Methionine-enkephalin and thyrotropin-stimulating hormone are intimately related in the human anterior pituitary.

The tissue distribution and function of opioid peptides in humans is incompletely defined. We report here that, unlike that in other species, the human anterior pituitary gland contains high concentrations of methionine-enkephalin (met-enkephalin). The met-enkephalin immunoreactive material was isolated and identified as authentic met-enkephalin by fast atom bombardment-mass spectrometry and Edman degradation sequencing. The met-enkephalin was localized in a large subpopulation of TSH immunoreactive cells (thyrotrophs). No other proenkephalin-derived opioid peptides were found in the pituitary, and there was no overlap between proopiomelanocortin and met-enkephalin immunoreactive cells. These results suggest that the human anterior pituitary gland contains a novel met-enkephalin precursor and a possible role for met-enkephalin in regulating human thyroid function.

Determination of tryptamine in rat brain by gas chromatography-mass spectrometry.

Tryptamine (TA) occurs in trace levels in the brain, but its role in the central nervous system is not clear. However, there is evidence that TA may be a neuromodulator since it binds to specific binding sites in the brain. TA was measured as a diheptafluorobutyryl derivative in rat whole brain by capillary gas chromatography-mass spectrometry using negative chemical ionization (NCI) and single ion monitoring (SIM). d(4)-TA was used as the internal standard. The ions m/z 532 and m/z 536 were monitored to identify TA and d(4)-TA, respectively and to calculate the concentration of TA in rat whole brain which was found to be 0.19 +/- 0.08 ng g(-1) (n = 8). The results confirm the earlier TA concentrations measured by GC-MS using positive electron impact ionization. However, NCI improved the signal/noise ratio of the method increasing its sensitivity for TA.

Peptide E and its products, BAM 18 and Leu-enkephalin, in bovine adrenal medulla and cultured chromaffin cells: release in response to stimulation.

Peptide E is a 25 amino acid opioid peptide which, if cleaved at the sole double basic (Lys-Arg) typical processing site, would generate two opioid fragments, the amino-terminal fragment BAM 18 and the carboxy-terminal fragment Leu-enkephalin. We have analysed extracts of bovine adrenal medulla in order to quantify these three opioid peptides (peptide E, BAM 18, and Leu-enkephalin). Here we present evidence that BAM 18 and Leu-enkephalin were present in similar amounts, whereas peptide E was present at a higher concentration. This is consistent with previous observations showing a preferential accumulation of larger peptides in the bovine adrenal, and also with the Lys-Arg bond being the principal site of cleavage of peptide E. However, when bovine adrenal chromaffin cells were maintained in culture for several days, Leu-enkephalin was found to be present in much greater amounts than was BAM 18-like immunoreactivity. The molar amounts of peptide E still exceeded the estimated levels of BAM 18 and Leu-enkephalin. We provide evidence that under conditions of basal release BAM 18 and peptide E were released, whereas Leu-enkephalin was released in much smaller amounts, if at all. On stimulation with nicotine results were consistent with an increased release of all three peptides with a preferential stimulation of Leu-enkephalin release. Under all conditions, the molar amounts of peptide E released apparently exceeded that of the other peptides. The results are discussed in terms of the regulation of partial proteolysis and the fate of peptide E.

Expression of opioid peptides in tumors.

We looked for opioid peptides and their precursors in 108 tumors of both neuroendocrine and nonneuroendocrine origin, using a monoclonal "pan-opioid" antibody, 3-E7, which recognizes the tetrapeptide Tyr-Gly-Gly-Phe (the sequence responsible for pharmacologic activity in all known opioid peptides), in conjunction with polyclonal antibodies directed against representative peptides of each of the three precursors (alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B). Using the avidin-biotin immunoperoxidase technique, we observed consistent cytoplasmic immunoreactivity (at least focally) in all of 15 adrenal pheochromocytomas, all of 6 thyroid medullary carcinomas, and all of 5 pituitary adenomas. Opioid staining was also observed in parathyroid adenomas (8 of 9), pancreatic islet-cell tumors (7 of 10), carcinoid tumors from various sites (18 of 26), and paragangliomas (1 of 2). There was no immunoreactivity in pulmonary small-cell carcinomas, Merkel-cell tumors of skin, neuroblastomas, or any of the non-neuroendocrine tumors examined. The expression of alpha-endorphin, [met]enkephalin-Arg-Gly-Leu, and dynorphin B varied from tumor to tumor; however, positive staining with the "pan-opioid" antibody was found in each tumor containing at least one of the three precursors. Opioid peptide immunoreactivity was also detected in non-neoplastic cells of the adrenal medulla, pancreatic islets, pituitary, intestinal and bronchial mucosa, and intestinal myenteric plexuses. We conclude that opioid expression within tumors is most likely due to enhanced expression of a normal cell product and that opioid peptides are useful markers of neuroendocrine differentiation in many tumors.

