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.     

Development of region-specific antisera for the C-terminal tetrapeptide of gastrin/cholecystokinin and their use in studies of immunoreactive forms of cholecystokinin in rat brain

Molecular forms of cholecystokinin in rat brain were studied by radioimmunoassay using two new antisera raised against the C-terminal tetrapeptide common to cholecystokinin and gastrin. Evidence is presented to show that one antiserum (L112) reacts at the C-terminus of the tetrapeptide, while the other antiserum (L131) reacts at its N-terminus. With antiserum L112 the predominant immunoreactive form of CCK found in extracts of rat brain corresponded to the C-terminal octapeptide; a minor immunoreactive form eluted from Sephadex G25 between the C-terminal octapeptide and the tetrapeptide. A similar pattern of molecular forms was found using a third antiserum (L48) previously shown to react well with the C-terminal octapeptide and poorly with the C-terminal tetrapeptide. Antisera L112 and L48 also revealed a quantitatively similar distribution of immunoreactive material in different regions of rat and cow brain. In contrast, antiserum L131 failed to demonstrate significant amounts of immunoreactive material in rat brain. It is concluded that the C-terminal octapeptide of cholecystokinin predominates in rat brain and that contrary to findings of previous workers there is little or no free C-terminal tetrapeptide present.     

The subcellular distribution of peptides immunoreactive for the amino-terminal of pro-cholecystokinin in rat brain

Antiserum 1942 raised against the synthetic peptide V-9-M is specific for the amino-terminus of pro-cholecystokinin (pro-CCK). It detects three major peptides in whole rat brain extracts with molecular weights of about 13 000 (peak 1), 8000 (peak 2) and 2700 (peak 3), of which the major one is peak 3. Rat brain was found to contain large quantities of these V-9-M-like peptides. Subcellular fractionation of whole rat brain was performed to determine what cellular component was enriched in these peptides. The molecular weight of the V-9-M-like and CCK-8-like peptides enriched in various subcellular fractions has been determined by Sephadex G-50 chromatography. Primary subcellular fractionation experiments indicated a significant enrichment of V-9-M-like peptides in the mitochondrial pellet (P2), a lesser amount in the microsomal pellet (P3), and a slight enrichment in the soluble fraction (S3). Further purification of the P2 fraction demonstrated an increase of V-9-M-like immunoreactivity in purified synaptosomes. With the exception of the enrichment in the soluble fraction, V-9-M-like peptides follow a similar distribution to that of CCK-8-like peptides. Sephadex chromatography of P2 and P3 fractions indicates that the major form of V-9-M present is the peak 3 (2700) form. This V-9-M-like peptide may represent an intermediate in the processing of CCK, and its presence in synaptosomes may indicate that the proteolytic cleavage of pro-CCK into CCK 58 and peak 3 takes place in synaptic vesicles.     

The distribution of corticotropin releasing factor-like immunoreactive neurons in rat brain

Using the indirect immunofluorescent technique, corticotropin releasing factor (CRF)-like immunoreactive nerve fibers and cell bodies were observed to be widely distributed in rat brain. A detailed stereotaxic atlas of CRF-like immunoreactive neurons was prepared. Large numbers of CRF-containing perikarya were observed in the nucleus paraventricularis, with scattered cells in the following nuclei: accumbens, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis hypothalami, amygdaloideus centralis, dorsomedialis, substantia grisea centralis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, vestibularis medialis, tractus solitarius and reticularis lateralis. The most intense staining of CRF-containing fibers was observed in the external lamina of the median eminence. Moderate numbers of CRF-like fibers were observed in the following nuclei: lateralis and medialis septi, tractus diagonalis, interstitialis stria terminalis, preopticus medialis, supraopticus, periventricularis thalami and hypothalami, paraventricularis, anterior ventralis and medialis thalami, rhomboideus, amygdaloideus centralis, habenulae lateralis, dorsomedialis, ventromedialis, substantia grisea centralis, cuneiformis, parabrachialis dorsalis and ventralis, tegmenti dorsalis lateralis, cerebellum, vestibularis medialis, reticularis lateralis, substantia gelatinosa trigemini and lamina I and II of the dorsal horn of the spinal cord. The present findings suggest that a CRF-like peptide may be involved in a neurotransmitter or neuromodulator role, as well as a hypophysiotropic role.     

Determination of cholecystokinin tetrapeptide and cholecystokinin octapeptide sulfate in different rat brain regions by high-pressure liquid chromatography with electrochemical detection

A method for the determination of cholecystokinins in biological material, based on high-pressure liquid chromatography with direct electrochemical detection (HPLC-EC), is described. Using this method, the levels of cholecystokinin tetrapeptide and octapeptide sulfate in rat brain cortex, hippocampus, striatum, and brain stem were measured and found to be comparable to those reported using radioimmunoassay methods. We show that HPLC-EC is sensitive enough to accurately determine neuropeptides in brain tissue without prior derivatization and is therefore, due to its simplicity, an attractive alternative to existing methods.     

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.     

Subcellular distribution of aromatic amino acid transaminases in rat brain

Abstract— The subcellular distributions of tyrosine transaminase, DOPA transaminase, tryptophan transaminase and 5-hydroxytryptophan (5-HTP) transaminase were studied in rat brain.

• 1 For all of these transaminases 60-81 per cent of the total activities were found in the crude mitochondrial fraction. Tyrosine transaminase was the most active enzyme.

