Voltage-clamp evidence of GABAA receptor subunit-specific effects: pharmacodynamic fingerprint of chlornordiazepam, the major active metabolite of mexazolam, as compared to alprazolam, bromazepam, and zolpidem.

BACKGROUND: Anxiolytic benzodiazepines, due to their clinical effectiveness, are one of the most prescribed drugs worldwide, despite being associated with sedative effects and impaired psychomotor and cognitive performance. Not every GABAA receptor functions in the same manner. Those containing α1 subunits are associated with sleep regulation and have a greater effect on the sedative-hypnotic benzodiazepines, whereas those containing α2 and/or α3 subunits are associated with anxiety phenomena and have a greater effect on the anxiolytic benzodiazepines. Therefore, characterization of the selectivity profile of anxiolytic drugs could translate into a significant clinical impact. METHODS: The present study pharmacodynamically evaluated chlornordiazepam, the main active metabolite of mexazolam, upon GABAA receptors containing α2 and/or α3, anxiety-related, and those containing an α1 subunit, associated with sleep modulation. RESULTS: As shown by whole-cell patch-clamp data, chlornordiazepam potentiated GABA-evoked current amplitude in α2 and α3 containing receptors without changing the current amplitude in α1 containing receptors. However, current decay time increased, particularly in GABAA receptors containing α1 subunits. In contrast, other anxiolytic benzodiazepines such as alprazolam, bromazepam, and zolpidem, all increased currents associated with GABAA receptors containing the α1 subunit. CONCLUSIONS: This novel evidence demonstrates that mexazolam (through its main metabolite chlornordiazepam) has a "pharmacodynamic fingerprint" that correlates better with an anxiolytic profile and fewer sedative effects, when compared to alprazolam, bromazepam and zolpidem, explaining clinical trial outcomes with these drugs. This also highlights the relevance of the pharmacological selectivity over GABAA receptor subtypes in the selection of benzodiazepines, in addition to their clinical performance and pharmacokinetic characteristics.

Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD.

It is well established that long-term depression (LTD) can be initiated by either NMDA or mGluR activation. Here we report that sustained activation of GluK2 subunit-containing kainate receptors (KARs) leads to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) endocytosis and induces LTD of AMPARs (KAR-LTDAMPAR) in hippocampal neurons. The KAR-evoked loss of surface AMPARs is blocked by the ionotropic KAR inhibitor UBP 310 indicating that KAR-LTDAMPAR requires KAR channel activity. Interestingly, however, blockade of PKC or PKA also reduces GluA2 surface expression and occludes the effect of KAR activation. In acute hippocampal slices, kainate application caused a significant loss of GluA2-containing AMPARs from synapses and long-lasting depression of AMPAR excitatory postsynaptic currents in CA1. These data, together with our previously reported KAR-LTPAMPAR, demonstrate that KARs can bidirectionally regulate synaptic AMPARs and synaptic plasticity via different signaling pathways.

Methadone: does it really have low efficacy at micro-opioid receptors?

There is confusion in the literature concerning the relative agonist efficacy of methadone at micro-opioid receptors (MOPrs). Here, we confirm that methadone is a full agonist in guanosine 5'-O-[gamma-thio]triphosphate (GTPgammaS) binding studies. Methadone, however, seems to have low efficacy in studies of MOPr activation of G-protein-gated potassium (GIRK) channels, but this is because it directly inhibits the GIRK channels. Methadone also inhibits alpha2-adrenoceptor-activated GIRK channels. Methadone is not a specific GIRK channel blocker. It also inhibits small conductance Ca2+-activated K+ (SK2) channels. We conclude that methadone is a full agonist at MOPrs that, as we and others have shown, induces MOPr desensitization and internalization.

C-terminal splice variants of the mu-opioid receptor: existence, distribution and functional characteristics.

The distribution of the mRNA of different C-terminal splice variants of the mu-opioid receptor in rat CNS was assessed by RT-PCR. The mRNA species for MOR1, MOR1A and MOR1B were readily detectable and distributed widely throughout the rat CNS, with levels of MOR1 and MOR1A mRNA being overall greater than for MOR1B. We did not find convincing evidence that significant levels of MOR1C, MOR1C1, MOR1C2 and MOR1D are present in rat CNS. To examine possible differences in the agonist-induced regulation of MOR1, MOR1A and MOR1B, we expressed these constructs in HEK293 cells along with G-protein-coupled inwardly rectifying K+ channel subunits and measured the rate and extent of desensitisation of (d-Ala2,N-Me-Phe4,glycinol5)-enkephalin (DAMGO)- and morphine-induced G-protein-coupled inwardly rectifying K+ currents. Morphine-induced desensitisation was rapid for all three splice variants (t1/2: 1.2-1.7 min) but DAMGO-induced desensitisation was significantly slower for MOR1B (t1/2 4.2 min). Inhibition of endocytosis by expression of a dynamin-dominant negative mutant increased the rate of DAMGO-induced desensitisation of MOR1B. These data show that some splice variants of mu-opioid receptor are widely expressed in rat CNS but question the existence of others that have been reported in the literature. In addition, whereas the rate of desensitisation of MOR1 and MOR1A is agonist-independent, that for MOR1B is agonist-dependent.

Agonist-selective mechanisms of mu-opioid receptor desensitization in human embryonic kidney 293 cells.

The ability of two opioid agonists, [d-Ala(2),N-Me-Phe(4),Gly(5)-ol]-enkephalin (DAMGO) and morphine, to induce mu-opioid receptor (MOR) phosphorylation, desensitization, and internalization was examined in human embryonic kidney (HEK) 293 cells expressing rat MOR1 as well G protein-coupled inwardly rectifying potassium channel (GIRK) channel subunits. Both DAMGO and morphine activated GIRK currents, but the maximum response to DAMGO was greater than that of morphine, indicating that morphine is a partial agonist. The responses to DAMGO and morphine desensitized rapidly in the presence of either drug. Expression of a dominant negative mutant G protein-coupled receptor kinase 2 (GRK2), GRK2-K220R, markedly attenuated the DAMGO-induced desensitization of MOR1, but it had no effect on morphine-induced MOR1 desensitization. In contrast, inhibition of protein kinase C (PKC) either by the PKC inhibitory peptide PKC (19-31) or staurosporine reduced MOR1 desensitization by morphine but not that induced by DAMGO. Morphine and DAMGO enhanced MOR1 phosphorylation over basal. The PKC inhibitor bisindolylmaleimide 1 (GF109203X) inhibited MOR1 phosphorylation under basal conditions and in the presence of morphine, but it did not inhibit DAMGO-induced phosphorylation. DAMGO induced arrestin-2 translocation to the plasma membrane and considerable MOR1 internalization, whereas morphine did not induce arrestin-2 translocation and induced very little MOR1 internalization. Thus, DAMGO and morphine each induce desensitization of MOR1 signaling in HEK293 cells but by different molecular mechanisms; DAMGO-induced desensitization is GRK2-dependent, whereas morphine-induced desensitization is in part PKC-dependent. MORs desensitized by DAMGO activation are then readily internalized by an arrestin-dependent mechanism, whereas those desensitized by morphine are not. These data suggest that opioid agonists induce different conformations of the MOR that are susceptible to different desensitizing and internalization processes.