Porcine Hypothalamus Research Articles

Identification of Neuropeptide W as the Endogenous Ligand for Orphan G-protein-coupled Receptors GPR7 and GPR8

The structurally related orphan G-protein-coupled receptors GPR7 and GPR8 are expressed in the central nervous system, and their ligands have not been identified. Here, we report the identification of the endogenous ligand for both of these receptors. We purified the peptide ligand from porcine hypothalamus using stable Chinese hamster ovary cell lines expressing human GPR8 and cloned the cDNA encoding its precursor protein. The cDNA encodes two forms of the peptide ligand with lengths of 23 and 30 amino acid residues as mature peptides. We designated the two ligands neuropeptide W-23 (NPW23) and neuropeptide W-30 (NPW30). The amino acid sequence of NPW23 is completely identical to that of the N-terminal 23 residues of NPW30. Synthetic NPW23 and NPW30 activated and bound to both GPR7 and GPR8 at similar effective doses. Intracerebroventricular administration of NPW23 in rats increased food intake and stimulated prolactin release. These findings indicate that neuropeptide W is the endogenous ligand for both GPR7 and GPR8 and acts as a mediator of the central control of feeding and the neuroendocrine system.

Distribution of galanin-like peptide in the rat brain.

Galanin-like peptide (GALP) is a novel galanin-like peptide isolated from the porcine hypothalamus. To determine the distribution of GALP in the rat brain, we performed immunohistochemical studies using a monoclonal antibody toward the N-terminal sequence of GALP. GALP-immunoreactive neuronal cell bodies were observed only in the arcuate nucleus (Arc), which was further confirmed by in situ hybridization studies using digoxigenin-labeled antisense GALP riboprobe. Additional immunostained cells were found in the median eminence and infundibular stalk. The GALP neurons found in the Arc were further characterized by double label immunohistochemistry. More than 85% of the GALP neurons were immunostained with leptin receptor antibody. However, the GALP neurons and fibers found in the Arc were not labeled with alpha-MSH, somatostatin, neuropeptide Y, agouti-related protein, or galanin antibodies, indicating that GALP is found in neurons other than these known Arc neurons. Dense staining of GALP-containing fibers was found in the anterior parvicellular part of the paraventricular hypothalamic nucleus, in the ventral part of the lateral septal nucleus, and in the bed nucleus of the stria terminalis. Relatively dense staining was noted in the medial preoptic area (MPA), and weak staining was noted in the periventricular hypothalamic nucleus. Detailed double labeling studies in the paraventricular hypothalamic nucleus demonstrated that GALP-containing fibers converged in a more rostral direction than did agouti-related protein-containing fibers. Furthermore, GALP-immunoreactive fibers were in close apposition with GnRH-immunoreactive fibers in the MPA and bed nucleus of the stria terminalis, and about 6% of GnRH-positive neurons in the MPA showed close contact with the GALP-immunoreactive fibers. Our findings indicate that GALP neurons, as leptin-responsive neurons, may participate in the regulation of feeding behavior and/or reproductive functions.

Distribution, regulation and role of hypothalamic galanin systems: renewed interest in a pleiotropic peptide family.

