Proc. Nati. Acad. Sci. USA Vol. 87, pp. 3299-3303, May 1990 Neurobiology
Calcitonin gene-related peptide stimulates proliferation of human endothelial cells (sensory neurons/angiogenesis/wound healing)
A. HA-GERSTRAND*t, C.-J. DALSGAARD**, B. JONZON§, 0. LARSSON¶,
AND
J. NILSSONII
Departments of *Anatomy and lPharmacology, Karolinska Institute, Stockholm, Sweden; §Department of Clinical Pharmacology, Huddinge University Hospital, Huddinge, Sweden; and Departments of tPlastic Surgery and IlMedicine, Karolinska Hospital, Stockholm, Sweden
Communicated by Viktor Mutt, December 29, 1989 (received for review November 2, 1989)
ABSTRACT The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. l I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cycdic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.
Several different factors have been shown to stimulate the formation of new vessels, angiogenesis, and/or to be mitogenic for cultured endothelial cells. These factors include polypeptide growth factors-i.e., basic and acidic fibroblast growth factor (bFGF, aFGF)-endothelial cell growth factor, which is a precursor form of aFGF, transforming growth factors a and /3, angiogenin, and tumor necrosis factor a (1). Angiogenic factors may be important when released locally by cells adjacent to the endothelium, possibly also produced by the endothelial cells themselves or as circulating factors in plasma. Indirect evidence has suggested trophic effects of peripheral neurons. In the newt, naturally occurring limb regeneration is prevented by damage to the peripheral nerve innervating the limb (2). Intact sensory innervation has been shown to be important for corneal wound healing in the rat (3), and in humans suffering from Parry-Romberg syndrome, a marked hemifacial dystrophia is observed in the area innervated by the trigeminal nerve (4). Although direct evidence from in vivo studies is essentially lacking, recent studies have shown that sensory neuropeptides regulate cell proliferation in vitro. Neurokinin A (NKA) and substance P (SP), which are structurally related, have been shown to induce proliferation of human fibroblasts and rat smooth muscle cells (5). This effect was shown to be parallel with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
increased phosphatidylinositol (PI) turnover (6). Furthermore, vasoactive intestinal polypeptide (VIP) has been shown to inhibit serum-induced proliferation of rat smooth muscle cells and to stimulate proliferation of human keratinocytes, both events paralleled with adenosine 3',5'-cyclic monophosphate (cAMP) formation (7, 8). In this study, the proliferative effect on cultured human umbilical vein endothelial cells (HUVECs) of the vasoactive neuropeptides calcitonin gene-related peptide (CGRP), NKA, neuropeptide Y (NPY), and VIP was examined by determining the increase in cell number and incorporation of [3H]thymidine. For comparison bFGF, an endothelial cell mitogen (9), was used. Since CGRP and bFGF both stimulated proliferation, theireffects on cAMP formation and PI turnover, two intracellular signal systems that frequently have been found to be involved in regulation of cell proliferation (10, 11), were examined. The proliferative effects of cholera toxin (CT), a potent inducer of cAMP formation (12), and thrombin, which induces breakdown of PI (13), were also investigated.
MATERIALS AND METHODS Cell Culture. HUVECs were isolated and cultured mainly as described by Jaffe et al. (14). Briefly, veins of fresh umbilical cords were rinsed with 50-100 ml of phosphatebuffered saline (PBS) and subsequently filled with a collagenase solution (0.1%; Worthington) and incubated at 370C for 20 min. Harvested cells were centrifuged at 800 x g for 5 min and resuspended in culture medium-i.e., medium 199 (M199; GIBCO) with addition of 20% fetal calf serum (FCS; GIBCO) and antibiotics (penicillin, 50 pug/ml; streptomycin, 50 units/ml) and cultured in gelatin-coated (0.2% gelatin in PBS at +40C for 30 min) culture flasks (Costar). Cell Proliferation Assays. Secondary and tertiary cultures were used for experiments. For determinations of increase in cell number and [3H]thymidine incorporation, HUVECs were transferred to gelatin-coated 4-well plates (1.9 cm2, 104 cells per well; Nunc) and 96-well plates (3 x 103 cells per well; Nunc), respectively. HUVECs were allowed to attach overnight in culture medium and were subsequently stimulated with CGRP, NKA, NPY, VIP (all 100 nM, Peninsula; CGRP was also kindly provided by I. MacIntyre), bFGF (1 ng/ml; kindly provided by California Biotechnology), CT (0.1 nM; Sigma), or thrombin (1 unit/ml; kindly provided by P. T. Larsson, Karolinska Institute) in assay medium-i.e., M199 containing 5% FCS and antibiotics. Cells used for studies of Abbreviations: HUVEC, human umbilical cord vein endothelial cell; CGRP, calcitonin gene-related peptide; SP, substance P; NKA, neurokinin A; VIP, vasoactive intestinal polypeptide; NPY, neuropeptide Y; bFGF, basic fibroblast growth factor; PI, phosphatidylinositol; InsP, inositol phosphate; CT, cholera toxin. tTo whom reprint requests should be addressed at: Department of Anatomy, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
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Neurobiology: Hagerstrand et al.
DNA synthesis were serum starved for 12 hr prior to 24 hr of stimulation in assay medium containing [3H]thymidine (3 puCi/ml; 1 Ci = 37 GBq; Amersham). A dose-response study on the proliferative effect of CGRP (0.001-100 nM) was performed. Stimulation of [3H]thymidine incorporation by CGRP-containing (0.01-1 nM) medium preincubated with a polyclonal CGRP antibody (2 hr at room temperature; antibody dilution, 1:100; Peninsula) was also examined. Cell number was determined after 4 days of stimulation (medium was renewed every 2nd day) by detachment of cells with a trypsin/EDTA (0.05%/0.01%) solution. Cells were suspended into single cells and counted in a cell counter (Analys Instrument, Stockholm). Incorporation of [3H]thymidine was examined in a Beckman scintillation counter after lysis with Triton X-100 (0.1% in distilled water). CGRP Receptor Binding Assay. Tertiary cultures were grown to subconfluence in 12-well plates (4 cm2 per well; Costar) in culture medium. The binding assay was performed mainly as described by Heldin et al. (15). Briefly, cells were rinsed twice with PBS containing 0.1% bovine serum albumin (Sigma) and triplicate cultures were incubated for 2 hr at +40C in PBS containing 1251-labeled CGRP (125I-CGRP) (1 nM; 70 Bq/fmol; kindly supplied by E. TheodorssonNorheim, Karolinska Hospital) alone or with the addition of unlabeled CGRP (0.001-1000 nM). Cells were rinsed repeatedly with PBS and lysed with Triton X-100 (1%) in distilled water. Radioactivity in lysates was determined in a y-counter
(LKB).
