Subcellular calcium oscillators and calcium influx support agonist-induced calcium waves in cultured astrocytes

We have analysed Ca2+ waves induced by norepinephrine in rat cortical astrocytes in primary culture using fluorescent indicators fura-2 or fluo-3. The temporal pattern of the average [Ca2+]i responses were heterogeneous from cell to cell and most cells showed an oscillatory response at concentrations of agonist around EC50 (200 nM). Upon receptor activation, [Ca2+]i signals originated from a single cellular locus and propagated throughout the cell as a wave. Wave propagation was supported by specialized regenerative calcium release loci along the length of the cell. The periods of oscillations, amplitudes, and the rates of [Ca2+]i rise of these subcellular oscillators differ from each other. These intrinsic kinetic properties of the regenerative loci support local waves when stimulation is continued over long periods of time. The presence of local waves at specific, invariant cellular sites and their inherent kinetic properties provide for the unique and reproducible pattern of response seen in a given cell. We hypothesize that these loci are local specializations in the endoplasmic reticulum where the magnitude of the regenerative Ca2+ release is higher than other regions of the cell. Removal of extracellular Ca2+ or blockade of Ca2+ channels by inorganic cations (Cd2+ and Ni2+) during stimulation of adrenergic receptors alter the sustained plateau component of the [Ca2+]i response. In the absence of Ca2+ release, due to store depletion with thapsigargin, agonist occupation alone does not induce Ca2+ influx in astrocytes. This finding suggests that, under these conditions, receptor-operated Ca2+ entry is not operative. Furthermore, our experiments provide evidence for local Ca2+ oscillations in cells which can support both wave propagation as well as spatially discrete Ca2+ signalling.