1. Galanin peptide and galanin receptor-binding sites are known to be widely distributed within the central nervous system, particularly in the hypothalamus in the preoptic area, the paraventricular (PVN) and supraoptic (SON) nuclei and the arcuate nucleus/median eminence. 2. The present brief review focuses on some recent studies of the regional and cellular localization of mRNA encoding galanin and two galanin receptor subtypes (GalR1 and GalR2) in the hypothalamus, regulation of galanin and/or galanin receptor expression in various nuclei by physiological stimuli, electrophysiological effects of galanin on hypothalamic neurons and the isolation and cloning of galanin-like peptide (GALP), a putative endogenous ligand for GalR2. 3. In situ hybridization studies in rat brain have demonstrated an abundance of GalR1 mRNA in SON, magnocellular (m) and parvocellular (p) PVN and dorsomedial, ventromedial and arcuate nuclei. In contrast, GalR2 mRNA is enriched in pPVN, but not mPVN, and is not detected in SON. In addition, GalR2 mRNA is present in the dorsomedial nucleus and is enriched in the arcuate nucleus compared with GalR1 transcripts, with numerous labelled cells in all subdivisions. 4. Neurons of the SON and PVN contain vasopressin and/or oxytocin, along with several other peptides, and the production and release of these hormones and peptides are modulated by various physiological stimuli. In relation to galanin systems, GalR1 and galanin expression is increased in magnocellular neurons by salt loading and is downregulated by lactation, consistent with an increased inhibition by galanin of vasopressin release following osmotic stimulation and a decreased inhibition of oxytocin release during lactation. 5. Powerful inhibitory effects of galanin on the electrical (and secretory) activity of magnocellular neurons and complex presynaptic actions of galanin on the synaptic release of glutamate in the arcuate nucleus in vitro suggest an active role for multiple galanin receptor subtypes in the regulation of these hypothalamic systems in vivo. 6. The recent isolation of a peptide from porcine hypothalamus (GALP-1-60) that is structurally related to galanin and appears to be selective for GalR2 over GalR1 and the subsequent cloning of GALP cDNA from pig, rat and humans should allow studies to help reveal the physiological role played by galanin receptor subtypes (especially GalR2) and their multiple ligands in the hypothalamus and other brain areas.

Galanin-Like Peptide mRNA in Neural Lobe of Rat Pituitary

Galanin-like peptide (GALP) was recently identified in the porcine hypothalamus, pituitary gland and gut, and has reported selectivity for the GalR2, c.f. the GalR1 receptor. GALP cDNAs have been cloned from pig, rat and human, and GALP mRNA expression is restricted to the arcuate nucleus in normal rat brain. This study examined the regional and cellular distribution of GALP mRNA in the rat pituitary gland, and subsequently determined the effect of osmotic stimulation on GALP transcript levels. GALP mRNA was not detected in the anterior or intermediate lobes, but moderate levels of GALP mRNA were present in the neural (posterior) lobe, in presumed pituicytes, of normal male and female rats. Osmotic stimulation by dehydration or salt loading produced a time-dependent increase in GALP mRNA levels in the neural lobe. Thus, dehydration for 4 days increased GALP mRNA 40-fold, while salt loading for 4, 7 or 10 days increased GALP levels 14-, 21- and 25-fold, respectively (p ≤ 0.001). Levels of vasopressin (VP) mRNA in the neural lobe were also increased by these treatments, consistent with previous reports. Galanin (GAL) and GalR2 receptor mRNAs were not detected in the neural lobe, under normal or osmotic stimulation conditions. In addition, GALP mRNA levels in the arcuate nucleus were not altered in dehydrated or salt-loaded rats; and GALP mRNA was not detected in magnocellular neurons of the supraoptic or paraventricular nucleus, despite the characteristic up-regulation of VP and GAL mRNA in these cells. In view of the established anatomy and function of VP/oxytocin neurons in the hypothalamo-neurohypophysial system and the role played by pituicytes in their regulation, the likely synthesis/release of GALP by these specialized astrocytes strongly suggests a role for this novel peptide in regulation of pituicyte morphology/function and/or neurohormone release.

An alternative approach to the synthesis of a recombinant galanin-like peptide (GALP)

GALP is a novel galanin-like peptide that has been isolated from porcine hypothalamus in our Research Division. In the present study, a large quantity of porcine GALP (pGALP) was synthesized with a high degree of purity using recombinant DNA technology. A basic fibroblast growth factor (bFGF) mutein, CS23, was used as a fusion partner and pGALP was released by chemical cleavage with cyanogen bromide (BrCN). Using various criteria such as HPLC, SDS–PAGE, amino acid analysis, amino acid sequence, relative-molecular-mass measurement by liquid secondary-ion mass spectrometry, as well as biological activity, the purified molecule was shown to be pGALP and was found to have the same biological activity as a chemically synthesized standard. The pGALP obtained here will be useful for elucidation of its physiological role in vivo. Furthermore, CS23ML76, a newly prepared bFGF mutein, was found to be more appropriate than CS23 as a fusion partner in this procedure.

