Characterization of complementary DNA encoding the rat neuromedin U precursor.

Neuromedin U (NmU), a peptide originally isolated from porcine spinal cord, is known for its ability to stimulate uterine smooth muscle contraction and to cause selective vasoconstriction. It was subsequently isolated from a number of species. Among the species studied, the five amino acids at the C-terminus of the peptide are totally conserved, suggesting that this region is of major importance. We have cloned and sequenced the cDNA encoding the rat NmU precursor protein using the anchor polymerase chain reaction technique. Sequence analysis revealed that NmU is synthesized as a 174-amino acid precursor. Like the precursors of most other small regulatory peptides, it has a hydrophobic signal peptide and a number of paired dibasic amino acids, which may serve as signals for enzymatic cleavage, to release NmU and a series of other peptides. These predicted flanking peptides of NmU show no significant homology with entries in the protein databases searched, and the cDNA likewise shows no homology with entries in the GenBank database. Northern blot analysis using total RNA extracted from different rat tissues shows high levels of NmU mRNA in the ileum, thyroid, and anterior pituitary. Southern blot analysis of rat genomic DNA demonstrates that NmU is a single copy gene.

Evidence for neuropeptide Y synthesis in the rat anterior pituitary and the influence of thyroid hormone status: comparison with vasoactive intestinal peptide, substance P, and neurotensin.

Neuropeptide Y (NPY), a 36-amino acid member of the pancreatic polypeptide family, was found to be present by RIA and immunocytochemistry in the rat anterior pituitary gland. NPY prohormone messenger RNA (mRNA) was identified in the pituitary by Northern blot analysis. The possible regulation of NPY was examined by determining the effects of thyroid hormone manipulation on peptide synthesis. Three other anterior pituitary neuropeptides, neurotensin (NT), substance P (SP), and vasoactive intestinal peptide (VIP), were studied for comparison. Hypothyroidism was found to significantly increase the pituitary content of NPY, SP, and VIP and their respective mRNAs but to decrease the quantity of NT. Immunocytochemistry revealed very weak NPY immunoreactivity in scattered cells in control rat anterior pituitaries, but in hypothyroid rats a greater number of positive cells were seen, and the staining was relatively intense. These positive cells were identified as a subset of thyrotropes. In T4-induced hyperthyroidism NPY, NT, and VIP levels were unaffected whereas SP concentrations fell considerably. TRH treatment produced a decrease in NT and had no effect on NPY, SP, or VIP. These changes were found only in the pituitary; no net change occurred in hypothalamic peptide and mRNA levels. Since the changes in pituitary peptide and mRNA levels occurred coordinately it appears that regulation by thyroid hormone status occurs, at least in part, directly at the level of gene transcription. The changes in these 4 regulatory peptides in hypothyroidism and their known powerful effects on pituitary function suggest that they may have a significant paracrine or autocrine influence in controlling the alterations in pituitary secretion.

The influence of thyroid hormone status on the hypothalamo-hypophyseal growth hormone axis.

Growth hormone (GH) synthesis is known to be impaired by either abnormally high or low levels of thyroid hormone. To determine the effects of these conditions on the central regulation of GH secretion, we have examined their effects on the hypothalamic regulatory peptides GH-releasing hormone (GH-RH) and somatostatin (SRIF). In thyroidectomized rat hypothalamus, a dramatic increase in GH-RH mRNA occurred in parallel with a decrease in peptide content. The significance of this phenomenon is uncertain and might possibly reflect some posttranscriptional derangement of GH-RH synthesis or an increased rate of GH-RH synthesis and release. In the hyperthyroid group, GH-RH showed significant decreases in both peptide and mRNA levels that might possibly reflect a decrease in GH-RH synthesis and secretion. No change was observed in SRIF peptide or mRNA levels in either thyroidectomized or T4-treated animals. As expected, GH mRNA levels in the anterior pituitary were dramatically decreased by thyroidectomy and unaffected by T4 treatment. In addition, in thyroidectomized pituitaries, the mature GH mRNA was observed to alter its structure, increasing in size by approximately 100 nucleotides. This increase in size was found to result from an increase in poly(A) tail length, the significance of which is as yet unclear.

The calcitonin-like sequence of the beta CGRP gene.

We have identified a region within the beta CGRP gene which has the potential to encode a novel calcitonin-like peptide. The gene is located on the short arm of chromosome 11 (11p 12-14.2) and we suggest that it resulted from a local duplication of the alpha gene. We have been unable to detect the corresponding mRNA in a variety of tissues which express alpha-calcitonin. It is not clear whether this sequence can be expressed in man.

Characterization of rat gastric inhibitory peptide cDNA.

Gastric inhibitory peptide (GIP) is a 42 amino acid gastrointestinal peptide which inhibits gastric acid secretion and stimulates pancreatic insulin secretion in the presence of glucose. Here we report the sequence of the cDNA encoding the rat GIP precursor. PreproGIP was 144 amino acids in length and comprised the GIP peptide itself, N- and C-terminal flanking peptides of 22 and 59 amino acids respectively and a typical hydrophobic signal peptide. The sequence indicated that GIP is released from its precursor by cleavage at single arginine residues. The C-terminal flanking peptide may have an important function since it was well conserved and contained a region of 16 amino acids with only a single, conservative replacement. Rat GIP mRNA was found in the duodenum and jejunum. Levels of GIP mRNA in the duodenum were increased twofold after a period of 2 days of starvation. There was no detectable expression of the GIP gene in other parts of the gastrointestinal tract or in other endocrine tissues. However, in pancreatic mRNA preparations, a larger mRNA was detected after low stringency hybridization. This could represent a further member of this gene family.

Extra-pancreatic expression of the rat islet amyloid polypeptide (amylin) gene.

Messenger RNA for rat islet amyloid polypeptide (IAPP) has been identified not only in the pancreas but also, in lesser amounts, in preparations from the stomach and dorsal root ganglia. In the stomach, insulin mRNA was not detectable, ruling out possible contamination by pancreatic tissue. Because IAPP and calcitonin gene-related peptide (CGRP) are related and CGRP is present in both stomach and dorsal root ganglia, it was possible that 'IAPP' signals were in fact due to cross-hybridization with CGRP mRNA. A second IAPP probe was constructed which does not cross-react. This probe also detected mRNA in both tissues, confirming the expression of IAPP in both tissues. The regional distribution of IAPP mRNA in the stomach did not parallel that of gastrin mRNA. IAPP mRNA was present in the antrum, centrum and pylorus and, like gastrin, the highest amounts were in the pylorus. However, the ratio between the pylorus and centrum was 3.6:1 for IAPP and 156:1 for gastrin. The effects of dietary manipulation were determined; a period of 48 h of starvation reduced pancreatic IAPP mRNA by approximately 60%, whereas in the stomach there was no significant reduction. If the action of IAPP was hormonal, pancreas and stomach would not be acting in concert. A paracrine role for gastric IAPP therefore seems more likely.

Depletion of islet amyloid polypeptide in the spontaneously diabetic (BB) Wistar rat.

Islet amyloid polypeptide (IAPP) in the pancreas of the spontaneously diabetic (BB) Wistar rat was examined by radioimmunoassay, and IAPP mRNA levels were determined by Northern blotting. IAPP-like immunoreactivity in the diabetic rat pancreas was found to be significantly depleted compared with control (non-diabetic) BB rats (85.9 +/- 5 pmol/g in control rats, n = 8, vs 8.97 +/- 0.9 pmol/g in diabetic rats, n = 5; mean +/- S.E.M.). A similar change in insulin concentrations was found, although insulin was present in approximately 100-fold greater amounts than IAPP. Chromatography of the IAPP immunoreactivity revealed a single molecular form, corresponding to synthetic IAPP. Northern blot analysis of pancreatic RNA (n = 4) revealed that IAPP mRNA in the diabetic group was depleted to 22% of the signal intensity in the control group. Insulin mRNA was dramatically reduced to only 4% of the control group and, in contrast, somatostatin was relatively unaffected, with the diabetic group retaining 86% of signal compared with the controls. This animal model of insulin-dependent diabetes results from severe autoimmune destruction of the beta cell. The extremely low levels of both insulin and its messenger RNA are in agreement with this. These results demonstrate that this pathological state is also associated with a loss of IAPP from the pancreas. Insulin-dependent diabetes is associated with a range of metabolic disturbances. It is possible that the concomitant depletion of IAPP may be a contributory factor in exacerbating the condition.

The interaction of CGRP and adrenomedullin with a receptor expressed in the rat pulmonary vascular endothelium.

An abundant, seven trans-membrane domain receptor related to the calcitonin receptor has been studied by a number of groups without identification of its ligand. A recent report claimed that the receptor was a type 1 CGRP receptor (Aiyar et al J. Biol. Chem. 271 11325-11329 (1996)). We have studied the equivalent rat sequence in transfected cells. When expressed in 293 cells the receptor interacts with CGRP and adrenomedullin with KD values of 1.2 nM for CGRP and 11 nM for adrenomedullin. Both ligands cause an elevation of intracellular cAMP with EC50 values of 4 nM and 20 nM respectively and these effects are inhibited by the antagonist CGRP8-37. The receptor is expressed at high levels in the pulmonary vascular endothelium. Both the pharmacological data and the localisation are consistent with the conclusion that the orphan receptor is a type J CGRP receptor. However, when expressed in COS-7 cells, no receptor activity could be demonstrated suggesting that 293 cells contain a factor necessary for functional receptor expression.

Distribution and developmental pattern of neuromedin U expression in the rat gastrointestinal tract.

This study has quantified, for the first time, the relative levels of neuromedin U (NmU) mRNA in the rat gastrointestinal tract using Northern blot analysis. NmU message was detected in all regions of the gastrointestinal tract from the oesophagus to the rectum. The greatest levels were found in the duodenum and jejunum, the principal sites for absorption, which were 2.5- and 3-fold respectively above ileal levels. Quantification of NmU mRNA and peptide contents in the duodenum, jejunum and ileum during postnatal development of the rat showed message and peptide levels to be greater in the maturing rat than in neonates. Message levels in the duodenum, jejunum and ileum showed 14-, 7- and 4-fold increases respectively between 1 and 56 days after birth, whilst the corresponding peptide levels in the duodenum, jejunum and ileum showed 33-, 14- and 25-fold increases respectively. Food deprivation caused a small, but significant, decrease in message levels in the jejunum and colon, but there was no change in the duodenum or ileum. This shows that the presence of food has little effect on NmU mRNA levels in the gut.

Expression of the rat amylin (IAPP/DAP) gene.

We have used the polymerase chain reaction with mixed sequence primers to generate a probe for rat amylin and have used this to detect expression in various rat tissues. Amylin mRNA is found in greatest concentrations in the pancreas where a single mRNA species can be detected giving a hybridisation signal intensity approximately 10% that of insulin mRNA. When the beta cell population was depleted with streptozotocin, both amylin and insulin mRNAs were reduced to a similar extent. Consistent with its supposed role in the control of carbohydrate metabolism, amylin mRNA was also found in the stomach. Unlike the related peptide, CGRP, amylin mRNA is not present in the thyroid and is not widely distributed in the central nervous system. The only nervous tissue in which it could be detected was the dorsal root ganglion. Surprisingly, amylin mRNA was also found in the lung though only at very low levels.

Differential expression of alpha-CGRP and beta-CGRP by primary sensory neurons and enteric autonomic neurons of the rat.

