Intracellular calcium level is an important factor influencing ion channel modulations by PLC-coupled metabotropic receptors in hippocampal neurons

Signaling pathways involving phospholipase C (PLC) are involved in various neural functions. Understanding how these pathways are regulated will lead to a better understanding of their roles in neural functions. Previous studies demonstrated that receptor-driven PLCβ activation depends on intracellular Ca2+ concentration ([Ca2+]i), suggesting the possibility that PLCβ-dependent cellular responses are basically Ca 2+ dependent. To test this possibility, we examined whether modulations of ion channels driven by PLC-coupled metabotropic receptors are sensitive to [Ca2+]i using cultured hippocampal neurons. Muscarinic activation triggered an inward current at -100 mV (the equilibrium potential for K+) in a subpopulation of neurons. This current response was suppressed by pirenzepine (an M1-preferring antagonist), PLC inhibitor, non-selective cation channel blocker, and lowering [Ca 2+]i. Using the neurons showing no response at -100 mV, effects of muscarinic activation on K+ channels were examined at -40 mV. Muscarinic activation induced a transient decrease of the holding outward current. This current response was mimicked and occluded by XE991, an M-current K+ channel blocker, suppressed by pirenzepine, PLC inhibitor and lowering [Ca2+]i, and enhanced by elevating [Ca 2+]i. Similar results were obtained when group I metabotropic glutamate receptors were activated instead of muscarinic receptors. These results clearly show that ion channel modulations driven by PLC-coupled metabotropic receptors are dependent on [Ca2+]i, supporting the hypothesis that cellular responses induced by receptor-driven PLCβ activation are basically Ca2+ dependent. © 2013 Elsevier B.V

Oyygen uptake of adriamycin resistant cells of Ehrlich ascites tumor

エールリッヒ腹水癌細胞を用いアドリアマイシンに対する耐性細胞（ADR耐性細胞）を樹立した。電子顕微鏡を用い撮影写真から細胞質当たりのミトコンドリア（MT）の割合を面積比で求めた。親株に比較して1μg/ml ADR耐性細胞では1.32倍、10μg/ml ADR耐性細胞では1.47倍であった。これらの細胞の呼吸を測定した。耐性細胞の内発呼吸は親株に比較して増加していた。1μg/ml ADR耐性細胞では1.45倍、10μg/ml ADR耐性細胞では1.49倍であり、MTの増加量とほぼ同じ割合であった。これらのことから、細胞が耐性になるとエネルギー消費が高まるために細胞内MTが増加し、その結果呼吸（酸素消費）が増加することが推察された。Adriamycin-resistant cells of Ehrlich ascites tumor cells were established in our laboratory. Using electron microscope, the area of mitochondria (MT) per cytoplasm of ADR-resistant cells were measured with planimeter. The values of wild-type cells, 1μg/ml ADR-resistant cells and 10μg/ml ADR-resistant cells were 39.3, 51.8 and 57.7 μ(2) per 1,000 μ(2) of cytoplasm, respectively. Oxygen consumption of 1 μg/ml ADR-resistant cells and 10 μg/ml ADR-resistant cells were 1.45-fold and 1.49-fold compared to that of wild-type cells, respectively. These results indicate that ADR-resistant cells require more energy to work efflux pump than wild-type cells

Usefulness of fecal calprotectin by monoclonal antibody testing in adult Japanese with inflammatory bowel diseases: a prospective multicenter study

Background/Aims Noninvasive objective monitoring is advantageous for optimizing treatment strategies in patients inflammatory bowel disease (IBD). Fecal calprotectin (FCP) is superior to traditional biomarkers in terms of assessing the activity in patients with IBD. However, there are the differences among several FCP assays in the dynamics of FCP. In this prospective multicenter trial, we investigated the usefulness of FCP measurements in adult Japanese patients with IBD by reliable enzyme immunoassay using a monoclonal antibody. Methods We assessed the relationship between FCP levels and disease or endoscopic activity in patients with ulcerative colitis (UC, n=64) or Crohn’s disease (CD, n=46) compared with healthy controls (HCs, n=64). Results FCP levels in UC patients strongly correlated with the Disease Activity Index (rs=0.676, P<0.0001) and Mayo endoscopic subscore (MES; rs=0.677, P<0.0001). FCP levels were significantly higher even in patients with inactive UC or CD compared with HCs (P=0.0068, P<0.0001). The optimal cutoff value between MES 1 and 2 exhibited higher sensitivity (94.1%). FCP levels were significantly higher in active UC patients than in inactive patients (P<0.001), except those with proctitis. The Crohn’s Disease Activity Index tended to correlate with the FCP level (rs=0.283, P=0.0565). Conclusions Our testing method using a monoclonal antibody for FCP was well-validated and differentiated IBD patients from HCs. FCP may be a useful biomarker for objective assessment of disease activity in adult Japanese IBD patients, especially those with UC