• 2 Tyrosine transaminase and DOPA transaminase had very similar distributions in all fractions, but the distribution of tryptophan transaminase and 5-HTP transaminase differed in the microsomal (Mic) and synaptic vesicle (M₂) fractions. Only 5-HTP transaminase was highly concentrated in the M₂ fraction.

• 3 DOPA transaminase was inhibited by dopamine and 5-HT, but these compounds had no effect on 5-HTP transaminase. Both enzymes were completely inhibited by m-hydroxybenzoyloxyamine.

     

Peptides comprising the bulk of rat brain extracts: isolation, amino acid sequences and biological activity.

Chromatographic separation of rat brain extracts followed by automatic Edman sequencing of the major individual components resulted in identification of 61 endogenous peptides derived from known functional proteins (hemoglobin, myelin basic protein, cytochrome-c oxidase, etc.) or unknown precursors. The results are compared with the data obtained earlier for bovine brain. Although the sequences of bovine and rat hemoglobin contain about 20% of amino acid substitutions, the families of structurally related peptides are very similar in both extracts. Several other proteins also give rise to identical or closely related peptide fragments in the two mammalian species. The outlined similarity extends almost exclusively to the most abundant peptides present in the extracts. The minor components show less overlap. Four hemoglobin-derived peptides isolated from rat brain were shown to be biologically active in tumor cells. Eleven are identical to bioactive peptides from other species. Ten structurally overlap with bioactive peptides from other sources. The data obtained show similar biosynthetic pathways of pool components in different species, the resultant peptides being aimed at fulfilling related functions.     

Distribution of immunoreactive cholecystokinin in the human hippocampus

The distribution of cholecystokinin immunoreactive (CCK-IR) nerve cell bodies and processes is reported in the human hippocampus by using the peroxidase-antiperoxidase technique of Sternberger. The CCK-immunoreactivity occurs in three major classes of interneurons: small (10-20 microns) horizontal multipolar neurons of the alveus and stratum oriens; small vertically oriented bipolar or multi-polar neurons in the stratum oriens and stratum pyramidale of Ammon's horn, layers II and III of the subicular system and the entorhinal area; large (20-35 microns) bipolar neurons in the hilus. Each region of the hippocampus is distinct in its CCK-IR nerve fibers content. Those fibers are particularly abundant around pyramidal cells of the CA2 and CA3 subfields of the Ammon's horn and around granular cells suggesting synaptic interaction between the CCK nerve terminals and glutamate neurons of these two regions. No CCK-IR fiber is detected in the fimbria and only a few number of CCK-IR beaded fibers are seen in the angular bundle. These anatomical data suggest that CCK interacts in the functional circuitry of the human hippocampus.     

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.     

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.     

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.     

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.     

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.     

Sulfated cholecystokinin octapeptide in the rat: pontomedullary distribution and modulation of the respiratory pattern.

We performed anatomical and physiological studies to determine the site and actions of sulfated cholecystokinin octapeptide (CCK8-S) on breathing. Peptide locations were determined by combined immunodetection of CCK8-S- containing synaptic varicosities and retrograde labeling of medullary neurons projecting to the ventral respiratory group. Retrogradely labeled neurons and CCK8-S immunolabeled varicosities overlapped within the nuclei of the solitary tract, ventral respiratory group, and the Kolliker-Fuse nucleus. Additional CCK8-S immunoreactive terminals were located in the rostroventrolateral medullary reticular nucleus, lateral paragigantocellular reticular nucleus, and the caudal pontine reticular nucleus. The respiratory effects of CCK8-S, which binds to CCK(A) and CCK(B) receptors, were examined by intravenous injection in adult rats and by bath application in the in vitro neonatal rat brainstem - spinal cord preparation. CCK8-S produced an increase in the mean amplitude of diaphragmatic electromyogram (EMG) of 28 +/- 35% (SD) and a decrease in mean respiratory interval of 13 +/- 4% in vivo. In vitro, CCK8-S significantly increased inspiratory duration and decreased respiratory interval, primarily by shortening expiratory duration. CCK8-unsulfated, a specific agonist for CCK(B) receptors, did not produce these effects. CCK8-S effects in the in vitro preparation were partially blocked by the CCK receptor antagonist lorglumide (final bath concentration 600 nM). These results suggest that CCK8-S modulates the respiratory rhythm via CCK(A) receptors within one or more medullary or pontine respiratory groups in both neonatal and adult rats.     

Tau polymerization: role of the amino terminus

The abnormal polymerization of the tau molecule into insoluble filaments is a seminal event in the neurodegenerative process underlying Alzheimer's disease. Previous experimentation has shown that the microtubule-binding repeat region of the molecule is vital for its ability to polymerize in vitro into filaments similar to those found in Alzheimer's disease. However, it is becoming clear that regions outside the microtubule-binding repeat, such as exons 2 and 3 and the carboxy-terminal tail, can greatly influence its polymerization. Since it has been previously postulated that the amino terminus of tau could be involved in generating pathological conformations in the disease state, its role in the polymerization process was investigated. This report demonstrates that the removal of the amino terminus greatly inhibits the polymerization of the tau molecule, reducing both the rate and extent of polymerization. These results support the hypothesis that the ability of tau to form specific conformations involving the amino terminus is an early event in the formation of tau polymers in the disease state. Furthermore, the mutation of arginine 5 to leucine ((R)5(L)), mimicking an amino-terminal tau mutation found in a single case of FTDP-17, enhances the polymerization of the tau molecule. Therefore, the amino terminus of the tau molecule, while largely overlooked in studies of its polymerization, is a significant contributor to the polymerization process.