Isolation of enzyme cDNA clones by enzyme immunodetection assay: isolation of a peptide acetyltransferase.

The biological activity of many proteins and peptides can be profoundly affected by enzyme-catalyzed covalent modifications such as acetylation, sulfation, glycosylation, or amidation. This article describes the cloning of such an enzyme, a peptide acetyltransferase from rat brain that catalyzes the amino-terminal acetylation of endorphins and perhaps other substrates in vivo. Blot-hybridization analysis suggests that the mRNA encoding the acetyltransferase is approximately 2.0 kilobases, is present in whole rat brain and rat hypothalamus, and is slightly larger in mouse AtT20 tumor cells. The acetyltransferase was cloned by using a strategy whereby a cDNA expression library was screened with a solid-phase enzyme-activity assay; this technique combines the use of the substrate coupled to a solid support and subsequent recognition of the product by using a specific antiserum. We have called this method the enzyme immunodetection assay (EIDA). The EIDA should prove useful in the isolation of other clones for proteins that possess enzymatic activity upon expression in bacterial hosts.

Small cell carcinoma cell lines contain opioid peptides and receptors.

Two human small cell carcinoma cell lines were assayed for total opioid and beta-endorphin-like immunoreactivity. Small cell carcinoma cell line NCI-H146 contained approximately 1.1 pmol/mg protein of total opioid immunoreactivity. This material was similar in size and immunoreactive determinants to C-terminally modified beta-endorphin. Small cell carcinoma cell line NCI-H187 contained approximately 0.2 pmol/mg protein total opioid immunoreactivity, which was of low molecular weight. NCI-H187 also contained approximately 1.2 pmol/mg protein of material similar in size and immunoreactive determinants to beta-lipotropin. The two small cell carcinoma cell lines were also examined for opioid receptors with the use of [3H]-etorphine as ligand. Both cell lines contained between 50 and 100 fmol/mg protein of specific, saturable, high-affinity opioid receptor binding sites. Together, these findings suggest a possible autocrine role for opioids in small cell carcinoma of the lung.

Constitutively expressed rat mRNA encoding a 70-kilodalton heat-shock-like protein.

A nearly full-length cDNA clone isolated from the rat pheochromocytoma cell line, PC12, revealed extensive nucleotide sequence similarity between the rat cDNA and the Drosophila melanogaster hsp70 gene. The rat recombinant clone encodes a 71,000-dalton protein that is 70% identical with the dipteran hsp70 protein. Remarkably, a truncated segment of this cDNA clone was originally isolated by immunoreactivity with antisera raised to catecholamine-synthesizing enzymes, suggesting that this heat shock protein and these catecholamine enzymes shared antigenic determinants. The rat hsp70-related mRNA is responsible for the production of a constitutive hsp70 protein, because it is present in abundant amounts in various tissues at normal growth temperatures and is only minimally induced by hyperthermia. The rat hsp70-related sequence is part of a multigene family that extends across species to mice and humans.

Co-localization and characterization of immunoreactive peptides derived from two opioid precursors in guinea pig adrenal glands.

