Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.

Agonist-evoked cytosolic Ca 2⍣ spikes in mouse pancreatic acinar cells are specifically initiated in the apical secretory pole and are mostly confined to this region. The role played by mitochondria in this process has been investigated. Using the mitochondria-specific fluorescent dyes MitoTracker Green and Rhodamine 123, these organelles appeared as a bright belt concentrated mainly around the secretory granule area. We tested the effects of two different types of mitochondrial inhibitor on the cytosolic Ca 2⍣ concentration using simultaneous imaging of Ca 2⍣ -sensitive fluorescence (Fura 2) and electrophysiology. When carbonyl cyanide m-chlorophenylhydrazone (CCCP) was applied in the presence of the Ca 2⍣ -releasing messenger inositol 1,4,5-trisphosphate (IP 3 ), the local repetitive Ca 2⍣ responses in the granule area were transformed into a global rise in the cellular Ca 2⍣ concentration. In the absence of IP 3 , CCCP had no effect on the cytosolic Ca 2⍣ levels. Antimycin and antimycin ⍣ oligomycin had the same effect as CCCP. Active mitochondria, strategically placed around the secretory pole, block Ca 2⍣ diffusion from the primary Ca 2⍣ release sites in the granule-rich area in the apical pole to the basal part of the cell containing the nucleus. When mitochondrial function is inhibited, this barrier disappears and the Ca 2⍣ signals spread all over the cytosol.

Glucose-inhibited neurons orchestrate behavior and metabolism according to body energy levels, but how glucose inhibits these cells is unknown. We studied glucose inhibition of orexin/hypocretin neurons, which promote wakefulness (their loss causes narcolepsy) and also regulate metabolism and reward. Here we demonstrate that their inhibition by glucose is mediated by ion channels not previously implicated in central or peripheral glucose sensing: tandem-pore K(+) (K(2P)) channels. Importantly, we show that this electrical mechanism is sufficiently sensitive to encode variations in glucose levels reflecting those occurring physiologically between normal meals. Moreover, we provide evidence that glucose acts at an extracellular site on orexin neurons, and this information is transmitted to the channels by an intracellular intermediary that is not ATP, Ca(2+), or glucose itself. These results reveal an unexpected energy-sensing pathway in neurons that regulate states of consciousness and energy balance.

The physiological signaling mechanisms that link normal variations in body energy status to the activity of arousal-and metabolismregulating brain centers are not well understood. The melanin-concentrating hormone (MCH) and orexin/hypocretin types of neurons of the lateral hypothalamus (LH) exert opposing effects on arousal and metabolism. We examined whether shifts in brain extracellular glucose that correspond to physiological changes in blood glucose can alter the electrical output of neurochemically and biophysically defined LH cells in mouse brain slices. Here, we show that physiologically relevant concentrations of glucose dose-dependently enhance the electrical excitability of MCH neurons by inducing depolarization and increasing membrane resistance. We also demonstrate that the same physiological shifts in glucose have the opposite effects on the electrical activity of orexin neurons. We propose that these direct actions of glucose on the arousal-and metabolism-regulating LH neurons play a key role in the translation of normal variations in body energy resources into appropriate changes in arousal and metabolism.