TABLE 1.

Receptor phosphorylation but not β-arrestin2 contributes to PKCϵ activation

WTMEF and BKOMEF cells were used. The AdOPRM1 and Ad3A were used to express receptor to about 0.5 pmol/mg protein. For ERK activation, cells were treated with 1 μm morphine, 10 nm etorphine, 10 nm fentanyl, and 1 μm DAMGO for 5 min. The amounts of phosphorylated ERK were normalized against those of total ERK. The results were further normalized against those under control condition. For receptor desensitization, cells were treated with 100 nm morphine, 1 nm etorphine, 1 nm fentanyl, and 100 nm DAMGO for 30 min. Normally ADP, but not the agonists, can induce [Ca²⁺]_i release. However, the agonists can potentiate the [Ca²⁺]_i release induced by ADP. Thus the abilities of agonists to induce this potentiation were used to indicate their abilities to activate receptor, and the percentage decrease in the potentiation was used to indicate the desensitization. The “% inh.” was calculated by using the formula: 100% - result in DMSO-treated cells/result in PKCϵi-treated cells. N/S suggests the effect of PKCϵi was not significant. N/A suggests the agonist did not induce signaling in DMSO group, therefore no decrease can be calculated.

ERK phosphorylation (% of basal level)
	WTMEF cells		BKOMEF cells
	AdOPRM1	Ad3A	AdOPRM1	Ad3A
Morphine
DMSO	178 ± 13	183 ± 7	176 ± 12	173 ± 8
PKCϵi	111 ± 7	97 ± 15	105 ± 9	103 ± 11
% Inh.	86	104	93	96
Etorphine
DMSO	183 ± 12	154 ± 13	113 ± 14	181 ± 16
PKCϵi	175 ± 14	116 ± 8	105 ± 12	118 ± 12
% Inh.	N/S	70	N/A	78
Fentanyl
DMSO	174 ± 8	184 ± 11	138 ± 10	174 ± 9
PKCϵi	185 ± 11	108 ± 13	123 ± 17	104 ± 7
% Inh.	N/S	90	N/A	95
DAMGO
DMSO	181 ± 10	153 ± 8	111 ± 7	183 ± 7
PKCϵi	174 ± 8	119 ± 6	109 ± 11	106 ± 11
% Inh.	N/S	64	N/A	93

Receptor Desensitization on[Ca2+]i (% decrease in basal activity)
	WTMEF cells		BKOMEF cells
	AdOPRM1	Ad3A	AdOPRM1	Ad3A
Morphine
DMSO	36 ± 12	43 ± 7	43 ± 7	38 ± 11
PKCϵi	6 ± 5	3 ± 4	2 ± 6	2 ± 7
% Inh.	83	93	95	95
Etorphine
DMSO	49 ± 7	26 ± 6	1 ± 7	45 ± 6
PKCϵi	42 ± 12	9 ± 7	9 ± 10	7 ± 3
% Inh.	N/S	65	N/A	84
Fentanyl
DMSO	45 ± 6	33 ± 12	17 ± 11	44 ± 5
PKCϵi	34 ± 10	11 ± 6	2 ± 5	5 ± 8
% Inh.	N/S	67	N/A	89
DAMGO
DMSO	37 ± 11	29 ± 12	6 ± 3	41 ± 14
PKCϵi	41 ± 9	9 ± 8	5 ± 9	8 ± 10
% Inh.	N/S	69	N/A	80