Effect of quercetin on the brain-derived neurotrophic factor gene expression in the rat brain.

INTRODUCTION: Quercetin is a ubiquitous flavonoid found in many plants. Neuroprotective effects of quercetin have been shown in several in vitro and in vivo studies, but its mechanism of action has not been fully defined yet. Brain-derived neurotrophic factor (BDNF) is a fundamental neurotrophin with vital functions in the survival of neuronal cells. In the present study, we aimed to investigate the effects of quercetin on expression of BDNF mRNA in the hippocampus of rat brain. METHODS: Male rats were daily gavaged with quercetin (10, 20 or 50 mg/kg·bwt) for 30 days. Hippocampal levels of the BDNF transcripts were assessed using quantitative (q) RT-PCR. RESULTS: Quercetin at doses of 20 and 50 mg/kg caused a significant increase in the mRNA expression of BDNF as compared with the control group. Quercetin treatment at a dose of 10 mg/kg failed to cause any significant changes in the levels of BDNF mRNA CONCLUSION: Our findings suggest that the neuroprotective effects of quercetin may be at least partly due to its inducing effects on the expression levels of the BDNF mRNA (Fig. 1, Ref. 40).

Quercetin potentiates transdifferentiation of bone marrow mesenchymal stem cells into the beta cells in vitro.

PURPOSE: Type 1 diabetes is an autoimmune disease caused by the destruction of ß-cells in the pancreas. Bone marrow mesenchymal stem cells are multipotent and easy accessible adult stem cells that may provide options in the treatment of type 1 diabetes. Injured pancreatic extract can promote the differentiation of rat bone marrow mesenchymal stem cells into ß-cells. We aimed to observe the effect of quercetin in differentiation and insulin secretion in ß-cells. METHODS: Bone marrow mesenchymal stem cells were obtained from the tibiae of rats. Cell surface markers were analyzed by flow cytometry. The cells were treated with rat injured pancreatic extract and quercetin for 2 weeks. Insulin secretion was measured by ELISA. Insulin expression and some islet factors were evaluated by RT-PCR. PDX1, a marker for ß-cell function and differentiation, was evaluated by both immunocytochemistry and Western blot. ß-cell count was determined by stereology and cell count assay. RESULTS: ELISA showed significant differences in insulin secretion in the cells treated with RIPE + 20 µM quercetin (0.55 ± 0.01 µg/L) compared with the cells treated with RIPE alone (0.48 ± 0.01 µg/L) (P = 0.026). RT-PCR results confirmed insulin expression in both groups. PDX1 protein was detected in both groups by Western blot and immunocytochemistry. Stereology results showed a significant increase in ß-cell number in the RIPE + quercetin-treated cells (47 ± 2.0) when compared with RIPE treatment alone (44 ± 2.5) (P = 0.015). CONCLUSIONS: Quercetin has a strengthening effect on the differentiation of rat bone marrow mesenchymal stem cells into ß-cells and increases insulin secretion from the differentiated ß-cells in vitro.

SKF 38393 and SCH 23390 inhibit reuptake of serotonin by rat hypothalamic synaptosomes.

BACKGROUND/AIMS: Both the dopamine receptor D(1) agonist SKF 38393 and the antagonist SCH 23390 are benzazepine derivatives that have been widely used as pharmacological tools and radioligands. Evidence suggests that behavioral effects of both compounds do not always correspond to their established receptor subtype selectivity. Here, we assessed the effects of SKF 38393 and SCH 23390 on the synaptosomal uptake of tritiated serotonin. METHODS: Uptake experiments were performed by using [(3)H]serotonin and synaptosomal fractions prepared from the hypothalamus of rat brain. RESULTS: Both SKF 38393 and SCH 23390 inhibited synaptosomal uptake of [(3)H]serotonin, with IC(50) values of 910 ± 60 nmol/l and 1,400 ± 80 nmol/l, respectively. Clomipramine, a known inhibitor of serotonin uptake, and (+)-amphetamine, a weak inhibitor, had IC(50) values of 14 ± 1 nmol/l and more than 10,000 nmol/l, respectively, under the same experimental conditions. The IC(50) values for SKF 38393 and SCH 23390 fall within the broad range of corresponding values for antidepressants that have been shown to inhibit the uptake of serotonin. This finding indicates that SKF 38393 and SCH 23390 can enhance the activity of the serotonergic system in the brain, a mechanism that may be responsible for some of the effects of these drugs. CONCLUSION: SKF 38393 and SCH 23390 are useful tools to differentiate D(1) from D(2) receptors, but their indirect effects on serotonergic mechanisms have to be considered.

