Chromatographic evidence for high-molecular-mass galanin immunoreactivity in pig and cat adrenal glands

Galanin was measured by radioimmunoassay in extracts of pig, cat and rat adrenals using non-C- and mid to C-terminally directed antibodies. The extracts were fractioned by gel chromatography and HPLC. The non-C-terminal galanin immunoreactivity in pig was 92.8 +/- 11.7 pmol/g, in cat 9.1 +/- 0.9 pmol/g and in rat less than 1 pmol/g. Two higher molecular forms of galanin have been identified in both pig and cat adrenal. One major large form behaves as if it was N-terminally extended (Kav pig 0.58, cat 0.48) and the other, a very high-molecular-mass form (Kav pig 0.10, 0.24, cat 0.10), as if it had both N- and C-terminal extensions.     

Galanin in porcine vagal sensory nerves: immunohistochemical and immunochemical study.

In this work, the presence of galanin was examined by immunohistochemistry, radioimmunoassay and high performance liquid chromatography (HPLC) in porcine nodose ganglia, mainly constituted of cell bodies from the vagal sensory neurons. Galanin-like immunoreactivity (Gal-LI) was revealed in 10 to 15% of the total cell bodies by the indirect immunofluorescent technique of Coons. For comparison, a positive staining was revealed in a few cell bodies of the submucous plexus and in fibers located in the different layers of the ileum. The extractable Gal-LI content in nodose ganglia was 7.2 +/- 0.8 pmol/g wet tissue, which represents a concentration about nine times lower than that found in the ileum. HPLC of extractable material revealed a predominant peak which coeluted with the synthetic peptide. We propose that, in pigs, galanin may play a role in the transmission of visceral information through the vagal afferences.     

Ontogeny of vasoactive intestinal peptide in the human fetal digestive tract

Immunocytochemistry and radioimmunoassay were used to assess the appearance time and tissue distribution of vasoactive intestinal peptide (VIP) in the digestive tract of the human fetus. By radioimmunoassay, VIP was measurable from 10 weeks of gestation. The peptide was abundantly distributed in the jejuno-ileum and colon, where the tissue peptide concentration rose from 9-14 weeks of gestation (18.4 +/- 4.4 and 22.0 +/- 5.0 pmol/g wet weight, respectively) to 15-21 weeks (83.0 +/- 21.1 and 98.6 +/- 36.4 pmol/g, respectively). Lower concentrations were recorded in pancreas from 9-14 weeks of gestation (4.3 +/- 0.8 pmol/g) to 15-21 weeks (13.9 +/- 3.7 pmol/g). The peptide concentration was 15.6 +/- 1.9 pmol/g in fundus and 25.5 +/- 3.2 pmol/g in antrum from 15 to 21 weeks of gestation. The highest concentration was recorded in duodenum from 15 to 21 weeks of gestation (118.4 +/- 40.8 pmol/g wet weight). Tissue VIP concentration and age were positively correlated in the jejuno-ileum. By immunofluorescence, immunoreactive VIP was localized in nervous fibers in the muscularis externa, in the submucosa and in the lamina propria. Scarce cell bodies were also found in the myenteric plexus. No immunofluorescent endocrine cells were observed. These results suggest: (1) the early appearance of immunoreactive VIP in gut, as early as 10 weeks of gestation; (2) the peptide, localized in nervous structures only, follows the same distribution pattern as that in adults; (3) the development of VIPergic structures is a continuous process, initiated during the 3rd month of pregnancy.     