Expression of the calcitonin gene-related peptide, alpha-calcitonin gene-related peptide (CGRP), and the homologous beta-CGRP were compared in sensory and enteric nerves of the rat. Analysis of CGRP-like immunoreactivity by cation exchange chromatography and radioimmunoassay showed that in the dorsal root ganglia, dorsal spinal cord and in those peripheral tissues where CGRP-like immunoreactivity is primarily localized to sensory fibres, alpha-CGRP concentrations were three to six times greater than beta-CGRP concentrations. In the intestine, however, beta-CGRP concentrations were up to seven times greater than alpha-CGRP concentrations. Only beta-CGRP was detected in the intestines of capsaicin-treated rats. Northern blot and in situ hybridization to alpha-CGRP- and beta-CGRP-specific probes showed that while both alpha-CGRP and beta-CGRP messenger ribonucleic acids occurred in the dorsal root ganglia, only beta-CGRP messenger ribonucleic acid occurred in the intestine, where it was localized to enteric neurons. Receptor binding sites on membranes of rat heart and colon had approximately equal affinities for alpha-CGRP and beta-CGRP. The two peptides were equipotent in increasing the rate and force of atrial contractions but alpha-CGRP was slightly (2.6 times) more potent than beta-CGRP in relaxing colonic smooth muscle. Thus, both alpha-CGRP and beta-CGRP occur in the rat nervous system and are both biologically active. Sensory neurons and enteric neurons have been identified as populations which preferentially express alpha-CGRP and beta-CGRP, respectively.

CGRP and adrenomedullin binding correlates with transcript levels for calcitonin receptor-like receptor (CRLR) and receptor activity modifying proteins (RAMPs) in rat tissues.

1. Putative receptors for CGRP and adrenomedullin have been investigated in the rat. Calcitonin Receptor-Like Receptor (CRLR), in combination with Receptor Activity Modifying Proteins (RAMPs) is hypothesized to bind either CGRP or adrenomedullin. The receptors known as RDC1 and L1 have also been shown to bind CGRP and adrenomedullin respectively. 2. In this study it is shown that rat CRLR cDNA specifies a CGRP receptor when co-transfected with RAMP-1 cDNA and an adrenomedullin receptor when co-transfected with either RAMP-2 or RAMP-3 cDNA in human embryonic kidney 293 cells. 3. CRLR, RAMP, RCD1 and L1 mRNA levels and CGRP and adrenomedullin receptor densities have been measured and correlated with each other in eight rat tissues selected for their distinctive patterns of CGRP and adrenomedullin binding. 4. The data are consistent with the predictions of the CRLR/RAMP model. CGRP binding correlates well with RAMP-1 mRNA levels (R=1.0, P=0.007), adrenomedullin binding shows a tendency to vary with RAMP-2 mRNA levels (R=0.85, P=0.14) and total binding is correlated with CRLR mRNA levels (R=0.94, P=0.03). The data do not support the hypothesis that RDC1 and L1 account for the majority of CGRP and adrenomedullin binding respectively.

The genomic breakpoint in a patient with Philadelphia-positive acute leukemia is 5' of the breakpoint cluster region.

We report a case of acute leukemia in which studies at presentation showed both myeloid and lymphoid cell surface markers. At relapse membrane markers studies were consistent with a leukemia of B-lymphoid lineage. However, immunoglobulin (Ig) and T cell receptor (TCR) beta chain genes were both found in a rearranged configuration. The majority of metaphases from the leukemic cells at presentation showed the Philadelphia chromosome, t(9;22)(q34;q11), whereas a minority were normal. At relapse both Ph-positive and -negative metaphases were still present in the bone marrow but some of the Ph-negative metaphases had acquired an additional chromosome #19 [47,XY, + 19]. Southern analysis of DNA from leukemic bone marrow cells at diagnosis showed no rearrangement of breakpoint cluster region (bcr). There was no bcr-abl chimeric mRNA typical of Ph-positive chronic myeloid leukemia (CML). However, the cells expressed an abl-related protein of Mr 190 kd with enhanced tyrosine kinase activity. Leukemic cell metaphases were studied by the technique of in situ hybridization with probes for C-lambda, sis, abl, and 5' bcr. The c-abl probe mapped to chromosome 22q11 in Ph-positive metaphases. The 5' bcr probe mapped to 9q+ in the Ph-positive metaphases and the C-lambda gene mapped to the Ph chromosome. Thus, the genomic breakpoint in this patient must lie upstream of the BCR defined by study of Ph-positive CML and downstream of the C-lambda gene locus. We speculate that the Ph-negative cells in this patient may represent a leukemic proliferation susceptible to acquisition of specific chromosomal changes.