Glucagon-like peptide-1 is a physiological incretin in rat.

Glucagon-like peptide-1 7-36 amide (GLP-1) has been postulated to be the primary hormonal mediator of the entero-insular axis but evidence has been indirect. The discovery of exendin (9-39), a GLP-1 receptor antagonist, allowed this to be further investigated. The IC50 for GLP-1 receptor binding, using RIN 5AH beta-cell membranes, was found to be 0.36 nmol/l for GLP-1 and 3.44 nmol/l for exendin (9-39). There was no competition by exendin (9-39) at binding sites for glucagon or related peptides. In the anaesthetized fasted rat, insulin release after four doses of GLP-1 (0.1, 0.2, 0.3, and 0.4 nmol/kg) was tested by a 2-min intravenous infusion. Exendin (9-39) (1.5, 3.0, and 4.5 nmol/kg) was administered with GLP-1 0.3 nmol/kg, or saline, and only the highest dose fully inhibited insulin release. Exendin (9-39) at 4.5 nmol/kg had no effect on glucose, arginine, vasoactive intestinal peptide or glucose-dependent insulinotropic peptide stimulated insulin secretion. Postprandial insulin release was studied in conditioned conscious rats after a standard meal. Exendin (9-39) (0.5 nmol/kg) considerably reduced postprandial insulin concentrations, for example by 48% at 15 min (431 +/- 21 pmol/l saline, 224 +/- 32 pmol/l exendin, P < 0.001). Thus, GLP-1 appears to play a major role in the entero-insular axis.

The effect of intra-cerebroventricular injection of insulin on nociception of formalin test in non-diabetic and short-term diabetic rat models.

Pain is a complex process in the central nervous system (CNS). Several factors can alter the pain threshold and insulin is one of them which is produced by the beta cells of pancreas and capable of crossing blood-brain barrier. The aim of this study was to evaluate the effects of intra-cerebroventricular (ICV) injection of insulin on the pain response to formalin in short-term induced diabetic and non-diabetic rats. Sixty-four Sprague-Dawley male rats (280 ± 30 g) were divided into non-diabetic and diabetic groups. Diabetes was induced with streptozotocin (STZ, 60 mg/kg, i.p) for elimination of peripheral insulin. After proving diabetes, insulin (5 mU/animal, 5 µL) was injected to the left lateral cerebral ventricle while equal volume of normal saline was injected in control groups. After 10 min, formalin test was performed. Present study showed that ICV injection of insulin possessed anti-nociceptive effect in non-diabetic rats in formalin test while in diabetic rats, it did not have this effect and even decreased pain threshold partially. In conclusion we showed that ICV injection of insulin in non-diabetic rats, in contrast with diabetic rats, has an anti-nociceptive effect in formalin test. In short-term diabetic rats, ICV injection of insulin was not able to reduce pain response and partially decreased pain threshold.

An abundant and specific binding site for the novel vasodilator adrenomedullin in the rat.

