Determination of KA values by controlled receptor expression in Xenopus oocytes.
1. In the present study we estimated the KA value of endothelin-1 (ET-1) for ETA-receptors by a new method in which the level of expression of ETA-receptors in Xenopus oocytes was altered in a controlled way. 2. Kvl.2 (a delayed rectifier type K channel) c RNA at the fixed concentration of 0.2 micro g micro l(-1) was mixed with ETA-receptor cRNA at various concentration ratios (10(-3)-3). Oocytes were examined 2-4 days after the injection of the cRNA mixtures. 3. In these oocytes, ET-1 suppressed the amplitude of Kvl.2 current in a dose-dependent manner in the range of 0.1-100 nM; the maximum inhibition produced by ET-1 was larger and the EC50 value for the inhibition by ET-1 was smaller as the mixture ratio was increased. Double-reciprocal plots of equiactive concentrations of ET-1 in 1/1- and 1/30-injected oocytes yielded a KA for ET-1 of 7.4 nM. The number of ETA-receptors in 1/30-injected oocytes was 13% of that in 1/1-injected oocytes, whereas the inhibition of the current in 1/30-injected oocytes was about 60% of that in 1/1-injected oocytes. This suggests the presence of spare receptors of ETA in the latter. 4. A saturation binding experiment estimated a KD value of 0.1 nM for ET-1 at ETA-receptors and the number of ETA-receptors in 1/30-injected oocytes was 23% of that in 1/1-injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and KD, which could be due to factors unique to the expression system employed in the present study.
Murakoshi
H
H
Department of Pharmacology, Tohoku University School of Medicine, Sendai, Japan.
Nunoki
K
K
Ishii
K
K
Taira
N
N
D000704:Analysis of Variance; D000818:Animals; D001667:Binding, Competitive; D004305:Dose-Response Relationship, Drug; D016232:Endothelins / Q000378:metabolism*; D005260:Female; D009865:Oocytes / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D018075:RNA, Complementary / Q000235:genetics / Q000378:metabolism; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000378:metabolism*; D014982:Xenopus laevis
D016232:Endothelins; D018075:RNA, Complementary; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin