Agonist-induced mitochondrial Ca2+ transients in smooth muscle.

We investigated the role of mitochondria (MT) in calcium signaling in a culture of rat aortic smooth muscle cells. We used targeted aequorin to selectively measure [Ca2+] in this organelle. Our results reveal that smooth muscle cell stimulation with agonists causes a large, transient increase in mitochondrial [Ca2+] ([Ca2+]m). This large transient can be blocked with inhibitors of the sarco-endoplasmic reticulum Ca2+-ATPase, suggesting a close relationship between the sarcoplasmic reticulum (SR) and the mitochondria. FCCP completely abolished the response to agonists, and targeted mitochondrial GFP revealed a vast tubular network of MT in these cells. When added before stimulation with ATP, IP3 inhibitors partially blocked the ATP-induced rise in mitochondrial Ca2+ release. The role of the Na+/Ca2+ exchanger (NCX) was examined by removing extracellular Na+. This procedure prevented the decrease in the [Ca2+]m transient normally seen on removal of extracellular Ca2+. We propose a functional linkage of MT and SR dependent on a narrow junctional space between the two organelles in which Ca2+ diffusion is restricted. Approximately half of the mitochondria appear to be associated with the superficial SR, which communicates with the extracellular space via NCX.

Pharmacological control of cellular calcium handling in dystrophic skeletal muscle.

Duchenne muscular dystrophy arises due to the lack of the cytoskeletal protein dystrophin. In Duchenne muscular dystrophy muscle, the lack of dystrophin is accompanied by alterations in the dystrophin-glycoprotein complex. We and others have found that the absence of dystrophin in cells of the Duchenne muscular dystrophy animal model, the mdx mouse, leads to elevated Ca(2+) influx and cytosolic Ca(2+) concentrations when exposed to stress. We have also shown that alpha-methylprednisolone, the only drug used successfully in the therapy of Duchenne muscular dystrophy, and creatine lowered cytosolic Ca(2+) levels in mdx myotubes. It is likely that chronic elevation of [Ca(2+)] in the cytosol in response to stress is an initiating event for apoptosis and/or necrosis in Duchenne muscular dystrophy or mdx muscle and that alterations in mitochondrial function and metabolism are involved. Other cellular signalling pathways (e.g. nitric oxide) might also be affected.

Stable transfection of cDNAs targeting specific steps of glycogen metabolism supports the existence of active gluconeogenesis in mouse cultured astrocytes.

In order to assess the participation of astrocytic gluconeogenesis in the synthesis of glycogen, mouse astrocytes were stably transfected with antisense cDNA of fructose-1,6-bisphosphatase (FBPase) and with sense and antisense cDNAs of glycogen synthase (GS). The antisenses of FBPase and GS have similar significant effect in decreasing astrocyte glycogen content by 60%, while sense GS significantly increased glycogen content by 100%. The FBPase activity was decreased by all three cDNAs used, while glycogen phosphorylase was not altered. The activity of GS was decreased by the antisense GS and increased by the sense GS. These data demonstrate that the gluconeogenesis in astrocytes is involved in the glycogenesis modulation.

In vivo and in vitro glycogenic effects of methionine sulfoximine are different in two inbred strains of mice.

We investigated the relationship between brain glycogen anabolism and methionine sulfoximine (MSO)-induced seizures in two inbred mouse strains that presented differential susceptibility to the convulsant. CBA/J was considered a MSO-high-reactive strain and C57BL/6J a MSO-low-reactive strain. Accordingly, the dose of MSO needed to induce seizures in CBA/J mice is lower than that in C57BL/6J mice, and CBA/J mice which had seizures, died during the first convulsion. In addition, the time--course of the MSO effect is faster in CBA/J mice than that in C57BL/6J mice. Analyses were performed in C57BL/6J and CBA/J mice after administration of 75 (subconvulsive dose) and 40 mg/kg of MSO (subconvulsive dose, not lethal dose), respectively. In the preconvulsive period, MSO induced an increase in the brain glycogen content of C57BL/6J mice only. Twenty-four hours after MSO administration, the brain glycogen content increased in both strains. The activity and expression of fructose-1,6-bisphosphatase, the last key enzyme of the gluconeogenic pathway, were increased in MSO-treated C57BL/6J mice as compared to control mice, at all experimental time points, whereas they were increased in CBA/J mice only 24 h after MSO administration. These latter results correspond to CBA/J mice that did not have seizures. Interestingly, the differences observed in vivo were consistent with results in primary cultured astrocytes from the two strains. This data suggests that the metabolism impairment, which was not a consequence of seizures, could be related to the difference in seizure susceptibility between the two strains, depending on their genetic background.