Adrenomedullin: receptor and signal transduction.

Adrenomedullin is a vascular tissue peptide and a member of the calcitonin family of peptides, which includes calcitonin, calcitonin-gene-related peptide (CGRP) and amylin. Its many biological actions are mediated via CGRP type 1 (CGRP(1)) receptors and by specific adrenomedullin receptors. Although the pharmacology of these receptors is distinct, they are both represented in molecular terms by the type II family G-protein-coupled receptor, calcitonin-receptor-like receptor (CRLR). The specificity here is defined by co-expression of receptor-activity-modifying proteins (RAMPs). CGRP(1) receptors are represented by CRLR and RAMP1, and specific adrenomedullin receptors by CRLR and RAMP2 or 3. Here we discuss how CRLR/RAMP2 relates to adrenomedullin binding, pharmacology and pathophysiology, and how chemical cross-linking of receptor-ligand complexes in tissue relates to that in CRLR/RAMP2-expressing cells. CRLR, like other type II family G-protein-coupled receptors, signals via G(s) and adenylate cyclase activation. We demonstrated that adrenomedullin signalling in cell lines expressing specific adrenomedullin receptors followed this expected pattern.

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.

Epithelial calcium transporter expression in human duodenum.

Calcium absorption in intestine and kidney involves transport through the apical membrane, cytoplasm, and basolateral membrane of the epithelial cells. Apical membrane calcium influx channels have recently been described in rabbit (epithelial calcium channel, ECaC) and rat (calcium transport protein, CaT1). We amplified from human duodenum a 446-base partial cDNA probe (ECAC2) having a predicted amino acid similarity of 97% to rat CaT1. Duodenum, but not ileum, colon, or kidney, expressed a 3-kb transcript. A larger transcript was also found in placenta and pancreas, and a different, faint transcript was found in brain. In duodenal biopsies from 20 normal volunteers, expression varied considerably but was not significantly correlated with vitamin D metabolites. This signal correlated with calbindin-D(9k) (r = 0.48, P < 0.05) and more strongly with the plasma membrane calcium ATPase PMCA1 (r = 0.83, P < 0.001). These data show that although individual variations in calcium channel transcripts are not vitamin D dependent, expression of genes governing apical entry and basolateral extrusion are tightly linked. This may account for some of the unexplained variability in calcium absorption.

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.

Glucagon-like peptide-2 increases sucrase-isomaltase but not caudal-related homeobox protein-2 gene expression.

To determine the effect of glucagon-like peptide-2 (GLP-2) on sucrase-isomaltase and caudal-related homeobox protein-2 (Cdx-2) gene expression, male Wistar rats were divided into total parenteral nutrition (TPN)-fed and GLP-2-treated, TPN-fed groups. TPN was given via a jugular line, inserted under anesthesia, for 7 days. The treatment group received 40 microg/day of GLP-2 intravenously with the TPN diet. The small intestine and colon were weighed and measured. Tissue was obtained from the jejunum, terminal ileum, and midcolon. RNA analysis, morphometry, and microdissection were performed. The weight of the small intestine of GLP-2-treated rats was greater than that of TPN-fed rats (P < 0.001). GLP-2 increased the mean metaphase arrests/crypt in both the jejunum and ileum (P < 0.001). Ileal expression of sucrase-isomaltase was increased by 1. 6-fold (P < 0.05). Jejunal expression was increased by a similar amount, although not significantly (P = 0.08). There was no change in Cdx-2 gene expression. Thus GLP-2 can maintain small intestinal morphology and function, but effects on gene expression are not mediated by gross changes in the level of the mRNA for the homeobox protein Cdx-2.

Factors involved in the duodenal expression of the human calbindin-D9k gene.

Calbindin-D9k is expressed in the cytoplasm of intestinal cells, where it is critical for dietary calcium absorption. Two striking aspects of the expression of this gene are its vitamin-D dependency and regional differences in expression, with high levels only in duodenum. We report studies of the human calbindin-D9k promoter. Differences between the reported sequences of the human calbindin-D9k promoter were first clarified before undertaking a functional analysis of this sequence. Studies of the rat gene have indicated that several transcription factors, including the caudal-related homeobox factor (CDX-2), hepatic nuclear factor-4 and CCAAT-enhancer-binding protein alpha (C/EBPalpha), could interact with elements in the promoter. Although these elements are conserved in the human gene, we show here that their intestinal distribution makes them unlikely to be critical positive factors. The calbindin-D9k gene contains multiple potential binding sites for homeobox transcription factors; one of these, known as IPF-1 or PDX-1, co-localizes in the intestine with calbindin-D9k. We show in gel-shift assays that the sequence within a putative vitamin-D-response element in the human calbindin-D9k promoter can bind expressed IPF-1/PDX-1 protein, although we cannot confirm binding of the vitamin-D-receptor protein. CDX-2 binds to the region around the TATA box, as in the rat gene, and may act as a negative factor in the distal intestine. Transfection studies in Caco-2 and MCF-7 cells with heterologous reporter vectors containing up to 1303 bp of the gene showed that this functioned as a weak promoter and indicated the presence of suppressor sequences, but did not show vitamin-D responsiveness. This indicates that other elements are also needed for the control of human calbindin-D9k expression.

