Biomedical subjects

J A Fischer

Publications and source records attributed to J A Fischer.

At least 37 records · Page 2Linked to original sources

PubMed1999

Molecular analysis of the klarsicht gene and its role in nuclear migration within differentiating cells of the Drosophila eye.

BACKGROUND: The temporally regulated, cell-type-specific transport of organelles has great biological significance, yet little is known about the regulation of organelle transport during development. The Drosophila gene klarsicht is required for temporally regulated lipid droplet transport in developing embryos and for the stereotypical nuclear migrations in differentiating cells of the developing eye. Klarsicht is thought to coordinate the function of several molecular motors bound to a single lipid droplet or to facilitate the attachment of dynein to the cargo, but it is not known whether Klarsicht affects motors directly or indirectly. RESULTS: Here, we have cloned the klarsicht gene and shown that it encodes a unique large protein. Drosophila klarsicht null mutants were viable, with obvious defects only in adult eye morphology. Epitope-tagged Klarsicht expressed in the eye from a transgene was perinuclear. In flies carrying transgenes that express markers for microtubule plus and minus ends, microtubules in differentiating cells of the eye were oriented with their plus ends apical and their minus ends at the nucleus. CONCLUSIONS: Drosophila klarsicht null mutants were viable and fertile, demonstrating that klarsicht is essential only for specific motor protein functions. Perinuclear localization of Klarsicht protein indicates that Klarsicht has a direct mechanical role in nuclear migration. Taken together with the finding that the minus ends of the microtubules are associated with the photoreceptor nuclei, the observation that Klarsicht is largely perinuclear supports the idea that Klarsicht associates with dynein, consistent with a model in which Klarsicht assists dynein in 'reeling in' the nucleus.

Animals↗

PubMed1999

Genetic analysis of the role of the drosophila fat facets gene in the ubiquitin pathway.

The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit the number of photoreceptors in each facet to eight. Ubiquitin is a small polypeptide that targets proteins for degradation by the proteasome. Deubiquitinating enzymes cleave ubiquitin-protein bonds. In order to investigate the role of FAT FACETS in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were assessed. In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets in the developing Drosophila eye and for their effects on eye morphology. The results of these experiments support the hypothesis that FAT FACETS activity antagonizes that of the proteolytic machinery. The implications of these results for the specificity of FAF and yeast UBPs are discussed as well.

Animals↗

PubMed1999

Maxadilan interacts with receptors for pituitary adenylyl cyclase activating peptide in human SH-SY5Y and SK-N-MC neuroblastoma cells.

Receptors for pituitary adenylyl cyclase activating peptide (PACAP) have been identified in human SH-SY5Y neuroblastoma cells with PACAP being 1000-fold more potent than vasoactive intestinal peptide (VIP) in [(125)I]PACAP binding inhibition and stimulation of cAMP accumulation. Maxadilan, a vasodilator peptide from the salivary gland of the sand fly Lutzomyia longipalpis also specifically bound to SH-SY5Y cells, and was equipotent to PACAP in [(125)I]PACAP and [(125)I]maxadilan binding inhibition, and stimulation of cAMP accumulation. Maxadilan and PACAP also increased the cytosolic free calcium concentration. In human SK-N-MC neuroblastoma cells PACAP, VIP and maxadilan equipotently stimulated cAMP accumulation. The maximal effects of VIP and maxadilan were additive and reached those of PACAP alone. In human T47D breast carcinoma cells PACAP and VIP were also equipotent in the stimulation of cAMP accumulation, but maxadilan was inactive. The results are consistent with the interaction of maxadilan with PACAP specific PAC(1)receptors in SH-SY5Y cells, but not with VPAC receptors, not differentiating between VIP and PACAP in T47D cells. Moreover, maxadilan is a PAC(1)receptor specific agonist which allows discrimination of co-expressed PAC(1)and VPAC receptors in SK-N-MC cells.

