Evidence for intrinsic regulation of met-enkephalin-immunoreactivity in gastroenteropancreatic tissues of the rat

In a study whether gastrointestinal endogenous opioids can be modified by vagal denervation or by pharmacological application of an opiate, we examined met-enkephalin-immunoreactivity in gastrointestinal tissue in rats with and without truncal vagotomy and with and without subcutaneously implanted morphine pellets. The immunoreactivity of the tissue extracts gave dose-response lines in the radioimmunoassay for met-enkephalin which were near parallel to that for the standard. On Sephadex chromatography the met-enkephalin immunoreactivity eluted at a position similar to synthetic met-enkephalin. Tissue concentration of met-enkephalin immunoreactivity was not significantly different from the respective control after vagotomy and after morphine treatment. Total gastric met-enkephalin immunoreactive content increased significantly after vagotomy in line with gastric hypertrophy occurring after vagotomy without a drainage procedure. From these results it is concluded that met-enkephalin immunoreactivity in the rat gastrointestinal tract is regulated intrinsically, it is neither altered by vagal denervation nor by exogenous opiate administration.     

Met-enkephalin: Rapid separation from brain extracts using high-pressure liquid chromatography,and quantitation by binding assay

Met-enkephalin can be rapidly separated with high recovery from leu-enkephalin, -endorphin, and enkephalin metabolites by high-pressure liquid chromatography on a Dupont SCX column. The technique permits separation of endogenous enkephalins from brain extracts for measurement by opiate-receptor binding assay, immunoassay, or for study of the incorporation of radiolabeled amino acids into enkephalin. Using this technique, met-enkephalin was separated from striatal extracts of rats killed by focused microwave irradiation and quantitated by the opiate-receptor binding assay. The concentration of met-enkephalin in the column eluate was 1.3 g/g tissue. This value is comparable to that obtained by radioimmunoassay without purification of the extracts.Pharmacology Research Associate, Pharmacology Research Associate Program, National Institute of General Medical Sciences.     

Characterization of opioid peptides in the rat stomach

A combination of several chromatographic and assay systems was used to characterize the opioid peptides in rat stomach extracts. Partial purification of opioid material in acetic acid extracts of the corpus plus antrum regions of the rat stomach was carried out by gel filtration chromatography on Sephadex G-50, followed by adsorption onto Amberlite XAD-2 resin. A single peak in opioid activity was determined by both radioreceptor assay (RRA) and bioassay. By high performance liquid chromatography, this peak was resolved into five distinct components, characterized by RRA and (or) radioimmunoassay, with retention times corresponding to methionine enkephalin (met-enk), leucine enkephalin, met-enk-arg6-gly7-leu8, met-enk-arg6-phe7, and dynorphin 1-13. Closer examination of the dynorphin component revealed the presence of dynorphins 1-17, 1-13, and 1-8. Trypsin digestion of the partially purified (Sephadex G-50 and Amberlite XAD-2 chromatographed) extract resulted in an overall increase in opioid activity, suggesting the presence of larger, possibly precursor forms.     

Immunohistochemical and biochemical evidence for the presence of the pentapeptide met-enkephalin and the heptapeptide met-enkephalin-Arg6-Phe7 but not the octapeptide met-enkephalin-Arg6-Gly7-Leu8 in amphibian chromaffin cells

Using specific antibodies to met-enkephalin, met-enkephalin-Arg⁶-Phe⁷ and met-enkephalin-Arg⁶-Gly⁷-Leu⁸, we have studied the distribution of these opioid peptides in the frog adrenal gland by means of the indirect immunofluorescence technique. Bright staining of all chromaffin cells was observed by application of met-enkephalin and met-enkephalin-Arg⁶-Phe⁷ antisera. No nerve endings could be detected. A few chromaffin cells were weakly stained by met-enkephalin-Arg⁶-Gly⁷-Leu⁸ antiserum. Using a specific radioimmunoassay for met-enkephalin, the dilution curve of frog adrenal extracts was parallel to that of synthetic met-enkephalin. The concentration of met-enkephalin-like material measured in crude acetic extracts of frog adrenals (2.31 ± 0.16 pmol/mg w. wt) was high when compared to those reported for most mammalian species. No leu-enkephalin and virtually no met-enkephalin-Arg⁶-Gly⁷-Leu⁸ were detected by the corresponding radioimmunoassays. Reverse phase HPLC analysis revealed that oxidized met-enkephalin was the main form of met-enkephalin detected in acetic extracts of frog adrenals. HPLC separation showed the presence of a peptide co-eluting with synthetic met-enkephalin-Arg⁶-Phe⁷. Higher molecular weight forms were also separated by HPLC. These results show the presence of both met-enkephalin and the heptapeptide met-enkephalin-Arg⁶-Phe⁷ and the lack of met-enkephalin-Arg⁶-Gly⁷-Leu⁸ in frog chromaffin cells.     