Rat adrenomedullin is a novel 50-amino acid peptide with structural similarities to the calcitonin family of peptides, calcitonin, calcitonin gene-related peptide (CGRP), and islet amyloid polypeptide (IAPP). Using rat [125I]adrenomedullin, specific binding sites were demonstrated in heart, lung, spleen, liver, soleus, diaphragm, gastrocnemius, and spinal cord membranes. The highest binding was present in heart and lung, which was further characterized. These sites exhibited saturation, dissociation, and competition. In rat lung, only rat (IC50 = 5.8 nM) and human (IC50 = 94 nM) adrenomedullin competed with [125I]adrenomedullin. However, in rat heart, rat (IC50 = 0.2 nM) and human (IC50 = 4.2 nM) adrenomedullin, IAPP (IC50 = 240 nM), and CGRP (IC50 = 1050 nM) all competed with [125I] adrenomedullin. Saturation analysis revealed binding capacities and dissociation constants of 2.8 +/- 0.3 pmol/mg protein and 1.3 +/- 0.3 nM, respectively, in lung and 0.47 +/- 0.11 pmol/mg protein and 0.41 +/- 0.14 nM in heart. Comparison with [125I]CGRP- and [125I]IAPP-binding sites in lung showed that rat adrenomedullin could potently inhibit at these sites (IC50 = 5 and 6 nM, respectively). Chemical cross-linking demonstrated a major band of 83,000 mol wt in lung, diaphragm, spleen, and liver and a band of 94,000 mol wt in heart, soleus, and gastrocnemius. Thus, [125I]adrenomedullin-binding sites in rat lung are abundant and can be differentiated from binding sites in rat heart, both pharmacologically and by mol wt.

Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung.

1. Angiotensin II (AII) binding density and the effect of chronic AII receptor blockade were examined in the rat model of hypoxia-induced pulmonary hypertension. 2. [125I]-[Sar1,Ile2]AII binding capacity was increased in lung membranes from rats exposed to hypoxia (10% fractional inspired O2) for 7 days compared to normal rats (Bmax 108 +/- 12 vs 77 +/- 3 fmol mg-1 protein; P < 0.05), with no significant change in dissociation constant. Competition with specific AII receptor subtype antagonists demonstrated that AT1 is the predominant subtype in both normal and hypoxic lung. 3. Rats treated intravenously with the AT1 antagonist, GR138950C, 1 mg kg-1 day-1 rather than saline alone during 7 days of exposure to hypoxia developed less pulmonary hypertension (pulmonary arterial pressure: 21.3 +/- 1.7 vs 28.3 +/- 1.1 mmHg; P < 0.05), right ventricular hypertrophy (right/left ventricle weight ratio: 0.35 +/- 0.01 vs 0.45 +/- 0.01; P < 0.05) and pulmonary artery remodelling (abundance of thick-walled pulmonary vessels: 9.6 +/- 1.4% vs 20.1 +/- 0.9%; P < 0.05). 4. The reduction in cardiac hypertrophy and pulmonary remodelling with the AT1 antagonist was greater than that achieved by a dose of sodium nitroprusside (SNP) that produced a comparable attenuation of the rise in pulmonary arterial pressure during hypoxia. 5. The data suggest that AII, via the AT1 receptor, has a role in the early pathogenesis of hypoxia-induced pulmonary hypertension in the rat.

Specific adrenomedullin binding sites and hypotension in the rat systemic vascular bed.

The potent vasodilator peptide, adrenomedullin, has been shown to be present in plasma, suggesting a physiological role in cardiovascular control. Here we investigated the hypotensive action of adrenomedullin in vivo, using the anaesthetised rat as the bioassay model, and adrenomedullin binding sites using ligand binding assays on rat blood vessel membranes. Rat alpha CGRP and both human and rat adrenomedullins induced dose-dependent, powerful and long-lasting hypotensive effects. At peptide doses used in this study (0.02-2 nmol/kg), the efficacy of both human and rat adrenomedullins was lower than that of rat alpha CGRP. The CGRP1-receptor antagonist, human CGRP(8-37) (200 nmol/kg) was able to completely inhibit the hypotensive effect of rat alpha CGRP (0.2 nmol/kg) but not that of rat adrenomedullin (2 nmol/kg), implying that the adrenomedullin action is independent of CGRP1-receptors. Ligand binding assays confirmed the presence of both CGRP and adrenomedullin binding sites in rat blood vessels. The 125I-rat adrenomedullin binding site has a Kd = 0.32 +/- 0.12 nM (n = 4) for rat adrenomedullin but has a Ki > 10(-6) M for rat alpha CGRP. Chemical cross-linking and SDS-PAGE analysis revealed theadrenomedullin binding protein to have a M(r) of 83000 with a minor band of M(r) = 99000. The results suggest that the hypotensive effect of adrenomedullin may be mediated via specific adrenomedullin binding sites, in vivo.