Gene expression in the forebrain of dexamethasone-treated pigs: effects on stress neuropeptides in the hypothalamus and hippocampus and glutamate receptor subunits in the hippocampus

Gene expression studies advance our understanding of the effects of stress and glucocorticoids on brain function and give a new direction to animal welfare research. In this context, the presence of messenger RNA s (mRNA s) for corticotrophin releasing hormone (CRH) and vasopressin (VP) in the porcine hypothalamus has recently been documented. This study investigated the expression of CRH, VP and ionotropic glutamate receptor (iGluR) subunit m RNA s in the brains of pigs treated with the synthetic glucocorticoid dexamethasone (Dex; 5 mg kg–1i.v.). In the hypothalamus, VP, but not CRH, m RNA was reduced 3 hours after Dex. In the hippocampus, expression of m RNA s for some iGluR subunits appeared to be differentially regulated 6 hours after Dex. In addition, CRH message was detected in the hippocampus and significantly upregulated in the CA1 region 3 hours after Dex. The relevance of these findings to stress neurobiology of the growing pig is discussed.

Galanin-Like Peptide (GALP) mRNA Expression Is Restricted to Arcuate Nucleus of Hypothalamus in Adult Male Rat Brain

Galanin-like peptide (GALP) is a novel 60-amino acid neuropeptide, isolated from porcine hypothalamus and subsequently identified in rats and humans, which has reported selectivity for the Gal-R2 galanin receptor [Ohtaki T et al: J Biol Chem 1999; 274: 37041–37045]. In the current study, the regional and cellular distribution of GALP mRNA in rat brain has been investigated by in situ hybridization of [³⁵S]-labelled oligonucleotide probes. In a thorough screening of adult male rat brain, GALP mRNA expression was detected only throughout the rostrocaudal extent of the arcuate nucleus (ARC) with the most abundant hybridization signal in the posterior, periventricular zones. GALP mRNA-positive neurons were mostly localized in the ventromedial division of the ARC, with many closely adjacent to the wall of the third ventricle. Smaller numbers of labelled neurons were also found in ventrolateral areas. The distribution of GALP mRNA was somewhat complementary to that for galanin (GAL) mRNA in the ARC, but contrasted with the broad distribution of this transcript throughout the hypothalamus. GAL mRNA was also distributed along the rostrocaudal extent of the ARC, but was most abundant in the anterior to middle levels and in ventrolateral regions. Interestingly, somatostatin mRNA expression appeared to overlap the distribution of GALP mRNA in posterior, ventromedial regions of the ARC. Thus, in adult rat brain GALP mRNA expression was restricted to a discrete subpopulation of neurons in the ARC, with a unique localization pattern unlike GAL or many other known peptide- and transmitter-containing cells in this region. GALP could, however, be co-expressed in sub-populations of other neuronal phenotypes (e.g. somatostatin neurons) or within cells that express Gal-R2 receptors. In view of the established anatomy and function of the ARC and the restricted localization of GALP mRNA, this novel peptide is likely to play a role in regulation of anterior pituitary hormone secretion, or in regulation of other hypothalamic peptide and transmitter systems.

Galanin-like peptide (GALP) is a target for regulation by leptin in the hypothalamus of the rat.

Galanin-like peptide (GALP), which was recently isolated from the porcine hypothalamus, shares sequence homology with galanin and binds with high affinity to galanin receptors. To study the distribution and regulation of GALP-expressing cells in the brain, we cloned a 120 base-pair cDNA fragment of rat GALP and produced an antisense riboprobe. In situ hybridization for GALP mRNA was then performed on tissue sections throughout the forebrain of adult ovariectomized female rats. We found GALP mRNA-containing cells in the arcuate nucleus (Arc), caudal dorsomedial nucleus, median eminence and the pituitary. Because GALP mRNA in the Arc appeared to overlap with the known distribution of leptin receptor mRNA, we tested the hypothesis that GALP expression is regulated by leptin. Using in situ hybridization, we compared the number of GALP mRNA-containing cells among groups of rats that were fed ad lib or fasted for 48 h and treated with either leptin or vehicle. Fasting reduced the number of identifiable cells containing GALP mRNA in the Arc, whereas the treatment of fasted animals with leptin produced a 4-fold increase in the number of cells expressing GALP message. The presence of GALP mRNA in the hypothalamus and pituitary and its regulation by leptin suggests that GALP may have important neuroendocrine functions, including the physiological regulation of feeding, metabolism, and reproduction.

