Anne Morinville

Up-regulation and trafficking of δ opioid receptor in a model of chronic inflammation: implications for pain control

&NA; Pharmacological and physiological evidence supports a role for delta (&dgr;) opioid receptors in the nociceptive mechanisms of inflammation. However, few data exist regarding &dgr; opioid receptor expression and localization in such conditions. In this study, we have assessed the distribution and function of &dgr; opioid receptors in the rat spinal cord following induction of chronic inflammation by intraplantar injection of complete Freunds adjuvant (CFA). Intrathecal administration of the selective &dgr; opioid receptor agonist, d‐[Ala2, Glu4] deltorphin, dose‐dependently reversed thermal hyperalgesia induced by CFA. In situ hybridization and Western blotting experiments revealed an increase in &dgr; opioid receptor mRNA and protein levels, respectively, in the dorsal lumbar spinal cord ipsilateral to the CFA injection site compared to the contralateral side and sham‐injected controls. By electron microscopy, immunopositive &dgr; opioid receptors were evident in neuronal perikarya, dendrites, unmyelinated axons and axon terminals. Quantification of immunopositive signal in dendrites revealed a twofold increase in the number of immunogold particles in the ipsilateral dorsal spinal cord of CFA‐injected rats compared to the contralateral side and to sham‐injected rats. Moreover, the relative frequency of immunogold particles associated with or in close proximity to the plasma membrane was increased in the ipsilateral dorsal spinal cord, indicating a more efficient targeting of &dgr; opioid receptors to neuronal plasma membranes. These data demonstrate that CFA induces an up‐regulation and increased membrane targeting of &dgr; opioid receptors in the dorsal spinal cord which may account for the enhanced antinociceptive effects of &dgr; opioid receptor agonists in chronic inflammatory pain models.

Immunohistochemical distribution of delta opioid receptors in the rat central nervous system : evidence for somatodendritic labeling and antigen-specific cellular compartmentalization

Many studies have reported on the distribution of delta opioid receptors (δOR) in the mammalian central nervous system (CNS) by using a variety of techniques. However, no general consensus has emerged with regards to the localization of this receptor due to inconsistencies in the immunohistochemical literature. In the present study, we analyzed the cellular and subcellular distribution of immunoreactive δOR in the rat CNS using two different antibodies (directed against a sequence in the C‐terminus or N‐terminus of the rat δOR). By using Western blotting, these two antibodies recognized similar forms of the δOR in COS‐7 cells transfected with this receptor, but distinct forms in membranes from the rat spinal cord. By using light microscopic immunohistochemistry, both antibodies recognized identical populations of nerve cell bodies throughout the CNS; the distribution of these cell bodies conformed to that of δOR mRNA‐expressing cells detected by in situ hybridization. However, whereas the C‐terminus‐directed antibody recognized predominantly perikarya and proximal dendrites, the N‐terminus–directed antibody also labeled extensively dendritic and terminal arbors. Furthermore, by using electron microscopy, the two antibodies were found not only to label differentially somatodendritic versus axonal compartments, but also plasma membrane versus cytoplasmic ones, suggesting that distinct immunological forms of the receptor are being targeted preferentially to different cellular and subcellular domains. J. Comp. Neurol. 440:65–84, 2001.

From Vanadis to Atropos: vanadium compounds as pharmacological tools in cell death signalling

Vanadium compounds exert a variety of biological responses, the most notable being their effects as insulin mimetics. More recently, they have been used as pharmacological tools to investigate signalling pathways. Some peroxovanadium compounds act as powerful protein tyrosine phosphatase inhibitors, modulating both the extent and duration of phosphotyrosine signals at the level of the transmembrane growth factor receptors and targets in the cytoplasm and nucleus. A brief history of vanadium compounds, selected chemical properties of vanadium compounds and the ability of peroxovanadium complexes to modulate the activities of protein tyrosine phosphatases and tyrosine kinases are presented in this review by Anne Morinville, Dusica Maysinger and Alan Shaver. From the range of biological activities of these compounds, this review focuses on cytotoxic effects and possible roles of mitogen-activated protein kinases in mediating the effects exerted by vanadium compounds.

