Effects of chronic morphine exposure on opioid inhibition of adenylyl cyclase in 7315c cell membranes: a useful model for the study of tolerance at mu opioid receptors.
The effects of prolonged morphine exposure on the mu opioid receptor in 7315c pituitary tumor cell membranes have been examined. Since a low concentration of naloxone reversed the inhibition of forskolin-stimulated adenylyl cyclase induced by the mu-selective agonist, Tyr-D-Ala-Gly-MePhe-Gly-ol (DAGO), and by high concentrations of [D-Pen2-D-Pen5]enkephalin (DPDPE), we suggest that these cells contain a homogeneous population of mu opioid receptors coupled to adenylyl cyclase via a guanyl nucleotide-binding protein. Studies measuring the ability of [D-Ala2-D-Leu5]enkephalin (DADLE), an opioid agonist, to inhibit adenylyl cyclase in cells that had been exposed to 100 microM morphine for varying periods of time, indicated that the agonist no longer inhibited enzyme activity after 5 hr of morphine exposure. Measurements of 3H-antagonist binding in membranes from cells exposed to morphine demonstrated a decreased receptor density after 24 hr of 100 microM morphine exposure with no change in the antagonist affinity. Computer analysis indicated a 20% decrease in the number of mu receptors labeled after 24 hr of morphine exposure and a 60% decrease after 72 hr of exposure. Computer analysis of agonist competition against 3H-antagonist binding confirmed the existence of one binding site with an affinity intermediate between the high and low apparent affinity states observed in membranes from untreated cells. Addition of 10 microM GTP gamma S did not affect the agonist affinity or receptor density in membranes from morphine-treated cells, suggesting that the receptors were uncoupled from G proteins, as observed in 7315c cell membranes that have been treated with pertussis toxin. Thus chronic morphine treatment induced a rapid loss of opioid mu receptor-mediated inhibition of adenylyl cyclase (desensitization), and a more slowly developing reduction in receptor number. The desensitization was accompanied by a loss of guanyl nucleotide regulation of agonist affinity. These findings are comparable to results reported for the delta opioid receptor and the beta-adrenergic receptor upon prolonged agonist exposure.
Puttfarcken
P S
PS
Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Werling
L L
LL
Cox
B M
BM
D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D004174:Diprenorphine / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology; D006150:Guanine Nucleotides / Q000494:pharmacology; D009020:Morphine / Q000494:pharmacology*; D009270:Naloxone / Q000494:pharmacology; D051381:Rats; D011957:Receptors, Opioid / Q000187:drug effects* / Q000378:metabolism; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu
D000067956:Adenylyl Cyclase Inhibitors; D006150:Guanine Nucleotides; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D017450:Receptors, Opioid, mu; D004174:Diprenorphine; D009270:Naloxone; D009020:Morphine; D019204:GTP-Binding Proteins