TY - JOUR
T1 - Coupling of the cloned rat κ-opioid receptor to adenylyl cyclase is dependent on receptor expression
AU - Hirst, R. A.
AU - Hirota, K.
AU - Grandy, D. K.
AU - Lambert, D. G.
PY - 1997/8/29
Y1 - 1997/8/29
N2 - This study describes the coupling of the recombinant rat κ-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4-7) and late (p14-17) passage after transfection. As passage increased the receptor numbers (B(max)) declined from 231 ± 24 (early) to 31 ± 2 fmol/mg protein (late) but the equilibrium dissociation constant (K(d)) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (Ki(H)) and low (Ki(L)) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% Ki(L):50% Ki(H)), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (±)bremazocine, ICI-204,448 and (±)trans-U-50488 methanesulfonate (1 μM), compounds with activity at κ-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not κ- agonist specific. Collectively, these data suggest that in CHO cells, κ- opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.
AB - This study describes the coupling of the recombinant rat κ-opioid receptor expressed in Chinese hamster ovary (CHO) cells to adenylyl cyclase and the effects of receptor density. The binding of [3H]diprenorphine ([3H]DPN) was dose dependent and saturable in membranes prepared from cells of early (p4-7) and late (p14-17) passage after transfection. As passage increased the receptor numbers (B(max)) declined from 231 ± 24 (early) to 31 ± 2 fmol/mg protein (late) but the equilibrium dissociation constant (K(d)) did not change. Spiradoline dose dependently displaced [3H]DPN from membranes prepared from early and late cells revealing both high (Ki(H)) and low (Ki(L)) affinity binding sites. There were no significant differences in the proportion of these sites (approximately 50% Ki(L):50% Ki(H)), and whilst spiradoline was generally less potent in late cells the differences were small and failed to reach statistical significance. In contrast, spiradoline produced a dose dependent inhibition of forskolin stimulated cAMP formation in whole cells with pIC50 of 8.62 and 8.00 in early compared with late cells. In addition, the maximum inhibition was dramatically reduced from 47 to 22%. Etorphine, (±)bremazocine, ICI-204,448 and (±)trans-U-50488 methanesulfonate (1 μM), compounds with activity at κ-receptors, produced a greater inhibition of cAMP formation in early (42.2, 45.8, 50.2 and 50.5%, respectively) than late (12.9, 11.8, 13.5 and 7.8%, respectively) cells, indicating that expression dependent inhibition of cAMP formation was not κ- agonist specific. Collectively, these data suggest that in CHO cells, κ- opioid receptor coupling to adenylyl cyclase is dependent on receptor expression levels.
KW - Adenylyl cyclase
KW - Chinese hamster ovary cells
KW - Recombinant κ-opioid receptor
KW - Spiradoline
KW - cAMP
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U2 - 10.1016/S0304-3940(97)00594-6
DO - 10.1016/S0304-3940(97)00594-6
M3 - Article
C2 - 9302101
AN - SCOPUS:0030848732
SN - 0304-3940
VL - 232
SP - 119
EP - 122
JO - Neuroscience Letters
JF - Neuroscience Letters
IS - 2
ER -