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Adrenomedullin increases intracellular Ca2+ and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C
Brain Research 786 Ž1998. 230–234
Short communication
Adrenomedullin increases intracellular Ca2q and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C Yasuhito Uezono a, ) , Izumi Shibuya b , Yoko Ueda a , Keiko Tanaka b , Yosuke Oishi c , Nobuyuki Yanagihara a , Susumu Ueno a , Yumiko Toyohira a , Toshitaka Nakamura c , Hiroshi Yamashita b , Futoshi Izumi a a
Department of Pharmacology, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan Department of 1st Physiology, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan Department of Orthopedic Surgery, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan b
c
Accepted 18 November 1997
Abstract The effects of adrenomedullin ŽAM., a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2q concentration ŽwCa2q x i . at concentrations greater than 10y7 M. Human calcitonin gene-related peptide ŽCGRP., a peptide structurally related to AM, also increased wCa2q x i with a potency similar to that of AM. AM increased wCa2q x i in the absence of extracellular Ca2q. Further, AM increased inositol 1,4,5-trisphosphate ŽInsŽ1,4,5.P3 . level in a concentration-dependent manner similar to that of AM-induced wCa2q x i , suggesting that AM-induced elevation of wCa2q x i is due to Ca2q release from InsŽ1,4,5.P3-sensitive stores. AM Ž10y9 to 10y6 M. increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the wCa2q x i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases wCa2q x i through a mechanism independent of cAMP. q 1998 Elsevier Science B.V. Keywords: Adrenomedullin; wCa2q x i ; Calcitonin gene-related peptide; Dual signaling; KG-1C; Oligodendrocyte
Adrenomedullin ŽAM., a hypotensive peptide originally isolated from human pheochromocytoma, is localized in various tissues such as adrenal medulla, lung, kidney and brain w8,21x. In the central nervous system ŽCNS., immunohistochemical study has shown that AM is expressed in several areas of the brain w16,20x. Owji et al. w13x have found that AM-sensitive receptors are expressed in the CNS by receptor binding assay, where AM exerts several behavioral and biological effects such as inhibition of drinking w11x, increase in regional cerebral blood flow and prevention of ischemic brain injury w3x. AM is structurally homologous to calcitonin gene-related peptide ŽCGRP., a hypotensive peptide known to be present in the CNS w15x. CGRP is reported to increase cAMP in the CNS w9x and AM is also reported to increase cAMP level in SK–N–MC neuroblastoma w24x, in NG )
Corresponding author. Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. Fax: q81-93601-6264; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 3 0 - 3
108-15 neuroblastoma= glioma hybrid cells and in rat astrocytes w25x. Recent report has shown that AM increased not only cAMP level but also the intracellular Ca2q concentration ŽwCa2q x i . in rat vascular endothelial cells w17x, suggesting dual signaling of AM in the tissue. On the other hand, AM increased only cAMP in most of the tissues examined so far including rat astrocytes w25x. KG-1C cells, derived from human glioma, have many characteristics of oligodendrocytes such as properties of producing S-100 protein and negative immunoreactivity of astrocyte specific glial fibrillary acidic protein ŽGFAP. as well as the resemblance of cell morphology with primary cultured oligodendrocytes w10x. Oligodendrocytes are equipped with a number of receptors for neuroligands and bioactive peptides such as histamine and bradykinin, which increase wCa2q x i and cAMP w14x. In the present study, we focused on the effects of AM on wCa2q x i and examined the signal transduction pathways in KG-1C cells. Here we report that functional AM receptors are present in KG-1C cells and AM increases wCa2q x i as well as cAMP in these cells.
