Photoaffinity labelling of dopamine receptors in molluscan smooth muscle

Relaxation of catch contraction of the anterior byssus retractor muscle of the sea mussel Mytilus edulis L. by dopamine is mediated through a dopamine receptor but not through adrenoceptors (Takayanagi et al 1981). Photoaffinity labelling is a technique widely used in biochemical in vitro studies to test an interaction between a ligand and its binding site. Therefore, we tried photoaffinity labelling of the dopamine receptor in order to study the dopamine receptor in the anterior byssus retractor muscle of M. edulis. Sea mussels, collected from the east coast of Tokyo Bay were stored in aerated seawater (NaCl 456, KCl 11, CaCl2 2H2O 11, MgCl2 6H2O 48 nM and Tris-HCl 25 mM; pH 7 . 8 to 8 . 0) at 10 degrees C and used within a week of collection. Muscle bundles (about 1 mm in diameter) were dissected from the anterior byssus retractor muscle and suspended in a 10 ml organ bath filled with artificial seawater bubbled with air and kept at 24 to 25 degrees C. Responses to drugs were recorded isotonically under a tension of 0 . 2 g. After the muscle had been exposed to acetylcholine (10(-4)M) for 2 min to induce catch contraction and washed with artificial seawater for 5 min, dopamine was applied. Relaxations following a 10 min exposure to various doses of dopamine were estimated. The response to 3 x 10(-7) M dopamine was considered as the maximum response to obtain dose-response curves (Takayanagi et al 1981). To irradiate the muscle, a Toshiba lamp FL-20E (wavelength: 270 to 350 nm) was used as a light source. The muscle, immersed in artificial seawater containing dopamine (10(-4)M), was irradiated (1 cm from the lamp) for 25 min and then washed with artificial seawater for 60 min (Takayanagi et al 1976). After the muscle was irradiated in the presence of dopamine (10(-4)M) for 25 min and washed for 60 min, the dose-response curve of dopamine was shifted in a parallel way towards doses about 8 times higher (Fig. 1). This inhibition of dopamine-induced responses continued for at least 2 h. The dose-response curve for dopamine was unaffected when the muscle was incubated with both dopamine (10(-4)M) and haloperidol (10(-4)M) for 25 min under the irradiation conditions (Fig. 1). However, the inhibitory action of dopamine was unaffected when the muscle was irradiated in the absence of dopamine and washed for 60 min, suggesting that 20 min irradiation did not influence mechanisms for relaxation of this smooth muscle by dopamine. Furthermore, when the muscle was incubated with dopamine (10(-4)M) or haloperidol (10(-4)M) for 25 min and washed with artificial sea water, the dose-response curve for dopamine was not influenced. When promethazine (10(-4)M), an antihistamine drug found to have no antidopaminergic action in this muscle (Yoshida et al 1981), was used instead of haloperidol (10(-4)M), the dose response curve for dopamine was shifted after irradiation (data not shown). These results indicate the possibility that dopamine is photolysed to a reactive compound which reacts irreversibly with the dopamine receptor.