Isolation and cDNA Cloning of a Novel Galanin-like Peptide (GALP) from Porcine Hypothalamus

Galanin is a widely distributed neuropeptide with a variety of physiological functions. Three galanin receptor subtypes, GALR1, GALR2, and GALR3, have been reported. We isolated a novel galanin-like peptide (GALP) from porcine hypothalamus by observing its activity for increasing [(35)S]GTPgammaS binding to a membrane preparation of GALR2-transfected cells. The peptide had 60 amino acid residues and a non-amidated C terminus. The amino acid sequence of GALP-(9-21) was completely identical to that of galanin-(1-13). A cloned porcine GALP cDNA indicated that GALP was processed from a 120-amino acid GALP precursor protein. The structures of rat and human GALP-(1-60) were deduced from cloned cDNA, which indicated that the amino acid sequences 1-24 and 41-53 were highly conserved between humans, rats, and pigs. Receptor binding studies revealed that porcine GALP-(1-60) had a high affinity for the GALR2 receptor (IC(50) = 0.24 nM) and a lower affinity for the GALR1 receptor (IC(50) = 4.3 nM). In contrast, galanin showed high affinity for the GALR1 (IC(50) = 0.097 nM) and GALR2 receptors (IC(50) = 0.48 nM). GALP is therefore an endogenous ligand that preferentially binds the GALR2 receptor, whereas galanin is relatively non-selective.

Expression of luteinizing hormone-releasing hormone and its receptor in porcine immune tissues

Luteinizing hormone-releasing hormone (LHRH) is the primary regulator of pituitary LH release. However, LHRH has also been identified in extrahypothalamic sites including immune tissues. Accordingly, immunomodulatory properties for LHRH have been suggested. We wanted to determine whether LHRH and its receptor are produced by immune tissues in the pig. First, a cDNA was cloned and sequenced from the porcine hypothalamus that showed 87.5% homology with the human LHRH gene. Internal primers were identified from this sequence for amplifying a 268 bp product by PCR. In addition to the hypothalamus, PCR products reflecting LHRH mRNA were amplified in porcine spleen, thymus, and peripheral blood lymphocyte (PBL) cDNA. LHRH mRNA was not detected in liver, cerebral cortex, or pituitary tissue samples. Primers were designed to amplify a 360 bp fragment of LHRH-receptor cDNA. PCR products reflecting LHRH-receptor mRNA were amplified in pig hypothalamus, pituitary, thymus, spleen and PBL cDNA samples. No such products were amplified in cortex and liver samples. In summary, we report the sequence of a cDNA coding for LHRH and Gonadotropin-RH associated peptide (GAP) in the pig hypothalamus. Additionally, we provide evidence that LHRH and its receptor are synthesized in porcine immune tissues. This leads us to speculate that LHRH may have local, immunomodulatory functions in pigs.

Catecholaminergic region A15 in the bovine and porcine hypothalamus

Magnocellular perikarya within the retrochiasmatic division of the supraoptic nucleus of bovine and porcine hypothalami were immunoreactive (ir) with antiserum against tyrosine hydroxylase (TH), but not dopamine-β-hydroxylase (DBH). Few cells in this region were also immunoreactive for vasopressin (VP) or oxytocin (OT). In contrast, the main division of the supraoptic nucleus contained numerous perikarya immunoreactive for VP and OT, but not TH nor DBH. Both the retrochiasmatic and principal divisions of the supraoptic nuclei contained TH- and DBH-ir fibers and varicosities. This region in bovine and porcine hypothalami corresponds to the ventral A15 catecholaminergic (dopamine-producing) cell group.

Distribution of interleukin 1β immunoreactivity within the porcine hypothalamus

The presence and localization of interleukin 1β immunoreactivity (IL 1β i.r.) was studied in the hypothalamus of four healthy, male pigs at 7 months of age, using immunocytochemical techniques on 100 μ vibratome and 10 μ paraffine sections. IL 1β i.r. was found in neuronal cell bodies and their processes within nuclei and fiber of the fiber tracts of the hyphotalamus as well as in varicose fibers, terminals and deposits within the median eminence. In addition, IL 1β i.r. was found in the walls of several, but not all, blood vessels and in very few glial cells.