Enantioselectivity of Candida Rugosa Lipase Toward Carboxylic Acids: A Predictive Rule from Substrate Mapping and X-Ray Crystallography

We used substrate mapping to develop a rule that predicts which enantiomer of chiral carboxylic acid esters reacts faster in hydrolyses catalyzed by lipase from Candida rugosa (CRL, triacylglycerol hydrolase, E. C. 3.1.1.3). This rule, based on the size of the substituents at the stereocenter, is not reliable for crude CRL. It predicts the favoured enantiomer for only 23 out of 34 examples, 68% reliability. However, this rule is completely reliable for purified CRL; it predicts the favoured enantiomer for all 16 examples correctly. The examples include arylpropanoicacids, aryloxypropanoic acids, α-halophenylacetic acids, mandelic acid and O-methylmandelic acid. Further, purified CRL did not catalyse the hydrolysis of N-CBZ-phenylalanine methyl ester and N-CBZ-norleucine methyl ester. These two substrates were exceptions to the rule with crude CRL as the catalyst. Besides eliminating several exceptions, purification also raised the enantioselectivity of CRL toward carboxylic acid esters. To provide a struc...

Mu-opioid receptor knockout prevents changes in delta-opioid receptor trafficking induced by chronic inflammatory pain.

&NA; Previous studies from our laboratory have demonstrated that both chronic inflammatory pain, induced by intraplantar injection of complete Freunds adjuvant (CFA), and prolonged (48 h) stimulation of mu‐opioid receptors (&mgr;OR) by systemic administration of a variety of selective agonists, resulted in enhanced plasma membrane targeting of delta‐opioid receptors (&dgr;OR) in neurons of the dorsal spinal cord. To determine whether &dgr;OR trafficking induced by chronic inflammation was dependent on the activation of &mgr;OR, we investigated by immunogold cytochemistry the effects of intraplantar CFA injection on the plasma membrane density of &dgr;OR in &mgr;OR knockout (KO) mice. In untreated wild‐type (WT) mice, only a small proportion of &dgr;OR was associated with neuronal plasma membranes in the dorsal horn of the spinal cord. The CFA‐induced inflammation produced a significantly higher ratio of plasma membrane to intracellular receptors, as well as a 75% increase in the membrane density of immunoreactive &dgr;OR, in dendrites of the ipsilateral dorsal horn as compared to untreated mice. This increase in the membrane density of &dgr;OR was likely due to a recruitment of receptors from intracellular stores since no difference in the overall &dgr;OR immunolabeling density was evident between CFA‐treated and untreated mice. Most importantly, the CFA‐induced changes in &dgr;OR plasma membrane insertion seen in WT animals were not present in the spinal cord of &mgr;OR KO mice. These results demonstrate that the integrity of &mgr;OR is necessary for CFA‐induced changes in &dgr;OR trafficking to occur and suggest that these changes could be elicited by stimulation of &mgr;OR by endogenous opioids released in response to chronic inflammatory pain.

Drug delivery to the nervous system

Delivery of drugs to the nervous system remains a challenge despite advances in our understanding of the mechanisms involved in the development of neurodegenerative disorders and the actions of neuroactive agents. Drug accessibility to the central nervous system is limited by the blood-brain barrier; although the peripheral nervous system is more accessible than the central nervous system, problems are still encountered, mainly owing to the poor stability and considerable side effects of many neuroactive compounds when administered systemically. Microencapsulation of neuroactive compounds and living cells producing such substances can overcome some of these shortcomings for delivery to the nervous system.

Phosphorylation of mitogen-activated protein kinase is altered in neuroectodermal cells overexpressing the human amyloid precursor protein 751 isoform

The aberrant expression or processing of the amyloid precursor protein (APP) is the only known genetic basis for presenile familial Alzheimers disease, and the molecular connection between APP and tau has been perplexing. Attention has focused on proline-directed serine/threonine kinases as mediating the cytoskeletal modifications of Alzheimers disease, and we show that overexpression of APP can influence the activation of a candidate kinase, the mitogen-activated protein kinase (MAPK). In murine embryonal carcinoma cells stably transfected with the human 751 isoform of APP, we observed steady-state hyperactivation of p42(MAPK) concomitant with APP overexpression 3 days after neuroectodermal differentiation. In more mature differentiated cells, immunocytochemical analysis revealed enhanced basal somatic and nuclear immunoreactivity for phosphorylated MAPK coupled with an attenuated phosphorylation response to growth factor stimulation. Our results suggest that APP can influence the MAPK signaling pathway in such a way that the absolute and time-dependent activation required for discrimination of the appropriate downstream response are compromised. Such an effect would have important consequences for the functioning of cells coincidentally expressing both proteins, a situation that occurs in neuronal populations vulnerable to Alzheimers disease pathology.

Activation of MAPK by potassium bisperoxo(1,10-phenanthroline)oxovanadate (V)

Potassium bisperoxo(1,10-phenantroline)oxovanadate (V) [bpV(phen)] is a potent protein tyrocine phosphatase inhibitor which mediates a variety of biological effects. The aim of these studies was to examine the role(s) of mitogen activated protein kinase (MAPK) pathways in PC12 cell proliferation and toxicity by bpV(phen). BpV(phen) exerts a bimodal effect in PC12 cells: proliferation at low and cell death at higher micromolar concentrations. Activation of MAPK by bpV(phen) depends on time and concentration. The phosphorylation pattern of extracellular regulated kinases (ERK 1/2), c-jun N-terminal activated kinases (JNK) and p38 in PC12 cells is strikingly different. Activation of JNK is sustained in PC12 cells. In contrast, ERK 1/2 activation is transient and treatment with PD98059 indicates that ERK activation by bpV(phen) is partly independent from the ras-MEK pathway. Stability studies of bpV(phen) in DMEM and PBS showed linear relationship with T1/2 about 6 h and 10 days in DMEM and PBS, respectively. Comparison between the time courses of MAPK activation and kinetics of bpV(phen) decomposition as assessed by 51V-NMR analysis show that the initial and maximal phosphorylation signals are produced in the presence of the complex bpV(phen) and not caused by the decomposition products of bpV(phen).

Bisperoxovanadium complex promotes dopamine exocytosis in PC12 cells

The effects of the peroxovanadium complex potassium bisperoxo(1,10-phenanthroline)-oxovanadate (bpV[phen]) have been studied on dopamine (DA) exocytosis in PC12 cells. Bisperoxo(1,10-phenanthroline)-oxovanadate does not elicit dopamine secretion in PC12 cells. However, treatment of PC12 cells with 30 microM bpV[phen] for 20 min significantly enhances the secretion induced by the Ca(2+)-ionophore A23187. The effects appear to be irreversible, and strikingly different from the transient and suppressing effects of orthovanadate, which, like bpV[phen], is also a protein tyrosine phosphatase inhibitor. Contrastingly, the short-lived peroxovanadates, formed in situ by the addition of hydrogen peroxide and orthovanadate, are relatively ineffective. The Ca(2+) chelating agent EGTA abolishes bpV[phen]-enhanced dopamine release. The extracellular-regulated protein kinases (ERK) and synaptophysin, proteins implicated in exocytosis, are both tyrosine-phosphorylated by bpV[phen] in a dose- and time-dependent manner, with a maximal effect at 30 microM. Pre-treatment of cells with PD98059 significantly reduced dopamine release (P<0.05). These results suggest that this peroxovanadium complex enhances dopamine exocytosis, at least in part, by ERK-mediated signaling pathway and synaptophysin-associated phosphatase(s).