Hyperresponsiveness to Extracellular Acidification-Mediated Contraction in Isolated Bronchial Smooth Muscles of Murine Experimental Asthma.

PURPOSE: Extracellular acidification is a major component of tissue inflammation, including airway inflammation. The extracellular proton-sensing mechanisms are inherent in various cells including airway structural cells, although their physiological and pathophysiological roles in bronchial smooth muscles (BSMs) are not fully understood. In the present study, to explore the functional role of extracellular acidification on the BSM contraction, the isolated mouse BSMs were exposed to acidic pH under contractile stimulation. METHODS AND RESULTS: The RT-PCR analyses revealed that the proton-sensing G protein-coupled receptors were expressed both in mouse BSMs and cultured human BSM cells. In the mouse BSMs, change in the extracellular pH from 8.0 to 6.8 caused an augmentation of contraction induced by acetylcholine. Interestingly, the acidic pH-induced BSM hyper-contraction was further augmented in the mice that were sensitized and repeatedly challenged with ovalbumin antigen. In this animal model of asthma, upregulations of G protein-coupled receptor 68 (GPR68) and GPR65, that were believed to be coupled with Gq and Gs proteins respectively, were observed, indicating that the acidic pH could cause hyper-contraction probably via an activation of GPR68. However, psychosine, a putative antagonist for GPR68, failed to block the acidic pH-induced responses. CONCLUSION: These findings suggest that extracellular acidification contributes to the airway hyperresponsiveness, a characteristic feature of bronchial asthma. Further studies are required to identify the receptor(s) responsible for sensing extracellular protons in BSM cells.

Glucosylsphingosine Causes Hematological and Visceral Changes in Mice-Evidence for a Pathophysiological Role in Gaucher Disease.

Glucosylceramide and glucosylsphingosine are the two major storage products in Gaucher disease (GD), an inherited metabolic disorder caused by a deficiency of the lysosomal enzyme glucocerebrosidase. The build-up of glucosylceramide in the endoplasmic reticulum and prominent accumulation in cell lysosomes of tissue macrophages results in decreased blood cell and platelet counts, and skeletal abnormalities. The pathological role of the deacylated form of glucosylceramide, glucosylsphingosine (lyso-Gb1), a recently identified sensitive and specific biomarker for GD, is not well investigated. We established a long-term infusion model in C57BL/6JRj mice to examine the effect of lyso-Gb1 on representative hallmark parameters of GD. Mice received lyso-Gb1 at a dosage of 10 mg·kg-1 per day as a continuous subcutaneous administration, and were routinely checked for blood lyso-Gb1 levels using liquid chromatography-multiple reaction monitoring mass spectrometry (LC/MRM-MS) measurements at four-weekly intervals throughout treatment. The C57BL/6JRj mice showed a stable increase of lyso-Gb1 up to->500-fold greater than the normal reflecting concentrations seen in moderately to severely affected patients. Furthermore, lyso-Gb1 accumulated in peripheral tissues. The mice developed hematological symptoms such as reduced hemoglobin and hematocrit, increased spleen weights and a slight inflammatory tissue response after eight weeks of treatment. The above findings indicate a measurable visceral and hematological response in treated mice that suggests a role for lyso-Gb1 in the development of peripheral signs of GD.

Increased TNF-alpha production by peripheral blood mononuclear cells in patients with Krabbe's disease: effect of psychosine.

BACKGROUND: Inflammatory and/or immune activation occurs both in animal models (twitcher mice) and in the brain of patients with Globoid cell leukodystrophy (GLD) or Krabbe's disease (KD). In this study we evaluated in vitro the cytokine profile of KD patients and the effect of psychosine, the toxic metabolite which plays a role in the demyelination process in these patients. MATERIALS AND METHODS: We studied cytokine production by peripheral blood mononuclear cells (PBMCs) isolated from four KD patients, diagnosed on the basis of clinical criteria. Cells were cultured and stimulated with appropriate agents and the supernatants collected before and after the addition of psychosine. Tumour necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8) and monocyte chemoattractant factor (MCP)-1) production was evaluated (ELISA method) and compared with a group of 10 normal subjects. RESULTS: We found a significant increase of TNF-alpha release by PBMCs of KD patients compared with healthy subjects; TNF-alpha production was significantly increased after LPS addition. Psychosine was able to induce a further significant increase (P < 0.05) only in cells obtained from KD patients and not from control subjects. No changes were found in IL-8 and MCP-1 production. CONCLUSIONS: The increased TNF-alpha production permits us to confirm the presence of an inflammatory-immune stimulus in KD patients, which may be induced and potentiated by the pathogenetic metabolite psychosine.