Low-speed subcellular fractionation method for determining noxious stimulus-evoked spinal neurokinin-1 receptor internalization.
Substance P release from nociceptive primary afferents activates post-synaptic neurokinin-1 (NK-1) receptors causing subsequent NK-1 receptor internalization. Fluorescent immunohistochemistry is typically used to quantify NK-1 receptor internalization, an indirect measure of substance P (SP) release. However, this technique entails several limitations that restrict its application. Using simple subcellular fractionation and immunoblotting methods, we demonstrate that intrathecal SP invokes a rapid and dose-dependent increase in dorsal horn cytoplasmic NK-1 receptors. We also show that hind paw compression and noxious thermal stimulation increase cytoplasmic NK-1 receptor, when compared to sham stimulations. Fluorescent immunohistochemistry confirmed that increases in cytoplasmic NK-1 corresponded with increased NK-1 receptor internalization. Herein, we report that low-speed centrifugation and Western immunoblotting provide NK-1 internalization results consistent with those obtained by more traditional methods. These data support previous findings demonstrating a role for spinal NK-1 receptors in nociceptive processing.
Jones
Toni L
TL
University of California-San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0818, USA. tljones@ucsd.edu
Hefferan
Michael P
MP
Marsala
Martin
M
Sorkin
Linda S
LS
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D004305:Dose-Response Relationship, Drug; D007839:Functional Laterality; D006930:Hyperalgesia / Q000139:chemically induced / Q000378:metabolism* / Q000503:physiopathology; D008297:Male; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000378:metabolism*; D013116:Spinal Cord / Q000378:metabolism* / Q000648:ultrastructure; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D013373:Substance P
D018040:Receptors, Neurokinin-1; D013373:Substance P