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.     

Chromatographic characterization of dynorphin and [Leu5]enkephalin immunoreactivity in guinea pig and rat testis

Tissues of the reproductive tract have been shown to contain mRNAs coding for pro-opiomelanocortin (POMC), pro-enkephalin and pro-dynorphin. However, the amounts of immunoreactive opioid peptides in these tissues are low, and in the case of the enkephalins and dynorphin, the molecular species responsible for the immunoreactivities have not been characterized. The chromatographic properties of dynorphin and enkephalin immunoreactivities in extracts of guinea pig and rat testis have therefore been determined. Dynorphin A and dynorphin B immunoreactivity was heterogeneous, with a significant amount attributable to high-molecular-weight forms. About 20% of the dynorphin A immunoreactivity, and about 40% of the dynorphin B immunoreactivity, in guinea pig testis extracts behaved as authentic dynorphin A or B, respectively during fractionation by ion exchange, gel filtration and high-performance liquid chromatography. Both high- and low-molecular-weight forms of [Leu5]enkephalin immunoreactivity were also present, with roughly 50-70% of the immunoreactivity attributable to low-molecular-weight forms. In extracts of guinea pig testis only a small part of this immunoreactivity eluted as authentic [Leu5]enkephalin during high-performance liquid chromatography. In rat testis most of the low-molecular-weight [Leu5]enkephalin immunoreactivity behaved as the authentic peptide. These results confirm that opioid peptides are produced in guinea pig and rat testis, and demonstrate that immunoreactive forms of the peptides similar to those found in brain and pituitary are present in the tissue.     

Gonadal steroids and anterior lobe dynorphin in the male rat

Immunoreactive (ir)-dynorphin levels were measured, and the species characterized by high performance liquid chromatography (HPLC), in the pituitary and hypothalamus of intact and castrate male rats. On HPLC, ir-dynorphin co-eluted with authentic dynorphin A 1-8, dynorphin A 1-17 and dynorphin 1-32 in the hypothalamus and intermediate lobe; in two different reversed phase (RP)-HPLC systems, anterior lobe ir-dynorphin co-eluted uniquely with dynorphin 32 (4K dynorphin). Anterior lobe levels of total ir-dynorphin were significantly lowered 7 days after castration, while HPLC profiles in all tissues remained unchanged. The change in anterior pituitary ir-dynorphin levels was reversed in a dose-related manner by dihydrotestosterone (15-500 micrograms/100 g b. wt/day); estradiol benzoate (3 micrograms/100 g/day) was without effect. The changes on castration and androgen administration suggest that gonadal steroids play a role in the regulation of dynorphin, as well as gonadotrophins and prolactin, within the anterior pituitary gland.     

Subcellular localization of immunoreactive dynorphin and vasopressin in rat pituitary and hypothalamus

Dynorphin is found mainly in the particulate fraction of rat pituitary gland and hypothalamus homogenates. Dynorphin-like immunoreactivity (DYN-LI) from neurointermediate lobe (NIL) homogenates migrates at the same rate as vasopressin-like immunoreactivity (AVP-LI), in sucrose density gradients, whereas DYN-LI from the hypothalamus appears to migrate principally in a less dense region of the gradient. This suggests that dynorphin and vasopressin from pituitary are present in organelles of similar size and density, while the bulk of the dynorphin in the hypothalamus appears to be stored in a different subcellular organelle. Anterior lobe (AL) dynorphin appears to migrate in two separate bands on density gradients: the less dense band (slower) migrates at a similar rate to that of dynorphin and vasopressin from NIL. When alpha-neo-endorphin was measured in sucrose gradients of NIL and hypothalamus, it was found to co-migrate with DYN-LI.     

Effects of dehydration on pro-dynorphin derived peptides in the neuro-intermediate lobe of the rat pituitary

Dehydration significantly reduced the concentration of immunoreactive dynorphin A(1-17), dynorphin A(1-8), alpha-neo-endorphin, beta-neo-endorphin, and leu-enkephalin in the rat pituitary posterior-intermediate lobe. A statistically significant increase in immunoreactive dynorphin A(1-8), alpha-neo-endorphin and leu-enkephalin was observed in the hypothalamus. Comparison of the molar ratios of dynorphin A(1-17): dynorphin A(1-8) and alpha-neo-endorphin: beta-neo-endorphin showed an altered profile of stored pro-dynorphin cleavage products in the posterior-intermediate lobe of the pituitary of dehydrated rats.     