Starvation-induced changes in secretin-like immunoreactivity of human plasma

Levels of secretin-like immunoreactivity in the plasma of 50 starved subjects were measured by radioimmunoassay and rose from 18 ± 3 (S.E.) pg/ml after 12 h, to 103 ± 12 pg/ml (P<0.005) after 36 h. The assay antibodies were found to be specific for a region of secretin located towards the C-terminal residue. Lactoperoxidase was used to label the secretin with ¹²⁵I and ion-exchange chromatography on SP-Sephadex C-25 was used to putify the labelled product.The plama immunoreactivity was purified by immunoaffinity chromatograpy on antibody-Sepharose conjugates and characterised by gel-filtration on Sephadex G-50 calibrated with molecular weight markers. After a 12-h fast, 10–20% of the immunoreactivity had a molecular weight of about 12 000, possibly due to precursors of secretin. Most of the remainder was smaller than secretin with molecular weight of less than 3000. This material comprised over 90% of the plasma immunoreactivity after a 36-h fast and may be due to degradation products.     

Radioimmunoassay of pancreatic polypeptide in mammalian and submammalian vertebrates using a carboxyl-terminal hexapeptide antiserum

Pancreatic polypeptide (PP) immunoreactivity in acid-ethanol extracts of the pancreas of representative species of mammals, birds, reptiles, amphibians, and fish was studied by a radioimmunoassay (RIA) that utilizes an antiserum which cross-reacts exclusively with the COOH-terminal hexapeptide of PP (CTPP). PP immunoreactivity in acid-ethanol extracts of rat nonpancreas tissues (stomach, duodenum, skeletal muscle, brain) was also examined. Significant concentrations of PP immunoreactivity were detected in the pancreatic extracts of all species, except fish. Appreciable quantities of PP immunoreactivity were also found in the stomach and duodenum of rats. In all cases, tissue extracts showed parallelism with reference PP (bovine) in the RIA. Gel chromatography (Sephadex G-50sf) of tissue extracts (rat, turtle) demonstrated a major peak of PP immunoreactivity, which eluted in the region of the reference PP. Salamander PP immunoreactivity eluted after bovine PP. In addition, the CTPP RIA can be applied to measure plasma levels of PP in rats, dogs, and humans. By using this PP RIA, we observed that plasma PP levels increase significantly in dogs (P less than 0.05) after intravenous administration of neurotensin. In rats, administration of intravenous bombesin resulted in a significant elevation of plasma PP.     

Cyanogen bromide cleavage of methionine residues as a control method for enkephalin immunocytochemistry

Serial semithin sections of rat neurohypophysis were immunostained with 2 antibodies to enkephalins using the peroxidase antiperoxidase method. One of the antibodies (R133) recognizes both met- and leu-enkephalin whereas the other (R26) reacts with met-enkephalin only. After cyanogen bromide pretreatment of the sections the antibody R133 stained only a subpopulation of nerve endings that were distinct from those stained with the latter antibody. R26-(met-enkephalin-like) immunoreactivity was totally abolished by cyanogen bromide pretreatment. This preincubation method which selectively interferes with the staining of met-enkephalin terminals may help to discriminate the two enkephalins in immunocytochemical preparations.     

Somatostatin radioimmunoassay with 125I-Nalpha-tyrosyl-somatostatin

Nalpha-Tyrosyl-somatostatin was synthesized and proved to be homogeneous. Radioiodination of this tyrosine-containing somatostatin analogue by either the lactoperoxidase method or the chloramine T method led to the formation of crude iodinated compound, which was purified by ion exchange chromatography on CM-Sephadex C-25 using a linear ammonium acetate buffer gradient. This purification process was found to be satisfactorily reproducible and suitable for the preparation of 125I-Nalpha-tyrosyl-somatostatin. Using the purified 125I-somatostatin analogue, radioimmunoassay for somatostatin was performed and the assay system was proved to be sensitive and specific for somatostatin. Immunoassays of hot-water extracts of porcine and tupaia brain, pancreas, stomach and various regions of the intestine in the system revealed that those tissues contained immunoreactive somatostatin at various concentrations. Of the results, it was remarkable that somatostatin immunoreactivity was found in the ileum, middle colon and rectum in both animals, although the concentration were lower when compared with those in the stomach, duodenum and jejunum.     

