Photoactivatable drugs for nicotinic optopharmacology.

Photoactivatable pharmacological agents have revolutionized neuroscience, but the palette of available compounds is limited. We describe a general method for caging tertiary amines by using a stable quaternary ammonium linkage that elicits a red shift in the activation wavelength. We prepared a photoactivatable nicotine (PA-Nic), uncageable via one- or two-photon excitation, that is useful to study nicotinic acetylcholine receptors (nAChRs) in different experimental preparations and spatiotemporal scales.

Altered nicotine reward-associated behavior following α4 nAChR subunit deletion in ventral midbrain.

Nicotinic acetylcholine receptors containing α4 subunits (α4ß2* nAChRs) are critical for nicotinic cholinergic transmission and the addictive action of nicotine. To identify specific activities of these receptors in the adult mouse brain, we coupled targeted deletion of α4 nAChR subunits with behavioral and and electrophysiological measures of nicotine sensitivity. A viral-mediated Cre/lox approach allowed us to delete α4 from ventral midbrain (vMB) neurons. We used two behavioral assays commonly used to assess the motivational effects of drugs of abuse: home-cage oral self-administration, and place conditioning. Mice lacking α4 subunits in vMB consumed significantly more nicotine at the highest offered nicotine concentration (200 µg/mL) compared to control mice. Deletion of α4 subunits in vMB blocked nicotine-induced conditioned place preference (CPP) without affecting locomotor activity. Acetylcholine-evoked currents as well as nicotine-mediated increases in synaptic potentiation were reduced in mice lacking α4 in vMB. Immunostaining verified that α4 subunits were deleted from both dopamine and non-dopamine neurons in the ventral tegmental area (VTA). These results reveal that attenuation of α4* nAChR function in reward-related brain circuitry of adult animals may increase nicotine intake by enhancing the rewarding effects and/or reducing the aversive effects of nicotine.

Method development and validation for ultra-high pressure liquid chromatography/tandem mass spectrometry determination of multiple prostanoids in biological samples.

Following oxygenation of arachidonic acid by cyclooxygenase to form prostaglandin H2 (PGH2), a variety of prostanoids can be generated with diverse physiologic effects on pain, inflammation, allergy, cardiovascular system, cancer, etc. To facilitate the quantitative analysis of prostanoids in human serum of cell culture, an ultra-high pressure LC (UHPLC)/MS/MS method was developed and validated for the measurement of six eicosanoids belonging to the cyclooxygenase pathway: PGE2, PGD2, 8-iso-PGF2alpha, PGF2alpha, 6-keto-PGF1alpha, and thromboxane B2 (TXB2). Selectivity, matrix effects, calibration model, precision, and accuracy (intraday and interday), lower limit of quantitation (LLOQ), recovery, stability, and sample dilution were evaluated. Fast UHPLC separation was carried out in only 0.5 min with isocratic elution, and each prostanoid was measured using negative electrospray ionization MS with collision-induced dissociation and selected reaction monitoring. UHPLC/MS/MS provided high throughput with peak widths of approximately 3 s and an LLOQ of 0.020 ng/mL for PGE2, 0.027 ng/mL for PGD2, 0.152 ng/mL for 8-iso-PGF2alpha, 0.179 ng/mL for PGF2alpha and 6-keto-PGF1alpha, and 0.013 ng/mL for TXB2.

Pivotal role of reactive oxygen species in differential regulation of lipopolysaccharide-induced prostaglandins production in macrophages.

Gram-negative bacterial endotoxin lipopolysaccharide (LPS) triggers the production of inflammatory cytokines, reactive oxygen species (ROS), and prostaglandins (PGs) by pulmonary macrophages. Here, we investigated if ROS influenced PGs production in response to LPS treatment in mouse bone marrow-derived macrophages (BMDM). We observed that pretreatment of BMDM with two structurally unrelated ROS scavengers, MnTMPyP and EUK-134, not only prevented LPS-induced ROS accumulation, but also attenuated the LPS-induced PGD(2), but not PGE(2), production. Conversely LPS-induced PGD(2), but not PGE(2), production, was potentiated with the cotreatment of BMDM with H(2)O(2). These data suggest that ROS differentially regulate PGD(2) and PGE(2) production in BMDM. In addition, selective inhibition of the ROS generator NADPH oxidase (NOX) using either pharmacologic inhibitors or its p47(phox) subunit deficient mouse BMDM also attenuated LPS-induced PGD(2), but not PGE(2) production, suggesting the critical role of NOX-generated ROS in LPS-induced PGD(2) production in BMDM. We further found that both hematopoietic PGD synthase (H-PGDS) siRNA and its inhibitor HQL-79, but not lipocalin PGDS (L-PGDS) siRNA and its inhibitor AT-56, significantly attenuated LPS-induced PGD(2) production, suggesting that H-PGDS, but not L-PGDS, mediates LPS-induced PGD(2) production in BMDM. Furthermore, data from our in vitro cell-free enzymatic studies showed that coincubation of the recombinant H-PGDS with either MnTMPyP, EUK-134, or catalase significantly decreased PGD(2) production, whereas coincubation with H(2)O(2) significantly increased PGD(2) production. Taken together, our results show that LPS-induced NOX-generated ROS production differentially and specifically regulates the H-PGDS-mediated production of PGD(2), but not PGE(2), in mouse BMDM.

