Intracellular calcium regulates many cell functions including contraction, secretion, respiration, etc. These functions are carried out inside discrete organelles (nucleus, mitochondria, …) or functional domains (subplasmalema, caveolae, …). As a consequence, studies on calcium signalling are evolving from the cellular to the subcellular environment. Classical calcium probes are difficult to target to organelles and often lack the proper affinity for calcium. The cloning of aequorin and determination of its structure enabled its targeting and fitting of its calcium affinity to monitor subcellular calcium signals. In addition, the use of ultrasensitive photon-counting photomultipliers (aequorimeters) or photon-counting cameras allows also recording of subcellular signals from individual cells. We have used targeted aequorins to monitor nuclear, cytosolic and mitochondrial calcium dynamics in single cells (Villalobos et al. 2001). Clonal GH3 cells and normal mouse anterior pituitary cells were infected with herpes virus delivering either nuclear, cytosolic or mitochondrial aequorins. After 24-48 h, cells were incubated for 1-2 h with coelenterazines to reconstitute the active enzyme. Then, cells were subjected to photon-counting imaging. With this methodology, we recorded spontaneous oscillations of calcium concentration in all three compartments. It is well established that pituitary cells display spontaneous cytosolic calcium oscillations secondary to electric activity. Calcium oscillations in all three compartments were inhibited by external calcium removal, blocking of calcium channels or plasma membrane hyperpolarization. Oscillations were, however, not affected by emptying of the intracellular calcium stores with thapsigargin. Thryrotropin releasing hormone (TRH), a hypophysiotrophic factor that enhances electric activity, increased oscillations in all three compartments. However, the frequency and/or amplitude of spontaneous calcium oscillations and its stimulation by TRH recorded in the cytosol markedly differed from those recorded in the nucleus or mitochondria. Specifically, whereas the signals in the nucleus were clearly dampened with respect to those in the cytosol, oscillations in the mitochondria were clearly enlarged severalfold. Thus, the same mechanism, namely electric activity, generates different patterns of calcium oscillations in the cytosol, the nucleus and mitochondria. In addition, we found evidence indicating that oscillations occur only in those mitochondria functionally coupled to plasma membrane calcium channels and sensing subplasmalema high calcium concentrations. The remaining mitochondria barely sensed nor underwent oscillations. Since calcium is known to regulate several mitochondrial dehydrogenases, our results indicate that spontaneous mitochondrial calcium oscillations regulate basal ATP synthesis. In support of this view, we found that abolition of oscillations by external calcium removal or calcium channel blockade decreased resting intracellular NAD(P)H levels. Moreover, since oscillations are only generated in a subpopulation of mitochondria, then we conclude that control of ATP synthesis is driven by electric activity in that population but not in the remaining mitochondria (Villalobos et al. 2001).

Regarding the nucleus, the dampening of nuclear calcium oscillations relative to those recorded in the core cytosol can be explained by either a permeability barrier at the nuclear envelope or, alternatively, by an increased buffer capacity of the nucleus (P. Chamero, C. Villalobos, M.T. Alonso & J. García-Sancho, submitted). At present, we do not have evidence to favour either of the two alternatives. However, whatever the mechanism, the functional consequence is that transmission of the cytosolic calcium signal to the nucleus depends on the signal’s naturel. Specifically, sustained calcium increases in the cytosol are faithfully transmitted into the nucleus whereas transient, oscillatory signals are strongly dampened when sensed by the nucleus. This may be a mechanism by which calcium may regulate differentially nuclear functions such as cell division or gene expression (P. Chamero, C. Villalobos, M.T. Alonso & J. García-Sancho, submitted).

Grants APC1999-011 and BFI 2001-2003 from the Spanish MCyT are gratefully acknowledged.