Y. Uezono et al.r Brain Research 786 (1998) 230–234
KG-1C cells were obtained from RIKEN ŽSaitama, Japan.. Chemicals were obtained from the following sources: Dulbecco’s modified Eagle’s medium ŽDMEM. and penicillin, streptomycin ŽGibco BRL, New York, USA.; fetal bovine serum ŽFBS., ŽBiocell, Rancho Dominguez, USA.; 3-isobutyl-1-methyl-xanthine ŽIBMX. ŽSigma, St. Louis, USA.; acetoxymethyl ester fura-2 Žfura2rAM. ŽDojindo, Kumamoto, Japan.; w 125 IxcAMP assay kit ŽYamasa, Chiba, Japan.; w 3 Hxinositol 1,4,5-trisphosphate ŽInsŽ1,4,5.P3 . assay kit ŽAmersham, Buckinghamshire, UK.. Peptides employed were all human-derived and obtained from Peptide Institute ŽOsaka, Japan.: adrenomedullin ŽAM., bradykinin, calcitonin gene-related peptide ŽCGRP.. All other chemicals were analytical grade from Nacalai Tesque ŽKyoto, Japan.. KG-1C cells were grown in DMEM supplemented with 10% FBS, 100 Urml penicillin, and 100 m grml streptomycin and cultured in a 35-mm dish ŽFalcon, Meylan Cedex, France.. Measurement of wCa2q x i was performed as reported previously w19x. Briefly, cultured KG-1C cells Ž2 = 10 3r35 mm glass bottom culture dish; MatTek, Ashland, USA. were plated and incubated in HEPES-buffered solution ŽHBS. containing Žin mM.: NaCl 140, KCl 5, CaCl 2 2, MgCl 2 1, HEPES 10 and glucose 10 ŽpH 7.4. with the addition of 5 m M fura-2rAM for 1 h. HBS containing 0.5% bovine serum albumin was pre-circulated before experiments to minimize the possibility that the peptides stick to the inner surface of the tubing of the perfusion system. The perfusion fluid was HBS warmed to 378C and cells were continuously perfused at a constant flow rate of 1.0 mlrmin. Ca2q-free solution was a modified HBS containing 1 mM EGTA without CaCl 2 and MgCl 2 . Fluorescence was measured with a Ca2q-imaging system equipped with an intensified CCD camera ŽQuanticellr700, JEOL, Japan. and wCa2q x i was calculated from the ratio of fluorescence intensities obtained with dual excitation at 340 and 380 nm. Cellular cAMP level in KG-1C cells was measured as reported previously w12x. Briefly, cells Ž2 = 10 5rdish. were washed with Krebs– Ringer Tris ŽKR–Tris. buffer containing Žin mM.: NaCl 154, KCl 5.6, CaCl 2 2.2, MgSO4 1.1, glucose 10, and Tris–HCl buffer 10 ŽpH 7.4., and incubated at 378C for 10 min with or without various concentrations of test compounds in KR–Tris containing 0.5 mM IBMX. cAMP was assayed by using w 125 IxcAMP radioimmunoassay kit. InsŽ1,4,5.IP3 was measured as reported previously w5x. In brief, cells Ž2 = 10 5rdish. were incubated in KR–Tris at 378C for 1 min with or without various concentrations of AM, bradykinin, or histamine. After the reaction, cells were scraped, sonicated, centrifuged, then the supernatants were used for InsŽ1,4,5.P3 assay. The cAMP and InsŽ1,4,5.P3 production were expressed in pmolsrmg protein and protein contents in the cells were determined according to the method of Bradford w2x. Data are expressed as mean " S.E.M. and are evaluated by analysis of variance. If significant F values were found, Scheffe’s test
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for multiple comparison was carried out. A level of ) p 0.05 was regarded as significant. As shown in Fig. 1A, AM at 10y7 or 10y6 M increased wCa2q x i in KG-1C cells. The wCa2q x i in response to AM were observed in 26 out of 40 cells examined Ž65.0%. and the peak increase in wCa2q x i from the base line Ž DwCa2q x i . with 10y7 M AM was 159.5 " 16.9 nM Ž n s 26.. AM at 10y8 M did not elicit wCa2q x i increase in all 18 cells examined. Bradykinin and histamine also increased wCa2q x i in a concentration-dependent manner ŽFig. 1B and C.. The responses to bradykinin and histamine at 10y7 and 10y5 M were observed in 27 out of 42 cells Ž64.3%. and in all 42 cells Ž100%., respectively, and the DwCa2q x i values at the same concentrations of the compounds were 1088 "
Fig. 1. Concentration-dependent increases in wCa2q x i induced by AM ŽA., histamine ŽB. or bradykinin ŽC. in KG-1C cells. AM, histamine or bradykinin was applied to the cells during the time indicated by the open horizontal bars. The traces are representative of 9 ŽA., 6 ŽB. and 6 ŽC. similar or identical experiments.
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Y. Uezono et al.r Brain Research 786 (1998) 230–234
180.1 nM Ž n s 27. and 607 " 62.0 nM Ž n s 42., respectively. AM increased wCa2q x i in the Ca2q-free HBS in 17 out of 22 cells examined Ž77.3%. ŽFig. 2A.. Histamine at 10y5 M, also increased wCa2q x i in the Ca2q-free HBS in all 14 cells Ž100%. ŽFig. 2B.. As shown in Fig. 2C, CGRP at 10y7 M increased wCa2q x i to almost same size as AM in all 8 cells Ž100%.. When AM or CGRP was applied to same cells at 5 to 10 min intervals, wCa2q x i responses did not show evident desensitization: the first and the second responses to AM were 189.0 " 40.0 and 180.0 " 25.0, respectively Ž n s 4. and those to CGRP were 152.0 " 29.0 and 164.0 " 61.0 nM, respectively Ž n s 4.. Forskolin at 10y5 M induced little or no increase in wCa2q x i in all 18 cells already responded to AM ŽFig. 2D.. Both AM and CGRP increased cAMP level in a concentration-dependent manner with EC 50 values of 1.8 = 10y8 and 0.9 = 10y8 M, respectively, whereas neither bradykinin nor histamine had significant effects on cAMP levels Ždata not shown.. Calcitonin and amylin, the other peptides structurally re-
lated to AM and CGRP, also increased cAMP, but to less extent than AM and CGRP ŽFig. 3A.. Forskolin at 10y6 M increased cAMP production, which are comparable with the responses to AM or to CGRP ŽFig. 3A.. AM significantly increased intracellular level of InsŽ1,4,5.P3 also in a concentration-dependent manner; it caused a 1.8-fold increase from the basal level at 10y7 M and no increase was observed at 10y8 M ŽFig. 3B.. The concentration-dependent increase in InsŽ1,4,5.P3 level induced by AM was well correlated with that in wCa2q x i induced by AM ŽFig. 1A.. Bradykinin and histamine also increased InsŽ1,4,5.P3 levels as previously reported in cultured oligodendrocytes ŽFig. 3C. w9x. To date, one study conducted in rat vascular endothelial cells has reported that AM increased both cAMP and wCa2q x i w17x. The present study has provided the second example for activation of the dual signaling pathways induced by AM. In rat astrocytes w24x and Swiss 3T3 fibroblast cells w22x, AM increased cAMP without increasing wCa2q x i through AM-specific receptors. Further, AM
Fig. 2. ŽA. and ŽB. Effects of removal of extracellular Ca2q on AM- or histamine-induced wCa2q x i increase. AM at 10y7 M ŽA. or histamine at 10y5 M ŽB. was applied to the cells in the Ca2q-free HBS for 90 s as indicated by the open horizontal bar. ŽC. and ŽD. Effects of CGRP or forskolin on the wCa2q x i in KG-1C cells. Cells were stimulated with 10y7 M AM followed by 10y7 M CGRP ŽC. or followed by 10y5 M forskolin for 90 s ŽD.. The traces are representative of 9 ŽA., 9 ŽB., 8 ŽC. and 18 ŽD. similar or identical experiments.
Y. Uezono et al.r Brain Research 786 (1998) 230–234
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Fig. 3. Effects of AM-related peptides and forskolin on cAMP level ŽA., and effects of AM ŽB., bradykinin or histamine ŽC. on InsŽ1,4,5.IP3 levels in KG-1C cells. ŽA. Cells were stimulated with a series of the structurally related peptides Ž10y7 M each. or forskolin Ž10y6 M. for 10 min in the presence of 0.5 mM IBMX and cAMP levels were measured. ŽB. Cells were stimulated with AM at concentrations from 10y8 to 10y6 M for 1 min at 378C and InsŽ1,4,5.IP3 was assayed. ŽC. Cells were stimulated with 10y7 M bradykinin or 10y5 M histamine for 1 min at 378C. Data are means" S.E.M. Ž n s 6. in ŽA. – ŽC.. ) p - 0.05 vs. basal value.