Regional concentration and chromatographic characterization of pituitary adenylate cyclase-activating polypeptide (PACAP) in the brain of the bullfrog,Rana catesbeiana

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a regulatory neuropeptide which functions as a hypothalamic factor for pituitary hormone release, and as a neurotransmitter, neuromodulator and neurotrophic factor in both frogs and mammals. This study examined the quantitative distribution and chromatographic characterization of immunoreactive PACAP in the central nervous system (CNS) of the bullfrog, Rana catesbeiana, using an enzyme immunoassay (EIA), named avidin-biotin complex detectable EIA for PACAP, and high-performance liquid chromatographic (HPLC) analysis. The brain of adult bullfrogs contained relatively high levels of immunoreactive PACAP (344.63 pmol/g wet weight of tissue). The average concentrations of immunoreactive PACAP in the regions of the telencephalon, diencephalon, tectum, cerebellum, rhombencephalon, and spinal cord were 213.84, 767.14, 524.94, 192.71, 237.67, and 362.04 pmol/g wet weight of tissue, respectively. The concentrations of immunoreactive PACAP increased with the brain development during metamorphosis, and the concentration of immunoreactive PACAP in the brain of tadpoles at the end of metamorphosis was approximately 200 pmol/g wet weight of tissue. The predominant form of immunoreactive PACAP in the CNS of adult and tadpole was eluted closely with synthetic PACAP38, but another smaller immunoreactivity also appeared in a the fraction, which corresponded to the retention time of synthetic PACAP27, as analyzed by reverse-phase HPLC.     

Distribution of neurokinin A-like and neurokinin B-like immunoreactivity in human peripheral tissues.

Using specific radioimmunoassay and immunocytochemistry for neurokinin A (NKA) and neurokinin B (NKB), distribution and localization of the two peptides in human peripheral tissues were studied. Both NKA-like immunoreactivity (NKA-LI) and NKB-like immunoreactivity (NKB-LI) were present in the walls of the gut and gall bladder and in the pancreas. In the gut, the values for NKA-LI were 0.56-35.73 pmol/g wet weight, while those in pancreas and gall bladder were 0.64-0.68 and 0.36 pmol/g wet weight, respectively. The values of NKB-LI were 0.45-2.66 pmol/g wet weight in the gut, 0.93-1.65 pmol/g wet weight in the pancreas, and 0.30 pmol/g wet weight in the gall bladder. The immunocytochemical reactivity to both peptides was localized to ganglia of the submucosal and myenteric nerve plexuses in the gut wall, and to neurons in the muscle layer and mucosa of the gut wall. Weak but positive NKA-LI appeared in nerve cells of the pancreas, while NKB-LI was not detectable in the pancreas. Conversely, in the gall bladder wall, NKA-LI was undetectable while a very faint NKB-LI was found in the muscle layer. The localization of NKA corresponded closely to that of NKB in the tissues although the relative concentrations of the peptides varied from organ to organ.     

Substance-P-related and neurokinin-A-related peptides from the brain of the cod and trout.

An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A-like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance-P-like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C-terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod. Gadus morhua contained only neurokinin-A-like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys-Pro-Arg-Pro-His-Gln-Phe-Phe-Gly-Leu-MetNH2) has the same amino acid sequence in its C-terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys-Pro-Arg-Pro-Gln-Gln-Phe-Ile-Gly-Leu-MetNH2), however, contains a substitution at position 8 (Phe----Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His-Lys-Ile-Asn-Ser-Phe-Val-Gly-Leu-MetNH2) and shows two substitutions (Thr3----Ile and Asp4----Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.     

Identification of mature nocistatin and nociceptin in human brain and cerebrospinal fluid by mass spectrometry combined with affinity chromatography and HPLC

Nocistatin (NST) and nociceptin/orphanin FQ (NCP) are two important bio-peptides derived from the precursor protein prepronociceptin (ppNCP), involved in several central nervous system (CNS) functions including pain transmission. Since the actual form of human NST in CNS is not fully characterized, we studied the structure of NST from human brain tissue and cerebrospinal fluid (CSF) samples. NST and NCP were isolated from human brain and CSF samples by affinity chromatography combined with HPLC. Mass spectrometry was used for the identification and characterization of the peptides. The total NST immunoreactivity was detected as 11.5+/-2.3 pmol/g tissue for the brain and 0.44 pmol/ml for the pooled CSF sample after the HPLC purification by radioimmunoassay. The presence of two different forms of mature nocistatin (NST-17 and NST-30) and a possible N-terminal methionine cleaved NST-29 were confirmed by both radioimmunoassay and mass spectrometry. Affinity chromatography, HPLC and mass spectrometry methods used in this study were highly sensitive and suitable for identification of actual chemical structures and quantification of very small amounts of peptides in biological samples. The present findings may help further for search for new treatment of neuropathic pain, which is often poorly managed by current therapies.     

