Publications

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.

The anticonvulsant gabapentin (GBP) has been shown effective for the treatment of neuropathic pain, although its mechanism of action remains unclear. A recent report has suggested that binding to the alpha(2)delta subunit of voltage-gated calcium channels contributes to its antinociceptive effect, based on the stereoselective efficacy of two analogs: (1S,3R)3-methylgabapentin (3-MeGBP) (IC(50) = 42 nM), which is effective in neuropathic pain models; and (1R,3R)3-MeGBP (IC(50) > 10,000 nM), which is ineffective (Field et al., 2000). The present study was designed to further examine the profiles of GBP and 3-MeGBP in rat models of acute and persistent pain. Systemic administration of GBP or (1S,3R)3-MeGBP inhibited tactile allodynia in the spinal nerve ligation model of neuropathic pain, whereas (1R,3R)3-MeGBP was ineffective. The antiallodynic effect of GBP, but not (1S,3R)3-MeGBP, was blocked by i.t. injection of the GABA(B) receptor antagonist [3-[[(3,4-dichlorophenyl)methyl]amino]propyl](diethoxymethyl)phosphinic acid (CGP52432). Systemic GBP or (1S,3R)3-MeGBP also inhibited the second phase of formalin-evoked nociceptive behaviors, whereas (1R,3R)3-MeGBP was ineffective. However, both (1S,3R)3-MeGBP and (1R,3R)3-MeGBP, but not GBP, inhibited first phase behaviors. In the carrageenan model of inflammatory pain, systemic GBP or (1R,3R)3-MeGBP failed to inhibit thermal hyperalgesia, whereas (1S,3R)3-MeGBP had a significant, albeit transient, effect. Systemic (1S,3R)3-MeGBP, but not GBP or (1R,3R)3-MeGBP, also produced an antinociceptive effect in the warm water tail withdrawal test of acute pain. These data demonstrate that GBP and 3-MeGBP display different antinociceptive profiles, suggesting dissimilar mechanisms of antinociceptive action. Thus, the stereoselective efficacy of 3-MeGBP, presumably related to alpha(2)delta binding, likely does not completely account for the mechanism of action of GBP.

A solvent-extraction-based radioassay for measuring sphingosine kinase (SKase) activity has been developed. The assay utilizes [3H]sphingosine substrate and differentially extracts the [3H]sphingosine-1-phosphate product. The extracted radioactivity is demonstrated to be primarily [3H]sphingosine-1-phosphate with less than 1% contamination by [3H]sphingosine. When assaying SKase activity in the soluble cell fraction, the extraction efficiency of the labeled sphingosine-1-phosphate product is a reproducible 78%, which allows for a simple back calculation to correct for the 22% extraction loss. With minor modification, the assay is also a reproducible procedure for determining SKase activity in subcellular membrane fractions. The assay is far more rapid than thin-layer chromatography and high-performance liquid chromatography methods, which makes it possible to do a large number of assays in a short period of time. The utility of the assay is demonstrated by using it to conduct a complete bisubstrate kinetic analysis of rat heart SKase.