ELSEVIER Regulatory Peptides 52 (1994) 97-110 Multiple molecular forms of tachykinins in rat spinal cord: a study comparing different extraction methods Ernst Brodin a,,, Annika Ros~n a, Elvar Theodorsson b, Alfred Jonczyk c, Bengt E.B. Sandberg d, Kerstin Brodin e aDepartment of Pharmacology, Karolinska Institutet, Box 60 400, S-104 01, Stockholm, Sweden b Department of Clinical Chemistry, Karolinska Hospital. Stockholm, Sweden CE. Merck, Darmstadt, Germany d H. Lundbeck A/S, Copenhagen, Denmark eDepartment of Clinical Pharmacology, Karolinska Hospital, Stockholm, Sweden (Received 14 July 1993; revised version received 22 March 1994; accepted 14 April 1994) Abstract Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of differ- ent tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromato- graphy (RP-HPLC) and radioimmunoassay with various C-terminally directed tachykinin antisera and a newly developed N-terminally directed substance P (SP)-antiserum (SPN 1) were used. Antiserum SPN 1 fully reacts with SP-analogues modified at the C-terminal end (SP free acid and SP-Gly-Lys) and also (77%) with SP(1-9) but not with C-terminal SP-fragments lacking 2 or more N-terminal amino acids. The highest levels of SP-like immunoreactivity (LI) and neu- rokinin A (NKA)-LI were measured after combined water and acetic acid extraction procedures. Also when measuring cholecystokinin-like immunoreactivity the highest level was obtained following this extraction procedure. RP-HPLC revealed a major component of SP-LI at the position of synthetic SP irrespectively of the extraction method and if the C- or N-terminally directed antiserum was used. Neutral water extracts contained a late eluting component detected with the C-terminally, but not with the N-terminally, directed antiserum. Acid and alkaline extracts, in contrast, contained components which could be detected with the N-terminally, but not with the C-terminally, directed SP-antiserum. Immunoreactive components eluting at the position of NKA and NKB were found in all types of extracts with NKA-, kassinin- and eledoisin-antisera. The NKB- and neuropeptide K (NPK)-components were more prominent in acid than in neutral and alkaline extracts. In conclusion, the present results indicate that rat spinal cord may contain molecular forms of tachykinin-like immunoreactivity in addition to those previously described and illustrate the importance of the choice * Corresponding author. Fax: + 46 8 317135. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSD1 0167-0 1 1 5(94)00036-W