Expression of genes involved in calcium absorption in human duodenum.

BACKGROUND: The role of vitamin D metabolites in the regulation of expression of genes involved in dietary calcium absorption by the human intestine remains poorly understood despite much work in animals. MATERIALS AND METHODS: To investigate this, we measured the expression of transcripts for two of these genes, calbindin-D9k and the basolateral membrane calcium pumping ATPase, PMCA1, in duodenal endoscopic biopsies from 40 subjects. Northern blots were hybridized with previously characterized probes, and the signal was quantified by phosphor imaging. These transcript levels were related to circulating vitamin D metabolites and also analysed according to the vitamin D receptor gene TaqI polymorphism determined in DNA from blood. RESULTS: Plasma 1,25-dihydroxycholecalciferol correlated significantly with calbindin-D9k RNA (r = 0.4, P < 0.02) but not with PMCA1. Plasma 25-OH-cholecalciferol was not correlated with transcripts for either gene and, furthermore, the mean levels of these transcripts did not differ significantly when grouped by vitamin D receptor genotype. CONCLUSION: In normal humans, 1, 25-dihydroxycholecalciferol has a small but significant relationship to duodenal expression of calbindin-D9k, but not to PMCA1 expression.

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.

Differences in expression of homeobox transcription factors in proximal and distal human small intestine.

BACKGROUND & AIMS: Different digestive enzymes and transporters are present in the duodenum, jejunum, and ileum, but the factors determining region-specific gene expression are not yet understood. Homeobox transcription factors are important in defining gradients of cellular differentiation. The aim of this study was to investigate whether their expression differs between proximal (duodenal) and distal (ileal) regions of human small intestine. METHODS: Intestinal RNA was prepared from surgical patients, and reverse-transcription polymerase chain reactions (PCRs) performed with mixed sequence oligonucleotide primers based on conserved regions. PCR products were identified by cloning and sequencing. Transcript abundance was determined by Northern blotting. RESULTS: The human homologues were identified as Cdx-1, Cdx-2 (or Cdx-3), Pdx-1 (previously named Islet/duodenal homeobox [Idx]-1, Ipf-1, or Stf-1), and 13 human homeodomain cluster genes, including HOXB3, HOXB4, and HOXA6. The relative abundance of some of these differed between duodenum and ileum. Pdx-1 transcripts were found only in duodenum, Cdx-2, Cdx-1, and HOXB3 were readily detectable in both regions, with Cdx-1 having a more marked distal expression. CONCLUSIONS: Many homeobox genes are expressed in human adult small intestinal mucosa, and some are found predominantly in one region.

The effect of gastrin-releasing peptide on gastrin and somatostatin messenger RNAs in humans infected with Helicobacter pylori.

BACKGROUND & AIMS: Gastrin-releasing peptide stimulates gastrin secretion but also inhibits its release via somatostatin. Exogenous gastrin-releasing peptide stimulates a greater increase in plasma gastrin concentrations in patients infected with Helicobacter pylori than in uninfected controls. Because this infection suppressed gastric mucosal somatostatin, we studied whether the increased gastrin response was a result of an abnormal response of the somatostatin cell. METHODS: Patients without dyspeptic ulcers received an infusion of either gastrin-releasing peptide or saline on separate occasions. Acid secretion was measured, and gastric biopsy specimens were taken for gastrin and somatostatin messenger RNA (mRNA) analysis and H. pylori diagnosis. RESULTS: In response to gastrin-releasing peptide, the increase in plasma gastrin concentrations in the infected patients was significantly higher than in the uninfected. Antral gastrin mRNA also increased significantly in the infected group but decreased significantly in the uninfected group. Basal somatostatin was lower in the infected group; gastrin-releasing peptide produced a significant increase in antral somatostatin mRNA concentration in infected, but not uninfected, patients. CONCLUSIONS: The somatostatin cell responds to gastrin-releasing peptide in H. pylori infection. Gastrin-releasing peptide normally inhibits gastrin mRNA expression, but inhibition is deficient in H. pylori infection, possibly because of low stimulated somatostatin levels.

