Molecular cloning, functional characterization of the porcine transient receptor potential V1 (pTRPV1) and pharmacological comparison with endogenous pTRPV1.
In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+]i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+]i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE >> NADA), low pH and noxious heat (>42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.
Ohta
Toshio
T
Laboratory of Pharmacology, Department of Biochemical Science, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. tohta@vetmed.hokudai.ac.jp
Komatsu
Ryuichi
R
Imagawa
Toshiaki
T
Otsuguro
Ken-Ichi
K
Ito
Shigeo
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D002211:Capsaicin / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017931:DNA Primers; D005727:Ganglia, Spinal / Q000166:cytology / Q000187:drug effects; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D018408:Patch-Clamp Techniques; D017386:Sequence Homology, Amino Acid; D013552:Swine; D050916:TRPV Cation Channels / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology*; D014162:Transfection
D017931:DNA Primers; D050916:TRPV Cation Channels; D002211:Capsaicin; D002118:Calcium