Research report Neuronal expression and regulation of CGRP promoter activity following viral gene transfer into cultured trigeminal ganglia neurons Paul L. Durham a , Penny X. Dong b , Kevin T. Belasco c , Jeffrey Kasperski b , William W. Gierasch b , Lars Edvinsson d , Donald D. Heistad c,e,f , Frank M. Faraci c,e , Andrew F. Russo b, * a Department of Biology, Southwest Missouri State University, Springfield, MO 65804, USA b Department of Physiology and Biophysics, University of Iowa, Iowa City, IA 52242, USA c Department of Pharmacology, University of Iowa, Iowa City, IA 52242, USA d Department of Internal Medicine, Lund University Hospital, S-221 85, Lund, Sweden e Department of Internal Medicine, University of Iowa, Iowa City, IA 52242, USA f Veterans Administration Medical Center, Iowa City, IA 52242, USA Accepted 10 November 2003 Abstract We have examined the regulation of calcitonin gene-related peptide (CGRP) promoter activity in primary cultures of rat trigeminal ganglia neurons. A viral vector was used to circumvent the potential complication of examining only a small subpopulation of cells in the heterogeneous cultures. Infection with high titers of recombinant adenovirus containing 1.25 kb of the rat CGRP promoter linked to the h- galactosidase reporter gene (AdCGRP – lacZ) yielded expression in about 50% of the CGRP-expressing neurons. The CGRP – lacZ reporter gene was preferentially expressed in neurons, with 91% co-expression with endogenous CGRP. In contrast, an adenoviral vector containing a CMV – lacZ reporter was predominantly expressed in non-neuronal cells, with only 29% co-expression with CGRP. We then asked whether the CGRP promoter in the viral vector could be regulated by serotonin receptor type 1 (5-HT 1 ) agonists. Promoter activity was decreased two- to threefold by treatment with five 5-HT 1B/D agonists, including the triptan drugs sumatriptan, eletriptan, and rizatriptan that are used for migraine treatment. As controls, CMV promoter activity was not affected, and 5-HT 1B/D receptor antagonists blocked the repression caused by sumatriptan and eletriptan. Thus, adenoviral gene transfer can be used in trigeminal ganglia neurons for studying the mechanisms of triptan drug action on CGRP synthesis. D 2003 Elsevier B.V. All rights reserved. Keywords: Calcitonin gene-related peptide; Adenovirus; Gene expression; Trigeminal ganglia; Migraine; 5-HT 1 receptor 1. Introduction Calcitonin gene-related peptide (CGRP) is a key neuro- peptide involved in regulating vascular tone in the peripheral and cerebral circulation [3,24,30]. The primary source of CGRP in the cerebral vasculature is the trigeminal ganglia [19,22]. These sensory neurons serve as a link between cerebral blood vessels and the brain stem. Activation of trigeminal neurons leads to elevated levels of CGRP and other peptides during the neurogenic inflammation that has been proposed as a cause of migraine pain [9,12,21]. Indeed, elevated CGRP levels have been documented in the external jugular outflow of migraine patients [11,12]. Interestingly, 5- HT 1 agonists such as sumatriptan, which are effective drugs for migraine and cluster headaches, have been reported to reduce serum CGRP levels, coincident with the alleviation of pain [9,21]. However, the mechanisms controlling CGRP expression in trigeminal ganglia neurons are largely not known. To study CGRP promoter regulation in primary cultures of trigeminal ganglia sensory neurons, we have constructed an adenovirus containing the lacZ reporter gene under control of the rat CGRP promoter (AdCGRP– lacZ). The CGRP promoter used in this study contains cell-specific responsive elements that drive expression in endocrine 0006-8993/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2003.11.005 * Corresponding author. Tel.: +1-319-335-7872; fax: +1-319-335- 7330. E-mail address: andrew-russo@uiowa.edu (A.F. Russo). www.elsevier.com/locate/brainres Brain Research 997 (2004) 103 – 110