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.

Cellular and regional expression of transcripts of the plasma membrane calcium pump PMCA1 in rabbit intestine.

The plasma membrane Ca(2+)-pumping adenosinetriphosphatase (PMCA) is the energy-dependent step in the active vitamin D-dependent absorption of dietary Ca2+ by the enterocyte. Studies of the various PMCA genes and splicing variants in humans and rats have indicated that the isoform known as PMCA1b is the predominant form expressed in small intestine. Using an oligonucleotide probe, we have studied the regional and cellular distribution of PMCA1 transcripts in rabbit intestinal tissues by in situ hybridization. On small intestinal RNA blots, this hybridized to species similar in size to those detected by PMCA1-specific cDNA probes; an additional larger transcript was present in rabbit than in rat or human. In situ hybridization signals were principally in the enterocyte population of the mucosa and were maximal in differentiating enterocytes on the lower part of the villus, a pattern similar to that previously demonstrated for other nutrient transporters. Reflecting the capacity of the different small intestinal segments to transport Ca2+, much higher levels of transcript were detected by both methods proximally (in duodenum) than distally (in jejunum and ileum) and were also higher in cecum and ascending colon mucosa than in descending colon. We conclude that as enterocytes differentiate in regions that absorb Ca2+, they express high levels of mRNA for PMCA1. These results confirm the importance of transcriptional regulation of this gene for active Ca2+ absorption.

Cytokine gene expression in Helicobacter pylori associated antral gastritis.

Infection of the gastric antrum by Helicobacter pylori is characterised by a cellular inflammatory infiltrate. Whether cytokines are involved in the pathogenesis of this gastritis has been investigated by studying the effect of eradicating H pylori on the expression of genes encoding the cytokines interleukin 8 (IL-8) and tumour necrosis factor alpha (TNF-alpha) in the antral mucosa. Gastric antral biopsy specimens were taken from nine patients with duodenal ulcers and cytokine transcripts were identified and quantified by northern blotting. After H pylori had been eradicated the chronic inflammatory infiltrate decreased in all the patients and the polymorphonuclear infiltrate virtually disappeared. Expression of genes also decreased. After eradication, the median TNF-alpha mRNA/rRNA fell to 48% (p = 0.02) and the median IL-8 mRNA/rRNA fell to 5% (p = 0.004) of initial values. These results support the role of increased synthesis of these cytokines in the pathogenesis of the gastritis.

Plasma-membrane calcium-pump isoforms in human and rat liver.

Plasma-membrane Ca(2+)-pumping ATPases (PMCAs) extrude Ca2+ from the cytoplasm of all cells. Some previous studies of ATP-dependent Ca2+ transport by liver membranes suggested there exist specific properties of the hepatic PMCA, including regulation by hormones which affect calcium signalling. Multiple PMCA isoforms are now known to result from expression of four different genes (known as PMCA 1-4) and alternative RNA splicing at three possible sites (A, B and C). We investigated which isoforms are expressed in adult human and rat liver RNA using reverse-transcription polymerase chain reaction with mixed primers designed to amplify parts of all the known PMCA transcripts. In human liver, products were identified by sequencing from PMCA1, PMCA2 and PMCA4, but not from PMCA3 or from any new gene. In rat liver, by contrast, only PMCA1 and PMCA2 were detectable, although we confirmed that the primers were able to amplify from rat lung a new sequence which is part of rat PMCA4. Of the alternatively spliced variants, at site A in the PMCA2 sequences, all the exons were included in both adult and fetal human liver. In human liver, the exon at site B was excluded in some products from PMCA1 and PMCA4, and at site C, only PMCA1b and one form of PMCA4 were found. Blots of human liver RNA showed PMCA1 and PMCA4 were abundantly expressed, unlike PMCA2. On blots of rat liver RNA, PMCA1 was more abundant than PMCA2, and purified rat parenchymal cell RNA gave similar findings. In summary, no new hepatic PMCA isoforms have been demonstrated, but differences between the predominant human and rat isoforms may have consequences for Ca2+ signalling or the response to liver cell injury.

Amylin/islet amyloid polypeptide expression in medullary carcinoma of the thyroid: correlation with the expression of the related calcitonin/CGRP genes.