Fc-mediated nonspecific staining of the porcine brain with rabbit antisera in immunocytochemistry is prevented by pre-incubation of the sera with proteins A and G.

Nonspecific staining was detected in immunocytochemical procedures on the porcine hypothalamus with rabbit antisera, irrespective of the antigen specificity of the sera, in magnocellular neurons of the paraventricular (PVN) and supraoptic nuclei (SON), and in the vasopressin- and oxytocin-containing nucleus (VON). The present study was designed to test the hypothesis that this staining is mediated by the Fc portion of rabbit immunoglobulins. Rabbit antisera against neuropeptides localized predominantly outside the PVN, SON, and VON were employed in combination with different detection methods. The intensity of the nonspecific staining varied depending on the antiserum and persisted after pre-absorption of the antisera with their homologous peptides. Nonspecific staining and antigen-specific staining were differentially affected by the method of tissue fixation. The nonspecific staining could be prevented by preincubation of the antisera with proteins A and G, which left the antigen-specific staining intact, whereas additional preabsorption with homologous peptide abolished all staining. These observations suggest that the Fc region of IgGs is indeed involved in the nonspecific staining. On press-blots of homogenates from SON tissue subjected to isoelectric focusing, one band in the low-pH region was found with all antisera. Pre-incubation of the antisera with protein A abolished the staining of this band but did not affect staining of antigen-specific bands. Pre-incubation with proteins A and G is proposed as a routine control to check for nonspecific staining mediated by the Fc region of IgGs in immunocytochemical procedures, particularly those that employ rabbit sera in porcine brain.

Isolation and characterization of two peptides with prolactin release-inhibiting activity from porcine hypothalami.

Two peptides with in vitro prolactin release-inhibiting activity were purified from stalk median eminence (SME) fragments of 20,000 pig hypothalami. Monolayer cultures of rat anterior pituitary cells were incubated with aliquots of chromatographic fractions and the inhibition of release of prolactin in vitro was measured by RIA in order to monitor the purification. The hypothalamic tissue extract was separated into 11 fractions by high-performance aqueous size-exclusion chromatography with one fraction showing a 4-fold increase in prolactin release-inhibiting factor (PIF) activity. This material was further purified by semipreparative reversed-phase (RP) HPLC. This process resulted in the separation of two distinct fractions that showed high PIF activity. These were further purified by semipreparative and analytical RP-HPLC to apparent homogeneity as judged by the UV absorbance profiles. Neither of the two peptides showed cross-reactivity with gonadotropin releasing hormone-associated peptide or with somatostatin-14 antibodies. Protein sequence analysis revealed that one of the PIF peptides was Trp-Cys-Leu-Glu-Ser-Ser-Gln-Cys-Gln-Asp-Leu-Ser-Thr-Glu-Ser-Asn-Leu-Leu- Ala-Cys - Ile-Arg-Ala-Cys-Lys-Pro, identical to residues 27-52 of the N-terminal region of the proopiomelanocortin (POMC) precursor (corresponding to amino acids 1-26 of the 16-kDa fragment). The sequence of the other PIF was Ala-Ser-Asp-Arg-Ser-Asn-Ala-Thr-Leu-Leu-Asp-Gly-Pro-Ser-Gly-Ala-Leu-Leu- Leu-Arg - Leu-Val-Gln-Leu-Ala-Gly-Ala-Pro-Glu-Pro-Ala-Glu-Pro-Ala-Gln-Pro-Gly-Val- Tyr, representing residues 109-147 of the vasopressin-neurophysin precursor. Synthetic peptides corresponding to the N-terminal region of POMC had significant PIF activity in vitro.

Permeability of the blood brain barrier and changes of the GnRH system within the porcine hypothalamus after active immunisation against GnRH

Permeability of the blood brain barrier and changes of the GnRH system within the porcine hypothalamus after active immunisation against GnRH

Permeability of the blood brain barrier and changes of the GnRH system within the porcine hypothalamus after active immunisation against GnRH

Permeability of the blood brain barrier and changes of the GnRH system within the porcine hypothalamus after active immunisation against GnRH

Poly(A) tail length of oxytocin- and lysine vasopressin-encoding mRNAs increases during development in the porcine hypothalamus

Lysine vasopressin- and oxytocin-encoding mRNAs have been analysed in the developing hypothalamus of the pig. The two hormone-encoding mRNAs were first detectable on fetal day 49 by Northern blot analysis. Whereas RNase mapping revealed identical transcripts throughout the developmental stages studied, Northern blots showed that the early transcripts appeared to be shorter (by 100-200 nucleotides) and more heterogeneous in size than those of later stages. This developmentally related length polymorphism was shown to be due to different poly(A) lengths and was abolished by removal of the poly(A) tails with RNase H. These results indicate that maturation of neurones in the developing porcine hypothalamus is accompanied by an increase in length of the poly(A) tail of vasopressin and oxytocin mRNAs.

Endothelin: A Novel Peptide in the Posterior Pituitary System

Endothelin (ET), originally characterized as a 21-residue vasoconstrictor peptide from endothelial cells, is present in the porcine spinal cord and may act as a neuropeptide. Endothelin-like immunoreactivity has now been demonstrated by immunohistochemistry in the paraventricular and supraoptic nuclear neurons and their terminals in the posterior pituitary of the pig and the rat. The presence of ET in the porcine hypothalamus was confirmed by reversed-phase high-pressure liquid chromatography and radioimmunoassay. Moreover, in situ hybridization demonstrated ET messenger RNA in porcine paraventricular nuclear neurons. Endothelin-like immunoreactive products in the posterior pituitary of the rat were depleted by water deprivation, suggesting a release of ET under physiological conditions. These findings indicate that ET is synthesized in the posterior pituitary system and may be involved in neurosecretory functions.

Hypothalamic prostaglandin E 2 receptors coupled to an adenylyl cyclase

We show that the effect of prostaglandin (PG) E 2 on luteinizing hormone-releasing hormone (LHRH) release involves a receptor-mediated process coupled to an adenylyl cyclase system. The adenylyl cyclase activity in rat hypothalamus synaptic membrane preparations was stimulated by PGE 2 and this stimulation was directly related to the presence of guanine nucleotide (GTP). PGE 2 specifically bound to P 2 membranes from rat and porcine hypothalami with similar characteristics. Computer-fitted saturation curves provided evidence for two binding components which may be two states of the same receptor (R H and R L). Experiments with Gpp(NH)p, a non-metabolizable analogue of GTP, suggested the interconversion of R H and R L. These results may reflect different states of the ternary complex (hormone-receptor-guanine binding protein). Magnesium (Mg 2+) can modify the R H and R L binding parameters, but seems to act directly on the PGE 2 receptor site.

Purification and characterization of peptides with corticotropin-releasing factor activity from porcine hypothalami.

Ten polypeptides that stimulated the release of corticotropin from superfused rat pituitary cells and that are structurally related to porcine corticotropin-releasing factor were isolated from porcine hypothalami. The purification was carried out by gel filtration followed by reversed-phase HPLC using trifluoroacetic acid or heptafluorobutyric acid as the ion-pairing agent in water/acetonitrile solvent systems. The purified peptides were homogeneous by chromatography and by sequence analysis. One major polypeptide was characterized. Its structure is -H-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Gl u-Val -Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys -Leu-Met-Glu-Asn-Phe-NH2 [Patthy, M., Horvath, J., Mason-Garcia, M., Szoke, B., Schlesinger, D. H. & Schally, A. V. (1985) Proc. Natl. Acad. Sci. USA 82, 8762-8766]. This 41-amino acid sequence is thought to represent porcine corticotropin-releasing factor. Based on automated gas-phase sequencing of the intact and CNBr-cleaved peptides, amino acid analysis, and carboxypeptidase Y digestion, the other nine polypeptides were found to be structurally similar to this 41-amino acid sequence. Modifications of this structure include deamidation of glutamine at position 26 or 29, oxidation of methionine at positions 21 and/or 38, a blocked N terminus, and deletion of phenylalanine amide at the C terminus. Eight of these nine modified peptides retained significant corticotropin-releasing factor activity as shown by the stimulation of corticotropin release from superfused rat and pig pituitary cells. Some of these peptides may be present in pig hypothalami, while the others could have been produced during the isolation.