Binding, Competitive↗

PubMed1999

A receptor activity modifying protein (RAMP)2-dependent adrenomedullin receptor is a calcitonin gene-related peptide receptor when coexpressed with human RAMP1.

Adrenomedullin (ADM) and alpha- and beta-calcitonin (CT) gene-related peptide (alpha-, betaCGRP) are structurally related vasodilatory peptides with homology to CT and amylin. An originally orphan human CT receptor-like receptor (hCRLR) is a Gs protein-coupled CGRP or ADM receptor when coexpressed with recently identified human single transmembrane domain receptor activity modifying proteins 1 (hRAMP1) or -2 (hRAMP2), respectively. Here, the function of the rat CRLR homologue (rCRLR) has been investigated in rat osteoblast-like UMR-106 cells and in COS-7 cells, in the absence and presence of hRAMP1 and -2 and combinations thereof. Transient expression of rCRLR in UMR-106 cells revealed an ADM receptor, and [125I]rat (r) ADM binding was enhanced with hRAMP2 and inhibited by 50% when hRAMP1 was coexpressed. Detectable [125I]h alphaCGRP binding required the presence of hRAMP1, and the expression of CGRP binding sites was unaffected by coexpressed hRAMP2. Specificity of ADM binding sites in [125I]rADM binding inhibition experiments was reflected by an over 1000-fold higher potency of rADM [half-maximal effective concentration = 0.19 +/- 0.05 nM (mean +/- SEM, n = 4)], compared with r alphaCGRP and r betaGRP, to induce a cAMP-responsive luciferase reporting gene (CRE-luc). In rCRLR and hRAMP1 cotransfected cells, expressing predominantly CGRP binding sites, r betaCGRP, r alphaCGRP, and rADM induced CRE-luc with half-maximal effective concentration of 0.27 +/- 0.17 nM, 0.37 +/- 0.27 nM, and 1.4 +/- 0.9 nM, respectively. In COS-7 cells, the results were comparable, but rCRLR required coexpressed hRAMP2 for ADM receptor function. This is consistent with higher levels of endogenous RAMP2 encoding messenger RNA in UMR-106, compared with COS-7 cells. In conclusion, the recognition of RAMP1 and -2 as mediators of CRLR expression as a CGRP or ADM receptor has been extended, with evidence that endogenous RAMP2 is sufficient to reveal an ADM receptor in UMR-106 cells. Inhibition of RAMP2-evoked ADM receptor expression by RAMP1 and generation of a CGRP receptor is consistent with competitive interactions of the different RAMPs with rCRLR.

Animals↗

PubMed1999

An amylin receptor is revealed following co-transfection of a calcitonin receptor with receptor activity modifying proteins-1 or -3.

Human receptor activity modifying proteins (RAMP) regulate the ligand specificity of the calcitonin-receptor-like-receptor (McLatchie et al., Nature 393:333-339 (1998)). Here we have investigated binding of [125I]-labeled human (h) calcitonin ([125I]hCT) and rat amylin ([125I]amylin) to rabbit aortic endothelial cells (RAEC) co-transfected with the hCT receptor isotype 2 (hCTR2) and RAMP1, -2 or -3. Specific binding of 125 pM [125I]hCT to cells transfected with hCTR2 alone was 6.7 +/- 0.7 fmol/50,000 cells (n=5), and was reduced by 45 +/- 2% and 86 +/- 3% (P < 0.001) in the presence of RAMP1 and -3, but remained unchanged with RAMP2. In the absence and presence of individual RAMPs [125I]hCT binding inhibition occured with similar IC50 of between 6 nM and 11 nM hCT, and human amylin was 24- to 54-fold less potent. Specific binding of 125 pM [125I]amylin to cells transfected with hCTR2 alone was 0.9 +/- 0.2 fmol/50,000 cells (n=6), and was increased by 262 +/- 48% (P < 0.005), 73 +/- 26% (P < 0.05) and 338 +/- 57% (P < 0.005) with RAMP1, -2 or -3, respectively. [125I]amylin binding was inhibited with IC50 of 3.1 +/- 0.5 nM and 4.0 +/- 0.8 nM human amylin in cells co-transfected with RAMP1 or -3, respectively, and hCT was 45 +/- 2- and 126 +/- 3-fold less potent. In conclusion, RAMP1 and -3 decrease calcitonin receptor expression in RAEC transfected with hCTR2 encoding cDNA and simultanously reveal an amylin receptor.

