Article date: October 1995
By: Hideyuki Murakoshi, Kazuo Nunoki, Kuniaki Ishii, Norio Taira, in Volume 116, Issue 3, pages 2062-2066
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.
Kvl.2 (a delayed rectifier type K channel) cRNA at the fixed concentration of 0.2 μg μl−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.
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.
A saturation binding experiment estaimated 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 1KD, which could be due to factors unique to the expression system employed in the present study.
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.
Kvl.2 (a delayed rectifier type K channel) cRNA at the fixed concentration of 0.2 μg μl−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.
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.
A saturation binding experiment estaimated 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 1KD, which could be due to factors unique to the expression system employed in the present study.
DOI: 10.1111/j.1476-5381.1995.tb16412.x
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