Determination of mu-, delta- and kappa-opioid receptors in forebrain cortex of rats exposed to morphine for 10 days: Comparison with animals after 20 days of morphine withdrawal
Abstract
Background Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold).
The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal.
Changes of downstream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on mu-, delta- and kappa-OR protein levels in FBC.
Methods Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group + M10) or 20 days (group + M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+ DTT) conditions, and analyzed for the content of mu-, delta- and kappa-OR by immunoblotting with C-and N-terminus oriented antibodies.
Results Significant down-regulation of delta-OR form exhibiting M-w approximate to 60 kDa was detected in PNS prepared from both (+ M10) and (+ M10/-M20) rats. However, the total immunoblot signals of mu-, delta- and kappa-OR, respectively, were unchanged.
Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats.
Conclusions In FBC, prolonged exposure of rats to morphine results in minor (delta-OR) or no change ( mu- and kappa-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal.