Peptides In Rat Brain Research Articles

The atypical chemokine receptor ACKR3/CXCR7 is a broad-spectrum scavenger for opioid peptides

Endogenous opioid peptides and prescription opioid drugs modulate pain, anxiety and stress by activating opioid receptors, currently classified into four subtypes. Here we demonstrate that ACKR3/CXCR7, hitherto known as an atypical scavenger receptor for chemokines, is a broad-spectrum scavenger of opioid peptides. Phylogenetically, ACKR3 is intermediate between chemokine and opioid receptors and is present in various brain regions together with classical opioid receptors. Functionally, ACKR3 is a scavenger receptor for a wide variety of opioid peptides, especially enkephalins and dynorphins, reducing their availability for the classical opioid receptors. ACKR3 is not modulated by prescription opioids, but we show that an ACKR3-selective subnanomolar competitor peptide, LIH383, can restrain ACKR3’s negative regulatory function on opioid peptides in rat brain and potentiate their activity towards classical receptors, which may open alternative therapeutic avenues for opioid-related disorders. Altogether, our results reveal that ACKR3 is an atypical opioid receptor with cross-family ligand selectivity.

Activation of Kappa Opioid Receptor Regulates the Hypothermic Response to Calorie Restriction and Limits Body Weight Loss.

Mammals maintain a nearly constant core body temperature (Tb) by balancing heat production and heat dissipation. This comes at a high metabolic cost that is sustainable if adequate calorie intake is maintained. When nutrients are scarce or experimentally reduced such as during calorie restriction (CR), endotherms can reduce energy expenditure by lowering Tb [1-6]. This adaptive response conserves energy, limiting the loss of body weight due to low calorie intake [7-10]. Here we show that this response is regulated by the kappa opioid receptor (KOR). CR is associated with increased hypothalamic levels of the endogenous opioid Leu-enkephalin, which is derived from the KOR agonist precursor dynorphin [11]. Pharmacological inhibition of KOR, but not of the delta or the mu opioid receptor subtypes, fully blocked CR-induced hypothermia and increased weight loss during CR independent of calorie intake. Similar results were seen with DIO mice subjected to CR. In contrast, inhibiting KOR did not change Tb in animals fed ad libitum (AL). Chemogenetic inhibition of KOR neurons in the hypothalamic preoptic area reduced the CR-induced hypothermia, whereas chemogenetic activation of prodynorphin-expressing neurons in the arcuate or the parabrachial nucleus lowered Tb. These data indicate that KOR signaling is a pivotal regulator of energy homeostasis and can affect body weight during dieting by modulating Tb and energy expenditure.

Effects of central RVD-hemopressin(α) administration on anxiety, feeding behavior and hypothalamic neuromodulators in the rat.

The endocannabinoid (eCB) system is strongly involved in the regulation of anxiety and feeding behavior. RVD-hemopressin(α) [RVD-hp(α)], a N-terminally extended form of hemopressin, is a negative allosteric modulator of the cannabinoid (CB) 1 receptor and a positive allosteric modulator of CB2 receptor which has been recently reported to exert anxiolytic/antidepressant and anorexigenic effects after peripheral administration in rats. Pharmacological evidences reported a possible link between brain hypocretin/orexin, monoamine and eCB systems, as regards appetite and emotional behavior control. Considering this, the aim of our work was to investigated the effects of RVD-hp(α) on anxiety like behavior and food intake after central administration and related it to monoamine levels and orexin-A gene expression, in the hypothalamus. We have studied the effects of central RVD-hp(α) (10nmol) injection on anxiety-like behavior and feeding using different behavioral tests. Hypothalamic levels of norepinephrine (NE), dopamine (DA) and serotonin (5-hydroxytryptamine, 5-HT) and gene expression of orexin-A and proopiomelanocortin (POMC) were measured by high performance liquid chromatography (HPLC) and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis, respectively. Central RVD-hp(α) administration decreased locomotion activity and stereotypies. Moreover, RVD-hp(α) treatment inhibited anxiogenic-like behavior and food intake, NE levels and orexin-A gene expression, in the hypothalamus. Concluding, in the present study we demonstrated that central RVD-hp(α) induced anxiolytic and anorexigenic effects possibly related to reduced NE and orexin-A and POMC signaling, in the hypothalamus. These findings further support the central role of the peptide in rat brain thus representing an innovative pharmacological approach for designing new anorexigenic drugs targeting eCB system.