Amyloid↗

PubMed1999

Islet amyloid polypeptide/amylin messenger RNA and protein expression in human insulinomas in relation to amyloid formation.

OBJECTIVE: Islet amyloid polypeptide (IAPP), also named amylin, is the predominant protein component of amyloid deposits in human islet beta cell tumours of the pancreas (insulinomas). IAPP is co-produced with insulin by islet beta cells. We investigated IAPP expression in relation to insulin expression and to amyloid formation in eleven insulinomas. DESIGN AND METHODS: RNA and protein extracts were prepared from the same pieces of tumour tissue, and from specimens of two normal human pancreata. IAPP and insulin mRNA and peptide content were quantified using Northern blot analysis and radioimmunoassay (RIA) respectively. Molecular forms of IAPP immunoreactivity were analysed by reversed-phase high-performance liquid chromatography (HPLC). The presence of islet hormones and of amyloid was assessed by (immuno)histochemical staining of paraffin sections. Plasma levels of IAPP and insulin prior to tumour resection were determined by RIA. RESULTS: IAPP and insulin mRNA and peptide content varied widely between the tumour specimens, and there was considerable intratumour heterogeneity of peptide content. HPLC analysis indicated correct proteolytic processing of the IAPP precursor protein. Amyloid deposits were detected only in the three tumours with the highest IAPP content. In contrast to insulin, plasma levels of IAPP were not elevated in the insulinoma patients. CONCLUSIONS: The spectrum of hormone production by insulinomas cannot be inferred from only a few tissue sections due to intratumour heterogeneity. Expression of the IAPP and insulin genes is not coupled in insulinomas, which produce properly processed mature IAPP. In addition to IAPP overproduction, additional factors such as intracellular accumulation of IAPP are involved in amyloidogenesis in insulinomas.

Adolescent↗

PubMed1998

Receptor activity modifying proteins regulate the activity of a calcitonin gene-related peptide receptor in rabbit aortic endothelial cells.

In Xenopus oocytes with an endogenous calcitonin gene-related peptide (CGRP) receptor, a receptor activity modifying protein (RAMP1) enhancing CGRP stimulated chloride currents of the cystic fibrosis transmembrane regulator was recently cloned [McLatchie, L.M. et al. (1998) Nature 393, 333-339]. Here, transient expression of RAMP1 in rabbit aortic endothelial cells (RAEC) brought about stimulation of cAMP accumulation by human (h) alphaCGRP with an EC50 of 0.41 nM. This was antagonized by a CGRP receptor antagonist alphaCGRP(8-37). Co-expression of RAMP3 together with RAMP1 reduced the maximal cAMP response to h alphaCGRP by 47% (P < 0.05). The cells also express RAMP2 encoding mRNA and an adrenomedullin (ADM) receptor coupled to stimulation of cAMP formation by hADM (EC50 0.18 nM). The latter was antagonized by an ADM receptor antagonist hADM(22-52). In conclusion, expression of a CGRP receptor in RAEC requires RAMP1. The same receptor presumably recognizes ADM making use of endogenous RAMP2. The results reveal competition between the different RAMPs in the regulation of CGRP/ADM receptor activity.

Adrenomedullin↗

PubMed1998

Parathyroid hormone responses of cyclic AMP-, serum- and phorbol ester-responsive reporter genes in osteoblast-like UMR-106 cells.

