Distribution of immunoreactive peptide B in the rat brain

Peptide B represents one of the most highly conserved sequences in proenkephalin. To investigate the potential presence of this peptide in the mammalian nervous system, an antiserum raised to this peptide was used to measure the distribution and molecular weight forms of immunoreactive Peptide B in the rat brain. Peptide B-immunoreactivity (ir) was found to be most concentrated in the hypothalamus and the striatum, with lower concentrations in the midbrain and medulla-pons. Characterization of Peptide B-ir by gel filtration demonstrated that the major immunoreactive peak in the hypothalamus corresponded to a peptide with the approximate molecular weight of Peptide B. The major immunoreactive peptide exhibited a retention time on HPLC indicative of a peptide slightly more hydrophilic than bovine Peptide B. The results suggest that proenkephalin in rat brain can be processed to peptides related to bovine Peptide B.     

Interleukin-2-like activity in injured rat brain

The lymphokine interleukin-2 (IL-2) promotes division and maturation of oligodendrocytes in culture (1). We now report that a IL-2-like activity was present in injured rat brain. The ion-exchange properties of this activity were similar to those of splenocyte IL-2 but its apparent molecular weight was higher. Brain IL-2-like activity was highest in the tissue immediately adjacent to the injury, reaching a maximal activity of about 8000 U/g tissue after 10 days postlesion. The mitogenic activity of injured-brain extracts on astrocytes and CTLL thymocytes was partially inhibited by monoclonal antibodies to murine IL-2 receptor. However, pure human IL-2 did not have mitogenic activity for cultured rat astrocytes. Purified astrocytes, alone or stimulated in a variety or ways, did not produce IL-2-like activity.     

FMRF-NH2-like mammalian peptide precipitates opiate-withdrawal syndrome in the rat

Yang et al. (14) have isolated from mammalian brain an octapeptide FLFQPQRF-NH2 (F-8-F-NH2) with certain antiopiate properties. Third ventricular injection of 2 micrograms of this peptide together with the aminopeptidase inhibitor bestatin precipitated an opiate-withdrawal syndrome in morphine-dependent but not in nondependent rats. Third ventricular injection in nondependent rats of 15 micrograms of the peptide together with bestatin induced a morphine-withdrawal-like behavioral syndrome. This syndrome was not produced by injection of bestatin or saline vehicle alone and was preventable by injection of 3.5 mg/kg morphine sulphate SC.     

Identification and characterization of a novel CCK-like immunoreactive peptide in pig CNS

Antibodies directed against the C-terminus of cholecystokinin octapeptide (CCK8) and caerulein were used to study immunoreactive peptides in pig brain. One antibody, a mouse monoclonal raised to caerulein (c.MAb), reacts equally with heptadecapeptide gastrin (G17), CCK8 and caerulein, the other raised to CCK8 (L48) shows 10 times lower immunoreactivity with caerulein compared with G17 and CCK8. Extracts were purified by adsorption to alginic acid, gel filtration chromatography and reversed phase HPLC. In addition to material with the expected properties of CCK33, 39 and 58 a novel peptide was identified that reacted 10 times better with c.MAb compared with L48. This material emerged in a similar position to CCK58 on Sephadex G50 but had a greater retention time on reversed phase HPLC. It had CCK-like bioactivity and digestion with trypsin gave a fragment showing a pattern of immunoreactivity similar to that of the parent compound. This pattern of activity is distinct from other known mammalian CCKs; the material may represent an addition to the gastrin-CCK family in mammals.     

Regional distribution of immunoreactive prolactin-releasing peptide in the human brain

Regional distribution of prolactin-releasing peptide (PrRP) in the human brain was studied by radioimmunoassay. The antiserum raised against human PrRP-31 in a rabbit was used in the assay, which showed 100% cross reaction with PrRP-20 and no significant cross reaction with other peptides. The highest concentrations of immunoreactive-PrRP were found in hypothalamus (912 +/- 519 fmol/g wet weight, n = 6, mean +/- SEM), followed by medulla oblongata (496 +/- 136 fmol/g wet weight) and thalamus (307 +/- 117 fmol/g wet weight). On the other hand, immunoreactive-PrRP was not detected in frontal lobe or temporal lobe (<50 fmol/g wet weight). Sephadex G50 column chromatography of the immunoreactive-PrRP in the hypothalamus and medulla oblongata showed three immunoreactive peaks; one peak eluting in the position of PrRP-20, one eluting in the position of PrRP-31 and one eluting earlier. Reverse phase high-performance liquid chromatography (HPLC) of these brain tissue extracts showed a peak eluting in the position of PrRP-20 and PrRP-31. The present study has shown for the first time the presence of immunoreactive-PrRP in the human brain. The immunoreactive-PrRP levels in the human hypothalamus were, however, lower than the levels of other neuropeptides with prolactin-releasing activity, such as thyrotropin-releasing hormone and vasoactive intestinal polypeptide.     

