We established two immortalized cell lines from cerebral cortex of normal (CNh) and trisomy 16 (CTb) mouse fetuses, an animal model of human trisomy 21. Those cells loaded with the fluorescent Ca2+ dyes, Indo-1 and Fluo-3, exhibited increments of intracellular Ca2+ ([Ca2+]i) in response to external glutamate, NMDA, AMPA and kainate. CTb cells exhibited higher basal Ca2+ concentrations and had higher amplitude and slower time-dependent kinetics in the decay than CNh cells, suggesting an impaired Ca2+ buffering capacity in the trisomy 16-derived cell line. Nicotine also induced increments of [Ca2+]i. The CTb cell line could represent a model for studying cellular alterations related to Down syndrome.
Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca 2+ dependent exocitosis of vasoactive compounds. Using the Ca 2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (BAMCECs) respond to acetylcholine (ACh) with a cytosolic Ca 2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23). The increase in cytosolic Ca 2+ induced by 10μM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 μM of hexamethonium. On the other hand, the increase in cytosolic Ca 2+ could be depressed by nifedipine (0.01-100 μM) or withdrawal of extracellular Ca 2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in BAMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca 2+ channels, influx of external Ca 2+ and liberation of vasoactive compounds.
Mechanisms involved in the association between hyperhomocysteinemia and vascular occlusive diseases remain unclear. Homocysteine (Hcy) may disturb calcium (Ca(2+) ) cytosolic regulation in endothelial cells, a process that can directly affect the synthesis of vasoactive substances, such as nitric oxide (NO). We have investigated the effect of acute and chronic incubation with high concentrations of Hcy (100 and 500 μmol/L) on the changes in the intracellular Ca(2+) concentration ([Ca(2+) ]i ) induced by ATP, using primary cultures of human umbilical vein endothelial cells (HUVEC). The changes in [Ca(2+) ]i , expressed as ΔFt /Fb , were measured using the microspectrofluorimetric technique with Fluo-3 as Ca(2+) indicator. HUVEC acutely exposed to Hcy did not produce significant effects on any of the parameters studied. However, chronic exposition (24 h) caused a significant decrease in the speed of store-mediated Ca(2+) entry, expressed as (ΔFt /Fb )/t (s(-1) ). Exposure of HUVEC to 100 and 500 µmol/L Hcy gave significantly lower values (0.019 ± 0.002 s(-1) , n = 5 and 0.021 ± 0.004 s(-1) , n = 6, respectively) compared to the controls (0.046 ± 0.004 s(-1) , n = 8, P < 0.003). This was detected only when the sustained phase of the ATP-induced [Ca(+2) ]i increase was isolated. These results demonstrate that high concentrations of Hcy can affect the mechanisms involved in [Ca(2+) ]i regulation of HUVEC, and that alteration occurs specifically in the sustained phase, which has been directly associated with NO synthesis.
Extensive proteolysis is observed when red blood cells are exposed to free radicals produced in the thermolysis of 2,2′‐azo‐bis(2‐amidinopropane). It is evaluated that nearly one amino terminal group is produced by each free radical introduced into the system. These groups are considered to arise mainly from band 3 fragmentation due to the action of red cell proteinases. Protein fragmentation takes place prior to significant hemolysis or lipid peroxidation, as evaluated by thiobarbituric acid‐reactive substances measurements.
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