increased cAMP but not wCa2q x i in COS cells expressing cloned AM receptors w1x. It appears that AM-specific receptors do not couple to the Ca2q-signaling pathway. By contrast, in rat pancreatic AR42J cells where CGRP receptors were expressed, CGRP increased both cAMP and wCa2q x i w18x. Moreover, AM as well as CGRP is able to bind to cloned CGRP receptors w7x. Our results showed that CGRP as well as AM increased wCa2q x i in KG-1C cells. One possible explanation is that AM may increase wCa2q x i through receptors that recognize both AM and CGRP rather than receptors specific for AM. Alternatively, one could expect that there expressed a large number of AM responsible receptors in KG-1C cells. As in the family of G protein-coupled receptors that activate both cAMP and phospholipase C pathways such as luteinizing hormone receptors w23x or parathyroid hormone receptors w6x, wCa2q x i increases are often observed in cells with large receptor densities, and the amplitudes of wCa2q x i response are proportional to the receptor density. Further characterization of the receptors responsible for AM-induced wCa2q x i response will await receptor binding assay with radiolabelled ligands for AM or CGRP, and pharmacological characterization of the receptors with the use of specific antagonists for either AM or CGRP receptors. Forskolin increased cAMP level whereas it failed to increase wCa2q x i in KG-1C cells. Moreover, bradykinin and histamine, which elicited significant increase in wCa2q x i , did not increase cAMP levels Ždata not shown., suggesting that the elevation of cAMP does not contribute to wCa2q x i increase in KG-1C cells. AM- as well as histamine-induced increase in wCa2q x i was observed in the absence of extracellular Ca2q, imply-
ing that the source of wCa2q x i increase in response to these compounds is intracellular but not extracellular in KG-1C cells. There are two intracellular Ca2q stores reported in various types of cells; the InsŽ1,4,5.P3-sensitive and ryanodine-sensitive stores Žsee review in Ref. w4x.. We showed that AM as well as histamine and bradykinin increased InsŽ1,4,5.P3 levels in parallel with the increment of wCa2q x i , suggesting that AM increases wCa2q x i from InsŽ1,4,5.P3sensitive Ca2q stores in KG-1C cells. In summary, AM increased wCa2q x i as well as cAMP, and the Ca2q-signaling pathway induced by AM is independent of cAMP, in human oligodendroglial KG-1C cells. AM may play a role in the regulation of oligodendrocytes through the dual signaling pathways.
Acknowledgements This research was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan ŽY.U.., and Japan Orthopedics and Traumatology Foundation ŽY.O...
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w15x D. Poyner, Pharmacology of receptors for calcitonin gene-related peptide and amylin, Trends Pharmacol. Sci. 16 Ž1995. 424–428. w16x F. Satoh, K. Takahashi, O. Murakami, K. Totsune, M. Sone, M. Ohneda, K. Abe, Y. Miura, Y. Hayashi, H. Sasano et al., Adrenomedullin in human brain, adrenal glands and tumor tissues of pheochromocytoma, ganglioneuroblastoma and neuroblastoma, J. Clin. Endocrinol. Metab. 80 Ž1995. 1750–1752. w17x Y. Shimekake, K. Nagata, S. Ohta, Y. Kambayashi, H. Teraoka, K. Kitamura, T. Eto, K. Kangawa, H. Matsuo, Adrenomedullin stimulates two signal transduction pathways, cAMP accumulation and Ca2q mobilization, in bovine aortic endothelial cells, J. Biol. Chem. 270 Ž1995. 4412–4417. w18x D.M. Shimeone, D.I. Yule, C.D. Logsdon, J.A. Williams, Ca2q signaling through secretagogue and growth factor receptors on pancreatic AR42J cells, Regulatory Peptides 55 Ž1995. 197–206. w19x K. Tanaka, I. Shibuya, T. Nagamoto, H. Yamashita, T. Kanno, Pituitary adenylate cyclase-activating polypeptide causes rapid Ca2q release from intracellular stores and long lasting Ca2q influx mediated by Naq influx-dependent membrane depolarization in bovine adrenal chromaffin cells, Endocrinology 137 Ž1996. 