat brain using in situ hybridization. ErbB2 and ErbB4 mRNAs were widely distributed within the germinal zones as early as E12. However, as development proceeded, ErbB2 mRNA was mainly present within the layers of cells immediately adjacent to the ventricular surface - the ventricular zone, while ErbB4 mRNA was predominantly expressed by subventricular zone cells, in the regions where these specialized germinal epithelia were present. ErbB3 mRNA distribution within germinal epithelia was more restricted, primarily confined to the diencephalon and rostral midbrain. Cultured neurospheres, which contain CNS stem cells, expressed ErbB2, ErbB4 and, to a lesser extent, ErbB3 protein as demonstrated by Western blot analysis. This expression declined during following differentiation. Heregulin-beta1, a neuregulin, had no effect on the proliferative capacity of neurospheres. Overall, our results indicate that ErbB2, ErbB3 and ErbB4 may play important and distinct roles in the genesis of the CNS. However, our in vitro data do not support a role for neuregulins in proliferation, per se, of CNS stem cells.     

Central interaction between endothelin and brain natriuretic peptide on pressor and hormonal responses

Interaction between intracerebroventricular (i.c.v.) administration of endothelin (ET) and brain natriuretic peptide (BNP) on pressor and hormonal responses was examined in unanesthetized, freely moving rats. I.c.v. administered ET (5, 20 or 40 pmol/2 microliters) dose-dependently increased arterial pressure. Plasma catecholamine levels were elevated by 40 pmol of ET, and plasma ACTH level was also elevated by centrally administered ET in a dose-dependent manner. I.c.v. administration of BNP (0.2, 1 nmol/3 microliters) dose-dependently attenuated central ET (40 pmol/2 microliter)-induced pressor response, plasma catecholamine and ACTH secretion. These results indicate that ET may be one of the neuropeptides which stimulate both sympathetic nervous system and hypothalamo-pituitary-adrenal axis, and that BNP and ET interact in the central nervous system (CNS) to regulate cardiovascular and hormonal functions. Furthermore, these results raise a possibility that BNP antagonizes the effect of not only angiotensin II but also other neuropeptides in the CNS.     

The development of rat brain bombesin-like peptides and their receptors

The development of bombesin-like peptides and their receptors was determined in 9 different rat brain regions. The concentration of immunoreactive bombesin increased dramatically in all brain regions examined during the third week after birth. In contrast, the density of [125I-Tyr4]bombesin binding sites was near maximal in all brain regions examined by the second postnatal week. These data indicate that the development of receptors precede bombesin-like peptide production.     

Transient hypoxia/hypoglycemia upregulates endothelin B receptors in cultured rat astrocytes

Endothelins are potent vasoactive peptides that bind to their specific receptors, playing an important role in the CNS under physiological and pathophysiological conditions. Astrocytes, which have been shown to express these receptors, also have a considerable role to play under physiological and pathophysiological conditions, particularly those involved in delayed neuronal death. We carried out in vitro receptor autoradiographic binding experiments using specific ligands for endothelin receptors on cultured rat astrocytes. On astrocytes, the specific binding sites for (125)I-PD151242 (a selective endothelin A receptor antagonist) and (125)I-IRL 1620 (a selective endothelin B receptor agonist) were detected. We also characterized the qualitative and quantitative changes of endothelin receptors 24 h after subjecting cultured rat astrocytes to a transient 4-h hypoxia/hypoglycemia insult, used as a model of delayed neuronal death. After transient hypoxia/hypoglycemia, the number of endothelin B receptors increased significantly on cultured astrocytes, but this did not occur among the endothelin A receptors. These findings suggest that the astrocytic effects associated with endothelin in delayed neuronal death include gliosis or the repair process or both, manifested primarily by an increase in the number of endothelin B receptors. This rise does not require interaction with other types of CNS cells. Endothelin A receptors might have a role taking their number into consideration on rat astrocytes under both physiological and pathophysiological conditions.