cAMP Formation. Tertiary cultures were grown to subconfluence in 12-well plates (4 cm2 per well; Costar) in culture medium. Cells were rinsed with serum-free M199 and stimulated with CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 nM) in 0.33 ml of serum-free M199 containing a phosphodiesterase inhibitor (Rolipram, 20 ,uM; Schering) for 15 min at 37°C. The effect of forskolin (1 uM) or forskolin (1 ,uM) plus CGRP (100 nM) on cAMP formation was also examined. Reactions were terminated by addition of ice-cold HCl04 to a final concentration of 0.4 M. After addition of HCl04, cells were frozen, thawed, and scraped, and the material was frozen until analysis. The insoluble material was spun down and aliquots from supernatants were neutralized with KOH and Tris base and used for cAMP analysis performed essentially as described by Brown et al. (16). PI Breakdown. Tertiary cultures of HUVECs were grown to subconfluence and incubated for 48 hr with myo-[3H]inositol (10 ACi/ml; specific activity, 14.3 Ci/mmol; NEN) in M199 with 5% FCS. Cells were washed with PBS containing Na2HPO4 (6.5 mM), KH2PO4 (1.5 mM), KCI (2.7 mM), NaCI (137 mM), CaCl2 (1 mM), MgCI2 (1 mM), and Hepes (2 mM). Cells were then incubated for 10 min with 0.6 ml of the same buffer containing 10 mM LiCl and were subsequently stimulated by the addition of 66-,A aliquots of the agonists, resulting in final concentrations of CGRP (0.1 ,uM), bFGF (1 ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). The reaction was terminated by addition of ice-cold HC104 to a final concentration of 0.27 M. The standard procedure for separation of inositol phosphates (InsP) was, with minor modifi-
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FIG. 1. Proliferation of HUVECs examined by means of increased cell number (a), and incorporation of [3H]thymidine (b), after stimulation with CGRP (e, 0.001-100 nM), bFGF (o, 1 ng/ml), or CT (i, 0.1 nM) in M199 with addition of 5% FCS. (a) Cell number was determined after 4 days of culture (medium was renewed twice) by detachment and counting of suspended single cells. (b) Cells were serum starved for 12 hr prior to 24 hr of stimulation with the addition of 3 ,uCi of [3H]thymidine per ml to the medium. Cells were lysed and radioactivity was determined in a scintillation counter. Data points are expressed as % increase [means ± SEM of triplicate (a) and quadruplicate (b) cultures] from control cultures. It was shown that CGRP increased cell number and [3H]thymidine incorporation with a maximum of 45% ± 3.3% (P < 0.01) and 53% ± 6.1% (P < 0.002) at 10 nM and 1 nM, respectively. It was also shown that the CGRP-induced increase in DNA synthesis was reduced by preincubation with a CGRP antibody (o). bFGF (1 ng/ml) increased cell number by 79% ± 6.8% (P < 0.001) and DNA synthesis by 103% ± 8.8% (P < 0.001), whereas CT (0.1 nM) induced a 58% ± 3.2% (P < 0.002) and 75% ± 11.3% (P < 0.001) increase in cell number and DNA synthesis, respectively. For statistical analysis, Student's t test was used. *, P < 0.01; **, P < 0.002; ***, P < 0.001.
Neurobiology: Hxgerstrand et al.
Proc. Natl. Acad. Sci. USA 87 (1990)
cations, carried out as described by Bone et al. (17). Radioactivity was measured in a scintillation counter (LKB).
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RESULTS Cell Proliferation. CGRP, but not NKA, NPY, or VIP (all 100 nM), increased the cell number after 4 days of stimulation. CGRP induced a concentration-dependent increase in cell number as compared to control cultures with a maximal effect of 45% ± 3.3% (P < 0.01) at 10 nM (Fig. la). bFGF (1 ng/ml) and CT (0.1 nM) increased cell number by 79% ± 6.8% (P < 0.001) and 58% ± 3.2% (P < 0.002), respectively (Fig. la), whereas thrombin (1 unit/ml) was ineffective (data not shown). Similar results were obtained when the cells were serum starved for 12 hr and subsequently stimulated in the presence of [3H]thymidine. CGRP induced a concentration-dependent stimulation of [3H]thymidine uptake with a maximum of 53% ± 6.1% (P < 0.002) at 1 nM (Fig. lb). NKA, VIP, and NPY (all 100 nM) were ineffective in stimulating [3H]thymidine incorporation. It was also shown that CGRP-induced [3H]thymidine uptake could be reduced by preincubating the CGRP-containing culture medium with a polyclonal CGRP antibody (Fig. lb). bFGF (1 ng/ml) induced a 103% ± 8.8% (P < 0.001) increase in DNA synthesis and CT (0.1 nM) induced an increase of 75% ± 11.3% (P < 0.001; Fig. lb), whereas thrombin (1 unit/ml) was ineffective in stimulating DNA synthesis. CGRP Receptor Binding. 1251-CGRP (1 nM) was shown to bind to cultured HUVECs (1408 ± 125 cpm per well, corresponding to 0.19 fmol per 105 cells). It was also shown that this binding was reduced in a dose-dependent manner when unlabeled CGRP (0.001-1000 nM) was added (Fig. 2). When a 1000-fold excess of unlabeled CGRP was added 125I-CGRP binding was reduced to 40% ± 5.1% of control binding. cAMP Formation. It was shown that CGRP stimulated cAMP formation in a concentration-dependent manner (Fig. 2). CT (0.1 nM) increased cAMP formation by 330%o ± 13.2% (P < 0.01), whereas bFGF was ineffective (Fig. 3). It was also shown that the effect of CGRP (10 nM), which increased cAMP formation by 168% ± 7.2% (P < 0.01), was potentiated by addition of forskolin (1 ,uM), a compound that commonly enhances receptor-mediated cAMP formation, to 703% + 3.7% of control cultures. Forskolin alone did not significantly
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CGRP, nM FIG. 3. Formation of cAMP by HUVECs after stimulation with CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 nM). Subconfluent HUVECs were stimulated in serum-free M199 for 15 min in the presence of agonists and the phosphodiesterase inhibitor Rolipram (20 uM). cAMP formation was determined on supernatants from lysed HUVECs and was assayed by protein saturation essentially as described by Brown et al. (16). Data are presented as means ± SEM of quadruplicate cultures. It was shown that CGRP (o) concentration dependently increased cAMP formation compared to control levels. CT (n, 0.1 nM) increased cAMP formation by 330%o ± 13.2% (P < 0.01), whereas bFGF (o, 1 ng/ml) was ineffective. cAMP formation in control cultures is indicated by the horizontal line. For statistical analysis, Student's t test was used. *, P < 0.01.