956–966. w20x Y. Ueta, K. Kitamura, T. Isse, I. Shibuya, N. Kabashima, S. Yamamoto, K. Kangawa, H. Matsuo, T. Eto, H. Yamashita, Adrenomedullin-immunoreactive neurons in the paraventricular and supraoptic nuclei of the rat, Neurosci. Lett. 202 Ž1995. 37–40. w21x H. Washimine, Y. Asada, K. Kitamura, Y. Ichiki, S. Hara, Y. Yamamoto, K. Kangawa, A. Sumiyoshi, T. Eto, Immunohistochemical identification of adrenomedullin in human, rat, and porcine tissue, Histochem. Cell Biol. 103 Ž1995. 251–254. w22x D.J. Withers, H.A. Coppock, T. Seufferlein, D.M. Smith, S.R. Bloom, E. Rozengurt, Adrenomedullin stimulates DNA synthesis and cell proliferation via elevation of cAMP in Swiss 3T3 cells, FEBS Lett. 378 Ž1996. 83–87. w23x X. Zhu, S. Gilbert, M. Birnbaumer, L. Birnbaumer, Dual signaling potential is common among Gs-coupled receptors and dependent on receptor density, Mol. Pharmacol. 46 Ž1994. 460–469. w24x U. Zimmermann, J.A. Fischer, R. Muff, Adrenomedullin and calcitonin gene-related peptide interact with the same receptor in cultured human neuroblastoma SK–N–MC cells, Peptides 16 Ž1995. 421– 424. w25x U. Zimmermann, J.A. Fischer, K. Frei, A.H. Fischer, R.K. Reinscheid, R. Muff, Identification of adrenomedullin receptors in cultured rat astrocytes and in neuroblastboma=glioma hybrid cells ŽNG108-15., Brain Res. 724 Ž1996. 238–245.
Short communication
Adrenomedullin increases intracellular Ca2q and inositol 1,4,5-trisphosphate in human oligodendroglial cell line KG-1C Yasuhito Uezono a, ) , Izumi Shibuya b , Yoko Ueda a , Keiko Tanaka b , Yosuke Oishi c , Nobuyuki Yanagihara a , Susumu Ueno a , Yumiko Toyohira a , Toshitaka Nakamura c , Hiroshi Yamashita b , Futoshi Izumi a a
Department of Pharmacology, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan Department of 1st Physiology, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan Department of Orthopedic Surgery, UniÕersity of Occupational and EnÕironmental Health, School of Medicine, Kitakyushu 807-8555, Japan b
c
Accepted 18 November 1997
Abstract The effects of adrenomedullin ŽAM., a hypotensive peptide, were investigated in cultured human oligodendroglial cell line KG-1C. Human AM increased the intracellular Ca2q concentration ŽwCa2q x i . at concentrations greater than 10y7 M. Human calcitonin gene-related peptide ŽCGRP., a peptide structurally related to AM, also increased wCa2q x i with a potency similar to that of AM. AM increased wCa2q x i in the absence of extracellular Ca2q. Further, AM increased inositol 1,4,5-trisphosphate ŽInsŽ1,4,5.P3 . level in a concentration-dependent manner similar to that of AM-induced wCa2q x i , suggesting that AM-induced elevation of wCa2q x i is due to Ca2q release from InsŽ1,4,5.P3-sensitive stores. AM Ž10y9 to 10y6 M. increased cAMP in a concentration-dependent manner. Forskolin also increased cAMP, but did not mimic the wCa2q x i-raising effect of AM. These findings suggest that functional AM receptors are present in oligodendroglial KG-1C cells and that AM increases wCa2q x i through a mechanism independent of cAMP. q 1998 Elsevier Science B.V. Keywords: Adrenomedullin; wCa2q x i ; Calcitonin gene-related peptide; Dual signaling; KG-1C; Oligodendrocyte
Adrenomedullin ŽAM., a hypotensive peptide originally isolated from human pheochromocytoma, is localized in various tissues such as adrenal medulla, lung, kidney and brain w8,21x. In the central nervous system ŽCNS., immunohistochemical study has shown that AM is expressed in several areas of the brain w16,20x. Owji et al. w13x have found that AM-sensitive receptors are expressed in the CNS by receptor binding assay, where AM exerts several behavioral and biological effects such as inhibition of drinking w11x, increase in regional cerebral blood flow and prevention of ischemic brain injury w3x. AM is structurally homologous to calcitonin gene-related peptide ŽCGRP., a hypotensive peptide known to be present in the CNS w15x. CGRP is reported to increase cAMP in the CNS w9x and AM is also reported to increase cAMP level in SK–N–MC neuroblastoma w24x, in NG )
Corresponding author. Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan. Fax: q81-93601-6264; E-mail: [email protected] 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 7 . 0 1 4 3 0 - 3
