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.     

Changes in the processing of pro-dynorphin end products in the substantia nigra during neonatal development

The steady-state levels of pro-dynorphin-related end products were measured in the substantia nigra of the rat at neonatal day 0, 7, 14 and in the adult. At neonatal day 0 there was evidence that pro-dynorphin had undergone posttranslational processing to yield dynorphin A-related products, dynorphin B(1-13) and alpha-neo-endorphin. At this stage the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) was 1:1 and a peak of 4 kilodalton dynorphin A-related immunoreactivity was detected. By neonatal day 7 the molar ratio of dynorphin A(1-17) to dynorphin A(1-8) resembled the adult processing pattern for the substantia nigra. The rapid maturation of the pro-dynorphin system in the substantia nigra is in contrast to the development of the pro-dynorphin system in the posterior pituitary where adult-like processing patterns are not observed until neonatal day 21 (11). Pro-enkephalin products have also been detected in the substantia nigra of the rat (15). At neonatal day 0 and neonatal day 7 the molar ratio of Met-enkephalin to Leu-enkephalin was 4:1. However, in the adult the molar ratio of Met-enkephalin to Leu-enkephalin was 1.4:1 in this terminal field. These results suggest that in the adult or perhaps late in neonatal development some pro-dynorphin end products undergo further proteolytic cleavage to yield Leu-enkephalin.     

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.     

Coexistence of multiple peptides in small intensely fluorescent (SIF) cells of inferior mesenteric ganglion of the guinea pig

Summary Coexistence of peptides in the small intensely fluorescent cells was demonstrated by immunocytochemistry for met-enkephalin-Arg-Gly-Leu, vasoactive intestinal polypeptide, somatostatin, neuropeptide Y and dynorphin. In the extreme example, a single cell was immunoreactive to all 5 peptides examined. Four peptides coexisted in 8% and three peptides in 13% of SIF cells. In 10% of SIF cells no peptide immunoreactivity could be detected. The most prevalent peptide was met-enkephalin (in 46% of cells), then vasoactive intestinal polypeptide (45%), somatostatin (39%), neuropeptide Y (31%) and dynorphin (24%). Met-enkephalin and vasoactive intestinal polypeptide coexisted most commonly (25%).     

Methionine5-enkephalin and opiate binding sites in the neurohypophysis of the bird, Gallus domesticus.

Uncertainties with respect to the cellular localization, binding characteristics and function of Met-enkephalin in the neurohypophysis of mammalian species prompted us to examine the neurohypophysis of a non-mammalian species for opioid material and opioid binding sites. In extracts of the neurohypophysis of the domestic fowl we found immunoassayable Met-enkephalin, but could not detect dynorphin(1-8)-like material. Met-enkephalin immunoreactivity was co-localized with mesotocin in the same nerve endings. Stereospecific opiate binding was specifically located in neurosecretosomes (isolated neurosecretory terminals) of the mesotocin type, as shown by autoradiography. Enkephalins therefore may modulate mesotocin release in an autocrine manner. The neurohypophysis of this common bird appears to be a favorable model for studies of enkephalin function in the absence of dynorphin.     

Beta-endorphin-immunoreactive components in human cerebrospinal fluid

Cerebrospinal fluid (CSF) from patients without neurological disorder was analyzed after Sep-Pak extraction for beta-endorphin (beta-EP)-immunoreactive components by combined reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay. A C-terminal directed antibody detected one major immunoreactive component, probably identical with beta-EP1-31. An N-terminal directed antibody detected several immunoreactive components. One co-eluted with beta-EP1-31 but the others are probably C-terminal truncated or otherwise modified forms of beta-EP1-31. However, they eluted differently from beta-EP1-16 (alpha-endorphin), beta-EP1-26, 1-27 and alpha,N-acetyl-beta-EP1-31. Alternatively, some of the fragments may represent C-terminal extended forms of pro-enkephalin A-derived Met-enkephalin. A Met-enkephalin antiserum detected several immunoreactive components probably representing N-terminal extended forms; neither of them were identical with the beta-EP-immunoreactive components. The results illustrate the heterogeneity of the beta-EP-immunoreactive components in CSF and the need to characterize the beta-EP radioimmunoassay before its application to biological extracts.     

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.     

Dynorphin A immunoreactivity in human cerebrospinal fluid

Dynorphin A immunoreactivity in human cerebrospinal fluid has been characterized. Large quantities of the fluid were fractionated by molecular sieving on a Sephadex G-50 column and analyzed by radioimmunoassay. Active fractions were further analyzed by means of HPLC and an enzyme radioimmunoassay procedure for identification of Leu-enkephalin-Arg6 sequences. Several dynorphin A-active components of varying sizes were identified. However, most immunoreactive material derived from species of higher molecular weight (Mr 3000 and 5000) than authentic dynorphin A (Mr 2000). The Leu-enkephalin core was found to reside in both these large structures.     