Alterations on Na+,K+-ATPase and Acetylcholinesterase Activities Induced by Amyloid-β Peptide in Rat Brain and GM1 Ganglioside Neuroprotective Action

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na(+),K(+)-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na(+),K(+)-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na(+),K(+)-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na(+),K(+)-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na(+),K(+)-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na(+),K(+)-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment.

Escitalopram Regulates Expression of TRH and TRH-Like Peptides in Rat Brain and Peripheral Tissues

Background: Escitalopram (eCIT) is a highly selective serotonin reuptake inhibitor (SSRI) that can be an effective treatment for a number of neuropsychiatric disorders including major depression. We, and others, have previously reported that thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH₂) and TRH-like peptides with the general structure pGlu-X-Pro-NH₂, where ‘X’ can be any amino acid residue, have neuroprotective, antidepressant, analeptic, arousal, and anti-epileptic effects that could mediate the neuropsychiatric and therapeutic effects of a variety of neurotropic agents. The present work explores the possible mediation of the therapeutic effects of eCIT by TRH and TRH-like peptides. Methods: In order to extend our understanding of the range of neurotransmitter systems that are modulated by and, in turn, influence the expression of TRH and TRH-like peptides, 16 male Sprague-Dawley rats were injected i.p. with eCIT (24 mg/kg BW) and the brain levels of TRH and TRH-like peptides in various brain regions involved in mood regulation and peripheral tissues with serotonergic innervation were measured 0, 2, 4, and 6 h later by combined HPLC and RIA. Results and Conclusion: Remarkable 3- to 25-fold increases in TRH and TRH-like peptide levels were observed 2 h after i.p. eCIT in the epididymis. This reproductive tissue has the highest level of serotonin found in most mammals. The acute (2 h) effect of eCIT in brain regions involved in mood regulation, particularly the nucleus accumbens and medulla oblongata, cerebellum, and striatum was to increase the levels of TRH-like peptides, most consistently Phe-TRH. An important exception was a decrease in the level of TRH in the nucleus accumbens. These responses, in general, were the opposite of those we have previously observed after acute restraint stress in this same rat strain. We conclude that some of the therapeutic effects of inhibition of serotonin reuptake are mediated by altered release of TRH and TRH-like peptides.

Rapid modulation of TRH-like peptides in rat brain by thyroid hormones

Recent identification of membrane receptors for T 4, T 3, 3,5-T 2, and 3-iodothyronamine that mediate rapid physiologic effects of thyroid hormones suggested that such receptors may supplement the regulation of TRH and TRH-like peptides by nuclear T 3 receptors. For this reason 200 g male Sprague–Dawley rats received daily i.p. injections of PTU or T 4. Levels of TRH and TRH-like peptides were measured 0, 2 h or 1, 2, 3, or 4 days later. Rapid increases or decreases in TRH and TRH-like peptide levels were observed in response to PTU and T 4 treatments in various brain regions involved in mood regulation. Significant effects were measured within 2 h of T 4 injection. Nuclear T 3 receptor-mediated changes in gene expression altering translation, post-translational processing and constitutive release of peptides require more than 2 h. We conclude that non-genomic mechanisms may contribute to the psychiatric effects of thyroid disease and thyroid hormone adjuvant treatment for major depression.