change cAMP formation. cAMP formation in control cultures was 0.55 ± 0.15 pmol per 105 cells. PI Breakdown. CGRP (100 nM), CT (0.1 nM), and bFGF (1 ng/ml) were all unable to significantly change InsP levels in HUVECs after 15 min of stimulation, whereas thrombin (1 unit/ml) increased InsP levels by 256% ± 32.2% (P < 0.001; Fig. 4). InsP formation in control cultures corresponded to 523 dpm per 105 cells. As previously shown (13), InsP2 and InsP3 levels were not changed from control levels after 15 min of stimulation (data not shown).
DISCUSSION CGRP was first identified as a polypeptide encoded by the calcitonin gene of the rat (18), and later a human counterpart was isolated and sequenced (19). In addition to thyroid tissue,
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CGRP, nM FIG. 2. Binding of 1251-CGRP to cultured HUVECs. Subconfluent tertiary cultures of HUVECs were incubated (for 2 hr, at +4°C) with 1251-CGRP alone (1 nM) or with the addition of unlabeled CGRP (0.001-1000 nM). Data are expressed as means SEM of triplicate cultures. It was shown that binding of 1251-CGRP in the absence of unlabeled CGRP (set as 100o) was 1408 + 125 cpm per well, corresponding to 0.19 fmol per 105 cells, and that this binding was dose dependently reduced by addition of unlabeled CGRP.
bFGF
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FIG. 4. Formation of InsP after stimulation with CGRP (100 nM), bFGF (1 ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). Subconfluent HUVECs were preincubated with LiCl (10 mM) in assay buffer for 10 min and were subsequently stimulated for 15 min after addition of agonists. PIs were analyzed in supernatants from lysed cells and were separated as described by Bone et al. (17). Data are presented as means ± SEM of quadruplicate cultures. It was shown that thrombin (1 unit/ml) increased InsP formation by 256% ± 32.2% (P < 0.001), whereas CGRP (0.1 AM), bFGF (1 ng/mI), and CT (0.1 nM) did not significantly change InsP formation. InsP formation in control cultures is indicated by the horizontal line. For statistical analysis, Student's t test was used. ***, P < 0.001.
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CGRP is widely distributed within nervous tissue (20). In the periphery, CGRP is present in sensory and autonomic nerve fibers, of which the sensory neurons show close relationship to blood vessels (21-23). CGRP-like immunoreactive nerve fibers have also been shown to be intimately related to capillary loops in the papilla of the dermis and also to be abundant in the highly vascularized endometrial stroma (24). Degradation of the basal membrane, proliferation, and migration of endothelial cells is required to complete the formation of new vessels, which is a crucial step in embryogenesis, tumor neovascularization, inflammation, and healing of injured tissue (1). A role of sensory neuropeptides in these events is implicated by several findings (25). CGRP is a potent vasodilator (26) and is released from sensory nerve fibers during ischemia (27). Experimental studies have shown that functionally intact sensory neurons and treatment with CGRP increase survival of ischemic surgical flaps (28, 29). Thus, CGRP released from peripheral nerve endings during ischemia may play a dual role by increasing the blood flow and by stimulating formation of new vessels. Sensory neurons have been shown to participate in both acute and chronic inflammatory events. Capsaicin pretreatment, which depletes the neuropeptide content in sensory neurons, attenuates joint injury in experimental arthritis in the rat (30). The severity of adjuvant-induced arthritis in the rat is increased by SP, which is costored with CGRP in sensory neurons. The severity is also correlated to the density of sensory innervation (31). Since inflammation is associated with both increased turnover of sensory neuropeptides and vessel formation, a release of CGRP may contribute to the vascularization of inflamed tissue. During wound healing, sprouting of sensory nerve fibers in relation to blood vessels, hair follicles, and sweat gland ducts toward the wound surface has been described (32). It was recently shown that SP stimulates monocytes-macrophages to produce tumor necrosis factor a (33), which has been shown to be angiogenic in vivo but does not stimulate proliferation in vitro (34). It is thus an intriguing possibility that sprouting sensory neurons may be important for both directional guidance, via activation of macrophages, and proliferation of endothelial cells. The intracellular mechanisms involved in stimulation of HUVEC proliferation are at present not known. In human dermal capillary cells, CT and the phosphodiesterase inhibitor isobutylmethylxanthine have been shown to stimulate cell proliferation (35). Activation of protein kinase C in capillary cells has been shown to induce elongation of the cells and to reduce the mitogenic response induced by growth factors (36). In this context, it is interesting to note that CGRP has been shown to activate adenylate cyclase in different cells, including HUVECs (37). In porcine aortic endothelial cells, however, endothelial cell growth factor and thrombin, which both stimulate PI breakdown, also appear to be mitogenic (38, 39). The present findings suggest that cAMP formation rather than PI breakdown is associated with proliferation of HUVECs. Whether increased cAMP formation per se stimulates proliferation or the effect is indirect by potentiating a stimulatory effect offactors in serum is not known. However, the presence of serum factors may be regarded as a natural environment for endothelial cells both in vivo and in vitro. CGRP has recently been shown to be trophic for development of the nicotinic receptor at the motor end plate (40) and also to act as a differentiating factor by inducing a dopaminergic phenotype in the mouse olfactory bulb (41). In addition to the previously shown effects of CGRP, our findings suggest that CGRP may also act as a local factor stimulating cell proliferation. The effect of CGRP on endothelial cells
Proc. Natl. Acad. Sci. USA 87 (1990)
