Simultaneous recordings of intracellular Ca2+([Ca2+]i) signal and extracellular DC potential were obtained from the CA1 region in 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)-ethane- N, N, N′, N′-tetraacetic acid penta-acetoxymethyl ester (Fura-2/AM)-loaded rat hippocampal slices. Superfusion with oxygen- and glucose-deprived medium (in vitro ischemia) for 5–6 min produced a rapid rise of the [Ca2+]i level in the stratum radiatum (rising phase of the [Ca2+]i signal), which occurred simultaneously with a rapid negative DC potential (rapid negative potential). When oxygen and glucose were reintroduced, the increased [Ca2+]i signal diminished rapidly (falling phase of the [Ca2+]i signal) during the generation of a slow negative DC potential (slow negative potential), which occurred within 1 min from the onset of the reintroduction. Thereafter, the [Ca2+]i signal partially and the slow negative potential completely returned to the preexposure level approximately 6 min after the reintroduction. The changes in [Ca2+]i signal during and after in vitro ischemia were very similar to the changes in the membrane potential of glial cells. The rising and falling phases of [Ca2+]i signal corresponded to the rapid depolarization and a depolarizing hump, respectively, in the repolarizing phase of glial cells. A prolonged application of in vitro ischemia or a reintroduction of either glucose or oxygen suppressed the falling phase after ischemic exposure. The application of ouabain (30 μM) generated both a rapid negative potential and a rapid elevation of [Ca2+]i, but no slow negative potential or rapid reduction in [Ca2+]i were observed. When oxygen and glucose were reintroduced to slices in the Na+-free or ouabain- or Ni2+-containing medium, the falling phase was suppressed. The falling phase was significantly accelerated in Ca2+- and Mg2+-free with EGTA-containing medium. In contrast, the falling phase was significantly slower in the Ca2+-free with high Mg2+- and EGTA-containing medium. The falling phase of the [Ca2+]isignal after ischemic exposure is thus considered to be primarily dependent on the reactivation of Na+, K+-ATPases, while the extrusion of cytosolic Ca2+ via the forward-mode operation of Na+/Ca2+ exchangers in glial cells is thought to be directly involved in the rapid reduction of [Ca2+]i after ischemic exposure.
Inhibition of protein synthesis in vitro by procollagen-derived fragments is associated with changes in protein phosphorylation.