Expression of spinal cord Fos protein in response to intrathecal adrenomedullin and CGRP in conscious rats.

Adrenomedullin (AM) immunoreactivity and mRNA, in addition to a large number of specific AM-binding sites, exist in the rat spinal cord. However, no phenotype has been reported for AM in the spinal cord. Here, expression of c-fos in response to intrathecal (i.t.) administration of AM, proadrenomedullin N-terminal 20 peptide (PAMP) and calcitonin gene-related peptide (CGRP) was examined in the thoracic, lumbar and sacral regions of spinal cord in conscious rats. Two hours after i.t. administration of either CGRP (2.5 and 10 microg) or AM (10 microg), the number of c-Fos immunoreactive nuclei was increased in all the spinal regions examined in this study, with the highest increase observed in the superficial dorsal horn. Few cells with c-fos immunoreactivity were found in the spinal cord of rats 2 h after i.t. injection of either saline or PAMP. Effects of AM (10 microg) and CGRP (2.5 microg) on c-fos expression were blocked when rats were pretreated with 40 microg of intrathecal CGRP8-37 (CGRP1 receptor antagonist). Fos-like immunoreactivity induced by i.t. CGRP and/or AM were also significantly abolished by i.t. administration of the nitric oxide (NO) inhibitor, l-NAME, indicating that endogenous NO is a necessary intermediary in CGRP and AM induced c-fos expression in the rat spinal cord. In conclusion, AM induces c-fos expression in rat spinal cord when administered intrathecally, with the pattern being similar to those produced by i.t. CGRP. Effects of the two peptides are sensitive to CGRP8-37 and l-NAME.

Effects of intracerebroventricular injection of glucagon like peptide-1 and its related peptides on serotonin metabolism and on levels of amino acids in the rat hypothalamus.

High concentrations of glucagon-like peptide-1 (7-36) amide (GLP-1) and its specific receptor (GLP-1R) have been found in the rat hypothalamus. In this study the actions of GLP-1 and its related peptides, exendin-4 (GLP-1R agonist), exendin (9-39) (GLP-1R antagonist) and GLP-1 (9-36) amide (the major GLP-1 metabolite) on levels of serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA) and amino acids (Glu, Asp, Gln, Gly, Tyr, Trp, GABA) in the hypothalamus were investigated. Intracerebroventricular (ICV) injection of GLP-1 (4 nmol) produced a significant reduction in levels of 5-HT (54%) and all measured amino acids (34 to 56%) compared with saline injected controls, whereas exendin (9-39) (4 nmol) was ineffective. ICV injection of exendin-4 produced a significant reduction in the levels of 5-HT, 5-HIAA, Trp, Glu, and Tyr. ICV injection of GLP-1(9-36) amide showed a statistically significant increase in the level of 5-HT, 5-HIAA and all the amino acids tested in this study. Prior administration of exendin (9-39) or GLP-1 (9-36) amide blocked the effects of GLP-1 on the levels of 5-HT and the amino acids. These data are consistent with exendin-4 being a GLP-1R agonist and exendin (9-39) being a specific GLP-1R antagonist. GLP-1 (9-36) amide, a primary metabolite of GLP-1, appears to act as an endogenous antagonist at the GLP-1R.

Spot urine 5-hydroxy indole acetic acid and acute appendicitis.