Isolation of immunoreactive beta-endorphin-related and Met-enkephalin-related peptides from the posterior pituitary of the amphibian, Xenopus laevis

Acid extracts of the posterior pituitary of the amphibian, Xenopus laevis, were analyzed with two heterologous region specific β-endorphin RIAs. Following gel filtration chromatography and cation exchange chromatography four peaks of immunoreactivity were detected. All four peaks were detected with a N-acetyl specific β-endorphin RIA. Peak I represented 92% of the total immunoreactivity isolated following cation exchange chromatography. This peak had a net positive charge at pH 2.5 of +1 and an apparent molecular weight of 1.4 Kd. Following reverse phase HPLC, Peak I fractionated into two peaks: Peak Ia and Peak Ib. Both peaks were detected with the N-acetyl specific β-endorphin RIA and a Met-enkephalin RIA, however, neither peak co-migrated with either Met-enkephalin or N-acetyl-β-endorphin(1–16). At present it is not clear whether Peak I is derived from pro-opiomelanocortin or one of the other opioid polyproteins. Peaks II, III, and IV represented 8% of the total immunoreactivity recovered following cation exchange chromatography. These peaks had net positive charges of +3, +4, and +5, respectively, and apparent molecular weights of 2.8, 3.2, and 3.5 Kd, respectively. These apparently N-acetylated β-endorphin-sized forms are minor end products of the pro-opiomelanocortin biosynthetic pathway.     

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.     

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.     

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.     

Atrial natriuretic peptide in the sheep

To study atrial natriuretic peptide (ANP) physiology in the chronically catheterized pregnant sheep model we developed a heterologous radioimmunoassay for ovine ANP using an antiserum raised against 1-28 human ANP. This antiserum (Tor I) is specific for the aminoterminus of the human ANP molecule and shows little cross reaction with any carboxyterminus ANP fragments. Ovine ANP immunoreactivity was characterized using this antiserum and a commercially available carboxyterminus ANP antiserum obtained from Peninsula Laboratories. Each antiserum detected 2 peaks of immunoreactivity in ovine atrial extracts chromatographed on a Biogel P-10 column. The minor peak migrated at a position close to 125I-human ANP whereas the major peak represented a larger molecular weight species of ANP. Examination of gel filtration eluates of ovine plasma extracts showed one immunoreactive ANP peak using the Tor I assay system and 2 peaks with the Peninsula Laboratories assay. Plasma immunoreactive ANP levels were determined in 9 sheep using both radioimmunoassay systems. Mean (+/- SEM) levels were similar using the Peninsula Laboratories and the Tor I assay systems (57 +/- 8 pg/ml versus 43 +/- 4 pg/ml, P greater than 0.05). Using the Tor I antiserum, fetal plasma immunoreactive ANP levels were found to be significantly higher than maternal levels (188 +/- 17 versus 48 +/- 8 pg/ml, P less than 0.01) whereas pregnant and nonpregnant adult sheep had similar plasma immunoreactive ANP levels (48 +/- 8 versus 43 +/- 4 pg/ml, P greater than 0.05). Disappearance curves of synthetic human ANP from the plasma of maternal and fetal sheep were assessed using both immunoassay systems and found to be similar.     

Western analyses of pregnancy-associated glycoprotein family (PAG) in placental extracts of various mammals

The present study was conducted in order to analyze the immunoreactivity of placental extracts of several animal species and humans against the following three groups of PAG antisera: anti-boPAG-I (R#497), -boPAG-II (R#435), and -caPAG (R#706). Placental proteins were obtained after extraction at neutral pH, followed by ammonium sulfate (A.S.) precipitation, dialysis, and lyophilization. The immunoreactivity of different placental extracts was revealed by the use of monodimensional SDS-PAGE, followed by blotting on nitrocellulose membrane and the identification of immunoreactive proteins after incubation with PAG antisera (Western blot technique). A strong immunoreactivity of proteins from synepitheliochorial placenta (cattle, sheep, goat, bison, buffalo, and deer) was demonstrated in both 20-50% and 50-80% A.S. fractions using the three antisera. Proteins from species with epitheliochorial placenta presented variable profiles of detected PAG-like proteins: in the sow, many immunoreactive forms were revealed by antisera boPAG-I and boPAG-II, whereas in the dromedary, only two forms were revealed by anti-boPAG-II. Concerning other species, our protocols showed for the first time a cross-reaction between PAG antisera with proteins extracted from dog, alpaca, dromedary, sea lion, and human placenta.     

An opioid system in connective tissue: a study of achilles tendon in the rat.

The occurrence of endogenous opioids and their receptors in rat achilles tendon was analyzed by immunohistochemistry (IHC), radioimmunoassay (RIA), and in vitro binding assays. The investigation focused on four enkephalins, dynorphin B, and nociceptin/orphanin FQ. Nerve fibers immunoreactive to all enkephalins (Met-enkephalin, Leu-enkephalin, Met-enkephalin-Arg-Gly-Lys, Met-enkephalin-Arg-Phe) were consistently found in the loose connective tissue and the paratenon, whereas dynorphin B and nociceptin/orphanin FQ could not be detected. The majority of enkephalin-positive nerve fibers exhibited varicosities predominantly seen in blood vessel walls. Measurable levels of Met-enkephalin-Arg-Phe and nociceptin/orphanin FQ were found in tendon tissue using RIA, whereas dynorphin B could not be detected. In addition to the endogenous opioids identified, delta-opioid receptors on nerve fibers were also detected by IHC. Binding assays to characterize the opioid binding sites showed that they were specific and saturable for [3H]-naloxone (Kd 7.01 +/- 0.98 nM; Bmax 23.52 +/- 2.23 fmol/mg protein). Our study demonstrates the occurrence of an opioid system in rat achilles tendon, which may be assumed to be present also in other connective tissues of the locomotor apparatus. This system may prove to be a useful target for pharmacological therapy in painful and inflammatory conditions by new drugs acting selectively in the periphery.     

Immunoreactive Met-enkephalin Arg6 in rat brain, and bovine brain, gut, and adrenal

Antibodies directed against the Met-enkephalin-related hexapeptide, Met-enk Arg6, have been used in radioimmunoassays in the characterization of material in rat brain, and bovine striatum, colon, and adrenal medulla. Met-enk Lys6 reacted 0.27 relative to Met-enk Arg6, but Leu-enk Arg6 and C-terminal extensions or deletions of Met-enk Arg6 showed less than 0.02 immunoreactivity. In rat brain, the concentration of Met-enk Arg6-like immunoreactivity was less than 20 pmol X g-1 in all regions, but after trypsinization of tissue extracts there were up to 80-fold increases in immunoreactivity as a result of cleavage of C-terminally extended forms. The tryptic product eluted as Met-enk Arg6 on gel filtration. In control extracts of rat brain there were at least three immunoreactive forms of Met-enk Arg6; one eluted in the position of the hexapeptide standard on gel filtration and HPLC while the others had properties of N-terminally extended forms. In bovine striatum and colon the hexapeptide-like material predominated; but in bovine adrenal extracts, there were relatively low concentrations of the hexapeptide and, instead, the dominant immunoreactive forms corresponded to two components that were probably N-terminally extended variants. Trypsin again produced marked increases in immunoreactivity. HPLC studies indicated that Met-enk Arg6Phe7- and Met-enk Arg6Gly7Leu8-like immunoreactive peptides were important substrates in bovine brain for the production of hexapeptide immunoreactivity after trypsin. The differences in the patterns of immunoreactive forms in bovine adrenal, colon, and brain are consistent with tissue variations in the pathways of posttranslational processing of the precursor molecules.     

Release of a dynorphin A (1-8) degrading enzyme from the spinal cord by intraventricular morphine in the rat

Intraventricular injection of morphine sulfate, 40 micrograms, released an enzyme from the spinal cord into the perfusate which degraded dynorphin A (1-8) and, to a lesser extent, dynorphin A (1-13) in urethane anesthetized rats. The enzyme did not degrade dynorphin A (1-17), Met-enkephalin, Leu-enkephalin, substance P and neurotensin. This dynorphin A (1-8) degrading enzyme was inhibited by aprotinin, thiorphan, and, to a lesser extent, by bacitracin but was not inhibited by bestatin. A kinetic study of the interaction between dynorphin A (1-8) and aprotinin with the enzyme indicated that it is competitive in nature. The pharmacological significance of the findings is still unknown.     

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.     

Immunochemical characterisation of tachykinin immunoreactivity in the nervous system of the garden snail,Helix aspersa

1. Circumoesophageal ganglia and foot muscle of the garden snail. Helix aspersa, were subjected to immunocytochemistry using antisera to the tachykinins, substance P (SP), neurokinin A (NKA), kassinin (KAS) and eledoisin (ELE).2. Immunoreactivity in neuronal somata and fibres was detected only with the SP antiserum.3. SP and NKA radioimmunoassays were performed on extracts of Circumoesophageal ganglia. In common with immunocytochemistry, immunoreactivity was only detected with the SP antiserum.4. Gel permeation chromatography of extracts resolved a single peak of immunoreactivity eluting slightly later than synthetic mammalian SP. Reverse-phase HPLC of immunoreactive fractions resolved two immunoreactive peptides representing oxidised and reduced forms of a single peptide.5. These data suggest that the nervous system of H. aspersa contains a single tachykinin with C-tenninal structural characteristics similar to mammalian SP.