Association between the ER and mitochondria has long been observed, and the formation of close contacts between ER and mitochondria is necessary for the ER-mediated sequestration of cytosolic calcium by mitochondria. Autocrine motility factor receptor (AMF-R) is a marker for a smooth subdomain of the ER, shown here by confocal microscopy to be distinct from, yet closely associated with the calnexin- or calreticulin-labeled ER. By EM, smooth ER AMF-R tubules exhibit direct interactions with mitochondria, identifying them as a mitochondria-associated smooth ER subdomain. In digitonin-permeabilized MDCK cells, the addition of rat liver cytosol stimulates the dissociation of smooth ER and mitochondria under conditions of low calcium. Using BAPTA chelators of various affinities and CaEGTA buffers of defined free Ca2+ concentrations and quantitative confocal microscopy, we show that free calcium concentrations <100 nM favor dissociation, whereas those >1 μM favor close association between these two organelles. Therefore, we describe a cellular mechanism that facilitates the close association of this smooth ER subdomain and mitochondria when cytosolic free calcium rises above physiological levels.

The effect of melatonin on the Ca(2+) signaling process in bovine aortic endothelial cells (BAE) and in primary cultured vascular endothelial cells from normotensive Sprague Dawley (SDR) and genetically hypertensive (SHR) rats was investigated using the Ca(2+) indicator Fura-2. Acute applications of melatonin failed to initiate a Ca(2+) response in the three cell types considered. However, preincubating SHR aortic endothelial cells with exposure to melatonin increased the internal Ca(2+) release triggered by bradykinin (BK) and ATP while stimulating the related agonist-evoked Ca(2+) entry. This effect appeared specific for SHR cells, as a similar incubation period failed to alter the Ca(2+) responses in BAE and SDR cells. Because of the known overproduction of free radicals in SHR cells, the effect of melatonin on Ca(2+) signaling was also tested in SDR and BAE cells exposed to the superoxide anion radical. Melatonin reversed the deleterious action of free radicals on Ca(2+) signaling in both cases, suggesting that its stimulatory effect in SHR was linked to its antioxidative properties. Finally, experiments where melatonin was applied between successive BK stimulation periods showed an enhancement of the agonist-evoked Ca(2+) entry in BAE and SDR cells. This effect appeared to be independent of the production of second messengers as no specific binding sites for melatonin, including MT1, MT2 and MT3 receptors, could be detected in BAE cells. We conclude that melatonin improves Ca(2+) signaling in dysfunctional endothelial cells characterized by an overproduction of free radicals while stimulating the agonist-evoked Ca(2+) entry in normal endothelial cells through a mechanism not related to its antioxidative properties.

The present work provides evidence that the Ca2+ signalling process in SHR endothelial cells is affected by an overproduction of free radicals, resulting in a depletion of releasable Ca2+ from IP3-sensitive and insensitive Ca2+ pools. These results point towards a beneficial action of antioxidants on Ca2+ signalling in endothelial cells from models of hypertension.