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Repeated administration of peroxisome proliferator-activated receptor gamma (PPARγ) agonists reduces neuropathic pain-like behavior and associated changes in glial activation in the spinal cord dorsal horn. As PPARγ is a nuclear receptor, sustained changes in gene expression are widely believed to be the mechanism of pain reduction. However, we recently reported that a single intrathecal (i.t.) injection of pioglitazone, a PPARγ agonist, reduced hyperalgesia within 30 minutes, a time frame that is typically less than that required for genomic mechanisms. To determine the very rapid antihyperalgesic actions of PPARγ activation, we administered pioglitazone to rats with spared nerve injury and evaluated hyperalgesia. Pioglitazone inhibited hyperalgesia within 5 minutes of injection, consistent with a nongenomic mechanism. Systemic or i.t. administration of GW9662, a PPARγ antagonist, inhibited the antihyperalgesic actions of intraperitoneal or i.t. pioglitazone, suggesting a spinal PPARγ-dependent mechanism. To further address the contribution of nongenomic mechanisms, we blocked new protein synthesis in the spinal cord with anisomycin. When coadministered intrathecally, anisomycin did not change pioglitazone antihyperalgesia at an early 7.5-minute time point, further supporting a rapid nongenomic mechanism. At later time points, anisomycin reduced pioglitazone antihyperalgesia, suggesting delayed recruitment of genomic mechanisms. Pioglitazone reduction of spared nerve injury-induced increases in GFAP expression occurred more rapidly than expected, within 60 minutes. We are the first to show that activation of spinal PPARγ rapidly reduces neuropathic pain independent of canonical genomic activity. We conclude that acute pioglitazone inhibits neuropathic pain in part by reducing astrocyte activation and through both genomic and nongenomic PPARγ mechanisms.

Indomethacin is a non-narcotic and non-steroidal anti-inflammatory drug used in the treatment of various inflammatory diseases such as rheumatoid arthritis, osteoarthritis, and ankylosing spondylitis. In neonates, it is also used for induction of closure of patent ductus arteriosus (PDA). Its mechanism of action is believed to be through the inhibition of cyclooxygenase. Due to narrow therapeutic window and number of side effects, it's monitoring, particularly in neonates, is recommended. In the gas chromatography method described here, the drug is extracted from serum or plasma using methylene chloride and phosphate buffer (pH 6). The methylene chloride phase containing drug is separated and dried under stream of nitrogen. The drug is derivatized using Bis-(Trimethylsilyl)trifluoroacetamide (BSTFA) with 1% TMCS (trimethylchlorosilane). The derivatized drug is analyzed using gas chromatography-mass spectrometry. Quantitation of the drug in a sample is achieved by comparing responses of the unknown sample to the responses of the calibrators using selected ion monitoring. Meclofenamic acid is used as an internal standard.

Ca2+ is an obligatory factor for both the extrinsic and intrinsic pathways of coagulation. In majority of in vitro studies, investigators use saturating concentrations of Ca2+ (5 to 10 mM) for FVIIa/tissue factor (TF) activation of factor IX (FIX), and factor X (FX) (extrinsic coagulation), as well as for the activation of FIX by FXIa, FX by FIXa/FVIIIa, and prothrombin by FXa/FVa (intrinsic coagulation). However, the concentration of Ca2+ in plasma is only 1.1 mM, which is considerably below the saturating concentration needed for optimal coagulation. Importantly, plasma also contains 0.6 mM Mg2+ that could compensate for subsaturating concentrations of Ca2+ in promoting coagulation. Previous studies have attempted to clarify this concept in FIX, FX and prothrombin activation. However, these studies are sparse and in virtually all cases not detailed. We have systematically examined the role of plasma concentration of Mg2+ (in addition to the plasma concentration of Ca2+) in promoting all Ca2+ dependent steps of extrinsic and intrinsic coagulation and compared it with the saturating concentration of Ca2+. The Km (~ 100 nM) for activation of FIX by FXIa was similar in the presence of plasma concentrations of Ca2+/Mg2+ or 5 mM Ca2+. Furthermore, the Km and Vmax for the activation of FX and FIX by FVIIa/TF were essentially similar for both conditions. The Km and Vmax for the activation of FX by FVIIIa/FIXa ± phospholipid, and prothrombin by FXa/FVa ± phospholipid were also indistinguishable in these two different metal ion conditions. Notably, when only plasma concentrations of Ca2+ (1.1mM or 1.7mM) were used in all reactions mentioned above, coagulation proceeded at suboptimal rates. In further studies, we used Biacore to investigate the binding of FXIa and FVIII to FIXa, soluble TF to FVIIa, and FVa to FXa. Soluble TF, dansyl-Glu-Gly-Arg (dEGR)-IXa and dEGR-Xa were coupled to CM5 chips in the presence of 10 mM Ca2+. At 5 mM Ca2+, the binding of FXIa to dEGR-IXa was characterized by a Kd of ~40 nM, binding of FVIII to dEGR-IXa by a Kd of ~100 nM, and FVa to dEGR-Xa by a Kd of ~120 nM. In the presence of plasma concentrations of Ca2+ and Mg2+, binding constants were similar to those obtained in the presence of 5 mM saturating Ca2+ concentration. Additional 45Ca2+ binding studies using equilibrium dialysis and prothrombin fragment 1, dEGR-VIIa and decarboxylated dEGR-VIIa, FIX and decarboxylated FIX, and FX and decarboxylated FX, indicated that in the g-carboxyglutamic acid (Gla) domain, 2-3 Ca2+ binding sites (Shikimoto, et al., J. Biol. Chem. 278, 24090-24094, 2003; Wang, et al., Biochemistry42, 7959-7966, 2003; Bajaj, et al., J. Biol. Chem.281, 24873-24888, 2006) out of seven core divalent ion binding sites (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992) could be replaced by Mg2+. Conversely, Mg2+ could not displace the Ca2+ binding sites in the epidermal growth factor-like domain 1 (EGF1) and protease domains of FIX or FX. Overall these studies indicate that (1) saturating concentrations of Ca2+ used in in vitro investigations are valid representations of coagulation studies, except for that Mg2+ compensates for suboptimal concentrations of Ca2+ under physiological conditions; (2) two of the Ca2+-binding sites in the Gla domain (numbers 1 and 7, per Tulinsky numbering (Soriano-Garcia et al., Biochemistry31, 2554-2566, 1992)), and possibly a third site (number 4) are specific for Mg2+ under physiologic conditions; and (3) the Ca2+-binding sites in the EGF1 and protease domains are specific for Ca2+ and can not be occupied by Mg2+ under physiologic conditions. In conclusion, Ca2+ and Mg2+ act in concert to promote optimal coagulation under physiologic conditions. Mg2+ alone does not promote coagulation since it cannot bind to the Ca2+ specific sites in the Gla domain necessary for folding of the Gla domain omega loop.