Peptides derived from both proenkephalin and prodynorphin have been identified in guinea pig adrenal medulla. In extracts of whole adrenal glands radioimmunoassays directed to the prodynorphin-derived peptides alpha-neoendorphin, dynorphin A, and dynorphin B detected high concentrations of immunoreactive material ranging from 113 to 216 pmol/gm. The concentrations measured by radioimmunoassays directed to the proenkephalin products met-enkephalin-Arg-Gly-Leu and met-enkephalin-Arg-Phe were 878 and 484 pmol/gm, respectively. No metorphamide or dynorphin(1-8) could be detected in the adrenals. Leucine-enkephalin immunoreactivity which can be generated from either prodynorphin or proenkephalin could also be measured in the extracts. Gel filtration showed the immunoreactive material, with the exception of that measured by the alpha-neoendorphin radioimmunoassay, to be predominantly of high molecular weight ranging from Mr = 3,000 to 12,000. Immunocytochemistry, using well characterized antisera to alpha-neoendorphin and met-enkephalin-Arg-Gly-Leu, demonstrated that the prodynorphin and proenkephalin products were present in the same cells in the medulla region of the gland. The results show that two opioid peptide precursors can be localized in the same cells and exhibit some common features in their processing. As a relatively homogeneous, localized system, the guinea pig adrenal gland should prove a valuable, in vivo model for the study of co-localized opioid precursors.

Isolation and characterization of substance P, substance P 5-11, and substance K from two metastatic ileal carcinoids.

Using an antiserum directed at the COOH-terminus of tachykinins, we have examined postmortem tissue from two cases of metastatic ileal carcinoid for the presence of tachykinin-like immunoreactivity. The vast majority of the immunoreactive tachykinin-like material eluted from a Sephadex G-50 column as two peaks at positions corresponding to molecular weights of 1300 and 850. The 1300 dalton peak was resolved by reverse-phase-HPLC into two components which by Edman sequencing, amino acid analysis, and fast atom bombardment (FAB)-mass spectrometry criteria, were identified as substance P and substance K. The 850 dalton peak was also resolved on RP-HPLC into two peaks which were resistant to Edman degradation but from amino acid analysis and FAB-mass spectrometry criteria were identified as pyro-Glu-substance P 5-11 and oxidized pyro-Glu-substance P 5-11. In control experiments substance P 5-11 was converted to pyro-Glu-substance P 5-11 during the extraction procedure. Both tumors also contained a minor immunoreactive peak which eluted from a Sephadex G-50 sizing column at a position corresponding to a molecular weight of 4000 which probably represents neuropeptide K. These results suggest that beta-preprotachykinin is preferentially expressed in carcinoid tumors and that substance K may also play a role in the carcinoid syndrome.

Gastrin-releasing peptide, a mammalian analog of bombesin, is present in human neuroendocrine lung tumors.

Several reports have indicated that the amphibian peptide bombesin is present in oat-cell carcinoma of the human lung. The recent observation that gastrin-releasing peptide (GRP), a 27-amino acid peptide isolated from porcine intestine, may be the mammalian analog of bombesin led the authors to look for this peptide in human pulmonary tumors. Examination of 36 human lung tumors (8 carcinoids, 8 oat-cell carcinomas, and 20 non-oat-cell carcinomas) by immunohistochemistry and radioimmunoassay demonstrated the presence of high, although variable, levels of GRP in neuroendocrine tumors, and not in other histologic types. These findings indicate that bombesin immunoreactivity in human lung tumors should be attributed to GRP or GRP-like molecules and that GRP may be a useful marker of neuroendocrine differentiation.

Gastrin-releasing peptide immunoreactivity in intestinal carcinoids.

Human neuroendocrine tumors are known to demonstrate immunoreactivity to the amphibian peptide bombesin. The recent observation that gastrin-releasing peptide (GRP), a 27 amino acid peptide first isolated from porcine intestine, may be the mammalian analog of bombesin led us to look for this peptide in intestinal carcinoid tumors. Formalin-fixed tissues from 20 of these tumors were examined by the immunoperoxidase technic, using specific antisera to the GRP molecule (1-27) and the GRP fragment (1-16). Intense diffuse cytoplasmic immunoreactivity was observed in carcinoids from the small intestine (7/10), appendix (4/5), and colon (1/5). In each positive case, the pattern of staining for GRP (1-16) and GRP (1-27) was identical. These findings indicate that bombesin-like immunoreactivity in human intestinal carcinoid tumors is attributable to GRP-like molecules and that GRP is a useful marker for neuroendocrine differentiation.

Regional distribution of metorphamide in rat and guinea pig brain.