BACKGROUND/AIMS: Appendectomy for suspected appendicitis cases is a common procedure. Its clinical diagnosis needs to be supported by accurate confirmatory tests. No single paraclinical test with a high degree of sensitivity and specificity is available for its diagnosis. The appendix contains numerous serotonin-producing cells (enterochromaffin cells). In the inflammatory process and subsequent cell injury, serotonin is released and converted to 5-HIAA (5-hydroxy indole acetic acid). We studied the elevation of 5-HIAA in the spot urine of acute appendicitis patients. METHODOLOGY: 5-HIAA was measured by high-performance liquid chromatography in the spot urine samples of 40 healthy individuals and 166 patients who presented to emergency units of the university hospitals with acute abdominal pain. The results of the urine concentrations were compared to the histopathology reports of the removed appendices and the final diagnosis of other diseases. RESULTS: From 80 cases with a presumptive diagnosis of appendicitis, 73 were operated on and seven cases discharged after a few hours observation. Sixty-five out of 66 documented appendicitis patients showed a striking increase of urinary spot 5-HIAA with significant differences vs. all cases of healthy control individuals (P < 0.001). The 5-HIAA values of all of the negative appendectomy cases (n = 7) and all of the discharged cases after the observation period (n = 7) were within healthy control ranges. The mean value of the appendicitis group (42.76 +/- 2.26 mumol/L) was also significantly higher vs. all other acute abdomens which could mimic acute appendicitis (P < 0.05) excepting gastroenteritis patients. Considering 20 mumol/L as the cutoff value sensitivity, specificity, positive and negative predictive values of this test for discriminating appendicitis in clinically suspected patients were 98%, 100%, 100% and 93%, respectively and in all acute abdomens were 98%, 71%, 69% and 98.6%, respectively. The patients with gastroenteritis also showed elevation of 5-HIAA (43.05 +/- 2.7 mumol/L) vs. other nonappendicitis groups (P < 0.05). CONCLUSIONS: We have concluded that measurement of 5-HIAA in spot urine is a highly reliable test supporting the clinical diagnosis of appendicitis and if it does not show an increase, appendicitis can be ruled out with a very high degree of confidence which helps to reduce unnecessary appendectomies. In clinically suspected appendicitis patients with diarrhea, an increase of 5-HIAA may not confirm the diagnosis.

Evaluation of dopamine receptor involvement in rat feeding behaviour.

1. The anorectic effect of dopamine agonists and antagonists were studied in rats. 2. Dopamine agonists bromocriptine, quinpirole or SKF 38393 treatment induced, a dose-dependent anorexia in rats. 3. Anorectic effect of bromocriptine was decreased in animals pretreated with pimozide (D-2 antagonist), but not by sulpiride (D-2 antagonist) or SCH 23390 (D-1 antagonist) pretreatment. 4. Anorexia induced by quinpirole was decreased by sulpiride or pimozide, but not by SCH 23390 administration. 5. While sulpiride and SCH 23390 failed to antagonize the anorectic response of SKF 38393, methergoline (5-HT antagonist) decreased anorexia induced by the drug. 6. A combination of quinpirole with SKF 38393 did not elicit potentiated anorectic response. 7. Decrease in food intake induced by bromocriptine, quinpirole or SKF 38393 was potentiated in reserpinized animals, although single administration of reserpine also induced a marked decrease in feeding. 8. Single administration of sulpiride, pimozide or methergoline did not change the feeding behaviour of rats, but SCH 23390 induced anorexia. 9. It is concluded that D-2 activation may induce inhibition of feeding and anorexia induced by SKF 38393 may be mediated through serotonergic mechanism(s).

A rat skeletal muscle cell line (L6) expresses specific adrenomedullin binding sites but activates adenylate cyclase via calcitonin gene-related peptide receptors.