Cyanogen bromide cleavage of methionin residues as a control method for enkephalin immunocytochemistry

Summary Serial semithin sections of rat neurohypophysis were immunostained with 2 antibodies to enkephalins using the peroxidase antiperoxidase method. One of the antibodies (R133) recognizes both met- and leu-enkephalin whereas the other (R26) reacts with met-enkephalin only. After cyanogen bromide pretreatment of the sections the antibody R133 stained only a subpopulation of nerve endings that were distinct from those stained with the latter antibody. R26-(met-enkephalin-like) immunoreactivity was totally abolished by cyanogen bromide pretreatment. This preincubation method which selectively interferes with the staining of met-enkephalin terminals may help to discriminate the two enkephalins in immunocytochemical preparations.     

Characterization of a Calcitonin Gene Related Peptide-Like Molecule in the Abalone,Haliotis tuberculata

Immunoreactive related CGRP molecules (ir-CGRP) were identified in the abalone, Haliotis tuberculata, mainly in mantle and cephalic part extracts. Ir-CGRP in both tissues accounted for 461 and 455.6 pg per mg of proteins, respectively. These CGRP-immunoreactive molecules were further analyzed for their ability to interact with the CGRP radioreceptor assay. In specific target tissues for CGRP (rat liver membranes), 50% inhibition of ¹²⁵I-labeled CGRP specific binding was observed with 4.7 μg and 21.1 μg of proteins from mantle and cephalic part extract, respectively. These molecules were submitted to gel-filtration chromatography on a Sephacryl S-100 column and were further analyzed in the radioreceptor assay specific for CGRP. The elution position of these molecules suggested a molecular weight close to that of synthetic salmon calcitonin.     

Possible evidence for the existence of a growth hormone fragment in porcine pituitary

In an attempt to search for growth hormone fragments in the pituitary, a radioimmunoassay was developed for a 55 residue S-amino-ethylated CNBr fragment (fragment B) of porcine growth hormone corresponding to residues 126–180 of human growth hormone. The assay was sensitive to 50 pg of fragment B whereas displacement of ¹²⁵I-labelled fragment B porcine growth hormone required a 10³ M excess and was non-parallel. In a homogolous porcine growth hormone radioimmunoassay, fragment B was non-reactive. Gel filtration of an extract of porcine pituitary on Sephadex G-75 revealed three peaks of fragment B immunoreactivity: peak I (29% of total immunoreactivity) eluted in the void volume, peak II (49%) eluted in the position of growth hormone, and peak III (12%) was more retarded than fragment B. Nearly all of the growth hormone immunoreactivity eluted as a single peak in the position of ¹²⁵I-labeled porcine growth hormone. The dilution curve of peak III but not of peaks I or II was parallel to that of fragment B. The results indicate the existence within porcine pituitary of material cross-reactive with a portion of the growth hormone molecule, possibly representing a growth hormone fragment.     

Culture of transformed horseradish roots as a source of fusicoccin-like ligands

Endogenous fusicoccin (FC) or related substances were sought in horseradish (Armoracia rusticana P.) roots. An actively growing root culture was derived from plants transformed with Agrobacterium rhizogenes. The presence of FC-like substances in ethanolic extracts from roots was established in a radioreceptor binding assay with plasmalemmal FC receptors and in radioimmune analysis with an antiserum specific for FC A. FC-like ligands were found in the tissue and medium of aseptically grown culture.Abbreviations FC fusicoccin - GC/MS gas chromatography/mass spectrometry - RIA radioimmunoassay - RRA radioreceptor analysis - BSA bovine serum albumin - Mes 4-morpholineethanesulfonic acid - HPLC high performance liquid chromatography     

Identification and characterization of arginine vasopressin-like substances in the rat testis