Heterogeneity of immunoreactive prolactin in the rat brain

Three immunoreactive prolactin proteins (24 Kd, 16 Kd, and 12 Kd) were identified in the rat brain using sodium dodecyl sulphate polyacrylamide gel electrophoresis, and western blot analyses. In male and female brains, the primary prolactin protein has a molecular weight of 24 Kd which is similar to that of pituitary prolactin. Two additional proteins with apparent molecular weights of 16 Kd and 12 Kd were also identified and were found in greater concentrations in the brain than in the pituitary, and were more predominant in the female brain. In addition, brain extracts proteolytically modify the 24K dalton PRL resulting in the formation of two fragments with apparent molecular weights of 16 and 8 Kd. These data indicate that the prolactin identified in the rat brain is similar to pituitary prolactin, and suggests, that like other PRL target tissues the brain may have the capacity to proteolytically modify prolactin.     

FMRF-NH2-like peptide is deficient in the pituitary gland of the Brattleboro rat

Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 (F-8-F-NH2), isolated from bovine brain, is an FMRF-NH2-like peptide with morphine-modulating activity. In the rat, F-8-F-NH2 immunoreactivity (IR) is highly localized in the neurohypophysis. In this study, F-8-F-NH2-IR was studied in the hypothalamo-neurohypophyseal system of an Arg8-vasopressin (AVP)-deficient animal, the Brattleboro (DI) rat, and the normal control Long-Evans (LE) strain. F-8-F-NH2-IR in the DI pituitary is below the level of detection in contrast to that in the LE (0.50 +/- 0.04 pmol/gland). Neuropeptide Y (NPY) levels are increased two-fold in the DI pituitary while AVP levels are below detection. The content of F-8-F-NH2-IR in the hypothalami and spinal cords of DI and LE rats is not statistically different, suggesting that the absence of F-8-F-NH2-IR in the Brattleboro pituitary is not due to a genetic defect in F-8-F-NH2 biosynthesis. The results of this study raise the question whether AVP could be involved in the regulation of F-8-F-NH2 immunoreactivity in the neurohypophysis.     

Localization of GRF-like immunoreactive neurons in the rat brain

The localization of human GRF1-44-immunoreactive neurons was studied in the rat brain. A dense accumulation of GRF-containing fibers was noted in the external layer of the median eminence. Cell bodies were observed in colchicine-treated rats. The most intensely fluorescent cluster of cells was contained in the arcuate nucleus. Other cells were seen on the base of the hypothalamus, within the median forebrain bundle, dorsal and ventral aspects of the ventromedial nucleus, zona incerta and dorsal part of the dorsomedial nucleus. These cells may influence the pulsatile release of pituitary growth hormone.     

Mammalian FMRF-NH2-like peptide in rat pituitary: decrease by osmotic stimulus.

Previous studies with the Brattleboro rat suggested a possible interaction at the pituitary level between AVP and the neuropeptide, F-8-F-NH₂. In order to test this hypothesis, we studied the effect of various osmotic stimuli on neurohypophyseal F-8-F-NH₂. In rats drinking 2% NaCl solution for two days, neural lobe AVP and F-8-F-NH₂ levels were equally reduced by 87%. After maximal depletion, pituitary levels of F-8-F-NH₂ and AVP rebounded in parallel when normal drinking water was reintroduced. Pituitary stalk transection depleted neurohypophyseal F-8-F-NH₂. The results of this study suggest that neurohypophyseal F-8-F-NH₂ originates from the hypothalamus and, furthermore, is coreleased along with AVP in response to hyperosmotic stimuli.     

Characterization of immunoreactive human C-type natriuretic peptide in brain and heart.