Parathyroid hormone (PTH) and PTH-related protein interact with a G protein-coupled receptor linked to the activation of adenylyl cyclase and phospholipase C signaling pathways. Regulation by PTH of the expression of three distinct, stably transfected luciferase reporter genes responsive to cAMP (CRE-luc), serum (SRE-luc) and phorbol ester (TRE-luc) has been studied in rat osteoblast-like UMR-106 cells. Maximal 43-fold induction of CRE-luc expression occurred in response to 100 nM rat (r)PTH(1-34) (EC50=0.44 nM), but SRE-luc and TRE-luc remained unaffected. Maximal 2.8- and 3.4-fold inductions of SRE-luc by 10 ng/ml EGF and 100 nM phorbol ester (PMA) were suppressed with 100 nM rPTH(1-34) (IC50=0.04 and 0.15 nM, respectively). Similarly, 7.3-fold induction of TRE-luc by 100 nM PMA was inhibited to 50% with 100 nM rPTH(1-34) (IC50=0.5 nM). Activation of mitogen-activated protein kinase by EGF and PMA was also suppressed by rPTH(1-34). 1 mM 8-Br-cAMP and 0.1 mM forskolin mimicked all the effects of rPTH(1-34). In conclusion, the regulation of target genes by PTH in osteoblast-like UMR-106 cells is mediated by the activation of the cAMP/protein kinase A signaling pathway.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1998

Differential expression of calcitonin and parathyroid hormone/parathyroid hormone-related protein receptors in P19 embryonic carcinoma cells treated with retinoic acid.

Mouse embryonic carcinoma P19 cell aggregates treated with retinoic acid (RA) sequentially differentiate into neurons and astrocytes, whereas attached cells develop a mesodermal phenotype. The expression of calcitonin (CT) and PTH/PTH-related protein (PTHrP) receptors was investigated in embryonic cells, and during neural and mesodermal differentiation. In embryonic P19 cells, specific binding of [125I]salmon (s) CT(1-32) ([125I]sCT(1-32)) was 56 fmol/mg protein, and of [125I]chicken (ch) [Tyr36]PTHrP(1-36) amide ([125I]chPTHrP(1-36)) < 0.5 fmol/mg protein. Correspondingly, cAMP was maximally stimulated 47-fold by sCT(1-32) (EC50 0.05 nM) and 3-fold by chPTHrP(1-36) (EC50 1.3 nM). Receptor autoradiography revealed specific binding of [125I]sCT(1-32) to the undifferentiated P19 cells, but not to RA induced neurons and astrocytes. At the same time, [125I]sCT(1-32) binding and cAMP accumulation by sCT were gradually decreased. But, specific binding of [125I]chPTHrP(1-36) was raised at least 6-fold compared with embryonic cells to 3 fmol/mg protein, in parallel with a 10-fold higher maximal cAMP accumulation. A similar, but delayed suppression of CT and stimulation of PTH/PTHrP receptor expression was observed during mesodermal cell differentiation. The results indicate that CT receptors are associated with undifferentiated P19 cells, whereas PTH/PTHrP receptors are expressed in RA induced neural and mesodermal cells.

Animals↗

PubMed1998

An inherited mutation associated with functional deficiency of the alpha-subunit of the guanine nucleotide-binding protein Gs in pseudo- and pseudopseudohypoparathyroidism.

Pseudohypoparathyroidism type Ia (PSP) is a disorder characterized by Albright's osteodystrophy, secondary hyperparathyroidism, lowered Gs activity, and resistance of the urinary cAMP excretion to exogenous PTH. The patients had raised basal serum levels of TSH and/or excessive TSH response to TRH. Here we have described a 38-bp deletion at the exon 1/intron 1 boundary of one Gs alpha allele in two mothers with pseudo-PSP and in six offsprings with PSP of a kindred with Albright's osteodystrophy. The deletion eliminates the splice donor site of exon 1. The pseudo-PSP patients presented decreased Gs activity, but normal urinary cAMP responses to PTH and normal TSH levels and responses to TRH. As monitored during 22 yr, they had normal serum levels of calcium and PTH. The findings demonstrate the same inherited functional defect of Gs alpha in two female patients with pseudo-PSP and in six of their offspring with PSP. The pathogenesis of clinical hypoparathyroidism remains to be clarified.

