Development Of Cellular Tolerance Research Articles

Chronic Treatment with Morphine Disrupts Acute Kinase-Dependent Desensitization of GPCRs

Based on studies using mutations of the µ-opioid receptor (MOR), phosphorylation of multiple sites on the C-terminus has been recognized as a critical step underlying acute desensitization and the development of cellular tolerance. The aim of this study is to explore which kinases mediate desensitization of MOR in brain slices from drug-naïve and morphine-treated animals. Whole-cell recordings from locus coeruleus neurons were made, and the agonist-induced increase in potassium conductance was measured. In slices from naïve animals, pharmacological inhibition of G-protein receptor kinase (GRK2/3) with compound 101 blocked acute desensitization. Following chronic treatment with morphine, compound 101 was less effective at blocking acute desensitization. Compound 101 blocked receptor internalization in tissue from both naïve and morphine-treated animals, suggesting that GRK2/3 remained active. Kinase inhibitors aimed at blocking protein kinase C and c-Jun N-terminal kinase had no effect on desensitization in tissue taken from naïve animals. However, in slices taken from morphine-treated animals, the combination of these blockers along with compound 101 was required to block acute desensitization. Acute desensitization of the potassium conductance induced by the somatostatin receptor was also blocked by compound 101 in slices from naïve but not morphine-treated animals. As was observed with MOR, it was necessary to use the combination of kinase inhibitors to block desensitization of the somatostatin receptor in slices from morphine-treated animals. The results show that chronic treatment with morphine results in a surprising and heterologous adaptation in kinase-dependent desensitization. SIGNIFICANCE STATEMENT: The results show that chronic treatment with morphine induced heterologous adaptations in kinase regulation of G protein coupled receptor (GPCR) desensitization. Although the canonical mechanism for acute desensitization through phosphorylation by G protein-coupled receptor kinase is supported in tissue taken from naïve animals, following chronic treatment with morphine, the acute kinase-dependent desensitization of GPCRs is disrupted such that additional kinases, including protein kinase C and c-Jun N-terminal kinase, contribute to desensitization.

Anti-opioid Effects of RFRP-3 on Magnocellular Neuron Activity in Morphine-naïve and Morphine-treated Female Rats.

Neuropeptide FF receptors (NPFFR1 and NPFFR2) have been proposed to possess anti-opioid properties, and be involved in the development of opiate tolerance and dependence. However, there is no evidence to date supporting such opioid effects at the cellular level in vivo. Using in vivo electrophysiological recordings from vasopressin and oxytocin neurons in the supraoptic nucleus, we aimed to determine the effects of NPFFRs on opiate inhibition, tolerance, and dependence at a cellular level. Both vasopressin and oxytocin neurons are acutely inhibited by opioids and develop opiate tolerance. Oxytocin neurons also develop cellular opiate dependence and undergo withdrawal hyperexcitation upon cessation of opiate administration. Here, the classical μ-opioid receptor agonist, morphine robustly inhibited the spontaneous firing rate of vasopressin and oxytocin neurons, and this inhibition was attenuated by pretreatment with the NPFFR1 agonist, RFamide-related peptide-3. In rats infused with morphine for 6 d, vasopressin neurons were unresponsive to morphine, indicating the development of cellular tolerance, but pretreatment with the NPFFR antagonist, GJ14, restored acute morphine inhibition. In morphine-infused rats, RFamide related peptide-3 did not induce withdrawal excitation in oxytocin neurons and GJ14 did not reverse naloxone-precipitated withdrawal excitation. This is the first evidence of anti-opioid effects of the NPFFR system at a cellular level in vivo. Our results suggest that the anti-opioid properties of the NPFFR system reduce morphine sensitivity during tolerance but that it is not involved in dependence.

Functional tolerance to ethanol in mice: relationship to lipid metabolism

Abstract The development of functional tolerance to ethanol was studied in young adult mice of the TO Swiss and C57BL strains. Ethanol administration was by intermittent inhalation for 7 h and the development of tolerance was assessed by estimating blood ethanol concentrations at successive losses of the righting reflex. The administration of (±)α-tocopherol at a dose previously shown to inhibit the effect of ethanol on peripheral phospholipid composition (750 mg kg−1 i.p.) was without effect on functional tolerance. Chronic administration of DL-carnitine, 7% w/w in diet for 10 days, a treatment previously shown to prevent ethanol-induced triglyceride accumulation, was also without effect on ethanol tolerance. Administration of the cholesterol synthesis inhibitor, diazacholesterol (30 mg kg−1 i.p.) 3 times per week for 3 weeks from weaning, markedly inhibited the subsequent development of functional tolerance. Administration of the inhibitor of protein synthesis, cycloheximide (300 μg kg−1 i.p.) greatly inhibited the development of ethanol tolerance whereas actinomycin D (100 μg kg−1 i.p.) was without effect. The results are discussed in relation to the hypothesis that ethanol cellular tolerance results from adaptation in the composition of the lipids of neuronal membranes. It is concluded that synthesis of some protein is involved in the development of cellular tolerance and also that cholesterol plays some part in the development of tolerance.

Genetic influences on the rate of development of ethanol tolerance and the ethanol physical withdrawal syndrome in mice.

Abstract A technique is described with which the development of cellular tolerance to ethanol can be followed in mice inhaling ethanol vapour. Cellular tolerance to ethanol's effects on either loss of righting reflex or loss of rotarod performance develops surprising rapidly (approximately 5 hours) in TO Swiss mice. When a comparison was made between strains, mice of the C57BL/6J strain were observed to show very rapid development of cellular tolerance, whereas mice of the DBA2 strain became tolerant to ethanol much more slowly. TO Swiss mice were intermediate. Groups of mice of each strain were then exposed to ethanol vapour for a period of 10 days, such that similar concentrations of ethanol were obtained in the blood of individuals of each strain. Ethanol was withdrawn after this time, and all mice investigated for signs of the ethanol physical withdrawal syndrome. Mice of the C57BL/6J strain showed a brief and mild withdrawal syndrome. DBA2 mice exhibited a severe and prolonged syndrome, whereas mice of the TO Swiss strain were again intermediate. It is suggested that the genetically determined capacity to adapt rapidly to the presence of ethanol (rapid cellular tolerance) is related to the ability to adapt rapidly to the removal of the drug, and that this limits the duration and severity of the ethanol withdrawal syndrome.