α-N-Acetyl β-Endorphins in the Pituitary: Immunohistochemical Localization Using Antibodies Raised Against Dynorphin(1-13)

Abstract: Intense immunohistochemical staining of the intermediate lobe of the pituitary was observed by using an antiserum raised against synthetic dynorphin(1-13) treated with a water-soluble carbodiimide (CDI). Subsequent studies showed that the immunostaining was blocked by preincubation of the antiserum with acetylated derivatives of both β-endorphin and dynorphin(1-13) as well as by CDI-treated dynorphin(1-13), but only weakly by authentic dynorphin(1-13). Neither nonacetylated β-endorphin nor any other fragments of the ACTH/endorphin precursor blocked the immunostaining of the intermediate lobe. Analysis of the CDI-treated dynorphin(1-13) used as an antigen showed that most of the peptide was acetylated at primary amino groups. CDI treatment of dynorphin(1-13) results in the formation of an acetyl derivative because the commercially available peptide is supplied as the acetate salt. The antibodies responsible for the intermediate lobe staining were isolated by affinity chromatography, using a column containing partially purified intermediate lobe extract linked to an affinity resin and a radioimmunoassay (RIA) was developed with CDI-treated dynorphin(1-13) used as a trace and as a standard. Competition studies showed 0.5-1% cross-reactivity with α-N-acetyl β-endorphin(1-31), α-N-acetyl β-endorphin(1-27), and totally acetylated β-endorphin(1-31). Nonacetylated β-endorphins did not cross-react. Posterior-intermediate lobe extracts from rat and beef were fractionated by gel filtration. Rat posterior-intermediate lobe extracts were also fractionated by cation-exchange chromatography. Fractionated extracts were analyzed by RIAs for β-endorphin, CDI-treated dynorphin(1-13), and authentic dynorphin(1-13). The results suggested that the peptides responsible for the intermediate lobe staining were mainly four different derivatives of β-endorphin bearing an acetyl group at the amino terminus. No immunostaining was seen in the posterior and anterior lobes of the pituitary. This suggests that the intermediate lobe is the main source of acetylated β-endorphins in the pituitary.     

Evidence for a Selective Processing of Proenkephalin B into Different Opioid Peptide Forms in Particular Regions of Rat Brain and Pituitary

The distribution of five major products of proenkephalin B [dynorphin1-17, dynorphin B, dynorphin1-8, alpha-neo-endorphin and beta-neo-endorphin] was studied in regions of rat brain and pituitary. The distribution pattern of immunoreactive (ir) dynorphin B (= rimorphin) was found to be similar to that of ir-dynorphin1-17, with the highest concentrations being present in the posterior pituitary and the hypothalamus. HPLC and gel filtration showed the tridecapeptide dynorphin B to be the predominant immunoreactive species recognized by dynorphin B antibodies in all brain areas and in the posterior pituitary. In addition, two putative common precursor forms of dynorphin B and dynorphin1-17 with apparent molecular weights of 3,200 and 6,000 were detected in brain and the posterior pituitary. The 3,200 dalton species coeluted with dynorphin1-32 on HPLC. In contrast with all other tissues, anterior pituitary ir-dynorphin B and ir-dynorphin1-17 consisted exclusively of the 6,000 dalton species. Concentrations of dynorphin1-8 were several times higher than those of dynorphin1-17 in striatum, thalamus, and midbrain while posterior pituitary, hypothalamus, pons/medulla, and cortex contained roughly equal concentrations of these two opioid peptides. No dynorphin1-8 was detected in the anterior pituitary. Concentrations of beta-neo-endorphin were similar to those of alpha-neo-endorphin in the posterior pituitary. In contrast, in all brain tissues alpha-neo-endorphin was found to be the predominant peptide, with tissue levels in striatum and thalamus almost 20 times higher than those of beta-neo-endorphin. These findings indicate that differential proteolytic processing of proenkephalin B occurs within different regions of brain and pituitary. Moreover, evidence is provided that, in addition to the paired basic amino acids -Lys-Arg- as the "typical" cleavage site for peptide hormone precursors, other cleavage signals also seem to exist for the processing of proenkephalin B.     

