pituitrin and fura-2-am

For better understanding of glial participation in cerebral ischemia, spectrofluorimetric analysis using the calcium indicator Fura-2AM was applied to examine the role of intracellular free Ca2+ ([Ca2+])i elevation induced by different neuroactive substances in cultured rat brain astrocytes. The activation by the general receptor agonist glutamate resulted in a biphasic cell response in [Ca2+]i. We couldn't observe N-methyl-D-aspartate-evoked [Ca2+]i response at all. Quisqualate triggered a complex [Ca2+]i response in astrocytes consisting of mobilization of Ca2+ from the intracellular stores and also Ca2+ influx from the extracellular space. Kainate elicited a markedly different Ca2+ signal an external Ca(2+)-dependent sustained [Ca2+]i rise resulting from the activation of the ionotropic glutamate receptor. According to our results two types of glutamate receptors, the quisqualate-specific metabotropic and kainate-specific ionotropic receptor, are involved in [Ca2+]i elevation in these cultures. We could monitor agonist-specific cell response to noradrenaline, serotonin, vasopressin and ATP as well in these cultured rat astrocytes.

Topics: Adenosine Triphosphate; Adrenergic alpha-Agonists; Animals; Animals, Newborn; Astrocytes; Brain Ischemia; Calcium; Cells, Cultured; Cytoplasm; Excitatory Amino Acid Agonists; Extracellular Space; Fluorescent Dyes; Fura-2; Glutamic Acid; Kainic Acid; N-Methylaspartate; Neurotransmitter Agents; Norepinephrine; Quisqualic Acid; Rats; Receptors, Glutamate; Serotonin; Spectrometry, Fluorescence; Vasoconstrictor Agents; Vasopressins

One of the major biological targets of free radical oxidations, prone, for anatomical reasons, to oxidative challenges, is the cardiovascular system. In the present paper the effect of hydrogen peroxide on intracellular ionized calcium ([Ca2+]i) homeostasis in smooth muscle cells (SMC) is studied, the major aim of the study being a better understanding of the protective effect of antioxidants and Ca2+ channel blockers. The exposure of SMC to 300 microM H2O2 induced a rapid increase of [Ca2+]i, followed by a decrease to a new constant level, higher than the basal before the oxidative challenge. When incubation medium was Ca2+ free, the pattern of [Ca2+]i change was different. The rapid increase was still observed, but it was followed by a rapid decrease to a level only slightly above the basal before the oxidative challenge. The involvement of intracellular Ca2+ stores was tested by using vasopressin, a hormone able to induce discharge of inositol 1,4,5-triphosphate-sensitive Ca2+ stores. When H2O2 was added after vasopressin no [Ca2+]i increase was observed. Treatment of cells, in which the stable increase of [Ca2+]i was induced by H2O2, with disulfide reducing compounds, induced a progressive decrease of [Ca2+]i toward the level observed before the oxidative challenge. Calcium channel blockers and antioxidants, on the other hand, effectively prevented the stabilization of [Ca2+]i at the high steady-state, after the internal Ca2+ release phase. Dihydropyridine Ca2+ channel blockers were by far more active than verapamil and among those the most active was lacidipine. Also the antioxidants trolox and N,N'-diphenyl-1,4-phenylenediamine both prevented the [Ca2+]i unbalance. These results suggest that Ca+ channel blockers and antioxidants, although inactive on oxidative stress-induced Ca2+ release from intracellular stores, prevent the increased influx apparently related to a membrane thiol oxidation.

Topics: Animals; Aorta; Calcium; Calcium Channel Blockers; Cell Line; Dihydropyridines; Egtazic Acid; Fluorescent Dyes; Fura-2; Homeostasis; Hydrogen Peroxide; Kinetics; Membrane Potentials; Muscle, Smooth, Vascular; Potassium Chloride; Rats; Spectrometry, Fluorescence; Vasopressins