We have previously demonstrated specific binding sites for adrenomedullin, a novel member of the calcitonin family of peptides, in rat muscles. It is unclear whether these receptors are vascular or muscular. Receptors for the structurally similar calcitonin gene-related peptide (CGRP) are present on myocytes and might be involved in the regulation of myocyte glucose metabolism and control by motor neurons. We investigated whether adrenomedullin binding sites were present on L6 myocytes. Specific [125I]adrenomedullin binding sites were demonstrated where adrenomedullin competed with an IC50 of 0.22 +/- 0.04 nM (mean +/- S.E.M.) and a concentration of binding sites (Bmax) of 0.95 +/- 0.19 pmol/mg of protein (mean +/- S.E.M.). CGRP and the specific CGRP receptor antagonist CGRP(8-37) competed weakly at this site (IC50 > 10 and 601 +/- 298 nM respectively). Binding studies with [125I]CGRP revealed a binding site for CGRP (IC50 = 0.13 +/- 0.01 nM; Bmax = 0.83 +/- 0.10 pmol/mg of protein) where both CGRP(8-37) and adrenomedullin competed with [125I]CGRP with IC50 values of 1.15 +/- 0.12 and 8.68 +/- 0.98 nM respectively. Chemical cross-linking showed the CGRP and adrenomedullin binding site-ligand complexes to have approximate molecular masses of 82 and 76 kDa respectively. Both CGRP and adrenomedullin increased adenylate cyclase activity with similar potencies. In both cases adenylate cyclase activation was blocked by CGRP(8-37). Stimulation with 10 nM adrenomedullin or CGRP caused an increase in the percentage of total activated cellular cAMP-dependent protein kinase from 38% in resting cells to 100% and 98% respectively. Therefore in L6 cells adrenomedullin can bind to CGRP receptors, activating adenylate cyclase and cAMP-dependent protein kinase.

Binding sites for islet amyloid polypeptide in mammalian lung: species variation and effects on adenylyl cyclase.

Islet amyloid polypeptide (IAPP) and calcitonin gene related peptide (CGRP) share a 47% sequence homology. IAPP can interact with adenylyl cyclase coupled CGRP receptors. We have examined [125I]IAPP binding in mouse, pig, and guinea pig lung membranes in competition with IAPP, CGRP, and CGRP(8-37). Three types of site were shown by order of potency: (i) mouse, IAPP > CGRP(8-37) >> CGRP; (ii) pig, CGRP > IAPP > CGRP(8-37); and (iii) guinea pig, CGRP = IAPP = CGRP(8-37). Chemical cross-linking of [125I]IAPP and [125I]CGRP binding sites in lung demonstrated that both sites had similar molecular weights in any one species but differed across species, i.e., mouse M(r) = 70,000 and 98,000; pig M(r) = 68,000, 56,000, and 47,000; and guinea pig M(r) = 106,000 and 56,000. Adenylyl cyclase activity was stimulated by forskolin and AlCl3-NaF in rat, mouse, pig, and guinea pig membranes. Only in mouse and pig were CGRP and IAPP able to stimulate adenylyl cyclase activity. In mouse lung CGRP and IAPP stimulated adenylyl cyclase activity with EC50 values of 642 +/- 222 nM (n = 4) and 325 +/- 115 nM (n = 4), respectively. In pig lung membranes EC50 values were 5.7 +/- nM (n = 4) for CGRP and 1230 +/- 1130 nM (n = 4) for IAPP. Thus IAPP either did not stimulate adenylyl cyclase activity in these lung membranes or did so with a low potency.

Adrenomedullin activity in chronically hypoxic rat lungs.

Adrenomedullin (AM) is a novel vasodilator with structural similarities to calcitonin gene-related peptide (CGRP). This study investigated AM activity in the rat lung during hypoxia-induced pulmonary hypertension. Both rat AM (0.2-10 nmol) and alpha-CGRP (0.2-2 nmol) produced dose-related reductions in pulmonary artery pressure in the isolated perfused lung ventilated with 2% O2. Pretreatment with alpha-CGRP, which demonstrated tachyphylaxis, or its antagonist, CGRP-(8-37), reduced the hypotensive response to AM, suggesting that part of the response to AM is mediated by CGRP receptors. 125I-labeled AM and 125I-labeled CGRP binding was significantly increased in lung membranes from 7-day hypoxic animals (AM from 1.94 +/- 0.3 to 3.36 +/- 0.4 and CGRP from 0.06 +/- 0.01 to 0.12 +/- 0.02 pmol/mg protein), with no change in dissociation constant. Moreover, the hypotensive response to both peptides was increased in the lungs of 7-day hypoxic rats. There was no significant change in lung immunoreactive AM concentrations (hypoxic 5.04 +/- 0.48 vs. control 6.28 +/- 0.76 pmol/g wet wt of tissue) or steady-state AM mRNA levels in 7-day hypoxic rats. Nonetheless, AM may be useful for the acute pharmacological manipulation of pulmonary artery pressure in hypoxia-induced pulmonary hypertension.

