Paradoxical relationship between RAVE (relative activity versus endocytosis) values of several opioid receptor agonists and their liability to cause dependence

Abstract

Aim: To examine the relationship between the RAVE (relative activity versus endocytosis) values of opiate agonists and their dependence liability by studying several potent analgesics with special profiles in the development of physical and psychological dependence.

Methods: The effects of (-)-cis-(3R,4S,2'R) ohmefentanyl (F9202), (+)-cis-(3R,4S,2'S) ohmefentanyl (F9204), dihydroetorphine (DHE) and morphine on [(35)S]GTP gamma S binding, forskolin-stimulated cAMP accumulation, and receptor internalization were studied in CHO cells stably expressing HA-tagged mu-opioid receptors (CHO-HA-MOR). cAMP overshoot in response to the withdrawal of these compound treatments was also tested.

Results: All four agonists exhibited the same rank order of activity in stimulation of [(35)S]GTP gamma S binding, inhibition of adenylyl cyclase (AC) and induction of receptor internalization: DHE>F9204>F9202>morphine. Based on these findings and the previous in vivo analgesic data obtained from our and other laboratories, the RAVE values of the four agonists were calculated. The rank order of RAVE values was morphine>F9202>F9204>DHE. For the induction of cAMP overshoot, the rank order was F9202>or=morphine>F9204>or=DHE.

Conclusion: Taken in combination with previous findings of these compounds' liability to develop dependence, the present study suggests that the agonist with the highest RAVE value seems to have a relatively greater liability to develop psychological dependence relative to the agonist with the lowest RAVE value. However, the RAVE values of these agonists are not correlated with their probability of developing physical dependence or inducing cAMP overshoot, a cellular hallmark of dependence.

Figure 1

The effects of F9202, F9204, DHE, and morphine on the stimulation of [³⁵S]GTPγS binding to membranes of CHO-HA-MOR cells. [³⁵S]GTPγS binding was performed as described under Materials and methods. Data obtained at each drug concentration were normalized to the percentage of the basal [³⁵S]GTPγS binding. Each data point represents the mean±SEM of at least three independent experiments conducted in triplicate. EC₅₀ values are shown in Table 1.

Figure 2

The effects of F9202, F9204, DHE, and morphine on forskolin-stimulated cAMP accumulation in CHO-HA-MOR cells. CHO-HA-MOR cells were assayed for the inhibition of cAMP accumulation using F9202, F9204, morphine, and DHE as described under Materials and methods. The forskolin-stimulated intracellular cAMP level in the absence of opioid agonists was defined as 100%, and the percentage of cAMP inhibition was calculated from the forskolin control value. Each data point represents the mean±SEM of at least three independent experiments conducted in triplicate. IC₅₀ values are shown in Table 1.

Figure 3

The effects of F9202, F9204, DHE, and morphine on μ-opioid receptors endocytosis. Flow cytometric analysis was employed as described under Materials and methods. The cells were treated with various concentrations of drugs for 1 h at 37 °C. Values are the mean±SEM of triplicate determinations in a representative experiment. EC₅₀ values are shown in Table 1.

Figure 4

The effects of chronic F9202, F9204, DHE, and morphine on cAMP overshoot in CHO-HA-MOR cells. CHO-HA-MOR cells were pretreated with morphine (1 μmol/L), F9202 (1 μmol/L), F9204 (1 μmol/L), and DHE (1 μmol/L) for 24 h at 37 °C. The intracellular cAMP levels were determined as described in the Materials and methods in the presence of 10 μmol/L naloxone. Forskolin-stimulated cAMP accumulation in the absence of opioid agonist was defined as 100%, and the percentage of intracellular cAMP level was calculated from the forskolin control value. Data are presented as means±SEM of at least three independent experiments performed in triplicate. ^cP<0.001 vs DHE-treated group. (one-way ANOVA, Tukey post tests).