A specific radioimmunoassay was developed for metorphamide, an endogenous, amidated opioid octapeptide, originally isolated from bovine brain and human pheochromocytoma tissues. The radioimmunoassay was used to determine the concentration of immunoreactive metorphamide in extracts from dissected regions of rat and guinea pig brain. Radioimmunoassay interfacing with Sephadex gel filtration and reverse phase high performance liquid chromatography confirmed that the immunoreactive substance measured corresponded to authentic metorphamide. Metorphamide was found to be widely distributed in brain regions from both species. However, the concentrations of immunoreactive metorphamide in regions from guinea pig brain were up to 5 times higher than the concentrations of immunoreactive metorphamide in rat brain regions. The results suggest that metorphamide is a specific processing product from proenkephalin in rodent brain.

Gastrin-releasing peptide-related peptides in a human malignant lung carcinoid tumor.

Recent immunohistochemical findings have indicated the presence of gastrin-releasing peptide in normal and pathological human lungs. Gastrin-releasing peptide is a 27-amino acid peptide isolated from porcine gut which bears considerable carboxyterminal homology with bombesin. We have characterized the gastrin-releasing peptide-like peptides present in a human malignant lung carcinoid tumor by gel chromatography and reverse-phase high-performance liquid chromatography. Our results show that this tumor did not contain bombesin; however, this tumor expressed a gastrin-releasing peptide-like compound, several amino-terminal fragments, and a carboxy-terminal fragment of gastrin-releasing peptide.

Regional distribution of dynorphin and neo-endorphin peptides in rat brain, spinal cord, and pituitary.

Five products of the dynorphin gene--alpha-neo-endorphin, beta-neo-endorphin, dynorphin A, dynorphin A-(1-8), and dynorphin B--were measured in various regions of rat brain and in rat spinal cord and pituitary. Specific antisera were used, supplemented by gel permeation analysis and high performance liquid chromatography, confirming the presence of dynorphin-32, dynorphin A, and dynorphin B in rat brain. In whole brain, alpha-neo-endorphin, dynorphin A-(1-8), and dynorphin B are present in much greater amounts than beta-neo-endorphin or dynorphin A. Although a general parallelism was found in the distribution of the five peptides, there were also noteworthy exceptions, suggesting that differential processing may occur.

Identification of pro-opiomelanocortin-derived peptides in the human adrenal medulla.

Extracts from adult human adrenals contained high concentrations of immunoreactive beta-endorphin and alpha-melanotropin. Lower quantities of immunoreactive adrenocorticotropic hormone could also be detected. Distribution studies showed the presence of pro-opiomelanocortin fragments in the adrenal medulla. No alpha-melanotropin, beta-endorphin, or adrenocorticotropic hormone could be found in adrenal extracts from several other mammalian species. Analysis of the beta-endorphin-like immunoreactivity using region specific radioimmunoassays interfacing with gel filtration and reverse-phase high-performance liquid chromatography showed the majority of the beta-endorphin-like material to exist as nonacetylated beta-endorphin-(1-31) with a small percentage of lipotropin-sized molecules. The alpha-melanotropin-like immunoreactivity cochromatographed on gel filtration and reverse-phase high-performance liquid chromatography with desacetyl alpha-melanotropin. The data suggest that pro-opiomelanocortin is expressed in the adrenal medulla of humans but is not detectable in the adrenal glands of many other mammalian species.

Identification of gastrin releasing peptide-related substances in guinea pig and rat brain.

Rat and guinea pig brain extracts were examined for the occurrence of gastrin-releasing peptide (GRP)-like substances by sequence specific radioimmunoassays interfaced with gel filtration and reversed phase high performance liquid chromatography (RP-HPLC). Tryptic digestion of the immunoreactive peptides followed by RP-HPLC was used to further characterize GRP-related peptides in brain. Using these analytical techniques it was found that guinea pig brain extracts contained a peptide with characteristics identical to authentic GRP (27 amino acid residues long). A carboxyterminal fragment with the characteristics of GRP(18-27) as well as a respective aminoterminal fragment with the characteristics of GRP(1-16) were also present in guinea pig brain extracts. The GRP(18-27) seems to correspond to the bombes in related material that has been described previously in mammalian brain extracts. Rat brain extracts also contained a peptide with the characteristics of GRP(18-27). The corresponding aminoterminal fragment, however, behaved differently on RP-HPLC from authentic GRP(1-16) and it was not recognized by antibodies directed to the aminoterminal tridecapeptide fragment of authentic GRP. Similarly the GRP-like peptide from rat brain did not comigrate on RP-HPLC with authentic GRP and was unreactive to antibodies directed toward the aminoterminus of GRP.

Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.

mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.