Lithium modulates expression of TRH receptors and TRH-related peptides in rat brain

Lithium is an established mood stabilizer and neuroprotective agent frequently used in the treatment of bipolar disorder and as an adjuvant in drug-resistant unipolar depression. The mechanisms underlying both the therapeutic efficacy of lithium and the exacerbation of symptoms following rapid withdrawal are not understood. From previous studies showing antidepressant and neuroprotective activities of thyrotropin releasing hormone (TRH) and TRH-related neuropeptides we hypothesized that lithium may have substantial effects on the expression and secretion of these peptides and/or their receptors in various rat brain regions involved in the regulation of mood. Chronic lithium effect on TRH receptor binding studies: The effect of 1 and 2 weeks of dietary lithium on [ 3H]3-Me-His-TRH binding to plasma membranes of nucleus accumbens, amygdala and pituitary of young adult male Wistar and the endogenously ‘depressed’ Wistar Kyoto (WKY) rats was measured by the method of Burt and Taylor [Burt, D.R., Taylor, R.L., Endocrinology 106 (1980) 1416–1423]. Acute, chronic and withdrawal effect of lithium on TRH and TRH-like peptide levels in young, adult male Sprague–Dawley rats: Rats were divided into four lithium treatment groups. Control animals received a standard laboratory rodent chow. The acute group received a single i.p. injection of 1.5 milli-equivalents of LiCl 2 h prior to killing. The chronic and withdrawal groups received standard rodent chow containing 1.7 g/kg LiCl for 2 weeks. Withdrawal rats were returned to standard chow 48 h prior to killing while the chronic animals continued on the LiCl diet. TRH, TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP (pGlu-Glu-Pro-NH 2, a TRH-like peptide with antidepressant activity) and Ps4 (a prepro-TRH-derived TRH-enhancing decapeptide) immunoreactivity (IR) were measured in 13 brain regions. The remaining samples were pooled and fractionated by high-pressure liquid chromatography followed by EEP radioimmunoassay. Chronic lithium treatment increased [ 3H]3Me-TRH binding in the nucleus accumbens and amygdala about two-fold in both Wistar and WKY rats but no change was observed in pituitary binding. The most widespread changes in TRH and TRH-related peptide levels were observed in the withdrawal group compared to the controls. The direction of change for the total IR was consistent for all TRH-IR and TRH-related peptide-IR within a given tissue. For example, withdrawal increased all peptide levels in the pyriform cortex and striatum but decreased these levels in the anterior cingulate and lateral cerebellum. Both acute injection and chronic treatment with LiCl decreased TRH and TRH-related peptide levels in the entorhinal cortex. Acute injection and withdrawal both increased EEP-IR in striatum by more than two-fold. The acute effects are most likely due to changes in the release of these peptides since 2 h is not sufficient time for alterations in peptide biosynthesis. Chronic treatment increased levels of pGlu-Phe-Pro-NH 2 levels in hippocampus, pGlu-Leu-Pro-NH 2, and peak ‘2’ in septum by more than four-fold. The present results are consistent with a component role for TRH and related peptides in the mood-altering effects of lithium administration and withdrawal frequently observed during treatment for depression and bipolar disorder.

