Multiple molecular forms of tachykinins in rat spinal cord: a study comparing different extraction methods

Various procedures for extraction at acid, neutral and alkaline pH were compared with regard to the yield of different tachykinins and tachykinin-like substances from rat spinal cord. Reverse phase high performance liquid chromatography (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 neurokinin 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 of extraction method in immunochemical studies. Combined extraction in water and acetic acid appears to be a suitable method when the content of peptides with different chemical properties are to be measured in a tissue sample.