108-15 neuroblastoma= glioma hybrid cells and in rat astrocytes w25x. Recent report has shown that AM increased not only cAMP level but also the intracellular Ca2q concentration ŽwCa2q x i . in rat vascular endothelial cells w17x, suggesting dual signaling of AM in the tissue. On the other hand, AM increased only cAMP in most of the tissues examined so far including rat astrocytes w25x. KG-1C cells, derived from human glioma, have many characteristics of oligodendrocytes such as properties of producing S-100 protein and negative immunoreactivity of astrocyte specific glial fibrillary acidic protein ŽGFAP. as well as the resemblance of cell morphology with primary cultured oligodendrocytes w10x. Oligodendrocytes are equipped with a number of receptors for neuroligands and bioactive peptides such as histamine and bradykinin, which increase wCa2q x i and cAMP w14x. In the present study, we focused on the effects of AM on wCa2q x i and examined the signal transduction pathways in KG-1C cells. Here we report that functional AM receptors are present in KG-1C cells and AM increases wCa2q x i as well as cAMP in these cells.
Y. Uezono et al.r Brain Research 786 (1998) 230–234
KG-1C cells were obtained from RIKEN ŽSaitama, Japan.. Chemicals were obtained from the following sources: Dulbecco’s modified Eagle’s medium ŽDMEM. and penicillin, streptomycin ŽGibco BRL, New York, USA.; fetal bovine serum ŽFBS., ŽBiocell, Rancho Dominguez, USA.; 3-isobutyl-1-methyl-xanthine ŽIBMX. ŽSigma, St. Louis, USA.; acetoxymethyl ester fura-2 Žfura2rAM. ŽDojindo, Kumamoto, Japan.; w 125 IxcAMP assay kit ŽYamasa, Chiba, Japan.; w 3 Hxinositol 1,4,5-trisphosphate ŽInsŽ1,4,5.P3 . assay kit ŽAmersham, Buckinghamshire, UK.. Peptides employed were all human-derived and obtained from Peptide Institute ŽOsaka, Japan.: adrenomedullin ŽAM., bradykinin, calcitonin gene-related peptide ŽCGRP.. All other chemicals were analytical grade from Nacalai Tesque ŽKyoto, Japan.. KG-1C cells were grown in DMEM supplemented with 10% FBS, 100 Urml penicillin, and 100 m grml streptomycin and cultured in a 35-mm dish ŽFalcon, Meylan Cedex, France.. Measurement of wCa2q x i was performed as reported previously w19x. Briefly, cultured KG-1C cells Ž2 = 10 3r35 mm glass bottom culture dish; MatTek, Ashland, USA. were plated and incubated in HEPES-buffered solution ŽHBS. containing Žin mM.: NaCl 140, KCl 5, CaCl 2 2, MgCl 2 1, HEPES 10 and glucose 10 ŽpH 7.4. with the addition of 5 m M fura-2rAM for 1 h. HBS containing 0.5% bovine serum albumin was pre-circulated before experiments to minimize the possibility that the peptides stick to the inner surface of the tubing of the perfusion system. The perfusion fluid was HBS warmed to 378C and cells were continuously perfused at a constant flow rate of 1.0 mlrmin. Ca2q-free solution was a modified HBS containing 1 mM EGTA without CaCl 2 and MgCl 2 . Fluorescence was measured with a Ca2q-imaging system equipped with an intensified CCD camera ŽQuanticellr700, JEOL, Japan. and wCa2q x i was calculated from the ratio of fluorescence intensities obtained with dual excitation at 340 and 380 nm. Cellular cAMP level in KG-1C cells was measured as reported previously w12x. Briefly, cells Ž2 = 10 5rdish. were washed with Krebs– Ringer Tris ŽKR–Tris. buffer containing Žin mM.: NaCl 154, KCl 5.6, CaCl 2 2.2, MgSO4 1.1, glucose 10, and Tris–HCl buffer 10 ŽpH 7.4., and incubated at 378C for 10 min with or without various concentrations of test compounds in KR–Tris containing 0.5 mM IBMX. cAMP was assayed by using w 125 IxcAMP radioimmunoassay kit. InsŽ1,4,5.IP3 was measured as reported previously w5x. In brief, cells Ž2 = 10 5rdish. were incubated in KR–Tris at 378C for 1 min with or without various concentrations of AM, bradykinin, or histamine. After the reaction, cells were scraped, sonicated, centrifuged, then the supernatants were used for InsŽ1,4,5.P3 assay. The cAMP and InsŽ1,4,5.P3 production were expressed in pmolsrmg protein and protein contents in the cells were determined according to the method of Bradford w2x. Data are expressed as mean " S.E.M. and are evaluated by analysis of variance. If significant F values were found, Scheffe’s test
