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Abstract: Mitogen-activated protein kinases (MAPKs) play critical roles in the central nervous system immune responses through glial function, which are regulated with relative selectivity (or preference) by MAPK phosphatases (MKP). Phosphorylated extracellular signal-regulated protein kinase (p-ERK) is preferentially dephosphorylated by MKP-3, which display little activity over p-p38 and p–c-Jun NH2-terminal kinases (p-JNK). It has been proposed that these substrate preferences may vary depending on tissue or functional cellular processes. Since astrocytes display a prominent activity of JNK>ERK under stressed or reactive phenotype, we hypothesize that MKP-3 possess a similar or differential substrate preference in astrocytes for JNK and ERK (ERK=JNK or JNK>ERK). We generated transient expression of MKP-3 by transfecting a specific cDNA in primary rat neonatal brain cortex astrocytes. Cells were stimulated with lipopolysaccharide (LPS), and MAPKs and downstream pro-inflammatory products were measured by Western blot and ELISA analyses. MKP-3 expression in primary astrocytes reduced LPS-induced p-ERK and p-p38 by ∼50%, and p-JNK by ∼75%, and moderately reduced nitrite oxide (NO), while completely blocked Interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). We confirmed MKP-3 specific activity by developing a BV-2 microglia cell line stably overexpressing MKP-3 and using a specific siRNA against MKP-3. Our data demonstrate MKP-3 has differential substrate preference in astrocytes compared to other cells types, since it preferentially dephosphorylated p-JNK over p-ERK. Our results indicate also that astrocytic immune functions can be modulated by MKP-3 induction, a strategy that could be beneficial in neurological conditions in which astrocytes play a pathophysiological role, i.e. persistent pain. [Copyright &y& Elsevier]

Abstract: Comparison of human, rat and mouse cannabinoid CB2 receptor primary sequences has shown significant divergence at the mRNA and protein sequence level, raising the possibility of species specific pharmacological properties. Additionally, given the importance of the dog as a non-rodent species for predicting human safety during the drug development process, we cloned the dog CB2 receptor gene and characterized its in-vitro pharmacological properties in a recombinant expression system. A 1.1kb dog peripheral cannabinoid receptor (dCB2) fragment encoding a 360 amino acid protein was cloned from dog spleen cDNA. Analysis of the cloned dCB2 polypeptide sequence revealed that it shares between 76 and 82% homology with rat, mouse, human and predicted chimpanzee cannabinoid CB2 receptors. The dog CB2 receptor expressed in CHO cells displayed similar binding affinities for various synthetic and endogenous cannabinoids as compared to those measured for the human and rat cannabinoid CB2 receptors. However, these ligands exhibited altered functional potencies and efficacies for the dog cannabinoid CB2 receptor, which was also found to be negatively coupled to adenylate cyclase activity. These complex pharmacological differences observed across species for the cannabinoid CB2 receptor suggest that caution should be exerted when analyzing the outcome of animal efficacy and safety studies, notably those involving cannabinoid CB2 receptor targeting molecules tested in the dog. [Copyright &y& Elsevier]