suggests a role in angiogenesis, including formation of new vessels in ischemia, inflammation, and wound healing. We thank Professor I. MacIntyre for providing human CGRP (founded by Bartos Foundation) and for valuable discussions; Dr. E. Theodorsson-Norheim for providing 125I-CGRP; Dr. P. T. Larsson for providing thrombin; Ms. H. Vrang, Ms. S. Mattisson, and Ms. M. Lind for excellent technical assistance; and Ms. E. Melander and colleagues at the Department of Gynecology and Obstetrics at the Karolinska Hospital for their help in collecting umbilical cords. This work was supported by grants from the Karolinska Institute, the Swedish Medical Research Council (Grants 7126 and 7464), SRA, Marcus and Amalia Wallenberg's Memorial Foundation, T. Nilsson's Foundation, and Knut and Alice Wallenberg's Foundation. 1. Folkman, J. & Klagsbrun, M (1987) Science 235, 442-447. 2. Singer, M. (1952) Q. Rev. Biol. 27, 169-200. 3. Rook, A., Wilkinson, D. S. & Ebling, F. J. G. (1968) Textbook of Dermatology (Davis, Philadelphia), pp. 475-488. 4. Converse, J. M. (1977) Plastic and Reconstructive Surgery (Saunders, Philadelphia), 2nd Ed., pp. 610-660. 5. Nilsson, J., von Euler, A. & Dalsgaard, C.-J. (1985) Nature (London) 315, 61-63. 6. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, C.-J. (1988) J. Cell. Physiol. 137, 141-145. 7. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, C.-J. (1988) Regul. Pept. 22, 267-274. 8. Hwgerstrand, A., Jonzon, B., Dalsgaard, C.-J. & Nilsson, J. (1989) Proc. NatI. Acad. Sci. USA 86, 5993-5996. 9. Oospodarowicz, D., Brown, K. D., Birdwell, C. R. & Zetter, B. R. (1978) J. Cell Biol. 77, 774-788. 10. Boynton, A. L. & Whitfield, J. F. (1983) Adv. Cyclic Nucleotide Res. 15, 193-294. 11. Berridge, M. (1986) in Oncogenes and Growth Control, eds. Kahn, P. & Graf, T. (Springer, Heidelberg), pp. 147-153. 12. O'Keefe, E. & Cuatrecasas, P. (1978) J. Membr. Biol. 42, 61-79. 13. Jaffe, E. A., Grulich, J., Weksler, B. B., Hampel, G. & Watanabe, K. (1987) J. Biol. Chem. 262, 8557-8565. 14. Jaffe, E. A., Nachmar, R. L., Becker, C. G. & Minick, R. (1973) J. Clin. Invest. 52, 2745-2756. 15. Heldin, C.-H., Backstrom, G., Ostman, A., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M. & Westermark, B. (1988) EMBO J. 4, 1387-1393. 16. Brown, B. C., Albano, J. B. M., Ekins, R. P., Sgherzi, A. M. & Tampion, W. (1971) Biochem. J. 121, 561-562. 17. Bone, E. A., Fretten, P., Palmer, S., Kirk, C. J. & Michell, R. H. (1984) Biochem. J. 221, 803-811. 18. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. & Evans, R. M. (1982) Nature (London) 298, 240-244. 19. Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. T. & MacIntyre, I. (1984) Nature (London) 308, 746-748. 20. Rosenfeld, M. G., Mermod, J.-J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. & Evans, R. M. (1983) Nature (London) 304, 129-135. 21. Hanko, J., Hardebo, J. E., Kahrstrom, J., Owman, C. & Sundler, F. (1985) Neurosci. Lett. 57, 91-95. 22. Lundberg, J. M., Franco-Cereceda, A., Hua, X., Hokfelt, T. & Fischer, J. A. (1985) Eur. J. Pharmacol. 108, 315-319. 23. Gibbins, I. L., Furness, J. B., Costa, M., MacIntyre, I., Hillyard, C.-J. & Girgis, S. (1985) Neurosci. Lett. 57, 125-130. 24. Kruger, L., Silverman, J. D., Mantyh, P. W., Sterini, C. & Brecha, P. W. (1989) J. Comp. Neurol. 280, 291-302. 25. Dalsgaard, C.-J., Hultgardh-Nilsson, A., Haegerstrand, A. & Nilsson, J. (1989) Regul. Pept. 25, 1-9. 26. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. & MacIntyre, I. (1985) Nature (London) 313, 54-56. 27. Franco-Cereceda, A., Saria, A. & Lundberg, J. M. (1989) Acta Physiol. Scand. 135, 173-187. 28. Kjartansson, J. & Dalsgaard, C.-J. (1987) Eur. J. Pharmacol. 142, 355-358. 29. Kjartansson, J., Dalsgaard, C.-J. & Jonsson, C.-E. (1986) Plast. Reconstr. Surg. 79, 218-221. 30. Levine, J. D., Dardick, S. J., Roizen, M. F., Helms, C. & Basbaum, A. I. (1986) J. Neurosci. 6, 3423-3429. 31. Levine, J. D., Clark, R., Devor, M., Helms, C., Moskowitz, M. & Basbaum, A. 1. (1984) Science 226, 547-549. 32. Hermansson, A., Lindblom, U., Dalsgaard, C.-J. & Bjorklund,
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Calcitonin gene-related peptide stimulates proliferation of human endothelial cells (sensory neurons/angiogenesis/wound healing)
A. HA-GERSTRAND*t, C.-J. DALSGAARD**, B. JONZON§, 0. LARSSON¶,
AND
J. NILSSONII
Departments of *Anatomy and lPharmacology, Karolinska Institute, Stockholm, Sweden; §Department of Clinical Pharmacology, Huddinge University Hospital, Huddinge, Sweden; and Departments of tPlastic Surgery and IlMedicine, Karolinska Hospital, Stockholm, Sweden
Communicated by Viktor Mutt, December 29, 1989 (received for review November 2, 1989)
ABSTRACT The effects of the vasoactive perivascular neuropeptides calcitonin gene-related peptide (CGRP), neurokinin A (NKA), neuropeptide Y (NPY), and vasoactive intestinal polypeptide (VIP) on proliferation of cultured human umbilical vein endothelial cells (HUVECs) were investigated. CGRP was shown to increase both cell number and DNA synthesis, whereas NKA, NPY, and VIP were ineffective. l I-labeled CGRP was shown to bind to HUVECs and this binding was displaced by addition of unlabeled CGRP, suggesting the existence of specific CGRP receptors. The effect of CGRP on formation of adenosine 3',5'-cycdic monophosphate (cAMP) and inositol phosphates (InsP), two intracellular messengers known to be involved in regulation of cell proliferation, was investigated. CGRP stimulated cAMP formation but was without effect on the formation of InsP. Proliferation, as well as cAMP formation, was also stimulated by cholera toxin. Basic fibroblast growth factor stimulated growth without affecting cAMP or InsP formation, whereas thrombin, which increased InsP formation, did not stimulate proliferation. We thus suggest that CGRP may act as a local factor stimulating proliferation of endothelial cells; that the mechanism of action is associated with cAMP formation; and that this effect of CGRP may be important for formation of new vessels during physiological and pathophysiological events such as ischemia, inflammation, and wound healing.
