Peptides In Rat Brain Research Articles

Binding sites for melanotropins and related peptides in rat brain

Binding sites for melanotropins and related peptides in rat brain

Binding of atrial and brain natriuretic peptides in brains of hypertensive rats

Displacement of bound [ 125I]α-atrial natriuretic peptide (α-ANP) by brain natriuretic peptide (BNP) was used to map receptors common to both peptides in rat brain by in vitro autoradiography. Both spontaneously hypertensive rats (SHR) and the normotensive Wistar-Kyoto control strain (WKY) were studied. In both strains, [ 125I]α-ANP bound densely to subfornical organ, choroid plexus and arachnoid mater. Binding at these sites in either strain was displaced similarly by 1 μM unlabelled α-ANP or BNP. However, no [ 125I]α-ANP was displaced by peptides unrelated to α-ANP or BNP. In WKY, both α-ANP and BNP competed with similarly high affinities for binding sites occupied by [ 125I]α-ANP. This was also true for SHR. However, SHR showed a substantial reduction in the maximum number of binding sites in the subfornical organ and choroid plexus which were competed for by the peptides. Therefore, BNP may be a significant high affinity ligand for brain receptors previously thought specific for atrial natriuretic peptides, including receptors which vary between WKY and SHR.

Autoradiographic localization of atrial and brain natriuretic peptide receptors in rat brain

Displacement of bound 125I-labeled atrial natriuretic peptide-(1-28) [alpha 125I-ANP-(1-28)] by alpha-ANP-(5-28) and porcine brain natriuretic peptide (BNP) was used to map receptors common to these peptides in rat brain by in vitro autoradiography. alpha-125I-ANP bound reversibly to subfornical organ, area postrema, median preoptic, supraoptic and paraventricular nuclei, and arachnoid mater. Binding at these sites was displaced similarly by 1 microM unlabeled alpha-ANP, alpha-ANP-(5-28), and BNP. Binding dissociation constants in the subfornical organ and arachnoid were 4.40 +/- 1.15 and 3.99 +/- 0.86 nM, respectively, for alpha-ANP, and 2.41 +/- 1.11 and 2.23 +/- 1.06 nM, respectively, for BNP. alpha-125I-ANP also bound to choroid plexus. Here 1 microM unlabeled alpha-ANP displaced significantly more radioligand than did 1 microM BNP, and the concentration displacing 50% of bound radioligand was 2.23 +/- 0.78 nM for alpha-ANP and 1.51 +/- 0.67 nM for BNP. alpha-ANP-(5-28) also displaced alpha 125I-ANP at all sites with significantly greater affinity than did unlabeled alpha-ANP. alpha-125I-ANP was not displaced by completely unrelated peptides. Therefore, both atrial and brain natriuretic peptides may be high-affinity ligands at common receptors in some cerebral localities.

Changes of proenkephalin and prodynorphin mRNAs and related peptides in rat brain during the development of deep prepyriform cortex kindling

The effects of deep prepyriform cortex (DPC) kindling on the amount of proenkephalin and prodynorphin mRNAs, Met 5-enkephalin (ME) and dynorphin (DYN) in rat brain were examined. Animals received electrical stimulation of the DPC until two consecutive stage 2 seizures (S2) or stage 5 seizures (S5) were attained. The proenkephalin mRNA and ME contents in the entorhinal cortex were increased 24 h after S2 and also 5 min and 24 h post S5. In the hippocampus, the proenkephalin mRNA level was reduced 24 h after S2 but increased 5 min and 24 h after S5. Elevated hippocampal ME concentration was observed 24 h after S2 and S5. Similarly, the ME level in the frontal cortex was increased 24 h after S2 and S5 but the proenkephalin mRNA content was only elevated at S5. In the striatum, the proenkephalin mRNA level was slightly increased 24 h after S2 and S5, but no change in ME content was found. The amount of prodynorphin mRNA in the hippocampus was attenuated only at 24 h after S5, whereas DYN concentration was reduced 5 min after S5. No change in striatal DYN concentration was observed despite a slight elevation of prodynorphin mRNA 24 h post S2 and S5. Six weeks after the last seizure, no difference in ME and DYN was found between kindled and control animals. These findings indicate that the enkephalin-containing perforant pathway in the entorhinal cortex-hippocampal region is particularly sensitive to electrical stimulations applied to the DPC. Its role and importance in the development of kindling are discussed.