Neuropeptide Y- and somatostatin-like immunoreactivities in ganglioneuroma, ganglioneuroblastoma and neuroblastoma

Neuropeptide Y (NPY)- and somatostatin (SS)-like immunoreactivities (LI) were investigated in tumor tissues of one ganglioneuroma (GN), 3 ganglioneuroblastomas (GNB) and one neuroblastoma (NB) by radioimmunoassay. NPY-LI was detected from all 5 tumor tissues (16.4-1247 pmol/g wet tissue). Sephadex G-50 column chromatography and reverse phase high performance liquid chromatography (HPLC) revealed that most of the NPY-LI in tumor extracts was eluted in an identical position to synthetic human NPY except one GNB (case 2). In this case, most of the NPY-LI was eluted in a higher molecular weight region than synthetic human NPY in Sephadex G-50 column chromatography and in a more hydrophobic position in HPLC. SS-LI was detected from 4 tumor extracts except one GNB (case 2) (21.3-787 pmol/g wet tissue). Sephadex G-25 column chromatography and reverse phase HPLC revealed that SS-LI in tumor extracts was eluted just after the void volume and then in the same positions as SS-28 and SS-14. These results suggest that NPY, SS-14 and SS-28 exist in tumor tissues of GN, GNB and NB, and most of the NPY-LI in one GNB was a higher molecular and more hydrophobic form of NPY-LI.     

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut, pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420-493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1-17 and 20-38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry. In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3 +/- 18 pmol GAWK 1-17/g wet weight of tissue (mean +/- SEM) and 12.4 +/- 2.9 pmol GAWK 20-38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1-17 2071.8 +/- 873.2 and GAWK 20-38 1292.7 +/- 542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B. Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.     

Quantitation of ryanodine receptor of rabbit skeletal muscle, heart and brain

The total number of high-affinity ryanodine receptor (RyR) binding sites present in skeletal and cardiac muscle and in brain tissue of the rabbit was determined by [3H]ryanodine binding to subfractions obtained by differential centrifugation of homogenates prepared in a low-ionic strength medium, containing 0.5% Chaps. In all three tissues at least 80% of [3H]ryanodine binding was recovered in the total membrane (TM) fraction obtained by centrifuging between 650 g for 10 min and 120,000 x g for 90 min. Skeletal muscle displayed higher contents of high-affinity RyR sites (about 49 pmol/g wet wt) than heart and brain (about 12 pmol and 3.5 pmol/g wet wt, respectively). The affinity for ryanodine, as well as the affinity for Ca2+, in the absence or presence of Ca2(+)-releasing drugs (caffeine and doxorubicin) of TM from skeletal muscle, were found to be identical to those of purified terminal cisternae. As low as 1 g of tissue was sufficient to perform several experiments.     

Distribution and molecular forms of peptides containing somatostatin immunodeterminants in extracts from the entire gastrointestinal tract of man and pig

Specimens from human porcine mucosal and muscular tissue from the entire gastrointestinal tract were extracted in acid ethanol, subjected to chromatography and analysed for somatostatin-like immunoreactivity by region-specific radioimmunoassays. The concentration of somatostatin-like immunoreactivity from man and pig ranged from 1.13 +/- 0.37 to 101.15 +/- 33.93 pmol/g wet weight, and from 7.64 to 159.48 +/- 23.79 pmol/g wet weight, respectively. In both species the highest concentrations were found in the jejunum. The immunoreactivity in intestinal mucosal extracts was distributed among four major peaks, two of which were identified by HPLC as somatostatin 1-28 and somatostatin 1-14, respectively. A peak of approx. 10 kDa was resolved by ion exchange plus HPLC into three components, two containing at least part of the somatostatin 1-14 sequence as well as (part of) the somatostatin 1-28(1-14) sequence (but differing in charge), the third containing only the 1-28(1-14) sequence. These peptides probably represent uncleaved and partially cleaved prosomatostatin. The fourth component to be identified by gel filtration was slightly larger than somatostatin 1-14. Extracts from the antrum, the pancreas and from muscular tissues contained almost exclusively somatostatin 1-14, and very little somatostatin 1-28, indicating that the somatostatin precursor is processed differently at these sites. Furthermore, extracts of porcine gastric antrum, analysed for somatostatin 1-28(1-14) immunoreactivity, showed two immunoreactive forms in the mucosa and three major forms in the muscular layers.     

Rat immunoreactive cholecystokinin (CCK): characterization using two chromatographic techniques

Acid and neutral extracts of rat cerebral cortex and upper small intestine were prepared and the endogenous concentrations of cholecystokinin-like immunoreactivity (CCK-LI) measured by three new CCK-specific radioimmunoassays. The characterization of the immunoreactive CCK molecular forms was undertaken using gel permeation chromatography in the presence of 6 M urea to minimise problems relating to peptide adsorption or aggregation. Reverse-phase high-performance liquid chromatography (HPLC) was also performed on the rat tissue extracts. Rat cortex contained 268 +/- 12 pmol/g CCK-LI, and over 90% resembled the sulphated CCK-8, which was preferentially extracted at neutral pH. In contrast, the rat upper small intestine (97 +/- 8 pmol/g of CCK-LI) contained less than 20% CCK-8, the majority of immunoreactive CCK being of larger molecular size and being preferentially extracted at acid pH. In the small intestine the predominant molecular form(s) was intermediate in size between CCK-33 and CCK-8. Large amounts of CCK-33 and of a molecular form larger than CCK-33 were also detected. It is concluded that post-translational cleavage of CCK differs in rat brain and gut.     

Galanin is co-localized with noradrenaline and neuropeptide Y in dog pancreas and celiac ganglion

Summary To visualize the localization and potential colocalization of noradrenaline and the putative pancreatic sympathetic neurotransmitters, galanin and neuropeptide Y (NPY), immunofluorescent staining for galanin, NPY and tyrosine hydroxylase (TH) was performed on sections of canine pancreas and celiac ganglion. In the pancreas, galanin-immuno-fluorescent nerve fibers were confirmed as densely and preferentially innervating the islets, whereas numerous NPY-positive nerve fibers were found in the exocrine parenchyma, the surrounding of the blood vessels and within the islets. Double-staining for the peptides and TH indicated that most galaninpositive nerve fibers were adrenergic, most NPY-positive nerve fibers were adrenergic, and many islet nerves contained both galanin and NPY, although some galaninpositive nerve fibers appeared to lack NPY. In the celiac ganglion, virtually all cell bodies were positive for both galanin and TH; a large subpopulation of these cells were also positive for NPY. Radioimmunoassay (RIA) of galanin in extracts of dog celiac ganglion revealed a very high content (256±33 pmol/g wet weight) of galanin-like immunoreactivity (GLIR), consistent with the dense staining observed. This GLIR behaved in a similar manner to synthetic porcine galanin in the RIA. In addition, the majority of the GLIR in ganglion extracts coeluted with the synthetic peptide upon gel filtration, although a minor peak of a larger apparent molecular weight was also observed, observations consistent with the presence of a precursor peptide. These findings suggest that galanin is a sympathetic post-ganglionic neurotransmitter in the canine endocrine pancreas and that NPY might serve a similar function.     