Several different factors have been shown to stimulate the formation of new vessels, angiogenesis, and/or to be mitogenic for cultured endothelial cells. These factors include polypeptide growth factors-i.e., basic and acidic fibroblast growth factor (bFGF, aFGF)-endothelial cell growth factor, which is a precursor form of aFGF, transforming growth factors a and /3, angiogenin, and tumor necrosis factor a (1). Angiogenic factors may be important when released locally by cells adjacent to the endothelium, possibly also produced by the endothelial cells themselves or as circulating factors in plasma. Indirect evidence has suggested trophic effects of peripheral neurons. In the newt, naturally occurring limb regeneration is prevented by damage to the peripheral nerve innervating the limb (2). Intact sensory innervation has been shown to be important for corneal wound healing in the rat (3), and in humans suffering from Parry-Romberg syndrome, a marked hemifacial dystrophia is observed in the area innervated by the trigeminal nerve (4). Although direct evidence from in vivo studies is essentially lacking, recent studies have shown that sensory neuropeptides regulate cell proliferation in vitro. Neurokinin A (NKA) and substance P (SP), which are structurally related, have been shown to induce proliferation of human fibroblasts and rat smooth muscle cells (5). This effect was shown to be parallel with The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
increased phosphatidylinositol (PI) turnover (6). Furthermore, vasoactive intestinal polypeptide (VIP) has been shown to inhibit serum-induced proliferation of rat smooth muscle cells and to stimulate proliferation of human keratinocytes, both events paralleled with adenosine 3',5'-cyclic monophosphate (cAMP) formation (7, 8). In this study, the proliferative effect on cultured human umbilical vein endothelial cells (HUVECs) of the vasoactive neuropeptides calcitonin gene-related peptide (CGRP), NKA, neuropeptide Y (NPY), and VIP was examined by determining the increase in cell number and incorporation of [3H]thymidine. For comparison bFGF, an endothelial cell mitogen (9), was used. Since CGRP and bFGF both stimulated proliferation, theireffects on cAMP formation and PI turnover, two intracellular signal systems that frequently have been found to be involved in regulation of cell proliferation (10, 11), were examined. The proliferative effects of cholera toxin (CT), a potent inducer of cAMP formation (12), and thrombin, which induces breakdown of PI (13), were also investigated.
MATERIALS AND METHODS Cell Culture. HUVECs were isolated and cultured mainly as described by Jaffe et al. (14). Briefly, veins of fresh umbilical cords were rinsed with 50-100 ml of phosphatebuffered saline (PBS) and subsequently filled with a collagenase solution (0.1%; Worthington) and incubated at 370C for 20 min. Harvested cells were centrifuged at 800 x g for 5 min and resuspended in culture medium-i.e., medium 199 (M199; GIBCO) with addition of 20% fetal calf serum (FCS; GIBCO) and antibiotics (penicillin, 50 pug/ml; streptomycin, 50 units/ml) and cultured in gelatin-coated (0.2% gelatin in PBS at +40C for 30 min) culture flasks (Costar). Cell Proliferation Assays. Secondary and tertiary cultures were used for experiments. For determinations of increase in cell number and [3H]thymidine incorporation, HUVECs were transferred to gelatin-coated 4-well plates (1.9 cm2, 104 cells per well; Nunc) and 96-well plates (3 x 103 cells per well; Nunc), respectively. HUVECs were allowed to attach overnight in culture medium and were subsequently stimulated with CGRP, NKA, NPY, VIP (all 100 nM, Peninsula; CGRP was also kindly provided by I. MacIntyre), bFGF (1 ng/ml; kindly provided by California Biotechnology), CT (0.1 nM; Sigma), or thrombin (1 unit/ml; kindly provided by P. T. Larsson, Karolinska Institute) in assay medium-i.e., M199 containing 5% FCS and antibiotics. Cells used for studies of Abbreviations: HUVEC, human umbilical cord vein endothelial cell; CGRP, calcitonin gene-related peptide; SP, substance P; NKA, neurokinin A; VIP, vasoactive intestinal polypeptide; NPY, neuropeptide Y; bFGF, basic fibroblast growth factor; PI, phosphatidylinositol; InsP, inositol phosphate; CT, cholera toxin. tTo whom reprint requests should be addressed at: Department of Anatomy, Karolinska Institute, Box 60400, S-104 01 Stockholm, Sweden.
