Effect of cold storage on cholinergic responses induced by electrical field stimulation in human bronchi Magnus Ba ¨ck a, * , Maria Costantino b , Charles Brink c , Xavier Norel c a Center for Molecular Medicine, Karolinska University Hospital, L8:03, 171 76 Stockholm, Sweden b Department of Experimental Medicine, 2nd University of Naples, via Costantinopoli 16-80138 Naples, Italy c INSERM u698, Centre Hospitalier Universitaire X Bichat, 46 rue Henri Huchard, 75870 Paris, France Received 27 February 2004; revised 9 February 2005; accepted 18 February 2005 Abstract The aim of the present study was to examine the effects of cold storage on the responses induced by electrical field stimulation (EFS) in human bronchial preparations. Responses induced by EFS and acetylcholine were studied in human bronchial rings mounted in organ baths, either on the day of surgery or after storage at 4 8C in Krebs–Henseleit solution for 24 and 48 h, respectively. The responses induced by EFS were studied at different voltages (20, 40 and 60 V) and at a range of frequencies (2, 4, 8, 10, 30 and 60 Hz). EFS induced a triphasic response, consisting of a cholinergic contraction, followed by a relaxation and subsequently a slow sustained contraction. The amplitude of the EFS- induced response was enhanced with increasing voltages and increasing frequencies. None of the three EFS-induced phases were significantly altered by cold storage at 24 h, whereas storage for 48 h significantly decreased the reactivity of the preparations. Likewise, the contractions induced by acetylcholine were unaltered after 24 h, but significantly depressed after 48 h. These results suggest that the reactivity of human bronchial preparations to EFS is not altered when tissues are conserved for 24 h, whereas prolonged storage should be avoided. q 2005 Elsevier Ltd. All rights reserved. Keywords: Electrical field stimulation; Cholinergic contractions; Human bronchi 1. Introduction Although cholinergic control of airway tone is the predominant regulator in both human and animal airways, there are other major species differences in the neural regulation of airway tone [1], making extrapolations of findings in animal studies to humans difficult. For example, adrenergic inhibitory nerves are present in the guinea pig trachea as well as in canine and feline airways, but not in humans [1–4]. Such species differences make human tissue essential for functional studies of airway pharmacology. However, human tissue is difficult to obtain for experimen- tal purposes and is often available in large quantities subsequent to pneumectomy. In order to take advantage of as much tissue as possible when available, a potential experimental procedure is to store part of the tissue for subsequent experiments. A limited number of studies have previously examined the effect of storage on physiological responses induced by different exogenous bronchoconstrictor agents in isolated human bronchi, showing that the sensitivity and contrac- tility to methacholine (storage at 4 8C for 55 h [5]), acetylcholine (ACh), histamine or anti-IgE (storage at 4 8C for 12 h; [6,7]) are not modified. Furthermore, storage of human bronchial preparations at K196 8C for 30 days in foetal calf serum and dimethyl sulfoxide preserves most of their functional (contraction/relaxation) properties after stimulation with exogenous agonists [8,9], whereas the response to antigen has been reported not to resist such cryopreservation [10]. Although these studies indicate that the contractile apparatus in human airways may resist storage, no investigations have addressed the effects of storage on endogenously released neurotransmitters in human airways. Pulmonary Pharmacology & Therapeutics 19 (2006) 297–302 www.elsevier.com/locate/ypupt 1094-5539/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2005.02.014 Abbreviations ACh, acetylcholine; ChE, cholinesterase; D0, Day 0; D1, Day 1; D2, Day 2; EFS, electrical field stimulation; NANC, non-adrenergic non-cholinergic. * Corresponding author. Tel.: C46 8 51 77 64 20; fax: C46 8 31 31 47. E-mail address: magnus.back@cmm.ki.se (M. Ba ¨ck).