Presence of calcitonin-like peptide in rat milk: possible physiological role in regulation of neonatal prolactin secretion.

Previous results have shown that salmon calcitonin (sCT), a peptide in rat brain and pituitary gland, inhibits basal and TRH-stimulated PRL release and reduces PRL mRNA levels in isolated anterior pituitary cells of adult rats in culture. Rat milk contains a variety of neuropeptides and hormones, some of which are absorbed in bioactive form to exert endocrine influences in the developing offspring. The present studies were undertaken to investigate whether a CT-like peptide is present in rat milk. Circulating PRL levels in neonatal rats are low, and there is an abrupt increase in the basal secretion of this hormone at weaning. A second objective was to examine whether CT plays a role in the regulation of PRL secretion in neonatal animals. A sensitive and specific RIA for sCT was developed and used to assay rat milk on various days of lactation for sCT-like immunoreactivity. sCT-like activity was present in the water-soluble (infranatant) fraction of milk throughout lactation in concentrations as high as 1589 pg/ml. There were no statistically significant differences in immunoreactive levels of the peptide in milk samples from different days of lactation. sCT-like immunoreactivity in rat milk infranatant coeluted with synthetic sCT on reverse phase HPLC, and these HPLC fractions inhibited basal PRL release when added to cultures of anterior pituitary cells. This inhibition of PRL release by the sCT-immunoreactive HPLC fractions was comparable to that exerted by equivalent concentrations of synthetic sCT. Newborn rats were injected sc with 10 microliters normal rabbit serum or anti-sCT serum from the day of birth until postpartum day 10. The rats were killed on day 11, and their sera were analyzed for PRL. Anti-sCT-injected rats showed a significant increase in serum PRL levels compared to those in untreated or normal serum-treated rats. These results demonstrate that a CT-like peptide, which is a potent inhibitor of PRL release, is present in rat milk throughout lactation, and that passive immunization with a highly specific anti-sCT serum leads to an increase in serum PRL levels in neonatal rats. CT, possibly of milk origin, may be a physiologically relevant PRL-inhibiting factor during the neonatal period.

Distribution of two distinct messenger ribonucleic acids encoding gastrin-releasing peptide in rat brain

There are two distinct mRNAs that encode the precursor to gastrin-releasing peptide (GRP) in rat brain. These two messages arise from separate transcription initiation sites located approximately 400 base pairs apart, which are presumably regulated by separate promoters. In the present study, we mapped the distribution of neurons containing GRP mRNAs by in situ hybridization using cRNA and synthetic DNA probes specific for the 1.5 kb GRP transcript and probes complementary to both the 1.5 kb and 1.1 kb transcripts. The distribution of neurons expressing GRP mRNA appears to be wider than that previously observed by immunohistochemical studies, suggesting an important functional role for this neuropeptide in a number of brain regions. We detected the 1.5 kb transcript only in cingulate cortex, Ammon's horn of the hippocampus and in subiculum. In contrast, the probe which hybridized to both GRP mRNAs labeled a broad range of brain areas, including those containing the 1.5 kb mRNA. These data suggest that the 1.5 kb mRNA encoding rat GRP is expressed only in specific parts of the limbic system, whereas the expression of the 1.1 kb GRP meassage is more widespread.

Identification and Characterization of TRH-Precursor Peptides

We have now characterized five cryptic peptides in rat brain together with two partially processed TRH-progenitor sequences. Our studies indicate that pro-TRH is processed at paired basic residues to a family of peptides, including TRH, and flanking and intervening sequences. These cryptic sequences may have evolutionary significance or have functions during mRNA processing, translation, and posttranslational processing, or may be biologically active peptides in their own right. We have recently identified a TRH producing-cell line and are characterizing cell lines stably transfected with expression vectors containing prepro-TRH cDNA. These models should provide further insights into the posttranslational processing of pro-TRH.

Pro-TRH Gene Expression and Precursor Peptides in Rat Brain:.

Pro-TRH Gene Expression and Precursor Peptides in Rat Brain:.