OBJECTIVE: The amylin/islet amyloid polypeptide (IAPP) gene is the third member of the calcitonin (CT)/calcitonin gene related peptide (CGRP) gene family, which includes the alpha CT/CGRP (CALC-I) and beta CGRP (CALC-II) genes. CT is predominantly expressed in thyroid C cells, alpha and beta CGRP (CGRP-I and II) in neural tissue and amylin/IAPP in pancreatic beta cells. Both the detailed tissue distribution and the physiological role of amylin are subjects of current research. We sought to characterize the RNAs transcribed from the IAPP gene in normal human pancreas and to investigate possible ectopic expression of this gene in neuroendocrine tumours. DESIGN AND TISSUES: RNA was extracted from normal human pancreas, five phaeochromocytomas and 12 medullary thyroid carcinomas (MTCs) and studied by Northern blotting. RESULTS: We found that in normal human pancreas the IAPP gene transcripts differ in size from those reported for human insulinoma. Expression of the amylin/IAPP gene was detected in seven of the MTCs, while it was not detected in phaeochromocytomas. There were no apparent clinical or histological differences between IAPP positive and IAPP negative MTCs. The relative expression levels of the four mRNAs of the CT/CGRP gene family varied between the different tumours. CONCLUSIONS: Our findings are consistent with the view that ectopic hormone production may occasionally be causally related to the common origin of related genes. The possibility that IAPP may constitute a minor component of MTC amyloid should be considered.

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.

Reciprocal changes in antral gastrin and somatostatin mRNA in pernicious anemia.

Somatostatin is involved in the regulation of gastrin by intragastric pH in animal models. To investigate whether this is so in man, we measured gastrin and somatostatin mRNA in endoscopic biopsies from six patients with hypergastrinemia and achlorhydria due to pernicious anemia and 12 age- and sex-matched controls. The pernicious anemia patients had significantly higher fasting plasma gastrin concentrations with a median (range) of 640 (420-3500) pmol/liter compared with 5 (2-58) pmol/liter, P < 0.001. The median gastrin mRNA/rRNA ratio was 10.4 (3.7-38.0) in the pernicious anemia patients compared with 1.7 (0.7-8.3) in the controls (P < 0.02), and it correlated strongly with the plasma gastrin concentration, r = 0.93, P < 0.0001. In contrast, the median somatostatin mRNA/rRNA ratio was lower in the pernicious anemia patients 0.84 (0.58-2.32) versus 2.04 (0.05-6.47) in the controls, P < 0.05. These findings suggest that in pernicious anemia gastric neutralization leads to hypergastrinemia through the modulation of antral gastrin synthesis by somatostatin.

Human and rat intestinal plasma membrane calcium pump isoforms.

The intestinal basolateral membrane Ca(2+)-transporting adenosinetriphosphatase is the energy-dependent step in the absorption of dietary Ca2+ by the vitamin D-dependent transcellular pathway. Multiple plasma membrane Ca(2+)-pump isoforms are produced from four genes (PMCA1 to 4) and alternative mRNA splicing. We have studied which isoforms are detectable in adult human and rat gastrointestinal tissues by polymerase chain reaction (PCR) amplification, sequencing, and blotting. PMCA1 was the predominant gene product amplified from human small intestinal mucosa, although a minor additional variant lacking the exon at splice site B was detected, which resembled that described for PMCA4. Of the variants described at site C, only the shortest transcript of PMCA1 was amplified; both previously described forms of PMCA4 were found, particularly in colon where PMCA4 predominated. From rat intestinal cDNA, mixed primer PCR amplified PMCA1 and a novel sequence, the rat PMCA4 homologue, which was expressed in many tissues including small intestinal muscle and colon. However, PMCA1 was overwhelmingly predominant in the mucosa of the small intestine, being most abundant in duodenum. These results suggest the involvement of the Ca(2+)-pump isoform PMCA1b in intestinal Ca2+ absorption.

A new calcitonin-receptor-like sequence in rat pulmonary blood vessels.

1. Two rat clones have been isolated which are similar to known calcitonin-receptor sequences. One of these does not have the distribution expected of a calcitonin receptor. It is widely distributed, with extremely high levels of expression in the lung, where it is associated with the blood vessels. 2. This rat sequence may represent the receptor for calcitonin-gene-related peptide or islet amyloid polypeptide. Both have binding activity in the lung and are potent vasodilators. The gene represented by this sequence may therefore play an important role in the maintenance of vascular tone.