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DNA synthesis were serum starved for 12 hr prior to 24 hr of stimulation in assay medium containing [3H]thymidine (3 puCi/ml; 1 Ci = 37 GBq; Amersham). A dose-response study on the proliferative effect of CGRP (0.001-100 nM) was performed. Stimulation of [3H]thymidine incorporation by CGRP-containing (0.01-1 nM) medium preincubated with a polyclonal CGRP antibody (2 hr at room temperature; antibody dilution, 1:100; Peninsula) was also examined. Cell number was determined after 4 days of stimulation (medium was renewed every 2nd day) by detachment of cells with a trypsin/EDTA (0.05%/0.01%) solution. Cells were suspended into single cells and counted in a cell counter (Analys Instrument, Stockholm). Incorporation of [3H]thymidine was examined in a Beckman scintillation counter after lysis with Triton X-100 (0.1% in distilled water). CGRP Receptor Binding Assay. Tertiary cultures were grown to subconfluence in 12-well plates (4 cm2 per well; Costar) in culture medium. The binding assay was performed mainly as described by Heldin et al. (15). Briefly, cells were rinsed twice with PBS containing 0.1% bovine serum albumin (Sigma) and triplicate cultures were incubated for 2 hr at +40C in PBS containing 1251-labeled CGRP (125I-CGRP) (1 nM; 70 Bq/fmol; kindly supplied by E. TheodorssonNorheim, Karolinska Hospital) alone or with the addition of unlabeled CGRP (0.001-1000 nM). Cells were rinsed repeatedly with PBS and lysed with Triton X-100 (1%) in distilled water. Radioactivity in lysates was determined in a y-counter
(LKB).
cAMP Formation. Tertiary cultures were grown to subconfluence in 12-well plates (4 cm2 per well; Costar) in culture medium. Cells were rinsed with serum-free M199 and stimulated with CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 nM) in 0.33 ml of serum-free M199 containing a phosphodiesterase inhibitor (Rolipram, 20 ,uM; Schering) for 15 min at 37°C. The effect of forskolin (1 uM) or forskolin (1 ,uM) plus CGRP (100 nM) on cAMP formation was also examined. Reactions were terminated by addition of ice-cold HCl04 to a final concentration of 0.4 M. After addition of HCl04, cells were frozen, thawed, and scraped, and the material was frozen until analysis. The insoluble material was spun down and aliquots from supernatants were neutralized with KOH and Tris base and used for cAMP analysis performed essentially as described by Brown et al. (16). PI Breakdown. Tertiary cultures of HUVECs were grown to subconfluence and incubated for 48 hr with myo-[3H]inositol (10 ACi/ml; specific activity, 14.3 Ci/mmol; NEN) in M199 with 5% FCS. Cells were washed with PBS containing Na2HPO4 (6.5 mM), KH2PO4 (1.5 mM), KCI (2.7 mM), NaCI (137 mM), CaCl2 (1 mM), MgCI2 (1 mM), and Hepes (2 mM). Cells were then incubated for 10 min with 0.6 ml of the same buffer containing 10 mM LiCl and were subsequently stimulated by the addition of 66-,A aliquots of the agonists, resulting in final concentrations of CGRP (0.1 ,uM), bFGF (1 ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). The reaction was terminated by addition of ice-cold HC104 to a final concentration of 0.27 M. The standard procedure for separation of inositol phosphates (InsP) was, with minor modifi-
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FIG. 1. Proliferation of HUVECs examined by means of increased cell number (a), and incorporation of [3H]thymidine (b), after stimulation with CGRP (e, 0.001-100 nM), bFGF (o, 1 ng/ml), or CT (i, 0.1 nM) in M199 with addition of 5% FCS. (a) Cell number was determined after 4 days of culture (medium was renewed twice) by detachment and counting of suspended single cells. (b) Cells were serum starved for 12 hr prior to 24 hr of stimulation with the addition of 3 ,uCi of [3H]thymidine per ml to the medium. Cells were lysed and radioactivity was determined in a scintillation counter. Data points are expressed as % increase [means ± SEM of triplicate (a) and quadruplicate (b) cultures] from control cultures. It was shown that CGRP increased cell number and [3H]thymidine incorporation with a maximum of 45% ± 3.3% (P < 0.01) and 53% ± 6.1% (P < 0.002) at 10 nM and 1 nM, respectively. It was also shown that the CGRP-induced increase in DNA synthesis was reduced by preincubation with a CGRP antibody (o). bFGF (1 ng/ml) increased cell number by 79% ± 6.8% (P < 0.001) and DNA synthesis by 103% ± 8.8% (P < 0.001), whereas CT (0.1 nM) induced a 58% ± 3.2% (P < 0.002) and 75% ± 11.3% (P < 0.001) increase in cell number and DNA synthesis, respectively. For statistical analysis, Student's t test was used. *, P < 0.01; **, P < 0.002; ***, P < 0.001.
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Proc. Natl. Acad. Sci. USA 87 (1990)
cations, carried out as described by Bone et al. (17). Radioactivity was measured in a scintillation counter (LKB).
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RESULTS Cell Proliferation. CGRP, but not NKA, NPY, or VIP (all 100 nM), increased the cell number after 4 days of stimulation. CGRP induced a concentration-dependent increase in cell number as compared to control cultures with a maximal effect of 45% ± 3.3% (P < 0.01) at 10 nM (Fig. la). bFGF (1 ng/ml) and CT (0.1 nM) increased cell number by 79% ± 6.8% (P < 0.001) and 58% ± 3.2% (P < 0.002), respectively (Fig. la), whereas thrombin (1 unit/ml) was ineffective (data not shown). Similar results were obtained when the cells were serum starved for 12 hr and subsequently stimulated in the presence of [3H]thymidine. CGRP induced a concentration-dependent stimulation of [3H]thymidine uptake with a maximum of 53% ± 6.1% (P < 0.002) at 1 nM (Fig. lb). NKA, VIP, and NPY (all 100 nM) were ineffective in stimulating [3H]thymidine incorporation. It was also shown that CGRP-induced [3H]thymidine uptake could be reduced by preincubating the CGRP-containing culture medium with a polyclonal CGRP antibody (Fig. lb). bFGF (1 ng/ml) induced a 103% ± 8.8% (P < 0.001) increase in DNA synthesis and CT (0.1 nM) induced an increase of 75% ± 11.3% (P < 0.001; Fig. lb), whereas thrombin (1 unit/ml) was ineffective in stimulating DNA synthesis. CGRP Receptor Binding. 1251-CGRP (1 nM) was shown to bind to cultured HUVECs (1408 ± 125 cpm per well, corresponding to 0.19 fmol per 105 cells). It was also shown that this binding was reduced in a dose-dependent manner when unlabeled CGRP (0.001-1000 nM) was added (Fig. 2). When a 1000-fold excess of unlabeled CGRP was added 125I-CGRP binding was reduced to 40% ± 5.1% of control binding. cAMP Formation. It was shown that CGRP stimulated cAMP formation in a concentration-dependent manner (Fig. 2). CT (0.1 nM) increased cAMP formation by 330%o ± 13.2% (P < 0.01), whereas bFGF was ineffective (Fig. 3). It was also shown that the effect of CGRP (10 nM), which increased cAMP formation by 168% ± 7.2% (P < 0.01), was potentiated by addition of forskolin (1 ,uM), a compound that commonly enhances receptor-mediated cAMP formation, to 703% + 3.7% of control cultures. Forskolin alone did not significantly
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CGRP, nM FIG. 3. Formation of cAMP by HUVECs after stimulation with CGRP (0.01-100 nM), bFGF (1 ng/ml), or CT (0.1 nM). Subconfluent HUVECs were stimulated in serum-free M199 for 15 min in the presence of agonists and the phosphodiesterase inhibitor Rolipram (20 uM). cAMP formation was determined on supernatants from lysed HUVECs and was assayed by protein saturation essentially as described by Brown et al. (16). Data are presented as means ± SEM of quadruplicate cultures. It was shown that CGRP (o) concentration dependently increased cAMP formation compared to control levels. CT (n, 0.1 nM) increased cAMP formation by 330%o ± 13.2% (P < 0.01), whereas bFGF (o, 1 ng/ml) was ineffective. cAMP formation in control cultures is indicated by the horizontal line. For statistical analysis, Student's t test was used. *, P < 0.01.