Pro-TRH Gene Expression and Precursor Peptides in Rat Brain:.

Pro-TRH Gene Expression and Precursor Peptides in Rat Brain:.

High-performance liquid chromatographic determination of substance p-like arginine-containing peptide in rat brain by on-line post-column fluorescence derivatization with benzoin

A high-performance liquid chromatographic method with fluorescence detection is described for the determination of substance P, one of the neuropeptides, in the hypothalamus tissue of rat brain. The detection is based on on-line post-column fluorescence derivatization selective for arginine-containing peptides. The endogenous substance P-like arginine-containing peptide extracted from the tissue and [D-Phe11]-neurotensin as an internal standard were separated from various interfering substances on a reversed-phase column (TSKgel ODS-120T) by gradient elution with acetonitrile-phosphate buffer (pH 2.3). The peptides in the eluate were then automatically converted into fluorescent derivatives for detection by reaction with benzoin. Arginine-containing fragments produced by the enzyme reaction of substance P in the chromatographic fraction with trypsin were also detected, for the identification of the endogenous substance P-like arginine-containing peptide. The method was sensitive enough to permit the quantitative determination of the peptide at a concentration as low as 580 fmol/mg of protein in the brain homogenate. The concentration values of the substance P-like arginine-containing peptide in the tissue were 9.45 +/- 1.50 pmol/mg of protein (six determinations).

High-performance liquid chromatographic determination of leucine-enkephalin-like peptide in rat brain by pre-column fluorescence derivatization involving formylation followed by reaction with 1,2-diamino-4,5-dimethoxybenzene

Fluorimetrically reactive leucine-enkephalin, one of the opioid peptides in rat brain tissues such as the striatum, cortex and hypothalamus, was assayed by reversed-phase high-performance liquid chromatography with fluorescence detection based on pre-column derivatization of the tyrosyl residue. The tyrosine-containing peptides extracted from the tissue were first formylated with chloroform in an alkaline medium, and the resulting aldehydes were then converted into highly fluorescent derivatives by reaction with 1,2-diamino-4,5-dimethoxybenzene. The derivative of leucine-enkephalin-like peptide in tissue was separated from variuos other fluorescent compounds on a reversed-phase column (TSK gel ODS-120T) by isocratic elution and detected by fluorimetry. The concentrations of the leucine-enkephalin-like peptide in the tissues were 20–245 pmol/g. The method is sensitive enough to permit the quantitative determination of the endogenous peptide at concentrations as low as 5.6 pmol/g in brain tissues.

Characterization of picomolar affinity binding sites for [125I]-human calcitonin gene-related peptide in rat brain and heart.

We have characterized picomolar affinity binding sites for human calcitonin gene-related peptide (CGRP) in rat brain and heart (atria and ventricle) membranes. By saturation analysis, apparent dissociation constant (KD) values of high affinity sites for [125I]-human CGRP are 9 approximately 15 pM (brain), 34 pM (ventricle) and 85 pM (atria). Low affinity sites with KD values of about 50 nM are found in rat brain and ventricle, but not in atria. Human and rat CGRP potently inhibited [125I]-human CGRP binding to these high affinity sites with apparent inhibition constant (Ki) values comparable to their KD values. Salmon calcitonin marginally inhibited these binding with Ki values between 0.1 microM and 1 microM. Extremely potent cardiovascular and gastrointestinal actions of CGRP might be mediated through CGRP binding sites with picomolar affinity which are similar to those we characterized in this study.

Localization of binding sites for calcitonin gene-related peptide in rat brain by in vitro autoradiography