Adolescent↗

PubMed1997

Parathyroid hormone-related protein (PTHrP) inhibits proliferation of Chinese hamster ovary cells stably transfected with a PTH/PTHrP receptor cDNA.

The effect of parathyroid hormone-related protein (PTHrP) on proliferation was examined in Chinese hamster ovary (CHO) cells transfected with a PTH/PTHrP receptor encoding cDNA. Treatment with chicken PTHrP(1-36) (chPTHrP) lowered the cell number to 49 +/- 2% of untreated controls after 6 days with a half-maximal effect at 1 nM. The effect was mimicked by human (h) PTH(1-34), Br-cAMP and forskolin, but not by the receptor antagonist hPTH(3-34). Reduction of cell number was accompanied by increased PTH/PTHrP receptor expression and persistently activated adenylyl cyclase, together with altered cell morphology from epithelial to spindle-like forms, clustered growth and increased phosphate uptake. chPTHrP increased [Ca2+]i, but failed to activate membrane bound protein kinase C (PKC). Pretreatment with chPTHrP did not affect phorbol ester stimulated PKC activity, and chPTHrP or serum evoked increases in [Ca2+]i. In conclusion, PTHrP induced inhibition of proliferation and altered cell morphology is mediated by increased adenylyl cyclase rather than by PKC-dependent mechanisms in CHO cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Genetic interactions with Rap1 and Ras1 reveal a second function for the fat facets deubiquitinating enzyme in Drosophila eye development.

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential very early in eye development, prior to facet assembly, to limit the number of photoreceptor cells in each facet of the compound eye to eight. The Fat facets protein facilitates the production of a signal in cells outside the developing facets that inhibits neural development of particular facet precursor cells. Novel gain-of-function mutations in the Drosophila Rap1 and Ras1 genes are described herein that interact genetically with fat facets mutations. Analysis of these genetic interactions reveals that Fat facets has an additional function later in eye development involving Rap1 and Ras1 proteins. Moreover, the results suggest that undifferentiated cells outside the facet continue to influence facet assembly later in eye development.

Amino Acid Sequence↗

PubMed1997

Tissue-specific mRNA expression of a calcitonin receptor-like receptor during fetal and postnatal development.

The distribution of calcitonin receptor-like receptor (CRLR) mRNA in developing rats was investigated by in situ hybridization. Signals were found in the piriform cortex, the central and basolateral amygdala and the amygdalostriatal transition area. Among peripheral organs, the CRLR was predominantly expressed in the lung. mRNA expression in blood vessels, liver, midgut, rectum and urethra was restricted to gestational days 16 and/or 20. The CRLR was thought to be a calcitonin gene-related peptide (CGRP) type 1 receptor (Aiyar et al., J. Biol. Chem., 271 (1996) 11325-11329). This contrasts with previously reported evidence that the CRLR is an orphan receptor with no identifiable interactions with CGRP and other related ligands (Flühmann et al., Biochem. Biophys. Res. Commun., 206 (1995) 341-347). In situ hybridization signals have not been detected in the cerebellum and the spleen known to present a high density of CGRP binding sites. The different regional distribution of CGRP receptor binding sites and CRLR mRNA implies the latter encoding a different CGRP receptor subtype.

Aging↗

PubMed1997

[Calcitonin, a proximal-tubular-acting diuretic: lithium clearance measurements in humans].

Lithium clearance was used to investigate the effect of calcitonin on renal sodium excretion. Two sequential renal function tests, calcitonin and placebo treated, were performed in 6 healthy students. Intravenous administration of 0.5 mg human calcitonin (hCT) (Cibacalcin, Ciba-Geigy, Basel, Switzerland) significantly raised fractional excretion of sodium (FENa), from 1.73% (0.97%) to 3.63% (0.89%) (p < 0.001), and lithium clearance (Cli), from 35.1 (5.4) to 53.9 (8.5) ml/ min x 1.73 m2 (p < 0.01). By using inulin and lithium clearance we calculated proximal (PFR), distal (DFR) and total excreted sodium (TAN). Our results clearly demonstrate that hCT has a proximal diuretic effect in humans and that a calcitonin induced proximal sodium loss is compensated by increased sodium reabsorption within 80 minutes after hCT application. Calcitonin increases the excretion of lithium and has a potential to be used in lithium intoxication.

Adult↗

PubMed1997

Mutagenesis screens for interacting genes reveal three roles for fat facets during Drosophila eye development.

The Drosophila fat facets gene encodes a deubiquitinating enzyme that regulates a cell communication pathway essential early during eye development to inhibit the determination of excess photoreceptors. Ubiquitin is a small polypeptide that tags proteins for degradation by a multisubunit proteolytic complex called the proteasome. The FAT FACETS protein is thought to be required to remove ubiquitin from a particular protein, thereby rescuing if from proteolysis. In order to identify the genes encoding the substrate of FAT FACETS and other components of the neural inhibition pathway, a mutagenesis screen for dominant enhancers of the fat facets mutant eye phenotype was performed. Several genes were identified, one of which is an excellent candidate for encoding a component of the pathway regulated by FAT FACETS. Three different eye phenotypes were observed when the fat facets mutants were dominantly enhanced by different mutations, suggesting that fat facets has other functions in addition to its critical role early in eye development.

Animals↗

PubMed1997

Biologically active human islet amyloid polypeptide/amylin in transgenic mice.

OBJECTIVE: Human islet amyloid polypeptide (hIAPP), also named amylin, is a pancreatic beta cell protein implicated in the pathogenesis of pancreatic islet amyloid formation and type 2 diabetes mellitus. To study the (patho)physiological roles of hIAPP, we have generated transgenic mice that overexpress hIAPP mRNA, in relation to endogenous mouse IAPP (mIAPP) mRNA, in pancreatic beta cells. The biological activity of human and mouse IAPP derived from pancreatic extracts was determined. METHODS: Pancreatic and plasma extracts of transgenic and control mice were analyzed by reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay, yielding a separation of hIAPP from mIAPP. Biological activity of immunoreactive human and mouse IAPP components derived from pancreatic extracts was assessed by calcitonin receptor-mediated stimulation of cyclic AMP accumulation in T47D human breast carcinoma cells. RESULTS: The predominant immunoreactive human and mouse IAPP gene products had the retention times on HPLC analysis of the corresponding synthetic peptides. The ratio of bioactive over immunoreactive hIAPP and mIAPP was 0.93 +/- 0.18 and 1.19 +/- 0.56 respectively. In extracts of two plasma pools from 4 transgenic animals, hIAPP was 4.6- to 7-fold more abundant than mIAPP. CONCLUSION: This study has shown that correctly processed hIAPP produced in transgenic mouse pancreatic beta cells exhibits full biological activity. The results validate these transgenic mice for the study of (patho)physiological roles of hIAPP in vivo.

Amyloid↗

PubMed1997

Coexistence of novel amylin-binding sites with calcitonin receptors in human breast carcinoma MCF-7 cells.