Amino acid sequence of human C-type natriuretic peptide (CNP) has recently been deduced to be identical to those of porcine and rat CNPs in the bioactive unit of C-terminal 22 residues (CNP-22) (1). Thus, tissue concentrations and molecular forms of immunoreactive (ir-) CNP in human brain and heart were determined or characterized using a radioimmunoassay established for porcine CNP. In human brain (hypothalamus and medullapons), ir-CNP was detected at a concentration of 1.04 pmol/g, being about 25 times or 70 times higher than ir-atrial (A-type) natriuretic peptide (ANP) or ir-brain (B-type) natriuretic peptide (BNP). CNP was present mainly as CNP-53, with CNP-22 as well as 13K CNP (presumed to be pro-CNP) as minor components. In heart, 1 approximately 5 pmol/g of ir-CNP was detected in both atrium and ventricle, but this ir-CNP was shown to be derived from crossreactivity of ANP. These results demonstrated that human CNP functions exclusively in the central nervous system in contrast to ANP and BNP which mainly function in the circulation system.     

Calcium-dependent pro-cholecystokinin V-9-M immunoreactive peptide release from rat brain slices and CCK-secreting rat medullary thyroid carcinoma cells in culture

The release of peptides immunoreactive for a synthetic peptide (V-9-M) contained in the amino-terminal of pro-CCK was examined. The potassium-evoked release of V-9-M immunoreactive peptides from rat cerebral cortical slices in vitro was calcium dependent. Cholecystokinin-secreting rat medullary thyroid carcinoma cells also secreted significant quantities of these peptides. Sephadex column chromatography of the release media from slices and cells showed two V-9-M immunoreactive peptides, one larger and one smaller than V-9-M itself. Previous behavioral studies have suggested that V-9-M has a distinct neuropharmacological profile. These results demonstrate that V-9-M-like peptides are released along with CCK-8 and are consistent with the hypothesis that V-9-M-like peptides may be neurotransmitters or neuromodulators or may be involved in the sorting or transport of CCK-8.     

Multiple forms of immunoreactive dynorphin in rat pituitary and brain

Multiple forms of immunoreactive dynorphin (I-dynorphin) in rat anterior pituitary (AP), intermediate-posterior pituitary (IP) and medial basal hypothalamus (MBH) were examined. Three I-dynorphin peaks were observed on gel filtration. The first peak (big form) was eluted near the position of beta-LPH, and was predominant in AP. The second peak (middle form) was eluted near the position of ACTH. The third peak (small form) was eluted at the position of dynorphin (1-13), ane was predominant in IP and MBH. The heterogeneity of this small form was examined by ion exchange chromatography and reverse phase high performance liquified chromatography (HPLC). I-dynorphin peaks were observed at the positions of dynorphin(1-17), (1-13), (1-11), (1-10) and other peptides. These results strongly suggest (i) the presence of dynorphin(1-17), (1-13), (1-11) and (1-10) in rat IP, (ii) dynorphin(1-11) and (1-10) as the major components in this small form, (iii) the difference of I-dynorphin processing in AP, IP and MBH.     

Localization of immunoreactive lamprey gonadotropin-releasing hormone in the rat brain

A highly specific antiserum against lamprey gonadotropin-releasing hormone (GnRH) was used to localize 1-GnRH in areas of the rat brain associated with reproductive function. Immunoreactive 1-GnRH-like neurons were observed in the ventromedial preoptic area (POA), the region of the diagonal band of Broca and the organum vasculosum lamina terminalis, with fiber projections to the rostral wall of the third ventricle and the organum vasculosum lamina terminalis. Another population of 1-GnRH-like neurons was localized in the dorsomedial and lateral POA, with nerve fibers projecting caudally and ventrally to terminate in the external layer of the median eminence. Other fibers apparently projected caudally and circumventrically to terminate around the cerebral aqueduct in the mid-brain central gray. By using a highly specific antiserum directed against mammalian luteinizing hormone-releasing hormone (m-LHRH), the localization of the LHRH neuronal system was compared to that of the 1-GnRH system. There were no LHRH neurons in the dorsomedial or the lateral region of the POA that contained the 1-GnRH neurons. As expected, there was a large population of LHRH neurons in the ventromedial POA associated with the diagonal band of Broca and organum vasculosum lamina terminalis. In both of these regions, there were many more LHRH neurons than 1-GnRH neurons and the LHRH neurons extended more dorsally and laterally than the 1-GnRH neurons. The LHRH neurons seemed to project to the median eminence in the same areas as those that were innervated by the 1-GnRH neurons. Absorption studies indicated that 1-GnRH cell bodies were eliminated by adding 1 microg of either 1-GnRH-I or 1-GnRH-III, but not m-LHRH to the antiserum before use. Fibers were largely eliminated by the addition of 1 microg 1-GnRH-III to the antiserum. No chicken GnRH-II neurons or nerve fibers could be visualized by immunostaining. Because the antiserum recognized GnRH-I and GnRH-III equally, we have visualized an 1-GnRH system in rat brain. The results are consistent with the presence of either one or both of these peptides within the rat hypothalamus. Because 1-GnRH-I has only weak nonselective gonadotropin-releasing activity, whereas 1-GnRH-III is a highly selective releaser of follicle-stimulating hormone, and because 1-GnRH neurons are located in areas known to control follicle-stimulating hormone release selectively, our results support the hypothesis that 1-GnRH-III, or a closely related peptide, may be mammalian follicle-stimulating hormone-releasing factor.     