Cocaine regulates TRH-related peptides in rat brain

Cocaine administration has previously been reported to alter the levels of prepro-TRH mRNA and TRH (pGlu-His-Pro-NH 2) in the limbic system of rats (J. Neurochem. 60 (1993) 1151). We have now demonstrated that a previously unrecognized family of TRH-like peptides is involved in the actions of cocaine. We treated young adult male Sprague-Dawley rats (five per group, 250 g body weight at sacrifice) for 2 weeks with either twice daily injections of saline (control group), twice daily injections of 15 mg/kg cocaine until sacrifice (chronic group), single injection of 15 mg/kg cocaine 2 h prior to sacrifice (acute group) or chronic cocaine injections replaced by saline injections 72 h prior to sacrifice (withdrawal group (WD)). Twelve different brain regions were dissected and immunoreactivity for TRH (TRH-IR), EEP (pGlu-Glu-Pro-NH 2; EEP-IR) and related peptides were measured by radioimmunoassay (RIA). High pressure liquid chromatography (HPLC) revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH, and other TRH-like peptides including EEP, pGlu-Val-Pro-NH 2 (Val 2-TRH), pGlu-Tyr-Pro-NH 2 (Tyr 2-TRH), pGlu-Leu-Pro-NH 2 (Leu 2-TRH), and pGlu-Phe-Pro-NH 2 (Phe 2-TRH). Following i.p. injection, these TRH-like peptides readily crossed the blood–brain barrier but cleared very slowly from brain tissues. Acute cocaine produced a 4.1-fold increase in Val 2-TRH level in medulla while Val 2-TRH and Tyr 2-TRH, increased 6.2- and 2.9-fold, respectively in pyriform cortex PYR. TRH and Leu 2-TRH, decreased 47 and 93%, respectively in the nucleus accumbens (AM) while other EEP-IR peaks decreased 50–100% consistent with the significant decrease in total EEP-IR in the AMs following acute cocaine treatment. Because 2 h is too short a time to alter levels of neuropeptides via changes in the rate of biosynthesis, the acute cocaine-induced elevation or reduction in TRH and related peptides is most likely due to suppression or stimulation, respectively, of the corresponding peptide secretion rate. Because TRH and TRH-like peptides have antidepressant, analeptic and euphorigenic properties, we conclude that these endogenous substances are potential mediators of both the cocaine “high” and withdrawal symptoms.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF-processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C-terminal rat VGF nonapeptide, thus indicating that they included the C-terminus of proVGF. Ectopic expression of the neuroendocrine-specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site-directed mutagenesis was used to identify the KRKRKK(488) motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR(555) site. By the combined use of high-resolution separation techniques, matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C-terminal region of proVGF.

Regulation of TRH and TRH-related peptides in rat brain by thyroid and steroid hormones

To investigate the possibility that TRH (pGlu-His-Pro-NH 2) and EEP (pGlu-Glu-Pro-NH 2) contribute to the behavioral and mood changes attending hypothyroidism, hyperthyroidism and hypogonadism, we have treated young, adult, male Sprague-Dawley rats (5/group, 250 g bw at time of sacrifice) for one week with either daily ip injections of saline, 5 μg T 4, 3 mg PTU or castration. Immunoreactivity for TRH (TRH-IR), TRH-Gly (pGlu-His-Pro-Gly, a TRH precursor), EEP and Ps4 (prepro-TRH-derived TRH-enhancing peptide) was measured in 8 brain regions by RIA. Castration reduced the Ps4-IR levels in hippocampus by 80%. High pressure liquid chromatography revealed that in many brain regions EEP-IR and TRH-IR consisted of a mixture of TRH and other TRH-like peptides including EEP, Val 2-TRH, Tyr 2-TRH, Leu 2-TRH and Phe 2-TRH. Transition from the hyperthyroid to the hypothyroid state increased the Val 2-TRH and Tyr 2-TRH levels in the accumbens by 10-fold and 15-fold, respectively, and the corresponding ratios for the pyriform cortex increased 9-fold and 12-fold, respectively. Hypothyroidism and castration reduced the levels of TRH and the majority of other TRH-like peptides in the entorhinal cortex. This is the first report that thyroid and steroid hormones alter the levels of TRH, prepro-TRH-derived peptides, and a newly discovered array of TRH-like neuropeptides in limbic brain regions.

Effects of electroconvulsive stimuli and MK-801 on neuropeptide Y, neurokinin A, and calcitonin gene-related peptide in rat brain.

