Spinal PKC activity and expression: role in tolerance produced by continuous spinal morphine infusion.

It has been hypothesized that spinal morphine tolerance results from protein kinase C (PKC) mediated phosphorylation. Chronic lumbar intrathecal (i.t.) infusion of morphine (20 nmol/microl/h) was shown to produce antinociception on day 1 (d1) that disappeared by d5 (tolerance). On d6, a bolus i.t. probe dose of morphine (60 nmol) produced a more profound antinociception in saline-infused rats than in morphine-infused rats. Coinfusion of morphine with a PKC inhibitor, chelerythrine, prevented tolerance to the probe morphine dose. Bolus i.t. chelerythrine or GF109203X (GF), another PKC inhibitor, on d5, but not the inactive homologue of GF Bisindolymaleimide V, also blocked development of tolerance after 24 h. I.t. morphine infusion, but not saline, produced a 2-fold increase in dorsal horn PKC phosphorylating activity and in the expression of PKCalpha/gamma. Bolus chelerythrine on d5 after spinal morphine infusion blocked upon an increase in PKC activity, confirming that at the behaviorally active dose the drug had the intended biochemical effect upon spinal PKC activity. PKC activity and protein expression did not change when assessed 1 h after bolus i.t. morphine in naive rats. Thus, tolerance produced by morphine infusion is dependent upon an increase in local phosphorylating activity by PKC. Blocking the PKC activity prevents expression of the morphine tolerance.

Differential distribution of PI3K isoforms in spinal cord and dorsal root ganglia: potential roles in acute inflammatory pain.

PI3-kinases (PI3Ks) participate in nociception within spinal cord, dorsal root ganglion (DRG), and peripheral nerves. To extend our knowledge, we immunohistochemically stained for each of the 4 class I PI3K isoforms along with several cell-specific markers within the lumbar spinal cord, DRG, and sciatic nerve of naive rats. Intrathecal and intraplantar isoform specific antagonists were given as pretreatments before intraplantar carrageenan; pain behavior was then assessed over time. The α-isoform was localized to central terminals of primary afferent fibers in spinal cord laminae IIi to IV as well as to neurons in ventral horn and DRG. The PI3Kß isoform was the only class I isoform seen in dorsal horn neurons; it was also observed in DRG, Schwann cells, and axonal paranodes. The δ-isoform was found in spinal cord white matter oligodendrocytes and radial astrocytes, and the γ-isoform was seen in a subpopulation of IB4-positive DRG neurons. No isoform co-localized with microglial markers or satellite cells in naive tissue. Only the PI3Kß antagonist, but none of the other antagonists, had anti-allodynic effects when administered intrathecally; coincident with reduced pain behavior, this agent completely blocked paw carrageenan-induced dorsal horn 2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl) propanoic acid (AMPA) receptor trafficking to plasma membranes. Intraplantar administration of the γ-antagonist prominently reduced pain behavior. These data suggest that each isoform displays specificity with regard to neuronal type as well as to specific tissues. Furthermore, each PI3K isoform has a unique role in development of nociception and tissue inflammation.

In silico dissection of cell-type-associated patterns of gene expression in prostate cancer.

Prostate tumors are complex entities composed of malignant cells mixed and interacting with nonmalignant cells. However, molecular analyses by standard gene expression profiling are limited because spatial information and nontumor cell types are lost in sample preparation. We scored 88 prostate specimens for relative content of tumor, benign hyperplastic epithelium, stroma, and dilated cystic glands. The proportions of these cell types were then linked in silico to gene expression levels determined by microarray analysis, revealing unique cell-specific profiles. Gene expression differences for malignant and nonmalignant epithelial cells (tumor versus benign hyperplastic epithelium) could be identified without being confounded by contributions from stroma that dominate many samples or sacrificing possible paracrine influences. Cell-specific expression of selected genes was validated by immunohistochemistry and quantitative PCR. The results provide patterns of gene expression for these three lineages with relevance to pathogenetic, diagnostic, and therapeutic considerations.