Pulmonary, Gastrointestinal and Urogenital Pharmacology Characterization of capsaicin induced responses in mice vas deferens: Evidence of CGRP uptake Majid Sheykhzade a , Saurabh Gupta b, ⁎, Tinne Sørensen a , Ole Aabling Sørensen a , Hans Koch a , Harrie C.M. Boonen a , Ole Back a , Bjarne Fjalland a a Department of Pharmacology and Pharmacotherapy, Faculty of Pharmaceutical Sciences, University of Copenhagen, DK-2100, Copenhagen, Denmark b Department of Neurology, Glostrup Research Institute, Glostrup Hospital, Faculty of Health Science, University of Copenhagen, DK-2600, Glostrup, Denmark abstract article info Article history: Received 26 April 2011 Received in revised form 7 June 2011 Accepted 15 June 2011 Available online 4 July 2011 Keywords: Capsaicin-induced response vas deferens (mouse) Calcitonin gene-related peptide (CGRP) uptake Receptor characterization Schild plot CGRP family of peptides Calcitonin gene-related peptide (CGRP) is extensively distributed in primary afferent sensory nerves, including those innervating the genitourinary tract. Capsaicin can stimulate the release of CGRP from intracellular stores of these nerves, but this phenomenon has not been investigated in-depth in isolated preparations. The present study sets out to study and characterize the capsaicin as well as CGRP-induced responses in isolated mouse vas deferens. The effects of capsaicin and CGRP family of peptides were studied on electrically-induced twitch responses in the absence or presence of transient receptor potential cation channel vanilloid subfamily member 1 (TRPV1) antagonist and CGRP receptor antagonists. Twitch responses were attenuated by capsaicin (1 nM–30 nM) and CGRP family of peptides. The potency order was CGRP N intermedin-long (IMDL) ~ [Cys(Et) 2,7 ]αCGRP ~ adrenomedullin (AM) N [Cys(ACM) 2,7 ]αCGRP N amylin (AMY). These responses were disinhibited by the CGRP receptor antagonists and TRPV1 antagonists. The addition of CGRP receptor antagonists caused a transient potentiation of the twitch response and this potentiation was blocked by pretreatment with capsaicin and enhanced by incubation with exogenous CGRP. During the second consecutive cumulative concentration-response curve with capsaicin, the first phase of concentration-response curve disappeared and this was partially restored when the mouse vas deferens was preincubated with CGRP, suggesting the uptake of exogenous CGRP by nerves. Besides showing capsaicin- induced CGRP releases this study shows that exogenous CGRP can be taken up in vas deferens and can be re- released. CGRP uptake will add another dimension in understanding the homeostasis of this neuropeptide. © 2011 Elsevier B.V. All rights reserved. 1. Introduction Calcitonin gene-related peptide (CGRP) is widely distributed both peripherally and centrally and is found in sensory nerves innervating smooth muscles of different peripheral organs, including the genitourinary tract (Jen et al., 1997; Saito et al., 1987). CGRP is stored in primary afferent sensory nerves and can be released from these stores by chemical or electrical stimulation. Capsaicin, a pungent extract from chili peppers, is an agonist at transient receptor potential cation channel vanilloid subfamily member 1(TRPV1), stimulates the release of glutamate and neuropeptides (CGRP, neurokinin A and substance P) (Gupta et al., 2010; Maggi et al., 1993; Wu et al., 2000). Capsaicin pharmacology is complex due to non-specific effects, and is further complicated by susceptibility of TRPV1 towards desensitiza- tion (Jung et al., 1998). Vas deferens is richly innervated with CGRP positive nerves and CGRP has been reported to inhibit twitch responses in isolated vas deferens across species (Filippelli et al., 1999; Tomlinson and Poyner, 1996). These functional studies have revealed the presence of both TRPV1 receptors and CGRP receptors (Calcrl+RAMP1) in vas deferens. Earlier studies report different functional potencies of CGRP and its peptide family in various tissues (Dennis et al., 1990; Dumont et al., 1997; Longmore et al., 1994; Wisskirchen et al., 1998), which may partially be attributed to the relative expression levels of receptor activity modifying protein (RAMPs 1–3) as well as the difference in receptor density. Analysis of the effects induced by capsaicin or by an individual endogenous peptide is often hampered by the co-release of multiple neuropeptides from sensory nerve endings in many tissues. This is particularly true for tachykinins which are co-released with CGRP and both these peptides induce similar biological effects such as vasodilatation. In this respect, vas deferens offers an advantage as CGRP inhibits and substance P potentiates electrically stimulated twitch responses in this isolated preparation (Al-Kazwini et al., 1986; Moritoki et al., 1987). A few studies have investigated the effect of CGRP in mouse vas deferens segments (Al-Kazwini et al., 1986) using the C-terminal fragment, CGRP 8–37 as a competitive antagonist at CGRP receptors European Journal of Pharmacology 667 (2011) 375–382 ⁎ Corresponding author at: Department of Neurology, Glostrup Research Institute, Glostrup Hospital, Faculty of Health Sciences, Ndr. Ringvej 69, DK-2600 Glostrup, Denmark. Tel.: +45 38 63 30 56; fax: +45 38 63 39 83. E-mail address: SAUGUP01@glo.regionh.dk (S. Gupta). 0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2011.06.031 Contents lists available at ScienceDirect European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar