Figure 3.

Superoxide that affects protein kinase activity is produced by mitochondria. A, B, High-frequency stimulation (100 Hz/18 sec) in the presence of rotenone- oligomycin, a mixture that inhibits mitochondrial respiration at complex I without significant ATP depletion, slightly enhanced [Ca²⁺]_i elevations (A) and completely inhibited mitochondrial O₂^- production (B). An essentially similar effect is seen in the presence of cyanide- oligomycin, which blocks respiration further down the chain at complex IV. C, Low-frequency stimulation (5 Hz/180 sec) with or without respiratory inhibitors had no effect on CaMKII autophosphorylation or pPKCα translocation. D, The normal increases in CaMKII and pPKCα activity that follow high-frequency stimulation (100 Hz/18 sec; compare white bars, black bars) were suppressed by either mixture of respiration blockers, indicating that Ca²⁺ entry-dependent mitochondrial O₂^- normally upregulates these kinases. E, The normal increases in CaMKII and pPKCα activity induced by strong Ca²⁺ entry after either high-frequency stimulation (100 Hz/18 sec) (right panel, black bars) or depolarization with 90 mm K⁺ (left panel, top row) were depressed in neurons overexpressing mitochondrial Mn-SOD/GFP (left panel, bottom row), which is expected because these cells scavenged O₂^- more efficiently (right panel, hatched bars). Symbols indicating statistical significance are as defined in Figure 2.