change cAMP formation. cAMP formation in control cultures was 0.55 ± 0.15 pmol per 105 cells. PI Breakdown. CGRP (100 nM), CT (0.1 nM), and bFGF (1 ng/ml) were all unable to significantly change InsP levels in HUVECs after 15 min of stimulation, whereas thrombin (1 unit/ml) increased InsP levels by 256% ± 32.2% (P < 0.001; Fig. 4). InsP formation in control cultures corresponded to 523 dpm per 105 cells. As previously shown (13), InsP2 and InsP3 levels were not changed from control levels after 15 min of stimulation (data not shown).
DISCUSSION CGRP was first identified as a polypeptide encoded by the calcitonin gene of the rat (18), and later a human counterpart was isolated and sequenced (19). In addition to thyroid tissue,
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CGRP, nM FIG. 2. Binding of 1251-CGRP to cultured HUVECs. Subconfluent tertiary cultures of HUVECs were incubated (for 2 hr, at +4°C) with 1251-CGRP alone (1 nM) or with the addition of unlabeled CGRP (0.001-1000 nM). Data are expressed as means SEM of triplicate cultures. It was shown that binding of 1251-CGRP in the absence of unlabeled CGRP (set as 100o) was 1408 + 125 cpm per well, corresponding to 0.19 fmol per 105 cells, and that this binding was dose dependently reduced by addition of unlabeled CGRP.
bFGF
CT
Thrombin
FIG. 4. Formation of InsP after stimulation with CGRP (100 nM), bFGF (1 ng/ml), CT (0.1 nM), or thrombin (1 unit/ml). Subconfluent HUVECs were preincubated with LiCl (10 mM) in assay buffer for 10 min and were subsequently stimulated for 15 min after addition of agonists. PIs were analyzed in supernatants from lysed cells and were separated as described by Bone et al. (17). Data are presented as means ± SEM of quadruplicate cultures. It was shown that thrombin (1 unit/ml) increased InsP formation by 256% ± 32.2% (P < 0.001), whereas CGRP (0.1 AM), bFGF (1 ng/mI), and CT (0.1 nM) did not significantly change InsP formation. InsP formation in control cultures is indicated by the horizontal line. For statistical analysis, Student's t test was used. ***, P < 0.001.
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CGRP is widely distributed within nervous tissue (20). In the periphery, CGRP is present in sensory and autonomic nerve fibers, of which the sensory neurons show close relationship to blood vessels (21-23). CGRP-like immunoreactive nerve fibers have also been shown to be intimately related to capillary loops in the papilla of the dermis and also to be abundant in the highly vascularized endometrial stroma (24). Degradation of the basal membrane, proliferation, and migration of endothelial cells is required to complete the formation of new vessels, which is a crucial step in embryogenesis, tumor neovascularization, inflammation, and healing of injured tissue (1). A role of sensory neuropeptides in these events is implicated by several findings (25). CGRP is a potent vasodilator (26) and is released from sensory nerve fibers during ischemia (27). Experimental studies have shown that functionally intact sensory neurons and treatment with CGRP increase survival of ischemic surgical flaps (28, 29). Thus, CGRP released from peripheral nerve endings during ischemia may play a dual role by increasing the blood flow and by stimulating formation of new vessels. Sensory neurons have been shown to participate in both acute and chronic inflammatory events. Capsaicin pretreatment, which depletes the neuropeptide content in sensory neurons, attenuates joint injury in experimental arthritis in the rat (30). The severity of adjuvant-induced arthritis in the rat is increased by SP, which is costored with CGRP in sensory neurons. The severity is also correlated to the density of sensory innervation (31). Since inflammation is associated with both increased turnover of sensory neuropeptides and vessel formation, a release of CGRP may contribute to the vascularization of inflamed tissue. During wound healing, sprouting of sensory nerve fibers in relation to blood vessels, hair follicles, and sweat gland ducts toward the wound surface has been described (32). It was recently shown that SP stimulates monocytes-macrophages to produce tumor necrosis factor a (33), which has been shown to be angiogenic in vivo but does not stimulate proliferation in vitro (34). It is thus an intriguing possibility that sprouting sensory neurons may be important for both directional guidance, via activation of macrophages, and proliferation of endothelial cells. The intracellular mechanisms involved in stimulation of HUVEC proliferation are at present not known. In human dermal capillary cells, CT and the phosphodiesterase inhibitor isobutylmethylxanthine have been shown to stimulate cell proliferation (35). Activation of protein kinase C in capillary cells has been shown to induce elongation of the cells and to reduce the mitogenic response induced by growth factors (36). In this context, it is interesting to note that CGRP has been shown to activate adenylate cyclase in different cells, including HUVECs (37). In porcine aortic endothelial cells, however, endothelial cell growth factor