Pituitary adenylate cyclase-activating polypeptide (PACAP)-like immunoreactivity in the brain of a teleost, Uranoscopus japonicus: immunohistochemical relationship between PACAP and adenohypophysial hormones

The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) could play a role in stimulating pituitary hormone release in fish brain. In this study, we used immunochemical techniques to examine the histological and quantitative distribution of PACAP in the central nervous system (CNS) of a teleost, the stargazer, Uranoscopus japonicus. In addition, high performance liquid chromatographic (HPLC) analysis was performed to characterize the form of PACAP present, while the relationship between PACAP and adenohypophysial hormones was also determined immunohistochemically. PACAP-like immunoreactive (LI) neuronal cell bodies and fibers were found not only in the hypothalamo-pituitary region but also in the midbrain and hindbrain regions. PACAP-LI fibers were identified in the neurohypophysis in close proximity to pituitary cells containing immunoreactive hormones such as somatolactin, the N-terminal peptide of proopiomelanocortin, and N-acetyl endorphin. The concentration of immunoreactive PACAP in whole brain tissue was approximately 300 pmol/g wet weight. The average concentrations of immunoreactive PACAP in regions of the telencephalon, diencephalon, tectum, cerebellum, and rhombencephalon were 217.53, 510.26, 83.30, 148.64, and 364.62 pmol/g, respectively. In reverse-phase HPLC experiments, the predominant form of immunoreactive PACAP eluted closely with synthetic stargazer PACAP38, while PACAP27-like immunoreactivity was negligible. These results suggest that PACAP38 is the predominant PACAP form in the stargazer CNS, and that PACAP acts not only as a hypophysiotropic factor for adenohypophysial hormone release but also as a neurotransmitter and neuromodulator in the CNS.     

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.     

Effects of solution of low pH and taurocholate on release of beta-endorphin-like immunoreactivity from human duodenal mucosa in vitro

The presence of beta-endorphin-like immunoreactivity (beta-EpLI) in human duodenum and its release were studied. beta-EpLI was detected in the duodenum (mucosa, 26.7 +/- 6.3 pmol/g wet weight, mean +/- SEM; remaining tissue 23.1 +/- 5.3 pmol/g wet weight) and the stomach (7.1 pmol/g wet weight). The two activities gave similar curves for inhibition of beta-Ep radioimmunoassay of synthetic beta-Ep. On gel-filtration chromatography of a duodenal extract, two components of beta-EpLI were separated. When human duodenal mucosa was perfused with a solution of pH2 or 1mM or 5mM taurocholate, the release of beta-EpLI from mucosa into the perfusate increased 2-4 fold. These results indicate that beta-EpLI present in human duodenal is released by the direct action of low pH or taurocholate on the duodenal mucosa and suggest that it may have a physiological role.     

Glycine-extended processing intermediate of proVIP: a new form of VIP in the rat

The biosynthesis of many peptides including vasoactive intestinal polypeptide (VIP) requires enzymatic alpha-carboxyamidation via a glycine-extended intermediate form. In an effort to identify and quantify glycine-extended VIP in rat tissue extracts a radio-immunoassay specific for this peptide was developed. The concentrations of glycine-extended VIP ranged from 1.3 pmol/g in the brain to 83.9 pmol/g in the small intestine. The identity of the peptide was substantiated by cation-exchange HPLC. The ratio of glycine-extended VIP to amidated VIP varied considerably being highest (63%) in the small intestine. The natural occurrence of glycine-extended VIP in connection with our recent demonstration of its biological activity suggest a physiological role for this biosynthetic intermediate VIP form.     

