3H-neurokinin A labels a specific tachykinin-binding site in the rat duodenal smooth muscle.
3H-Neurokinin A (3H-NKA) with high specific activity (75 Ci/mmol) was synthesized to study NKA (NK-2)-binding sites on membrane preparations of various tissues in the rat, including brain, spinal cord, duodenum, vas deferens, and ileum. The binding capacity of 3H-NKA (0.9 nM) was very low in membrane preparations of different central nervous system regions and the ileum smooth muscle (0.2-2 fmol/mg of protein). In contrast, relatively high specific binding was found in membrane suspensions of the rat duodenal smooth muscle (18 fmol/mg of protein) and the vas deferens (8 fmol/mg of protein). 3H-NKA-binding sites were further characterized on the rat duodenal smooth muscle. The specific binding of 3H-NKA was shown to be temperature dependent, saturable, reversible, and increased in parallel with the protein concentration. Scatchard analyses and Hill plots of equilibrium binding studies in the concentration range of 0.40-30 nM revealed that 3H-NKA bound to a single class of noninteracting binding sites (Bmax = 270 fmol/mg of protein, KD = 13.3 nM). Displacement of 3H-NKA with different tachykinin-related peptides gave the following rank order of potencies: NKA greater than NKA (4-10) greater than kassinin greater than eledoisin greater than NKB much greater than substance P greater than physalaemin, which suggests that the binding site labeled by 3H-NKA is different from substance P (NK-1)-and NKB (NK-3)-binding sites. The biological activities of tachykinins and related peptides were tested by measuring their contractile effects on the rat duodenum and rabbit pulmonary artery, two tissues known to be sensitive for NKA. Ki values were correlated with the EC50 obtained in biological assays. The results revealed a significant correlation (r = 0.86, p less than 0.01) between Ki and EC50 values obtained in the isolated rabbit pulmonary artery, whereas there was no significant correlation between binding affinities and biological responses on the rat duodenum (r = 0.62, p greater than 0.05).
Bergström
L
L
Chaire de Neuropharmacologie, INSERM U.114, Collège de France.
Beaujouan
J C
JC
Torrens
Y
Y
Saffroy
M
M
Glowinski
J
J
Lavielle
S
S
Chassaing
G
G
Marquet
A
A
D'Orleans-Juste
P
P
Dion
S
S
D000818:Animals; D004386:Duodenum / Q000032:analysis* / Q000187:drug effects; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D007700:Kinetics; D009130:Muscle, Smooth / Q000032:analysis*; D015288:Neurokinin A; D009479:Neuropeptides / Q000378:metabolism* / Q000494:pharmacology; D011651:Pulmonary Artery / Q000187:drug effects; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D017981:Receptors, Neurotransmitter / Q000032:analysis*; D018039:Receptors, Tachykinin; D013535:Suspensions; D015320:Tachykinins; D014316:Tritium
D007457:Iodine Radioisotopes; D009479:Neuropeptides; D017981:Receptors, Neurotransmitter; D018039:Receptors, Tachykinin; D013535:Suspensions; D015320:Tachykinins; D014316:Tritium; D015288:Neurokinin A