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for multiple comparison was carried out. A level of ) p 0.05 was regarded as significant. As shown in Fig. 1A, AM at 10y7 or 10y6 M increased wCa2q x i in KG-1C cells. The wCa2q x i in response to AM were observed in 26 out of 40 cells examined Ž65.0%. and the peak increase in wCa2q x i from the base line Ž DwCa2q x i . with 10y7 M AM was 159.5 " 16.9 nM Ž n s 26.. AM at 10y8 M did not elicit wCa2q x i increase in all 18 cells examined. Bradykinin and histamine also increased wCa2q x i in a concentration-dependent manner ŽFig. 1B and C.. The responses to bradykinin and histamine at 10y7 and 10y5 M were observed in 27 out of 42 cells Ž64.3%. and in all 42 cells Ž100%., respectively, and the DwCa2q x i values at the same concentrations of the compounds were 1088 "
Fig. 1. Concentration-dependent increases in wCa2q x i induced by AM ŽA., histamine ŽB. or bradykinin ŽC. in KG-1C cells. AM, histamine or bradykinin was applied to the cells during the time indicated by the open horizontal bars. The traces are representative of 9 ŽA., 6 ŽB. and 6 ŽC. similar or identical experiments.
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180.1 nM Ž n s 27. and 607 " 62.0 nM Ž n s 42., respectively. AM increased wCa2q x i in the Ca2q-free HBS in 17 out of 22 cells examined Ž77.3%. ŽFig. 2A.. Histamine at 10y5 M, also increased wCa2q x i in the Ca2q-free HBS in all 14 cells Ž100%. ŽFig. 2B.. As shown in Fig. 2C, CGRP at 10y7 M increased wCa2q x i to almost same size as AM in all 8 cells Ž100%.. When AM or CGRP was applied to same cells at 5 to 10 min intervals, wCa2q x i responses did not show evident desensitization: the first and the second responses to AM were 189.0 " 40.0 and 180.0 " 25.0, respectively Ž n s 4. and those to CGRP were 152.0 " 29.0 and 164.0 " 61.0 nM, respectively Ž n s 4.. Forskolin at 10y5 M induced little or no increase in wCa2q x i in all 18 cells already responded to AM ŽFig. 2D.. Both AM and CGRP increased cAMP level in a concentration-dependent manner with EC 50 values of 1.8 = 10y8 and 0.9 = 10y8 M, respectively, whereas neither bradykinin nor histamine had significant effects on cAMP levels Ždata not shown.. Calcitonin and amylin, the other peptides structurally re-
lated to AM and CGRP, also increased cAMP, but to less extent than AM and CGRP ŽFig. 3A.. Forskolin at 10y6 M increased cAMP production, which are comparable with the responses to AM or to CGRP ŽFig. 3A.. AM significantly increased intracellular level of InsŽ1,4,5.P3 also in a concentration-dependent manner; it caused a 1.8-fold increase from the basal level at 10y7 M and no increase was observed at 10y8 M ŽFig. 3B.. The concentration-dependent increase in InsŽ1,4,5.P3 level induced by AM was well correlated with that in wCa2q x i induced by AM ŽFig. 1A.. Bradykinin and histamine also increased InsŽ1,4,5.P3 levels as previously reported in cultured oligodendrocytes ŽFig. 3C. w9x. To date, one study conducted in rat vascular endothelial cells has reported that AM increased both cAMP and wCa2q x i w17x. The present study has provided the second example for activation of the dual signaling pathways induced by AM. In rat astrocytes w24x and Swiss 3T3 fibroblast cells w22x, AM increased cAMP without increasing wCa2q x i through AM-specific receptors. Further, AM
Fig. 2. ŽA. and ŽB. Effects of removal of extracellular Ca2q on AM- or histamine-induced wCa2q x i increase. AM at 10y7 M ŽA. or histamine at 10y5 M ŽB. was applied to the cells in the Ca2q-free HBS for 90 s as indicated by the open horizontal bar. ŽC. and ŽD. Effects of CGRP or forskolin on the wCa2q x i in KG-1C cells. Cells were stimulated with 10y7 M AM followed by 10y7 M CGRP ŽC. or followed by 10y5 M forskolin for 90 s ŽD.. The traces are representative of 9 ŽA., 9 ŽB., 8 ŽC. and 18 ŽD. similar or identical experiments.