The distribution of binding sites for calcitonin gene-related peptide (CGRP) in rat brain were studied using in vitro autoradiography. In a radioreceptor assay using [ 125I]human calcitonin gene-related peptide as the radioligand, with cerebellar cortical membranes, rat calcitonin gene-related peptide had a binding affinity constant of1.16 ± 0.23 × 10 10 M −1 and a site concentration of43.4 ± 3.4 fmol mg protein. In this system, human calcitonin gene-related peptide had a binding affinity constant of3.9 ± 0.7 × 10 9 M −1 whereas salmon calcitonin was very weak with a binding affinity constant of only6.8 ± 4.0 × 10 5 M −1. CGRP binding localized by in vitro autoradiography, using [ 125I]rat calcitonin gene-related peptide. had a characteristic distinct distribution in the rat brain. There were high concentrations of binding found over the accumbens nucleus, the organum vasculosum of the lamina terminalis, ventral caudate putamen, median eminence, the arcuate nucleus, lateral amygdaloid nucleus and lateral mammillary nucleus, the superior and inferior colliculi, pontine nuclei, molecular and Purkinje cell layers of the cerebellar cortex. the nucleus of the solitary tract, the inferior olivary nuclei, hypoglossal complex and the vestibular and cochlear nuclei. The distribution of these binding sites suggests multiple roles for CGRP in the central nervous system including auditory, visual, gustatory and somatosensory processing, and in neuroendocrine control.

A new immunization procedure for obtention of anti-leucine-enkephalin antibodies: Part II. Effects of Olfactory Bulb Removal on pro-enkephalin related peptides in rat brain

Bilateral Olfactory Bulb Removal (OBR) induced both complex behavioral alterations and a decrease of many neurotransmitter levels. We studied brain levels of the pro-enkephalin related peptides 45 days after OBR. Opioid levels were studied using three different highly specific antisera exhibiting very high affinities in radioimmunoassays in striatum, hypothalamus, hypophysis, brain stem and cortex. Methionine enkephalin levels increase significantly in striatum (42%), hypophysis (94%) and hypothalamus (25%) and non-significantly in the other areas. Leucine-enkephalin levels tended to increase in all dissected structures but a significant increase only occured in striatum (42%). Octapeptide levels (Methionine-enkephalin-Arg-Gly-Leu) significantly increase in striatum (22%) and decrease in hypophysis (97%) and in brain stem (76%). All these results are partially consistent with the decrease of opiate binding described previously after OBR and suggest a complex imbalance in neurotransmitters after such a sensorial deprivation. It is suggested that the modifications of enkephalinergic neurotransmission might be related to the stressful state induced by OBR.

Neuromedin B-like peptides in rat brain: Biochemical characterization, mechanism of release and localization in synaptosomes

Neuromedin B-like peptides were characterized in the rat brain. A rabbit antisera was utilized which recognized neuromedin B but not bombesin or GRP. Using gel filtration and HPLC techniques, a major and minor peak of immunoreactivity was present in rat brain extracts. In both cases the main peak of immunoreactivity coeluted with synthetic neuromedin B. The density of neuromedin B-like peptides ranged 50-fold being greatest in the olfactory bulb and hypothalamus, intermediate in the hippocampus, spinal cord, medulla/pons, pituitary, midbrain, thalamus, striatum and cortex and lowest in the cerebellum. Release studies indicated that neuromedin B-like peptides were secreted from hypothalamic, olfactory bulb and thalamic slices in a Ca ++-dependent manner when KCl (75 mM) was present. Also, the neuromedin B-like peptides in the rat brain were localized to synaptosomes. These data indicate that neuromedin B-like peptides may function as regulatory peptides in the CNS distinct from bombesin/GRP.

The endogenous tripeptide Tyr-Gly-Gly as a possible metabolite of opioid peptides in rat brain: Identification, regional distribution, effects of lesions and formation in depolarized slices

Using a sensitive radioimmunoassay, the tripeptide Tyr-Gly-Gly (YGG) which corresponds to the N-terminal sequence of opioid peptides was detected in rat brain and identified by HPLC. Its regional distribution paralleled that of (Met 5)enkephalin (YGGFM), a marker of enkephalin neurons. Ablation of these neurons in the striato-pallidal pathway by intrastriatal kainate, induced a significant decrease in YGG levels in caudateputamen and globus pallidus (−49%), consistent with the hypothesis that YGG originates from enkephalin neurons. When pallidal slices were incubated under various conditions, YGG was mainly found in the incubation medium indicating a predominantly extracellular localization. Depolarization of these slices by a K +-stimulus elicited a release of YGGFM accompanied by a marked increase in YGG levels. Bestatin and amastatin further enhanced YGG levels, reflecting the participation of aminopeptidases in the metabolism of the tripeptide and its precursor. Captopril, an inhibitor of the angiotensin-converting enzyme (ACE) showed no effect on the recovery of YGGFM and YGG. In contrast, the formation of YGG was completely prevented by Thiorphan (IC 50 value=9 nM) and phosphoramidon, two inhibitors of “enkephalinase” (EC 3.4.24.11; membrane metallo-endopeptidase), thus identifying the latter as the YGG-forming enzyme. The K +-induced increase in YGG + YGGFM levels in medium containing bestatin exceeded by about 60% the amount of YGGFM released from tissues, suggesting that YGG was mainly formed by extracellular hydrolysis of the various opioid fragments of the proenkephalin molecule. In vivo, YGG levels of cerebral regions were also markedly reduced in rats treated with acetorphan, a parenterally active “enkephalinase” inhibitor. All data suggest that YGG levels constitute an index of opioid peptide release.