Hypothalamo-posterior pituitary system in Brattleboro rats: immunoreactive levels of leucine-enkephalin, dynorphin (1-17), dynorphin (1-8) and alpha-neo-endorphin

The levels of immunoreactive leucine-enkephalin, alpha-neo-endorphin, dynorphin (1-17) and dynorphin (1-8) have been determined in the hypothalamus and posterior pituitary from male and female Brattleboro rats homozygous (unable to produce vasopressin) and heterozygous (producing vasopressin) for diabetes insipidus, and from male and female Long Evans rats. In the hypothalamus we found no significant differences in the levels of these peptides while there were great differences in extracts from the posterior pituitary: female homozygous animals have greatly reduced levels in all four peptides compared to the heterozygous controls. In male homozygous animals the differences in the dynorphin (1-17) and leucine-enkephalin levels were small whereas the concentrations of alpha-neo-endorphin and dynorphin (1-8) showed a significant decrease compared to the male heterozygous controls. The results indicate a reduction in opioid peptides linked to the vasopressin deficiency in a partially sex dependent manner.     

Existence of different forms of adrenocorticotropin in rat hypothalamus

Adrenocorticotropin (ACTH)-like immunoreactivity and bioactivity were extracted from rat hypothalamus and fractionated by high pressure liquid chromatography. Analysis of the fractions either by radioimmunoassay or bioassay (corticosteroid production from rat adrenal cells) revealed several peaks of immunoreactivity and bioactivity. Only 20-25% of total ACTH-like immunoreactivity and bioactivity eluted with the same retention time as authentic ACTH 1-39. The results suggest that different forms of ACTH exist in rat hypothalamus.     

Stress-induced changes in brain Met-enkephalin, Leu-enkephalin and dynorphin concentrations.

Methionine-enkephalin (Met-enkephalin), leucine-enkephalin (Leu-enkephalin) and dynorphin A (1-17) (dynorphin A) concentrations in discrete brain areas were determined in the mice showing behavioral changes induced by stress using radioimmunoassay (RIA). In the present experiment, we used environment-induced conditioned suppression of motility and forced swimming-induced immobility. In the environment-induced conditioned suppression of motility, Met-enkephalin concentration in the striatum and hypothalamus significantly decreased. Leu-enkephalin concentration in the hypothalamus also decreased. Dynorphin A concentration in the striatum decreased, but significantly increased in the hypothalamus and pituitary. In the forced swimming-induced immobility, Met-enkephalin concentration in the striatum significantly decreased. Leu-enkephalin concentration in the hypothalamus and pituitary significantly decreased. Dynorphin A concentration in the pituitary decreased, but significantly increased in the hypothalamus. Our results indicated that the concentrations of Met-enkephalin, Leu-enkephalin and dynorphin A in the discrete brain areas changed in two different stressful situations. These findings suggested that these peptides might modulate the behavioral changes induced by stressors.     

Regional distribution of α-neo-endorphin in rat brain and pituitary

α-Neo-endorphin was isolated as the first form of “big” Leu-enkephalin and its complete amino acid sequence has recently been established. Using an antiserum raised against synthetic α-neo-endorphin, a highly sentitive and specific radioimmunoassay was developed. The antiserum practically possesses no cross-reactivity to Leu-enkephalin, dynorphin[1–13] and PH-8P, and very little to β-neo-endorphin. Distribution of α-neo-endorphin has been determined in rat brain and pituitary by the use of the highly specific antiserum. The highest concentration was observed at posterior lobe of pituitary. Furthermore, immunoreactive α-neo-endorphin was characterized by gel-filtration and high performance liquid chromatography, and shown to be identical with authentic α-neo-endorphin.     