Y. Uezono et al.r Brain Research 786 (1998) 230–234
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Fig. 3. Effects of AM-related peptides and forskolin on cAMP level ŽA., and effects of AM ŽB., bradykinin or histamine ŽC. on InsŽ1,4,5.IP3 levels in KG-1C cells. ŽA. Cells were stimulated with a series of the structurally related peptides Ž10y7 M each. or forskolin Ž10y6 M. for 10 min in the presence of 0.5 mM IBMX and cAMP levels were measured. ŽB. Cells were stimulated with AM at concentrations from 10y8 to 10y6 M for 1 min at 378C and InsŽ1,4,5.IP3 was assayed. ŽC. Cells were stimulated with 10y7 M bradykinin or 10y5 M histamine for 1 min at 378C. Data are means" S.E.M. Ž n s 6. in ŽA. – ŽC.. ) p - 0.05 vs. basal value.
increased cAMP but not wCa2q x i in COS cells expressing cloned AM receptors w1x. It appears that AM-specific receptors do not couple to the Ca2q-signaling pathway. By contrast, in rat pancreatic AR42J cells where CGRP receptors were expressed, CGRP increased both cAMP and wCa2q x i w18x. Moreover, AM as well as CGRP is able to bind to cloned CGRP receptors w7x. Our results showed that CGRP as well as AM increased wCa2q x i in KG-1C cells. One possible explanation is that AM may increase wCa2q x i through receptors that recognize both AM and CGRP rather than receptors specific for AM. Alternatively, one could expect that there expressed a large number of AM responsible receptors in KG-1C cells. As in the family of G protein-coupled receptors that activate both cAMP and phospholipase C pathways such as luteinizing hormone receptors w23x or parathyroid hormone receptors w6x, wCa2q x i increases are often observed in cells with large receptor densities, and the amplitudes of wCa2q x i response are proportional to the receptor density. Further characterization of the receptors responsible for AM-induced wCa2q x i response will await receptor binding assay with radiolabelled ligands for AM or CGRP, and pharmacological characterization of the receptors with the use of specific antagonists for either AM or CGRP receptors. Forskolin increased cAMP level whereas it failed to increase wCa2q x i in KG-1C cells. Moreover, bradykinin and histamine, which elicited significant increase in wCa2q x i , did not increase cAMP levels Ždata not shown., suggesting that the elevation of cAMP does not contribute to wCa2q x i increase in KG-1C cells. AM- as well as histamine-induced increase in wCa2q x i was observed in the absence of extracellular Ca2q, imply-
ing that the source of wCa2q x i increase in response to these compounds is intracellular but not extracellular in KG-1C cells. There are two intracellular Ca2q stores reported in various types of cells; the InsŽ1,4,5.P3-sensitive and ryanodine-sensitive stores Žsee review in Ref. w4x.. We showed that AM as well as histamine and bradykinin increased InsŽ1,4,5.P3 levels in parallel with the increment of wCa2q x i , suggesting that AM increases wCa2q x i from InsŽ1,4,5.P3sensitive Ca2q stores in KG-1C cells. In summary, AM increased wCa2q x i as well as cAMP, and the Ca2q-signaling pathway induced by AM is independent of cAMP, in human oligodendroglial KG-1C cells. AM may play a role in the regulation of oligodendrocytes through the dual signaling pathways.
Acknowledgements This research was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan ŽY.U.., and Japan Orthopedics and Traumatology Foundation ŽY.O...
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