and thrombin, which both stimulate PI breakdown, also appear to be mitogenic (38, 39). The present findings suggest that cAMP formation rather than PI breakdown is associated with proliferation of HUVECs. Whether increased cAMP formation per se stimulates proliferation or the effect is indirect by potentiating a stimulatory effect offactors in serum is not known. However, the presence of serum factors may be regarded as a natural environment for endothelial cells both in vivo and in vitro. CGRP has recently been shown to be trophic for development of the nicotinic receptor at the motor end plate (40) and also to act as a differentiating factor by inducing a dopaminergic phenotype in the mouse olfactory bulb (41). In addition to the previously shown effects of CGRP, our findings suggest that CGRP may also act as a local factor stimulating cell proliferation. The effect of CGRP on endothelial cells
Proc. Natl. Acad. Sci. USA 87 (1990)
suggests a role in angiogenesis, including formation of new vessels in ischemia, inflammation, and wound healing. We thank Professor I. MacIntyre for providing human CGRP (founded by Bartos Foundation) and for valuable discussions; Dr. E. Theodorsson-Norheim for providing 125I-CGRP; Dr. P. T. Larsson for providing thrombin; Ms. H. Vrang, Ms. S. Mattisson, and Ms. M. Lind for excellent technical assistance; and Ms. E. Melander and colleagues at the Department of Gynecology and Obstetrics at the Karolinska Hospital for their help in collecting umbilical cords. This work was supported by grants from the Karolinska Institute, the Swedish Medical Research Council (Grants 7126 and 7464), SRA, Marcus and Amalia Wallenberg's Memorial Foundation, T. Nilsson's Foundation, and Knut and Alice Wallenberg's Foundation. 1. Folkman, J. & Klagsbrun, M (1987) Science 235, 442-447. 2. Singer, M. (1952) Q. Rev. Biol. 27, 169-200. 3. Rook, A., Wilkinson, D. S. & Ebling, F. J. G. (1968) Textbook of Dermatology (Davis, Philadelphia), pp. 475-488. 4. Converse, J. M. (1977) Plastic and Reconstructive Surgery (Saunders, Philadelphia), 2nd Ed., pp. 610-660. 5. Nilsson, J., von Euler, A. & Dalsgaard, C.-J. (1985) Nature (London) 315, 61-63. 6. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, C.-J. (1988) J. Cell. Physiol. 137, 141-145. 7. Hultgirdh-Nilsson, A., Nilsson, J., Jonzon, B. & Dalsgaard, C.-J. (1988) Regul. Pept. 22, 267-274. 8. Hwgerstrand, A., Jonzon, B., Dalsgaard, C.-J. & Nilsson, J. (1989) Proc. NatI. Acad. Sci. USA 86, 5993-5996. 9. Oospodarowicz, D., Brown, K. D., Birdwell, C. R. & Zetter, B. R. (1978) J. Cell Biol. 77, 774-788. 10. Boynton, A. L. & Whitfield, J. F. (1983) Adv. Cyclic Nucleotide Res. 15, 193-294. 11. Berridge, M. (1986) in Oncogenes and Growth Control, eds. Kahn, P. & Graf, T. (Springer, Heidelberg), pp. 147-153. 12. O'Keefe, E. & Cuatrecasas, P. (1978) J. Membr. Biol. 42, 61-79. 13. Jaffe, E. A., Grulich, J., Weksler, B. B., Hampel, G. & Watanabe, K. (1987) J. Biol. Chem. 262, 8557-8565. 14. Jaffe, E. A., Nachmar, R. L., Becker, C. G. & Minick, R. (1973) J. Clin. Invest. 52, 2745-2756. 15. Heldin, C.-H., Backstrom, G., Ostman, A., Hammacher, A., Ronnstrand, L., Rubin, K., Nister, M. & Westermark, B. (1988) EMBO J. 4, 1387-1393. 16. Brown, B. C., Albano, J. B. M., Ekins, R. P., Sgherzi, A. M. & Tampion, W. (1971) Biochem. J. 121, 561-562. 17. Bone, E. A., Fretten, P., Palmer, S., Kirk, C. J. & Michell, R. H. (1984) Biochem. J. 221, 803-811. 18. Amara, S. G., Jonas, V., Rosenfeld, M. G., Ong, E. S. & Evans, R. M. (1982) Nature (London) 298, 240-244. 19. Morris, H. R., Panico, M., Etienne, T., Tippins, J., Girgis, S. T. & MacIntyre, I. (1984) Nature (London) 308, 746-748. 20. Rosenfeld, M. G., Mermod, J.-J., Amara, S. G., Swanson, L. W., Sawchenko, P. E., Rivier, J., Vale, W. W. & Evans, R. M. (1983) Nature (London) 304, 129-135. 21. Hanko, J., Hardebo, J. E., Kahrstrom, J., Owman, C. & Sundler, F. (1985) Neurosci. Lett. 57, 91-95. 22. Lundberg, J. M., Franco-Cereceda, A., Hua, X., Hokfelt, T. & Fischer, J. A. (1985) Eur. J. Pharmacol. 108, 315-319. 23. Gibbins, I. L., Furness, J. B., Costa, M., MacIntyre, I., Hillyard, C.-J. & Girgis, S. (1985) Neurosci. Lett. 57, 125-130. 24. Kruger, L., Silverman, J. D., Mantyh, P. W., Sterini, C. & Brecha, P. W. (1989) J. Comp. Neurol. 280, 291-302. 25. Dalsgaard, C.-J., Hultgardh-Nilsson, A., Haegerstrand, A. & Nilsson, J. (1989) Regul. Pept. 25, 1-9. 26. Brain, S. D., Williams, T. J., Tippins, J. R., Morris, H. R. & MacIntyre, I. (1985) Nature (London) 313, 54-56. 27. Franco-Cereceda, A., Saria, A. & Lundberg, J. M. (1989) Acta Physiol. Scand. 135, 173-187. 28. Kjartansson, J. & Dalsgaard, C.-J. (1987) Eur. J. Pharmacol. 142, 355-358. 29. Kjartansson, J., Dalsgaard, C.-J. & Jonsson, C.-E. (1986) Plast. Reconstr. Surg. 79, 218-221. 30. Levine, J. D., Dardick, S. J., Roizen, M. F., Helms, C. & Basbaum, A. I. (1986) J. Neurosci. 6, 3423-3429. 31. Levine, J. D., Clark, R., Devor, M., Helms, C., Moskowitz, M. & Basbaum, A. 1. (1984) Science 226, 547-549. 32. Hermansson, A., Lindblom, U., Dalsgaard, C.-J. & Bjorklund,
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