Comparison of structural requirements of α-MSH and ACTH for inducing excessive grooming and pigment dispersion

α-MSH and ACTH-like peptides are known to play an important role in the adaptation of many vertebrates to a new environment. These peptides induce pigment dispersion in amphibian melanophores through a receptor-mediated mechanism. In this study we compared the structural requirements of these peptides for melanotropic activity on Xenopus laevis melanophores with those for inducing excessive grooming in the rat. With the exception of ACTH 1–24 there is a close resemblance in structure-activity relationships of the fragments and analogs tested in the two bioassays. [Nle 4,-D-Phe 7]-α-MSH is extremely active in both assays. Weak agonists such as [Leu 9]-α-MSH did not possess antagonistic properties either in the melanophore assay or in the excessive grooming test. The data suggest that the mechanism of action of α-MSH-like peptides in rat brain is receptor-mediated like their action on melanophores.

Cholecytokinin (CCK) gene-related peptides: distribution and characterization of immunoreactive pro-CCK and an amino-terminal pro-CCK fragment in rat brain

An antiserum specific for the amino terminus of pro-CCKK detects 3 major peptides in rat brain with molecular weithts of 13, 000, 8000 and 2700 daltons. These peptides are abundant both in CCK terminal-field regions and regions rich in CCK perikarya. The two high molecular weight peptides probably represent CCK precursor molecules. The smaller peptide is probably the amino-terminal fragment of pro-CCK and may be stored and released along with CCK-8, and have a potential role in synaptic transmission.

Peptides in rat brain immunoreactive for the carboxyl terminal extension of cholecystokinin: Distribution and chromatography

Utilizing an antiserum raised against a peptide fragment identical to part of the carboxyl terminal extension of cholecystokinin (CCK) predicted by the sequence of CCK mRNA [7], an antiserum has been generated which does not detect CCK 39, CCK 33, CCK 8, CCK 4 or gastrin 171. This antiserum detects several peptides in rat brain, one similar in size to CCK 33 and another slightly larger than CCK 8. These peptides may represent carboxyl-terminally extended forms of CCK, though their chemical structure has not been determined. These peptides are present in all brain regions where CCK 8 can be detected. The abundance of these peptides, their localization in CCK terminal regions, and their enrichment in synaptosome preparations [1] imply that the tryptic cleavage and amidation reaction occur late in the processing of CCK (as has been observed for other biologically active peptides), and probably occur in the synaptic vesicle.

Diazepam-binding inhibitor: a neuropeptide located in selected neuronal populations of rat brain.

An endogenous polypeptide of rat brain has been identified that is capable of displacing 1,4-benzodiazepines and the esters of the 3-carboxylic acid derivatives of beta-carbolines from their specific synaptic binding sites. This polypeptide was termed diazepam-binding inhibitor (DBI). Previous studies have shown that DBI injected intraventricularly in rodents elicits "proconflict" responses and antagonizes the "anticonflict" action of benzodiazepines. An antiserum to this peptide, directed toward an immunodeterminant near its amino terminus, makes it possible to detect, measure, and study the neuronal location of this peptide in rat brain. In the rat cerebral cortex, DBI immunoreactivity is located in neurons that are not GABAergic (GABA, gamma-aminobutyric acid); in the cerebellum and hippocampus, however, it might be present also in GABAergic neurons.