Tachykinin Neuropeptide Research Articles

Identification of zymogen and mature forms of human carboxypeptidase H A processing enzyme for the synthesis of peptide hormones

Carboxypeptidase H (CPH) is one of several processing enzymes required for the conversion of peptide hormone precursors into their smaller active forms. In this study, high levels of CPH activity was found in a liver metastasis of a human ideal carcinoid which expresses β-preprotachykinin mRNA and the tachykinin neuropeptides, substance P and substance K. This human CPH showed properties of a zinc-metallopeptidase that is structurally similar to bovine and rat CPH. Immunoblots of the human ileal carcinoma with anti-bovine CPH showed that CPH activity is represented by two proteins of apparent molecular masses 57 and 55 kDa. Cell-free translation of poly(A) + RNA followed by immunoprecipitation with anti-bovine CPH showed that human CPH mRNA encodes a precursor protein of apparent molecular mass 75 kDa. These data demonstrate that human CPH is synthesized as a zymogen, prepro-CPH, which must be cleaved to form catalytically active CPH.

Tachykinin precursors and receptors: Molecular genetic studies

Neuropeptides play an important role for providing the specificity and complexity of interneuronal communications. To understand the function and regulation of peptidergic neurons, it is necessary to elucidate detailed cellular mechanisms of peptidergic neurons, including peptide production, receptor function and signal transduction. Our studies concern some of these mechanisms by investigating molecular aspects of mammalian tachykinin neuropeptides and their receptors. In this article, we describe some characteristic features of gene expressions of the tachykinin precursors and discuss our recent studies concerning the molecular nature of the tachykinin receptors.

Receptor binding sites for substance P, but not substance K or neuromedin K, are expressed in high concentrations by arterioles, venules, and lymph nodules in surgical specimens obtained from patients with ulcerative colitis and Crohn disease.

Several lines of evidence indicate that tachykinin neuropeptides [substance P (SP), substance K (SK), and neuromedin K (NK)] play a role in regulating the inflammatory and immune responses. To test this hypothesis in a human inflammatory disease, quantitative receptor autoradiography was used to examine possible abnormalities in tachykinin binding sites in surgical specimens from patients with inflammatory bowel disease. Surgical specimens of colon were obtained from patients with ulcerative colitis (n = 4) and Crohn disease (n = 4). Normal tissue was obtained from uninvolved areas of extensive resections for carcinoma (n = 6). In all cases, specimens were obtained less than 5 min after removal to minimize influences associated with degradation artifacts and were processed for quantitative receptor autoradiography by using 125I-labeled Bolton-Hunter conjugates of NK, SK, and SP. In the normal colon a low concentration of SP receptor binding sites is expressed by submucosal arterioles and venules and a moderate concentration is expressed by the external circular muscle, whereas SK receptor binding sites are expressed in low concentrations by the external circular and longitudinal muscle. In contrast, specific NK binding sites were not observed in any area of the human colon. In colon tissue obtained from ulcerative colitis and Crohn disease patients, however, very high concentrations of SP receptor binding sites are expressed by arterioles and venules located in the submucosa, muscularis mucosa, external circular muscle, external longitudinal muscle, and serosa. In addition, very high concentrations of SP receptor binding sites are expressed within the germinal center of lymph nodules, whereas the concentrations of SP and SK binding sites expressed by the external muscle layers are not altered significantly. These results demonstrate that receptor binding sites for SP, but not SK or NK, are ectopically expressed in high concentrations (1000-2000 times normal) by cells involved in mediating inflammatory and immune responses. These data suggest that SP may be involved in the pathophysiology of inflammatory bowel disease and might provide some insight into the interaction between the nervous system and the regulation of inflammation and the immune response in human inflammatory disease.