Splicing-active nuclear extracts from rat brain

In the nervous system, alternative pre-mRNA splicing generates the diverse protein machineries needed for cell excitation and synaptic communication. Yet, many questions remain about how these mechanisms are regulated by RNA binding proteins in the environment of differentiated cells and tissues. Here, we describe the preparation and use of splicing active nuclear extracts derived from the cerebellum and cerebral cortex regions of rat brain as a resource for in vitro studies. These tissue-specific extracts promote the neuron-specific pathway of splicing, and display characteristic changes in hnRNP protein function and expression. These extracts can be used in combination with affinity selection and depletion/complementation assays to identify regulatory factors and to characterize their interactions and effects on spliceosome assembly. The ability to prepare extracts from brain regions at a range of postnatal ages provides opportunities to address related questions as a function of cell differentiation. These neuronal extracts may also be valuable for the development of in vitro assays to elucidate other neuron-specific RNA processing pathways, such as 3' end formation, RNA editing, or miRNA maturation.     

Fate of (D-Ala2)-deltorphin-I-like immunoreactive neurons in 6-hydroxydopamine lesioned rat brain

The use of a polyclonal antiserum specific to C-terminal tetrapeptide amide of (D-Ala2)deltorphin-I, a naturally occurring amphibian skin opioid peptide, has already demonstrated the presence of immunoreactive neurons in rat midbrain. Double immunostaining identified these neurons as a subpopulation of the mesencephalic dopaminergic neurons that were also tyrosine hydroxylase-immunopositive and calbindin-D28kD- negative, namely, the neurons predominantly affected in Parkinson disease. We followed the fate of these neurons after a monolateral injection of 6-hydroxy-dopamine into rat brain. Almost all the immunopositive neurons and their nigrostriatal, mesolimbic and mesocortical projections on the side ipsilateral to the lesion disappeared. Only a few scattered immunopositive neurons within the substantia nigra, pars compacta, and those of supramammillary nucleus remained unaffected. The consistent overlap of dopamine and this new molecule provides a further key to identifying the mammalian counterpart of these amphibian skin opioid peptides.     

Immunohistochemical demonstration of a dermorphin-like peptide in the rat brain

Summary The new opioid heptapeptide dermorphin, first isolated from frog skin, has been localized immunohistochemically in nerve cell bodies and nerve fibers of the arcuate-periarcuate region, around the organum vasculosum of the lamina terminalis (OVLT) and in the subfornical organ of the rat brain. A role of dermorphin in modulating LHRH release is suggested.     

Occurrence of a novel nucleotide, Zpp5'A2'p, in rat liver extracts

The nucleotide Zpp5'A2'p has been isolated from rat liver. Z stands for an unknown compound, probably a nucleoside. The preliminary structure of Zpp5'A2'p has been elucidated by treatment with phosphodiesterase and/or alkaline phosphatase and analysis of the products of the reaction by high pressure liquid chromatography. The following ultraviolet absorption spectral characteristics were determined at pH 7.0: Zpp5'A2'p (lambda max = 265 nm; A250/A260 = 0.76; A280/A260 = 0.83); Zp (lambda max = 280 nm; A250/A260 = 0.88; A280/A260 = 1.46). The molar extinction coefficient found for Zp, at 280 nm, was (7.5 + 0.9) X 10(3) M-1 cm-1. The base of Zp could correspond to an indole derivative.