We have previously demonstrated the suppression of Leydig cell steroidogenesis by arginine vasopressin (AVP) in vitro. Since the circulating level of AVP is too low to mediate a testicular action of this peptide, we have conducted studies to identify testicular AVP-like substances. The supernatant of homogenized, acid-extracted rat testes was found to contain AVP immunoreactivity which showed parallelism with synthetic AVP in a specific radioimmunoassay for AVP. Chromatography of this extract on a Sephadex G-25 column produced three peaks of AVP immunoreactivity. The largest peak eluted close to the column void volume, a second smaller peak eluted at the total column volume, while a third peak co-eluted with synthetic AVP. Following acetone precipitation, ether extraction, and octadecylsilica (C18) adsorption chromatography of the acid extract, the third peak of AVP immunoreactivity (about 600 pg/testis) was fully retained by C18 chromatography and showed parallelism with synthetic AVP in both radioimmuno- and radioreceptor assays. This substance also co-migrated with synthetic AVP on both Sephadex G-25 and reverse-phase thin layer chromatography (RPTLC). The second peak was only partially retained by C18 adsorption chromatography, dilution curves were not parallel with synthetic AVP in radioimmuno- or radioreceptor assay, and this material failed to co-migrate with synthetic AVP on Sephadex G-25 and RPTLC. The first peak of apparent AVP immunoreactivity was associated with an enzyme(s) that degraded labeled AVP. This enzymatic activity, as well as the immunoreactivity, could be eliminated by heating the extract to 90 degrees C. These results demonstrate, by a number of independent criteria, that rat testes contain a substance which behaves like authentic AVP. Due to its high concentration, the AVP-like peptide may be synthesized or concentrated by testis cells. In addition, testis tissue contains enzymatic activity which degrades AVP and could represent a site of regulation of AVP action. Coupled with the previously demonstrated testis action of AVP, these results suggest a paracrine or autocrine role of the neurohypophysial hormone at the testis level.     

Met-enkephalin immunoreactivity in blood platelets

Picogram amounts (50–150 pg/mg protein) of immunoreactive met-enkephalin material (met-enkephalin in IR) were detected by radioimmunoassay in human, rat and rabbit platelets. Characterization of this material by thin-layer chromatography, gel filtration chromatography and high-pressure liquid chromatography indicated that it behaves identically with synthetic met-enkephalin. No high molecular weight met-enkephalin IR could be detected in the platelet extracts, even after trypsin hydrolysis, using two antisera which are able to recognize some of the putative met-enkephalin precursors present in the adrenal gland or striatum. $\text{In vitro}$, thrombin released platelet met-enkephalin in IR concomitantly with 5-hydroxytryptamine (5-HT), suggesting a common subcellular localization, i.e. the 5-HT storing organelles, for met-enkephalin IR and the amine. $\text{In vivo}$, platelet met-enkephalin IR in the Sprague-Dawley rat was affected neither by adrenalectomy nor by hypophysectomy. Thirteen- and 18-week-old spontaneous hypertensive rats (SHR) had lower platelet concentrations of met-enkephalin in IR than age matched normotensive Wistar-Kyoto rats.     

Distribution of CRF-like immunoreactivity in the rabbit

CRF-like immunoreactivity was measured by radioimmunoassay in the brains and gastroenteropancreatic tract of normal rabbits. It was detected in the brain, with the highest concentration being found in the ventral hypothalamus. The distribution of immunoreactivity was much more limited in the rabbit brain than in the rat brain, with substantial amounts of peptide detected only in areas of close proximity to the hypothalamus, e.g., thalamus, preoptic area, midbrain and amygdala. In addition, the extrahypothalamic immunoreactivity was slightly retarded on Sephadex G-50 chromatography relative to rat CRF-like immunoreactivity and synthetic ovine CRF. No apparent CRF-like immunoreactivity was detected in boiling water extracts of lung, pancreas, duodenum or antrum. These data in conjunction with a previous report of void volume immunoreactivity on Sephadex G-50 only in the hypothalamus suggest that CRF is synthesized only in the hypothalamus and is not a member of the class of peptides found throughout the gastroenteropancreatic tract and the central nervous system.     

β-lipotropin-like material in human pancreas and pyloric antral mucosa

Evidence for the presence of the endogenous opioid precursor β-lipotropin has been found in human pyloric antral mucosa and pancreas by radioimmunochemical displacement curves, Sephadex chromatography, ion exchange chromatography, and immunocytochemistry. Endogenous opioids are not only present in the pituitary gland and nervous tissue but also in the stomach and pancreas, supporting the concept of a common neuro-endocrine system present in brain and gut.     

