Molecular characterization and functional expression of a substance P receptor from the sympathetic ganglion of Rana catesbeiana.
Substance P is an important neuropeptide neurotransmitter in the central, autonomic and enteric nervous systems. In sympathetic ganglia, substance P is thought to play a role in modulating synaptic transmission. Release of substance P by neuronal stimulation or direct application of substance P to ganglionic neurons increases neuronal excitability. An amphibian substance P receptor complementary DNA has been cloned and characterized from bullfrog, Rana catesbeiana, sympathetic ganglion complementary DNA libraries. The deduced primary structure contains features indicative of a seven transmembrane domain G-protein-coupled receptor. The deduced protein sequence shows 69% identity to previously cloned mammalian substance P receptors. In situ hybridization analysis performed on bullfrog sympathetic ganglia using digoxigenin-labelled complementary RNA probe demonstrated that approximately 75% of the principal neurons displayed reaction product above background levels. Radioligand binding studies were performed on stably transfected cells with [(125)I]Tyr-1-substance P as the ligand. Substance P had an IC50 of 16 nM and the agonist potency profile was substance P>neurokinin A >> neurokinin B. The order of potency for three tachykinins to increase intracellular calcium when applied to a stably transfected clonal cell line was substance P>neurokinin A >> neurokinin B. This order of agonist potency also held for inhibition of the M-type potassium current in intact bullfrog sympathetic neurons. The non-peptide substance P antagonists CP-96345 and RP-67580 at concentrations that block mammalian substance P receptors had little or no effect on the responses to substance P at the bullfrog receptor. Overall, these results demonstrate that the cloned sequence has the features consistent with and characteristic of a substance P receptor. The results are discussed with reference to the established pharmacology of the bullfrog substance P receptor and known structure activity relationships of mammalian tachykinin receptors.
Simmons
M A
MA
Department of Pharmacology, Marshall University School of Medicine, Huntington, West Virginia, USA.
Brodbeck
R M
RM
Karpitskiy
V V
VV
Schneider
C R
CR
Neff
D P
DP
Krause
J E
JE
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects; D006224:Cricetinae; D004305:Dose-Response Relationship, Drug; D005728:Ganglia, Sympathetic / Q000187:drug effects / Q000378:metabolism*; D017403:In Situ Hybridization; D008297:Male; D008969:Molecular Sequence Data; D011869:Radioligand Assay; D011892:Rana catesbeiana; D018040:Receptors, Neurokinin-1 / Q000187:drug effects* / Q000235:genetics*; D013373:Substance P / Q000494:pharmacology*
D018040:Receptors, Neurokinin-1; D013373:Substance P