Detection of Met-enkephalin and Leu-enkephalin in the posterior pituitary of the holostean fish, Amia calva

Immunohistochemical analysis of the pituitary of the holostean fish, Amia calva, indicated that enkephalin-related immunoreactivity was restricted to the pars nervosa, and was not detected in other regions of the pituitary. Fractionation of acid extracts of posterior pituitaries by reverse phase HPLC followed by RIA analysis indicated the presence of immunoreactive Met-enkephalin and Leu-enkephalin. No immunoreactive forms were detected with RIAs specific for either Met-enkephalin-RF or Met-enkephalin-RGL. The molar ratio of Met- to Leu-enkephalin in this terminal field was 3:1 (n = 4). HPLC fractions were also digested with trypsin and carboxypeptidase B to test for C-terminally extended forms of Met-enkephalin. A novel modified form of Met-enkephalin was detected. Extracts of the posterior pituitary, forebrain, midbrain, hypothalamus and hindbrain were screened with RIAs specific for the Pro-dynorphin end products, alpha-neo-endorphin, dynorphin A(1-17), dynorphin A(1-8) and dynorphin B(1-13). The results of these analyses were negative. Collectively, these data suggest that a Pro-enkephalin-like molecule is present in holostean fish. The holostean enkephalin precursor contains at least Met-enkephalin and Leu-enkephalin. However, Pro-dynorphin-related end products with antigenic determinants similar to mammalian dynorphin A(1-17), dynorphin A(1-8), dynorphin B(1-13) and alpha-neo-endorphin could not be detected in the brain or pituitary of this species.     

A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

Presence of immunoreactive dynorphin in human cerebrospinal fluid

Immunoreactive dynorphin (I-dynorphin) was measured by radioimmunoassay in human cerebrospinal fluid (CSF). I-dynorphin concentration in CSF was 30 +/- 2 pg/ml. Sephadex G-25 column chromatography showed the main peak eluted at the position of dynorphin-(1-17). HPLC elution profile of this major peak from gel filtration showed a large peak corresponding to the position of dynorphin-(1-17) and small peaks corresponding to the positions of dynorphin-(1-13), dynorphin-(1-12) and other unknown peptides.     

Top concentrations of dynorphin-like immunoreactivity in fractions of rat anterior pituitary cells enriched in gonadotrophs

A possible relationship between anterior pituitary cells containing luteinizing hormone (LH) and those containing the endogenous opioid dynorphin and other proenkephalin B-derived peptides was examined. Gonadotroph-enriched and gonadotroph-depleted cell fractions were prepared from cell suspensions of adult female rat anterior pituitary glands by centrifugal elutriation. Fractions with high or low concentrations of LH contained also high or low concentrations of dynorphin-like immunoreactivity. A positive correlation was found between the content in the eluted cell fractions of LH and dynorphin-like immunoreactivity with a correlation coefficient and a slope of the regression line close to one. Therefore, LH-containing and dynorphin-containing cells of the rat adenohypophysis exhibit almost the same characteristics under the conditions of centrifugal elutriation. This is consistent with the suggestion that dynorphin and other proenkephalin B-derived peptides may be colocalized with LH and/or follicle-stimulating hormone in at least some of the gonadotrophs of the rat anterior pituitary gland.     

Expression of adrenomedullin2/intermedin in human brain, heart, and kidney

Adrenomedullin2/intermedin (AM2/IMD) is a novel member of the calcitonin/calcitonin gene-related peptide (CGRP) family. In the present study, we developed a specific radioimmunoassay of human AM2/IMD. Expression of AM2/IMD was studied in the human brain, pituitary, heart and kidney obtained at autopsy by radioimmunoassay and immunocytochemistry. Immunoreactive-AM2/IMD was detected by radioimmunoassay in human brains (range; 0.163-1.495 pmol/g wet weight), pituitaries (4.46+/-0.689 pmol/g wet weight, mean+/-S.E.M, n=3), left ventricles of hearts (0.251+/-0.0321 pmol/g wet weight, n=4), kidneys (3.49+/-1.18 pmol/g wet weight, n=5), and plasma obtained at healthy subjects (24.7+/-1.78 pmol/l, n=3). Reverse-phase high performance liquid chromatography showed that immunoreactive-AM2/IMD in human brain, kidney and plasma extracts were eluted in the position of authentic AM2/IMD. Additional peaks eluted earlier were found in the brain tissue and plasma. Immunocytochemistry showed that immunoreactive-AM2/IMD was localized in paraventricular and supraoptic nuclei of hypothalamus, anterior and posterior lobes of pituitary, cardiomyocytes, pericardial adipocytes, vascular endothelial cells of pericardial veins, and vascular smooth muscle cells of coronary arteries and renal arterioles as well as in renal tubular cells. The present study has shown expression of AM2/IMD in various types of cells in the central nervous system and the cardiovascular system, and suggested possible (patho)physiological roles of AM2/IMD in these systems.     