Calcitonin gene-related peptide-like immunoreactivity in the central nervous system and peripheral organs of rats

The concentrations of rat calcitonin gene-related peptide-like immunoreactivity (rCGRP-LI) in various organs of male rats as well as the molecular heterogeneity of rCGRP-LI in tissue extracts was examined using a specific radioimmunoassay (RIA) for rCGRP and gel-filtration chromatography. rCGRP-LI was high in extracts of the spinal cord (202 +/- 22.6 pg/mg wet wt. of tissue; mean +/- S.E.M.) and of the thyroid (229 +/- 62.3 pg/mg). rCGRP-LI was detectable in the brainstem, hypothalamus, stomach, duedenum, pancreas and kidney. The elution pattern of the extracts on a Sephadex G-50 column showed 3 peaks of rCGRP-LI irrespective of organs and tissues. The first peak corresponded to authentic rCGRP-(1-37). The second and third rCGRP-LI peaks probably consisted of C-terminal fragments of rCGRP, because they had a lower molecular weight than rCGRP-(1-37) and because our antiserum cross-reacts with a synthetic C-terminal fragment. The ratio of 3 rCGRP-LI molecules, however, differed between neural tissue extracts and others. The main component of rCGRP-LI in neural tissue was authentic rCGRP-(1-37), while the smaller fragments of rCGRP were chiefly contained in other tissues like the stomach, pancreas and thyroid. The relative ratio of rCGRP-LI molecules with different size in respective tissue extracts was not changed after leaving the dissected tissues for 2 h at room temperature. These findings indicate that rCGRP-LI is abundantly present in the thyroid as well as the spinal cord and it is detected in lower amounts in the alimentary tract and central nervous system. rCGRP-LI in the extracts consists of 3 different components, the proportions of which vary from one tissue to another, probably reflecting tissue-specific differences in the processing of CGRP.     

High affinity binding sites for somatostatin to rat pituitary

Binding sites for somatostatin (SS) are described in rat pituitary membranes using either [¹²⁵I-Tyr¹¹]-SS-14 or [Leu⁸, D-Trp²², ¹²⁵I-Tyr²⁵]-SS-28 as radioligands; in each case saturable and high affinity binding sites with K_D's for SS of 1.09 and 0.95 nM respectively have been characterized. The binding capacity is 100 f mols/mg protein. The potencies of various SS analogs measured in the radioreceptor assay are in agreement with the potencies in a bioassay measuring inhibition of growth hormone release; in particular, SS-28 is slightly less potent than SS-14. A comparison of these data with those describing SS binding in brain and pancreas suggests that some pharmacological differences may exist between pituitary, brain and pancreas binding sites for SS.     

Identification by specific radioimmunoassay of two novel peptides derived from the C-terminus of porcine preprogastrin

A radioimmunoassay has been developed using antibodies to a synthetic analogue of the C-terminal hexapeptide sequence of the porcine gastrin precursor. Boiling water extracts of porcine antral mucosa contained immunoreactive material that diluted in parallel with standard peptide. Concentrations of immunoreactivity were 5.5 +/- 0.8 nmol X g-1 (mean +/- S.E.M.) in antral mucosa and were closely similar to those of C-terminal heptadecapeptide gastrin immunoreactivity (5.0 +/- 0.6 nmol X g-1). Approximately 30-fold lower concentrations were found in porcine duodenum. A similar distribution was found in ferret, but human, rat and chicken antrum did not contain significant quantities of immunoreactivity. Gel filtration of porcine antral extracts on Sephadex G-50 revealed a single peak of immunoreactivity eluting in a similar position to G17, but on anion-exchange chromatography two peaks of immunoreactive material were separated. These also differed in their retention time on reverse phase HPLC. Both peptides are probably derived by tryptic cleavage at the C-terminus of porcine preprogastrin. No evidence was found to suggest that there are significant quantities of unprocessed preprogastrin in hog antral mucosa. The precise chemical difference between the two immunoreactive peptides identified here remains to be established; together, however, they provide specific markers for progastrin synthesis.     

Distribution and characterization of cyclo (His-Pro)-like immunoreactivity in anuran (frog) skins

The distribution of cyclo (His-Pro)-like immunoreactivity in frog skins from seven frog species was examined. The chromatographic elution profile of cyclo (His-Pro)-like immunoreactivity in amphibian skins measured by radioimmunoassay corresponded precisely to that of [³H-Pro]-cyclo (His-Pro) after DEAE-Cellulose, Sephadex G-25 and high-pressure liquid chromatography. The concentrations of cyclo (His-Pro) in frog skins were much higher than the concentrations of TRH previously observed in skin and the concentrations of cyclo (His-Pro) in both brain and gastrointestinal tract.