Rats were pretreated with 0.9% NaCl, or 0.1 or 1.0 mg/kg MK-801, an anticonvulsant and a psychotomimetic drug, and 60 minutes later given ECS or sham ECS. After six sessions the animals were sacrificed and neuropeptide Y (NPY-), neurokinin A (NKA-), and calcitonin gene-related peptide (CGRP-) like immunoreactivity (-LI) measured with radioimmunoassays. ECS increased NPY-LI in frontal cortex, striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex and hippocampus. MK-801 increased CGRP in a dose-response manner in frontal cortex, and NKA-LI in occipital cortex. Although the higher MK-801 dose reduced seizure duration by 50%, the ECS induced NPY-LI increase in striatum, occipital cortex and hippocampus, and NKA-LI in occipital cortex was not diminished. In contrast, there was a parallel decrease in seizures and NPY-LI and NKA-LI changes in frontal cortex and hippocampus, respectively. Investigation of neuropeptides in brain may contribute to understanding of the mechanisms of action of antidepressive and antipsychotic treatments and of psychotomimetic drugs.

The α4 Subunit of the GABAA Receptors from Rat Brain and Retina

A novel anti-α4 antibody has been used for the purification and characterization of the α4-containing GABAA receptors in the rat brain and for studying the immunocytochemical distribution of the α4 subunit peptide in rat brain and retina. The anti-α4 antibody recognized a 66 kDa peptide in brain membranes and immunoprecipitated 10–28% of the brain GABAA receptors in various brain regions as determined by [3H]muscimol binding. The highest immunoprecipitation values were obtained in the thalamus and the lowest in the cerebellum. Surprisingly, the receptors immunoprecipitated by anti-α4 showed little or no diazepam-insensitive or diazepam-sensitive [3H]Ro15-4513 binding sites in any brain region. In the cerebellum, where 25% of the [3H]Ro15-4513 binding is diazepam-insensitive, much of the latter was immunoprecipitated by an anti-α6 antibody but not by the anti-α4 antibody. Immunoblots of immunoaffinity-purified GABAA receptors from the cerebral cortex on immobilized anti-α4 revealed molecular colocalization of α4 and γ2. However, the absence of significant benzodiazepine binding in these GABAA receptors suggests that the assembly of the α4 and γ2 subunits in the cerebral cortex and in other brain regions is such that they do not normally form diazepam-insensitive [3H]Ro15-4513 binding sites. This result contrasts with the presence of diazepam-insensitive [3H]Ro15-4513 binding sites in the GABAA receptors expressed in heterologous systems resulting from the combination of α4, γ2 and β2 subunits. Immunocytochemistry has revealed the abundance of α4 peptide immunoreactivity in the thalamus and dentate gyrus (mainly in the hilar neurons and the inner third of the granule cell layer). The α4 immunoreactivity is also present in the external plexiform layer of the olfactory bulb and in all layers of the neocortex and pyriform cortex. In the retina, α4 is concentrated on ganglion cells (including some giant ganglion cells), the inner plexiform layer and to a lesser extent in the outer plexiform layer. Copyright © 1996 Elsevier Science Ltd.

Processing of prodynorphin-derived peptides in striatal extracts. Identification by electrospray ionization mass spectrometry linked to size-exclusion chromatography

Proteolytic processing of prodynorphin-derived peptides in rat brain was studied with the help of high performance size exclusion chromatography (SEC) connected to electrospray ionization mass spectrometry. Extracts from rat striatum were incubated with individual synthetic dynorphin peptides. Dynorphin A was the most resistant to proteolytic cleavage, converting slowly to Leu-enkephalin (0.3 pmol/min), whereas dynorphin B was processed to this pentapeptide at a 10 4-fold higher rate. Minor cleavage was also observed between Arg 6 - Arg 7. Alphaneoendorphin was also rapidly metabolized to Leu-enkephalin (6 nmol/min) and, to a lesser extent, to Leu-enkephalinArg 6. This new strategy for studying peptidases can easily be adapted to identification of components present in body fluids.

