The ability of two opioid agonists, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5 -ol]-enkephalin (DAMGO) and morphine, to induce -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.

Specific hapten-binding B cells were identified in the splenic marginal zones following immunization with hapten-protein conjugates. Hapten binding by marginal zone B cells does not appear to be due to passive absorption of anti-hapten antibody. For double immunization with two haptens, 2,4-dinitrophenyl (DNP) and 2-phenyloxazalone (Ox) each conjugated to hemocyanin, resulting in the appearance of discrete DNP-binding cells and Ox-binding cells in the marginal zone. Very few cells were identified which bound both haptens. The hapten-binding cells in the marginal zones have a phenotype characteristic of other marginal zone B cells. They express surface IgM but not IgD. Occasional cells also have surface IgG2c. All hapten-binding cells possessed the antigen recognized by the monoclonal antibody HIS 14 but lacked those identified by HIS24 and HIS22. Hapten-binding B cells were shown to have been in cell cycle shortly before entering the marginal zone but were no longer in cell cycle after arriving at that site. Once in the marginal zone hapten-binding cells were shown to remain in that site for upwards of 2 weeks. Following reimmunization with DNP-hemocyanin, DNP-binding but not Ox-binding cells were lost from the marginal zone. At the same time DNP-binding cells arrived in the periarteriolar lymphocytic sheath and to a lesser extent the follicles. These cells were in active cycle and appeared to give rise both to plasma cells and marginal zone hapten-binding cells. It is concluded that hapten-binding cells found in the marginal zones are memory B cells i.e. they have been derived from B cells which have undergone antigen-driven proliferation, they are no longer in cell cycle but can be induced to re-enter cell cycle by subsequent exposure to antigen. Good antibody responses were obtained following immunization with hapten-polysaccharides; however, no hapten-binding cells appeared in the marginal zones in response to these T cell-independent type 2 antigens.

Background and purpose:The ability of an agonist to induce desensitization of the m-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). Experimental approach: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)a knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). Key results: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCa, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCa knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. Conclusions and implications: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCa-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.

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Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of brain serotonergic systems. These effects could result from genetic influences, adverse early life experiences, or acute stressful life events, all of which can alter serotonergic neurotransmission and have been implicated in determining vulnerability to neuropsychiatric disorders. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on neuronal tryptophan hydroxylase 2 (tph2) mRNA expression, we investigated in rats the effects of maternal separation (separation from the dam for 180 min/day from postnatal day 2-14; MS180, a model of vulnerability to a depression-like syndrome), neonatal handling (separation from the dam for 15 min/day from postnatal day 2-14; MS15, a model of decreased stress sensitivity), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on tph2 mRNA expression in the dorsal raphe nucleus (DR). Among rats exposed to social defeat, MS180 rats had increased tph2 mRNA expression in the DR, while MS15 rats had decreased tph2 mRNA expression compared to AFR rats. Social defeat increased tph2 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR, a subdivision of the DR that is part of a sympathomotor command center. Overall, these data demonstrate that early life experience and stressful experience during adulthood interact to determine tph2 mRNA expression. These changes in tph2 mRNA expression represent a potential mechanism through which adverse early life experiences and stressful life experiences during adulthood may interact to increase vulnerability to stress-related psychiatric disease.