Lipopolysaccharide-induced expression of microsomal prostaglandin E synthase-1 mediates late-phase PGE2 production in bone marrow derived macrophages.

Cyclooxygenase (COX)-2 expression and release of prostaglandins (PGs) by macrophages are consistent features of lipopolysaccharide (LPS)-induced macrophage inflammation. The two major PGs, PGE(2) and PGD(2), are synthesized by the prostanoid isomerases, PGE synthases (PGES) and PGD synthases (PGDS), respectively. Since the expression profile and the individual role of these prostanoid isomerases-mediated inflammation in macrophages has not been defined, we examined the LPS-stimulated PGs production pattern and the expression profile of their synthases in the primary cultured mouse bone marrow derived macrophages (BMDM). Our data show that LPS induced both PGE(2) and PGD(2) production, which was evident by ∼8 hrs and remained at a similar ratio (∼1∶1) in the early phase (≤12 hrs) of LPS treatment. However, PGE(2) production continued increase further in the late phase (16-24 hrs); whereas the production of PGD(2) remained at a stable level from 12 to 24 hrs post-treatment. In response to LPS-treatment, the expression of both COX-2 and inducible nitric oxide synthase (iNOS) was detected within 2 to 4 hrs; whereas the increased expression of microsomal PGES (mPGES)-1 and a myeloid cell transcription factor PU.1 did not appear until later phase (≥12 hrs). In contrast, the expression of COX-1, hematopoietic-PGDS (H-PGDS), cytosolic-PGES (c-PGES), or mPGES-2 in BMDM was not affected by LPS treatment. Selective inhibition of mPGES-1 with either siRNA or isoform-selective inhibitor CAY10526, but not mPGES-2, c-PGES or PU.1, attenuated LPS-induced burst of PGE(2) production indicating that mPGES-1 mediates LPS-induced PGE(2) production in BMDM. Interestingly, selective inhibition of mPGES-1 was also associated with a decrease in LPS-induced iNOS expression. In summary, our data show that mPGES-1, but not mPGES-2 or c-PGES isomerase, mediates LPS-induced late-phase burst of PGE(2) generation, and regulates LPS-induced iNOS expression in BMDM.

Competitive enzymatic interactions determine the relative amounts of prostaglandins E2 and D2.

Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI(2) and PGF(2α) after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH(2) metabolism might be redistributed when the PGE(2) pathway is blocked. To address the determinants that govern the relative amounts of PGs, we developed an in vitro cell-free method, based on liquid chromatography-tandem mass spectrometry, to measure the exact amounts of these PGs formed in response to the addition of recombinant isomerases and their selective inhibitors. Our in vitro cell-free assay results were confirmed in cells using bone marrow-derived macrophage. Initially, we determined the in vitro stability of PGH(2) and noted that there was spontaneous nonenzymatic conversion to PGD(2) and PGE(2). mPGES-1 markedly increased the conversion to PGE(2) and decreased conversion to PGD(2). Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD(2) and decrease of PGE(2). A detailed titration study showed that the ratio of PGE(2)/PGD(2) was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH(2) metabolism is redistributed by the presence of distal isomerases or by blocking the major metabolic outlet, which could determine the relative benefits and risks resulting from interdiction in nonrated-limiting components of PG synthesis pathways.

Salvage of amputated upper extremities with temporary ectopic implantation followed by replantation at a second stage.

Salvage of the complex amputation of extremities, such as combined with devastating segmental injuries, extensive soft tissue defect, and multiple important organ injuries, continues to be a challenge for plastic surgeons. Temporary ectopic implantation of the amputated part to a healthy recipient site allows the patient to recover from critical combined injuries, radical debridements, and soft tissue repair. In this article, the authors report two cases of temporary ectopic implantation of complexly amputated forearms, followed by successful replantation to their anatomic positions at a second stage. The contralateral upper extremity is an acceptable recipient site for temporary ectopic implantation. In secondary replantation, a cross-arm flap can be designed to carry the vascular pedicle from the ectopic implantation recipient to improve blood supply to the replanted part when the second blood supply is established. The authors validated that temporary ectopic implantation of amputated parts provides an alternative procedure for the salvage of amputated extremities under special circumstances.

Temporary ectopic implantation for salvage of amputated lower extremities: case reports.

Two cases of temporary ectopic implantation of a complex amputated foot, followed by replantation to its anatomic position, are reported. Both cases of amputated foot were complicated by devastating soft-tissue injuries in the proximal stump of the amputation, fracture of the femur, and hemorrhagic shock, which ruled out the possibility of primary foot replantation. Both feet were temporarily ectopically implanted onto the contralateral legs, with microvascular anastomoses of the vessels to the recipient posterior tibial artery and saphenous vein. When the patient's general condition allowed, and the soft-tissue defects were repaired, the ectopic implanted feet were replanted to their anatomic positions. Both feet survived the temporary ectopic implantation and second-stage replantation. The length of the injured legs was maintained, and the feet regained their function in 4- and 6-month follow-ups. We conclude that temporary ectopic implantation of amputated parts provides an innovative procedure for the salvage of amputated extremities under special circumstances. A contralateral healthy extremity is an ideal recipient site for temporary ectopic implantation. The temporary ectopic implantation and second-stage replantation of an amputated foot and distal leg with indications can obtain satisfactory results.