The calcitonin gene-related peptide (CGRP) antagonist CGRP(8-37) blocks vasodilatation in inflamed rat skin: involvement of adrenomedullin in addition to CGRP.

The neuropeptide calcitonin gene-related peptide (CGRP) is a potent microvascular vasodilator in rat skin and effects are antagonised by CGRP(8-37). In this study, CGRP(8-37) significantly (P<0.05) inhibited the time-dependent (3-5 h) increase in skin blood flow measured in the anaesthetised rat, after intradermal administration of the inflammatory cytokine interleukin-1beta (3 pmol/site), indicating the involvement of CGRP1 receptors. The CGRP-related peptide adrenomedullin (ADM) is also a potent vasodilator in rat skin, with effects antagonised by CGRP(8-37). We show that ADM mRNA expression is increased in rat skin after treatment with IL-1beta and that the IL-1beta-induced blood flow is blocked by a selective ADM antibody (P<0.05). Thus ADM is expressed locally in the inflamed cutaneous microvasculature where it can, in addition to, or as an alternative to CGRP, contribute to IL-1beta-induced vasoactive effects.

Calcitonin gene-related peptide messenger RNA is expressed in sensory neurones of the dorsal root ganglia and also in spinal motoneurones in man and rat.

Calcitonin gene-related peptide (CGRP) mRNA was localised to neurones of the dorsal root ganglia and motoneurones of the ventral horn in man and rat. Presence of alpha- and beta-CGRP mRNA was confirmed by Northern blot analysis of rat tissues which showed alpha-CGRP was the predominant gene. The distribution of CGRP gene transcripts corresponded with neurones displaying CGRP immunoreactivity in the ganglia of both species and in the rat ventral horn. In man few motoneurones were immunoreactive despite many expressing CGRP mRNA. In situ hybridisation revealed not only sensory but also motor neurones are sites of CGRP manufacture. Thus in conjunction with other evidence the present study reinforces the proposed muscle trophic role for this peptide.

Lack of correlation of free deoxypyridinoline excretion with Taq1 restriction length polymorphisms in the vitamin D receptor gene in males.

An association between allelic variants in the vitamin D receptor gene and bone mineral density has been previously described. A bimodal variation in the rate of bone resorption (as measured by urinary deoxypyridinoline excretion rate) has also been reported. We have recruited male volunteers, to minimise variation associated with ovarian function, to investigate a possible connection between these observations. Allelic variants in the vitamin D receptor gene were identified as Taq1 restriction fragment length polymorphisms. The ratio of variants TT:Tt:tt occurred with a frequency of 34%:47%:17%. Excretion rates of urinary free deoxypyridinoline, measured by immunoassay, were compared in age-matched males from each genetic group. There were no significant differences based on the paired Student's t-test. Excretion rates declined with age (P = 0.04) and the best fit model fits the same regression line to each group. Genetic variation in the vitamin D receptor is not linked with differences in bone resorption rates.

Altered calcitonin gene in a young patient with osteoporosis.

To assess whether calcitonin is important in maintaining the integrity of bone the calcitonin gene of a young male patient with osteoporosis and no detectable plasma concentrations of calcitonin was studied. Genomic Southern blots with various restriction enzymes showed no large abnormalities in his calcitonin gene. Genomic clones representing his calcitonin gene were then analysed. His gene encoded normal precursor polypeptides for calcitonin and calcitonin gene related peptide; the only abnormality identified was a single base insertion in the intron separating exons IV and V of the gene. The affected sequence is homologous with an intron sequence from beta globin that is implicated in splicing and forming a crucial intermediate structure during the maturation of messenger RNA. The change observed may be responsible for the patient's calcitonin deficiency and consequently for his condition, suggesting that calcitonin is important in preventing bone loss.