CDNA eloping of bovine substance-K receptor through oocyte expression system

The neuropeptide receptors which are present in very small quantities in the cell and are embedded tightly in the plasma membrane have not been well characterized. Mammals contain three distinct tachykinin neuropeptides, substance P, substance K and neuromedin K, and it has been suggested that there are multiple tachykinin receptors. By electrophysiological measurement, we have previously shown that Xenopus oocytes injected with brain and stomach mRNAs faithfully express mammalian substance-P and substance-K receptors, respectively. Here we report the isolation of the cDNA clone for bovine substance-K receptor (SKR) by extending this method to develop a new cloning strategy. We constructed a stomach cDNA library with a cloning vector that allowed in vitro synthesis of mRNAs and then identified a particular cDNA clone by testing for receptor expression following injection of the mRNAs synthesized in vitro into the oocyte system. Because oocytes injected with exogenous mRNAs can express numerous receptors and channels, our new strategy will be applicable in the general molecular cloning of these proteins. The result provides the first indication that the neuropeptide receptor has sequence similarity with rhodopsin-type receptors (the G-protein-coupled receptor family) and thus possesses multiple membrane-spanning domains.

Substance P precursor and kininogen: their structures, gene organizations, and regulation

Substance P precursor and kininogen: their structures, gene organizations, and regulation

Malignant Carcinoid Tumors An Analysis of 103 Patients with Regard to Tumor Localization, Hormone Production, and Survival

In a prospective study of 103 patients with carcinoid tumors consecutively referred for medical treatment, the most common sites of the primary tumors were the ileum (73%), bronchi (7%), and jejunum (4%). All patients had local metastases, and 96 (93%) also had liver metastases. The most common initial symptoms were diarrhea (32%), ileus (25%), and flush (23%). The overall frequency of diarrhea was 84% and of flush was 75%. Heart insufficiency caused by cardiac valve disease was seen in 33% of the patients. The carcinoid syndrome, including flush, diarrhea, and elevated urinary 5-hydroxyindole acetic acid (5-HIAA) concentrations, was manifested by 69 patients (67%), 64 of whom (93%) had carcinoid tumors of mid-gut origin. Elevated urinary 5-HIAA was found in 91 patients (88%), of which 89 displayed liver metastases. The plasma concentration of the tachykinin neuropeptide K (NPK) was elevated in 67 patients (66%), 63 of whom had tumors of the mid-gut region. Serum pancreatic polypeptide (PP) and human chorionic gonadotrophin alpha levels were elevated in 43% and 28% of the patients, respectively, and the highest levels were found in patients with metastatic bronchial carcinoid tumors. Thirty-nine of the 103 patients are now dead; 18 died of tumor progression, whereas 14 patients died of heart failure secondary to a carcinoid tricuspidal valve insufficiency. The estimated median survival from the time of histologic diagnosis was 14 years, and from the time of carcinoid syndrome was 8 years.

Structure and gene organization of bovine neuromedin K precursor.

cDNA and genomic DNA clones for the precursor of a mammalian neuropeptide tachykinin, neuromedin K, have been isolated and characterized by molecular cloning and sequence analysis. The deduced amino acid sequence indicates that the bovine neuromedin K precursor (preprotachykinin B) consists of 126 amino acid residues including a putative signal peptide. There are two preprotachykinin B mRNAs that differ only at the 5' extremity of the untranslated regions. The major mRNA species is encoded by seven exon sequences, while the minor species includes two extra 5' exon sequences and lacks the 5' terminus of the first exon sequence for the major mRNA species. The above gene organization for the major preprotachykinin B mRNA closely resembles that for the preprotachykinin A mRNA encoding the precursor for substance P and substance K. This structural resemblance strongly suggests that the two preprotachykinin genes have evolved from a common ancestor gene. Furthermore, we have found that the preprotachykinin A and B mRNAs markedly differ in their major expression sites. The results described thus indicate that the diversity of the mammalian tachykinin system has been acquired through various cellular mechanisms including gene duplication and differential expression of duplicated genes.

The Cost of Mating

The Cost of Mating