Amylin, calcitonin (CT) and calcitonin gene-related peptide (CGRP) share limited structural homology including amino-terminal ring structures linked by a disulfide bridge and amidated carboxy-termini. Here, we have compared [125I]Bolton-Hunter-[Lys1] rat amylin ([125I]amylin) binding and the stimulation of cyclic AMP accumulation by human (h) amylin, hCT and hCGRP-I in the human breast carcinoma cell lines MCF-7 and T47D, which predominantly express hCT1a and hCT1b receptor isoforms (hCTR1a, hCTR1b) at a similar total number of hCT-binding sites. In MCF-7 cells, half-maximal inhibition (IC50) of [125I]amylin binding by human amylin was observed at 3.6 +/- 0.8 nM (n = 6). hCT and hCGRP-I displaced [125I]amylin binding with 22 and 66 times higher IC50. [125I]hCT binding was inhibited by hCT with an IC50 of 8.1 +/- 1.9 nM (n = 5), and human amylin and hCGRP-I were over 100 times less potent. In T47D cells, on the other hand, specific binding of [125I]amylin was not observed, but hCT inhibited [125I]hCT binding with an IC50 of 3.2 +/- 0.4 nM (n = 3), and human amylin and hCGRP-I had over 200 times higher IC50. In MCF-7 cells, half-maximal stimulation (EC50) of cyclic AMP accumulation by human amylin, hCT and hCGRP-I occurred at 1.4 +/- 0.2, 1.7 +/- 0.4 and 6.3 +/- 1.3 nM respectively. In T47D cells, the EC50 of hCT was 0.32 +/- 0.02 nM (n = 3), and 30- and 1900-fold higher with human amylin and hCGRP-I. In conclusion, the expression of hCTR1a and hCTR1b and [125I]hCT binding were indistinguishable in MCF-7 and T47D cells. Yet, [125I]amylin binding was only recognized in MCF-7 cells, consistent with a distinct amylin receptor.

Amyloid↗

PubMed1996

Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma x glioma hybrid cells (NG108-15).

Adrenomedullin (ADM) is a hypotensive peptide with structural homology, including a ring structure linked by a disulfide bridge, to calcitonin gene-related peptide (CGRP), calcitonin and amylin. ADM is predominantly synthesized in the adrenal medulla, but immunoreactive ADM has also been detected in the human brain. Here we have characterized ADM binding sites in cultured rat astrocytes using human [125I]ADM(1-52) as radioligand. Half-maximal inhibition of [125I]ADM(1-52) binding by intact rat ADM(1-50) amounted to 0.27 +/- 0.03 nM (n = 15). The related peptides rat alpha-CGRP, rat amylin and salmon calcitonin displaced [125I]ADM(1-52) at 85-, 148-, and > 4000-fold higher concentrations. Half-maximal stimulation of cAMP accumulation by rat ADM(1-50) was obtained with 1.00 +/- 0.12 nM (n = 16). Rat alpha-CGRP was 214-fold, and rat amylin and salmon calcitonin were > 1000-fold less potent. Concerning cAMP accumulation the results were indistinguishable in mouse neuroblastoma x rat glioma hybrid cells (NG108-15), but here rat alpha-CGRP was > 1000-fold less potent than rat ADM(1-50). Human ADM(22-52) and human CGRP-I(8-37), which lack the ring structure, failed to stimulate cAMP accumulation, but they antagonized rat ADM(1-50) stimulated cAMP accumulation with inhibitory constants of 365 +/- 93 nM and 92 +/- 2 nM In astrocytes, and 45 +/- 3 nM and 1300 +/- 500 nM in NG108-15 cells. Rat ADM(1-50) did not raise cytosolic free calcium concentrations in astrocytes and NG108-15 cells. In conclusion, we have identified novel ADM receptors coupled to cAMP formation in cultured rat astrocytes and NG108-15 cells. Different interactions with the homologous peptide CGRP as well as truncated receptor antagonists ADM(22-52) and CGRP(8-37) in rat astrocytes and neuroblastoma x glioma hybrid cells are consistent with ADM receptor isotypes in the brain.

Animals↗