Leukocyte opioid receptors mediate analgesia via Ca(2+)-regulated release of opioid peptides.

Opioids are the most powerful analgesics. As pain is driven by sensory transmission and opioid receptors couple to inhibitory G proteins, according to the classical concept, opioids alleviate pain by activating receptors on neurons and blocking the release of excitatory mediators (e.g., substance P). Here we show that analgesia can be mediated by opioid receptors in immune cells. We propose that activation of leukocyte opioid receptors leads to the secretion of opioid peptides Met-enkephalin, ß-endorphin and dynorphin A (1-17), which subsequently act at local neuronal receptors, to relieve pain. In a mouse model of neuropathic pain induced by a chronic constriction injury of the sciatic nerve, exogenous agonists of δ-, µ- and κ-opioid receptors injected at the damaged nerve infiltrated by opioid peptide- and receptor-expressing leukocytes, produced analgesia, as assessed with von Frey filaments. The analgesia was attenuated by pharmacological or genetic inactivation of opioid peptides, and by leukocyte depletion. This decrease in analgesia was restored by the transfer of wild-type, but not opioid receptor-lacking leukocytes. Ex vivo, exogenous opioids triggered secretion of opioid peptides from wild-type immune cells isolated from damaged nerves, which was diminished by blockade of Gαi/o or Gßγ (but not Gαs) proteins, by chelator of intracellular (but not extracellular) Ca(2+), by blockers of phospholipase C (PLC) and inositol 1,4,5-trisphosphate (IP3) receptors, and was partially attenuated by protein kinase C inhibitor. Similarly, the leukocyte depletion-induced decrease in exogenous opioid analgesia was re-established by transfer of immune cells ex vivo pretreated with extracellular Ca(2+) chelator, but was unaltered by leukocytes pretreated with intracellular Ca(2+) chelator or blockers of Gαi/o and Gßγ proteins. Thus, both ex vivo opioid peptide release and in vivo analgesia were mediated by leukocyte opioid receptors coupled to the Gαi/o-Gßγ protein-PLC-IP3 receptors-intracellular Ca(2+) pathway. Our findings suggest that opioid receptors in immune cells are important targets for the control of pathological pain.

Human glutamate carboxypeptidase II inhibition: structures of GCPII in complex with two potent inhibitors, quisqualate and 2-PMPA.

Human glutamate carboxypeptidase II (GCPII) occurs in the central nervous system as well as in human prostate (where it is called prostate-specific membrane antigen; PSMA). Inhibitors of the enzyme have been shown to provide neuroprotection, but may also be useful for the detection, imaging and treatment of prostate cancer. Crystal structures were determined of the extracellular part of GCPII (amino-acid residues 44-750) in complex with two potent inhibitors, quisqualate and 2-PMPA (the strongest GCPII inhibitor to date), at resolutions of 3.0 and 2.2 A, respectively. In addition, models were constructed for binding of the inhibitors willardiine, homoibotenate, L-2-amino-4-phosphonobutanoic acid and L-serine-O-sulfate to the S1' site of the enzyme. The common denominator for high-affinity binding to the S1' site is the formation of two strong salt bridges.

Cellular target genes of Epstein-Barr virus nuclear antigen 2.

Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) is a key determinant in the EBV-driven B-cell growth transformation process. By activating an array of viral and cellular target genes, EBNA-2 initiates a cascade of events which ultimately cause cell cycle entry and the proliferation of the infected B cell. In order to identify cellular target genes that respond to EBNA-2 in the absence of other viral factors, we have performed a comprehensive search for EBNA-2 target genes in two EBV-negative B-cell lines. This screen identified 311 EBNA-2-induced and 239 EBNA-2-repressed genes that were significantly regulated in either one or both cell lines. The activation of most of these genes had not previously been attributed to EBNA-2 function and will be relevant for the identification of EBNA-2-specific contributions to EBV-associated malignancies. The diverse spectrum of EBNA-2 target genes described in this study reflects the broad spectrum of EBNA-2 functions involved in virus-host interactions, including cell signaling molecules, adapters, genes involved in cell cycle regulation, and chemokines.