Murine Peripheral Tissues Research Articles

Sulphide quinone reductase contributes to hydrogen sulphide metabolism in murine peripheral tissues but not in the CNS

Hydrogen sulphide (H(2) S) is gaining acceptance as a gaseous signal molecule. However, mechanisms regarding signal termination are not understood. We used stigmatellin and antimycin A, inhibitors of sulphide quinone reductase (SQR), to test the hypothesis that the catabolism of H(2) S involves SQR. H(2) S production and consumption were determined in living and intact mouse brain, liver and colonic muscularis externa using gas chromatography and HPLC. Expressions of SQR, ethylmalonic encephalopathy 1 (Ethe1) and thiosulphate transferase (TST; rhodanese) were determined by RT-PCR and immunohistochemistry. In the colonic muscularis externa, H(2) (35) S was catabolized to [(35) S]-thiosulphate and [(35) S]-sulphate, and stigmatellin reduced both the consumption of H(2) (35) S and formation of [(35) S]-thiosulphate. Stigmatellin also enhanced H(2) S release by the colonic muscularis externa. In the brain, catabolism of H(2) (35) S to [(35) S]-thiosulphate and [(35) S]-sulphate, which was stigmatellin-insensitive, partially accounted for H(2) (35) S consumption, while the remainder was captured as unidentified (35) S that was probably bound to proteins. Levels of mRNA encoding SQR were higher in the colonic muscularis externa and the liver than in the brain. These data support the concept that termination of endogenous H(2) S signalling in the colonic muscularis externa occurs via catabolism to thiosulphate and sulphate partially via a mechanism involving SQR. In the brain, it appears that H(2) S signal termination occurs partially through protein sequestration and partially through catabolism not involving SQR. As H(2) S has beneficial effects in animal models of human disease, we suggest that selective inhibition of SQR is an attractive target for pharmaceutical development.

Apolipoprotein E influences amyloid-beta clearance from the murine periphery.

The relationship between amyloid-beta protein (Abeta) metabolism and Alzheimer's disease is currently poorly understood. While it is well known that the generation of Abeta results from enzymatic cleavage of its parent molecule, the amyloid beta protein precursor (AbetaPP), there is little information available regarding its in vivo clearance. The E4 isoform of apolipoprotein E (apoE) has been associated with poor clearance of Abeta under in vitro conditions. This is thought to be due to its poor ability to bind Abeta compared with the other common isoforms, apoE2 and apoE3. Although cell culture studies support the notion that Abeta clearance depends upon apoE isoform, validation of these findings requires Abeta clearance studies in vivo. In this study, we examined the clearance of Abeta in vivo from the periphery in mice that expressed apoE (C57BL/6J) or lacked apoE (APOE knockout). We measured the clearance of peripherally injected Abeta over time and additionally, the quantities sequestered by peripheral organs. Western blot analysis of the murine plasma indicated that the half-life of Abeta in the periphery was approximately 15 minutes. The livers of the C57BL/6J mice were found to have sequestered approximately 40% of the total injected Abeta at 90 minutes post-injection, whilst their kidneys contained 5% of the total injected Abeta. In contrast, the livers and kidneys of the APOE knockout animals were found to contain no detectable Abeta. These findings indicate that Abeta is rapidly removed from the plasma by murine peripheral tissues and the rate of its clearance is affected by apoE.

Expression of the purported sigma 1 (σ 1) receptor in the mammalian brain and its possible relevance in deficits induced by antagonism of the NMDA receptor complex as revealed using an antisense strategy

Sigma (σ) receptors have generated a great deal of interest on the basis of their possible role in psychosis, neuroprotection and various other behaviors including learning processes. The existence of at least two classes of σ receptor binding sites (σ 1 and σ 2) is now well established. The recent cloning of the mouse, guinea pig and human σ 1 receptors has allowed the study of the discrete distribution of the σ 1 receptor mRNA in rodent and human brain tissues using in situ hybridization. Overall, the sites of expression of specific σ 1 receptor mRNA signals were in accordance to the anatomical distribution of σ 1 receptor protein first established by quantitative receptor autoradiography. Specific σ 1 receptor hybridization signals were found to be widely, but discretely distributed, in mouse and guinea pig brain tissues. The highest levels of transcripts were seen in various cranial nerve nuclei. Lower, but still high hybridization signals were observed in mesencephalic structures such as the red nucleus, periaqueductal gray matter and substantia nigra, as well as in some diencephalic structures including such as the habenula and the arcuate, paraventricular and ventromedial hypothalamic nuclei. Superficial (I–II) and deeper (IV–VI) cortical laminae were moderately labeled in the mouse brain. Moderate levels of σ 1 receptor mRNA were also found in the pyramidal cell layer and the dentate gyrus of the hippocampal formation. Other structures such as the thalamus and amygdaloid body also expressed the σ 1 receptor mRNA although to a lesser extent. In murine peripheral tissues, strong hybridization signals were observed in the liver, white pulp of the spleen and the adrenal gland. In the postmortem human brain, moderate levels of σ 1 receptor mRNA, distributed in a laminar fashion, were detected in the temporal cortex with the deeper laminae (IV–VI) being particularly enriched. In the hippocampal formation, the strongest hybridization signals were observed in the dentate gyrus while all other subfields of the human hippocampal formation expressed lower levels of the σ 1 receptor mRNA. Antisense oligodeoxynucleotides against the purported σ 1 receptor were used next to investigate the possible role of this receptor in dizocilpine (MK-801)/NMDA receptor blockade-induced amnesia. Following a continuous intracerebroventricular infusion of a specific σ 1 receptor antisense into the third ventricle (0.4 nmol/h for 5 days), σ 1/[ 3H](+)pentazocine binding was significantly reduced in mouse brain membrane homogenates while a scrambled antisense control was without effect. Moreover, the σ 1 receptor antisense treatments (5 nmol/injection, every 12 h×3 or 0.4 nmol/h for 5 days) attenuated (+)MK-801/NMDA receptor blockade-induced cognitive deficits in the treated mice while a scrambled antisense control had no effect. Taken together, these results demonstrate the widespread, but discrete, distribution of the σ 1 receptor mRNA in the mammalian central nervous system. Moreover, antisense treatments against the purported σ 1 receptor gene reduced specific σ 1/[ 3H](+)pentazocine binding and modulated cognitive behaviors associated with NMDA receptor blockade providing further evidence for the functional relevance of the cloned gene.

Localization of Leptin Receptor mRNA Splice Variants in Murine Peripheral Tissues by RT-PCR andin SituHybridization

Expression of leptin receptor splice variants, including the long form variant (Ob-Rb), has been examined in murine peripheral tissues. RT-PCR indicates that the leptin receptor, Ob-R, and in particular the Ob-Ra splice variant are expressed in a wide range of tissues. Expression of the Ob-R receptor was localized byin situhybridization to specific sites in the spleen, testes, kidney, liver, lung, and adrenal. However, the long form leptin receptor, Ob-Rb, was only expressed at significant levels in the medulla of the adrenal and the inner zone of the medulla of the kidney. The specific sites of expression of different splice variants within peripheral tissues has important implications with regard to the function of leptin.