Characterisation and molecular identification of adrenomedullin binding sites in the rat spinal cord: a comparison with calcitonin gene-related peptide receptors.

Calcitonin gene-related peptide (CGRP) and its receptors are found in mammalian spinal cord. We show, for the first time, binding sites for the novel related peptide adrenomedullin in rat spinal cord microsomes. 125I-Adrenomedullin binding showed high affinity (KD = 0.45 +/- 0.06 nM) and sites were abundant (Bmax = 723 +/- 71 fmol/mg of protein). CGRP, amylin, and calcitonin did not compete at these sites (Ki > 10 microM). High-affinity CGRP binding sites (KD = 0.18 +/- 0.01 nM) were much less numerous (Bmax = 17.7 +/- 2.4 fmol/mg of protein) and showed competition by unlabeled adrenomedullin (Ki = 34.6 +/- 2.4 nM). Chemical cross-linking revealed a major band for 125I-adrenomedullin of M(r) = 84,400 +/- 1,200 and a minor band of M(r) = 122,000 +/- 8,700. 125I-CGRP cross-linking showed bands of lower molecular weight (M(r) = 74,500 +/- 5,000 and 61,000 +/- 2,200). Enzymic deglycosylation of the adrenomedullin binding site showed a considerable carbohydrate content. Neither adrenomedullin nor CGRP was able to increase cyclic AMP in spinal cord. Adrenomedullin mRNA was present in spinal cord, at one-third of its level in lung, and adrenomedullin immunoreactivity was present, at a low concentration (40 fmol/g of tissue). Thus, the presence of abundant binding sites and adrenomedullin mRNA and immunoreactivity anticipate an as yet undefined function for this peptide in spinal cord.

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.

Rat-2 fibroblasts express specific adrenomedullin receptors, but not calcitonin-gene-related-peptide receptors, which mediate increased intracellular cAMP and inhibit mitogen-activated protein kinase activity.

Rat-2 fibroblasts demonstrate specific binding of 125I-labelled rat adrenomedullin (KD=0.43 nM; Bmax=50 fmol/mg of protein) in the absence of 125I-labelled calcitonin-gene-related peptide (CGRP) binding. Therefore Rat-2 cells were used to examine the pharmacology and signal transduction pathways of adrenomedullin receptors. We examined the effects of adrenomedullin, the CGRP receptor antagonist CGRP-(8-37) and the amylin antagonists AC187 and AC253 on receptor binding and cAMP production. AC253, AC187 and CGRP-(8-37) inhibited 125I-adrenomedullin binding, with respective IC50 values of 25+/-8, 129+/-39 and 214+/-56 nM. Adrenomedullin dose-dependently increased intracellular cAMP (approximate EC50=1.0 nM). CGRP-(8-37), AC253 and AC187 antagonized adrenomedullin-stimulated cAMP production at micromolar concentrations. Using kinase-substrate assays, Mono Q FPLC and 'phospho-specific' Western blotting, we found that adrenomedullin alone abolished basal mitogen-activated protein kinase (MAPK) activity and dose-dependently inhibited platelet-derived-growth-factor-stimulated MAPK activity. Radioimmunoassay for adrenomedullin of media from Rat-2 cells showed a linear release of adrenomedullin-like immunoreactivity of 3.1 fmol/h per 2x10(6) cells. Gel-filtration chromatography showed that this adrenomedullin-like immunoreactivity co-eluted with synthetic rat adrenomedullin. Northern blotting with a rat adrenomedullin cDNA probe was used to confirm the presence of adrenomedullin mRNA. However, neither Northern blotting nor reverse transcriptase-PCR showed the presence of the cloned adrenomedullin receptor (L1). We conclude that the Rat-2 cell line expresses a specific adrenomedullin receptor (coupled to cAMP production and regulation of MAPK) and secretes adrenomedullin, which may participate in a regulatory control loop.