In vivo cleavability of a disulfide-based chimeric opioid peptide in rat brain.

Brain delivery of systemically administered neuropeptide drugs may be achieved by the synthesis of chimeric peptides, wherein the peptide is coupled to transport vectors via avidin-biotin technology. The present study focuses on factors that optimize the linkage of drugs to transport vectors. The vector is the OX26 monoclonal antibody to the transferrin receptor, and the model peptide used in these studies is [Lys7]dermorphin (K7DA). The K7DA is monobiotinylated at the epsilon-amino group of the Lys7 residue with either a cleavable linker, e.g., disulfide, using NHS-SS-biotin, or a noncleavable linker, e.g., amide, using NHS-XX-biotin. Disulfide cleavage of the biotinylated derivative yields the desbiotinylated peptide, which is thiolated. Structures of the K7DA analogues were confirmed by secondary ion mass spectrometry. The biotinylated peptides were coupled to a thiol-ether conjugate of the OX26 antibody and either neutral avidin (NLA) or streptavidin. The binding constants (Ki) of the K7DA, the biotinylated K7DA (bio-XX-K7DA), the desbiotinylated K7DA, and the bio-XX-KD7A conjugated to NLA-OX26 were 0.62 +/- 0.14, 1.59 +/- 0.27, 1.24 +/- 0.24, and > 10 nM, respectively, and were determined with a mu-opioid peptide radioreceptor assay. Comparable results were obtained with in vivo tail-flick analgesia testing following intracerebroventricular (icv) injection of opioid chimeric peptides. Reversibility of pharmacologic action of thiolated peptide was demonstrated by icv naloxone administration. The cleavability of the disulfide linker in vivo in rat plasma and brain was assessed with gel filtration HPLC and internal carotid artery perfusion of labeled opioid chimeric peptides.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the tachykinin neurokinin B precursor peptide in rat brain by immunocytochemistry and in situ hybridization

Tachykinins exert a broad range of actions in the mammalian nervous system. While much is known about the localization of peptides derived from one of the two mammalian tachykinin genes (substance P- and neurokinin A-encoding preprotachykinin), little has been reported on the localization of peptides derived from a second tachykinin gene encoding neurokinin B. Using an antiserum raised against a 30-residue peptide fragment (Peptide 2) of the protein precursor to neurokinin B, we have mapped the distribution of Peptide 2 by immunocytochemistry. Peptide 2 antiserum specificity was determined by western blot analysis (which showed antibody cross-reactivity to a neurokinin B fusion protein from a cloned neurokinin B-encoding complementary DNA) and by the elimination of immunoreactive product in brain tissue sections upon preabsorption with a 10 μM concentration of Peptide 2 peptide. In addition, we report on the distribution of neurokinin B-messenger RNA with a full-length complementary RNA probe to localize cells that express the neurokinin B precursor. Peptide 2 immunoreactivity and neurokinin B-messenger RNA-positive cells were found, in some instances, paralleling the distribution of substance P and in other cases existing separately from substance P. Peptide 2 immunoreactivity as well as neurokinin B-messenger RNA-positive cells were found in the main olfactory bulb, cortex, olfactory tubercle, nucleus accumbens, hippocampus, bed nucleus of the stria terminalis, amygdala, medial habenula, periaqueductal gray, superior and inferior colliculus, and nucleus of the spinal trigeminal tract. Whereas substance P is found throughout the rat brain, neurokinin B appears to be partitioned more to forebrain than to brainstem structures. The marked differences in the distribution of both tachykinins in the rat central nervous system suggests that neurokinin B may play an important role in olfactory, gustatory, visceral, and neuroendocrine processing of information.