The distribution of GAWK-like immunoreactivity in neuroendocrine cells of the human gut,pancreas, adrenal and pituitary glands and its co-localisation with chromogranin B

Summary GAWK is a recently discovered peptide isolated from extracts of human pituitary gland and subsequently shown to be identical to sequence 420–493 of human chromogranin B. The distribution of this peptide was studied in human gut, pancreas, adrenal and pituitary glands using antisera to two portions of the 74 amino acid peptide (sequences 1–17 and 20–38). In addition, the co-existence of GAWK immunoreactivity with other peptides and chromogranin B was investigated using comparative immunocytochemistry.In the gut, GAWK was localised mainly to serotonin-containing cells of the mucosal epithelium, where electron microscopy showed it to be stored in typical electron-dense (250 nm diameter) granules, and to a moderate population of nerve fibres in the gut wall. Considerable quantities of GAWK-like immunoreactivity were measured in the gut, up to 36.3±18 pmol GAWK 1–17/g wet weight of tissue (mean±SEM) and 12.4±2.9 pmol GAWK 20–38/g. Chromatography of gut extracts revealed several GAWK-like immunoreactive peaks. GAWK-like immunoreactivity was also detected in endocrine cells of pancreas, pituitary gland and adrenal medulla, where the highest concentrations of GAWK-like immunoreactivity were measured (GAWK 1–17 2071.8±873.2 and GAWK 20–38 1292.7±542.7 pmol/g). Endocrine cells containing GAWK-like immunoreactivity were found also to be immunoreactive for chromogranin B.Our results define a discrete distribution of GAWK immunoreactivity in human endocrine cells and nerves and provide morphological support for the postulated precursor-product relationship between chromogranin B and GAWK. Details of the functions of this peptide are awaited.     

Pharmacological characterization of the binding of [3H]bremazocine in guinea-pig brain: evidence for multiplicity of the kappa-opioid receptors

In guinea-pig brain, [3H]bremazocine has a binding capacity of 27.2 pmol/g wet tissue, which is statistically different from that of [3H]ethylketazocine (14.7 pmol/g wet tissue) or the sum of the individual binding capacities of mu-, delta-, and kappa-selective ligands (15.0 pmol/g wet tissue). Saturation studies of [3H]bremazocine performed in the presence of unlabelled mu-, delta-, and kappa-blockers still reveal a homogeneous population of binding sites. [3H]Bremazocine under suppressed conditions displays at these sites a Kd of 2.51 nM with a binding capacity of 9.15 pmol/g wet tissue. We have performed the pharmacological characterization of these additional opioid binding sites. Displacement curves measured with a number of opioid substances were all best fitted to a one-site model. The stereoselectivity of these additional sites was demonstrated by using two groups of stereoisomers. Oripavine and benzomorphan opioids were among the most potent drugs at the [3H]bremazocine sites (mu + delta + kappa suppressed). Diprenorphine, bremazocine, cyclazocine, and ethylketazocine displayed apparent affinities constants (1/Ka) of 8.66, 7.57, 21.4, and 38.0 nM, respectively at those sites. The kappa-selective drugs U50488, U69593, PD117302, and tifluadom were inhibitors of the binding of [3H]bremazocine at these sites with apparent affinities of 113, 268, 76.9, and 47.9 nM. All mu- or delta-selective drugs tested in this study have caused weak or no inhibition of the binding. Correlation analyses were done between the different affinities measured at the [3H]bremazocine sites (mu + delta + kappa suppressed) and those observed at the known mu-, delta-, and kappa-sites of the guinea-pig brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit neuromedin U-25: lack of conservation of a posttranslational processing site

The rabbit small intestine contains neuromedin U-like immunoreactivity (22 pmol/g wet tissue weight) that was resolved into a single major molecular form by reversed-phase HPLC. The primary structure of the peptide was established as: Phe-Pro-Val-Asp-Glu-Glu-Phe-Gln-Ser-Pro10-Phe-Gly-Ser-Arg-Ser-Arg- Gly-Tyr-Phe- Leu20-Phe-Arg-Pro-Arg-Asn.NH2. In rabbit neuromedin U, the Arg16-Arg17 dibasic residue processing site that is found in pig and dog neuromedin U-25 is replaced by Arg-Gly, but this potential monobasic processing site is not utilized by cleavage enzyme(s) in the intestine.