小鼠神经系统内强啡肽B免疫活性物质及其高压液相层析(HPLC)分析

强啡肽B是一种新发现的阿片肽。本工作以自制的兔抗强啡肽 B血清建立灵敏的放射免疫测定法,测定了小鼠神经系统与垂体内强啡肽 B免疫活性物质的含量,其中以垂体、下丘脑含量最高。 放射免疫测定结合 Sephadex C-50 层析和HPLC分析的结果表明,小鼠脑内强啡肽B免疫活性物质的主要成分是强啡肽 B,另外也包括了一定量的强啡肽-32和另一未知的分子量更大的成分,但不像大鼠还含有强啡肽 B-29。这种种属特异性的意义有待进一步研究。     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

Heterogeneity of immunoreactive dynorphin B-like material in human, rat, rabbit and guinea-pig heart.

Immunoreactive dynorphin B-like material (ir-dyn B) was detected in acetic acid extracts of human atrial specimens and of rat, rabbit and guinea-pig atria and ventricles by a validated radioimmunoassay. Levels were high in rabbit atrium (66.76 +/- 7.04 pmol/g) but lower and superimposable in human and rat atria (28.18 +/- 3.20 and 30.22 +/- 2.45 pmol/g, respectively). Gel permeation chromatography revealed ir-dyn B eluting close to column exclusion and in forms with an apparently higher molecular weight than authentic dyn B in human and rat samples. In contrast, almost all the immunoreactivity from rabbit and guinea-pig acetic extracts eluted as a single peak in the region of standard dyn B. Reverse-phase high performance liquid chromatography of the pooled gel chromatography fractions of this peak showed up a molecular form with the same retention time as authentic dyn B and a second minor peak of unknown immunoreactive material eluting three fractions earlier. Digestion with carboxypeptidase B excluded the hypothesis that this latter could be dyn B-Arg14. Therefore, it might be a metabolite of endogenous dyn B recognized by the antibody used in this study.     

The effect of steroids on the size heterogeneity of pituitary prolactin in castrate male rats

Male Sprague-Dawley rats were castrated and given daily sc. injections of estradiol (E2, 10 micrograms/day), testosterone propionate (TP, 1.0 mg/day), dihydrotestosterone (DHT, 1.0 mg/day) or sesame oil (SO, 0.2 ml/day). A group of sham castrate males received daily sc. injections of SO (0.2 ml/day). On day 8 of steroid treatment animals were decapitated and anterior pituitaries were removed and hemisected. Each half was homogenized in PBS buffer (0.01 M Na2HPO4-NaH2PO4; 0.14 M NaCl; 0.1% bovine serum albumin) at either pH 7.6 or 10.6. Homogenates were chromatographed on Sephadex G-100 columns and eluted fractions were assayed for prolactin (PRL) by RIA. Four immunoreactive forms of PRL, designated as "void volume," "big big," "big" and "little," were eluted from the pituitary homogenates of each experimental group. Homogenates obtained at pH 7.6 contained a greater percentage of PRL in the "void volume" and less activity in the "big" and "little" forms than pH 10.6 homogenates in all experimental groups. Pituitaries from SO- and TP-treated castrate animals contained significantly greater percentages of activity in the "void volume" at pH 7.6 compared to the other groups. At pH 10.6, the pituitary homogenates from the E2-treated group eluted a significantly greater percentage of "big" PRL and a smaller percentage of "little" PRL compared to all other groups. These findings suggest that androgenic and estrogenic steroids may play a role in the pituitary PRL molecular size profile of the male rat.