Interleukin-1 is a primary mediator of immune responses to injury and infection, but the mechanism of its cellular release is unknown. IL-1 exists as two agonist forms (IL-1α and IL-1β) present in the cytosol of activated monocytes/macrophages. IL-1β is synthesized as an inactive precursor that lacks a signal sequence, and its trafficking does not use the classical endoplasmic reticulum-Golgi route of secretion. Using primary cultured murine peritoneal macrophages, we demonstrate that P2X7 receptor activation causes release of IL-1β and IL-1α via a common pathway, dependent upon the release of Ca2+ from endoplasmic reticulum stores and caspase-1 activity. Increases in intracellular Ca2+ alone do not promote IL-1 secretion because a concomitant efflux of K+ through the plasmalemma is required. In addition, we demonstrate the existence of an alternative pathway for the secretion of IL-1α, independent of P2X7 receptor activation, but dependent upon Ca2+ influx. The identification of these mechanisms provides insight into the mechanism of IL-1 secretion, and may lead to the identification of targets for the therapeutic modulation of IL-1 action in inflammation.

In the present work, we investigated the relationship between mitochondrial function and Ca 2ϩ homeostasis in brain slices obtained from mice that aged normally. In acute preparations, the cerebellar neurons had similar values for intracellular free Ca 2ϩ ([Ca 2ϩ ] i ) regardless of their age (range, 6 weeks to 24 months). However, compared with the young slices, the aged neurons (20-24 months) showed an enhanced rate of [Ca 2ϩ ] i increases as a function of the time the slices were maintained in vitro. When slices were stimulated (KCl depolarization), there were significant differences in the patterns of [Ca 2ϩ ] i signal displayed by the young and old cerebellar granule neurons. More importantly, the aged neurons showed a significant delay in their capacity to recover the resting [Ca 2ϩ ] i . The relationship between [Ca 2ϩ ] i and mitochondrial membrane potential was assessed by recording both parameters simultaneously, using fura-2 and rhodamine-123. In both young and aged neurons, the cytosolic [Ca 2ϩ ] i signal was associated with a mitochondrial depolarization response. In the aged neurons, the mitochondria had a significantly longer repolarization response, and quantitative analysis showed a direct correlation between the delays in mitochondrial repolarization and [Ca 2ϩ ] i recovery, indicating the causal relationship between the two parameters. Thus, the present results show that the reported changes in Ca 2ϩ homeostasis associated with aging, which manifest principally in a decreased capacity of maintaining a stable resting [Ca 2ϩ ] i or recovering the resting [Ca 2ϩ ] i values after stimulation, are primarily attributable to a metabolic dysfunction in which the mitochondrial impairment plays an important role.

1. In isolated dorsal root ganglion cells (DRG neurons)

The mitochondrial membrane potential (psimito) is an important parameter not only of mitochondrial but also of cellular status. Prolonged mitochondrial depolarization is associated with various forms of neuronal death. Assessment of mitochondrial depolarization can take advantage of the specific properties of the lipophilic dyes that distribute in a potentiometrically determined ratio across membranes. Using conventional imaging, we showed that rhodamine 123 accumulated in the mitochondria, generating a highly heterogeneous pattern of spatial distribution of fluorescence across the cell body. Collapse of the psimito following exposure to a protonophore, carbonylcyanide p-chloromethoxyphenylhydrazone (CCCP), released rhodamine 123 from mitochondria into the cytosol. Under acutely changed conditions, this increased the overall intensity of the fluorescence signal and significantly decreased the degree of spatial heterogeneity of the signal. If mitochondrial depolarization was sustained chronically, the intensity of the signal decreased, but the increase in the spatial homogeneity of the fluorescent signal was maintained. Image analysis showed that the level of spatial heterogeneity of the signal can be assessed by calculating, for each individual neurone, the spread of pixel intensities values around the mean. This spread is defined by the coefficient of variation (CV), which is a measure of the standard deviation normalized to the average, and was inversely related to mitochondrial depolarization measured under different conditions. Thus, the degree of spatial heterogeneity of the rhodamine 123 signal measured from a neurone is a reliable indicator for the assessment of mitochondrial depolarization and can be used in experiments to monitor psimito over shorter or longer periods.

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