Determination of vasoactive intestinal peptide in rat brain by high-performance capillary electrophoresis

A high-performance capillary electrophoresis (HPCE) method for determining vasoactive intestinal peptide (VIP) in rat brain was developed. Cerebral cortex was first extracted by solid-phase extraction and purified by reversed-phase high-performance liquid chromatography. The VIP-rich fraction was further analysed by capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography using a commercial HPCE instrument with UV detection. The identity of the peak of endogenous VIP was confirmed by performing multiple CZE analyses at different pH values. This HPCE method allows VIP to be detected and measured with good molecular specificity and could represent a reference method to validate data obtained by radioimmunoassay.

P-380 - On the neurotransmitter release induced by ouabain to interact with gangliosides and coexisted peptide in rat brain

P-380 - On the neurotransmitter release induced by ouabain to interact with gangliosides and coexisted peptide in rat brain

Processing of TRH precursor peptides in rat brain and pituitary is zinc dependent

The enzymes responsible for the posttranslational processing of precursor proteins to form alpha-amidated peptide hormones require the availability of several cofactors, including zinc, copper and ascorbate ions. Major changes in the availability of these cofactors, as well as the rate of hormone precursor conversion to active hormone, occur during neonatal development, aging and caloric restriction. The effects of 6 weeks of a zinc-deficient (ZD1) diet, pair feeding (PF) and partial zinc deficiency (ZD6) compared to a control diet on the enzymatic cleavage and processing of prepro-TRH to form TRH have been studied in the hypothalamus, brain, and pituitary of young adult male Sprague-Dawley rats. Reverse phase high pressure liquid chromatography (HPLC) revealed that TRH was the major TRH-IR component of the hypothalamus, brain and pituitary. The effect of zinc deficiency on the TRH-Gly-IR HPLC profile of rat brain was to reduce selectively the area of the peaks for TRH-Gly and other low molecular weight pro-TRH peptide fragments with a C-terminal Gly compared to the corresponding TRH-Gly-IR peaks of the control group. We conclude that the processing of prepro-TRH to form TRH is zinc dependent via posttranslational processing enzymes such as carboxypeptidase H.

Pre-columm fluorescence derivatization high-performance liquid chromatography of opioid peptides in rat brain and its use for enzymatic peptide characterization.

A high-performance liquid chromatographic method involving fluorescence derivatization followed by separation on a reversed-phase polymer (octadecylated polyvinylalcohol copolymer gel) column is described for the determination of opioid peptides in rat brain tissues. The peptides extracted from brain tissues were converted into fluorescent derivatives by reaction with hydroxylamine, cobalt(II) ion and borate. The derivatives were separated on an Asahipak ODP-50 column by gradient elution of acetonitrile in the mobile phase containing borate buffer (pH 9.5). The detection limits (S/N = 3) for the peptides were 0.33-1.21 pmol per 100 microliters injected. The method actually permit the determination of leucine enkephalin, methionine enkephalin, methionine enkephalin-Arg-Phe and methionine enkephalin-Arg-Gly-Leu in the tissues. The method is also applied to the characterization of the peptides in the tissues by means of enzymatic degradations with carboxypeptidase A and trypsin.

Determination of Seven Opioid Peptides in Rat Brain by High Performance Liquid Chromatography with On-Line Postcolumn Fluorescence Derivatization

A high performance liquid chromatographic method with on-line postcolumn fluorescence derivatization is described for a simple and sensitive determination of opioid peptides, including enkephalins in rat brain. The peptides were separated on a reversed-phase column (Asahipak ODP-50) by gradient elution of acetonitrile in the mobile phase containing a borate buffer (pH 10.0). They were then automatically converted into fluorescent derivatives by reactions with hydroxylamine, cobalt(II) ion and borate. This derivatization was specific for N-terminal tyrosine-containing peptides. It was successfully used not only for the simultaneous quantification of the endogenous seven opioid peptides at the picomole level in brain tissue, but also for their identification by means of enzymatic degradation using carboxypeptidase A and trypsin. The detection limits (S/N=3) for the synthetic peptides were 0.5–1.5 pmol in the injection volume.