Two mechanisms of calcium oscillations in adipocytes

In non-excitable cells, several kinds of agonist-induced oscillations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i) are known which differ in their form and generation mechanism. The oscillation source is, as a rule, the regulation of Ca²⁺ mobilization from intracellular stores through inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) and in some cases through ryanodine receptors (RyR). In the present work, oscillations in single mature adipocytes of mice epididymal fat on the ninth day of cultivation are studied. Cells were stimulated by acetylcholine (ACh) or by fetal bovine serum (FBS). ACh at a concentration of 0.1–5 μM evoked a rise in [Ca²⁺]_i to a peak and subsequent oscillations whose peaks and troughs declined along with increasing amplitude while frequency decreased. In most cells oscillations lasted less than 5 min. The new constant or interspike level exceeded the initial one or was equal to it (at 1 μM ACh). The removal of ACh stopped oscillations immediately. An inhibitor of phospholipase C (U73122) or of IP₃R (Xestospongin C) did not affect the pattern of responses, which means that the generation of oscillations does not depend on IP₃. At the same time, suppression of responses by ryanodine, which blocks RyR, was observed. Besides, oscillatory responses were abolished by inhibitors of phosphatidylinositol 3-kinase, NO synthase, and cGMP-dependent protein kinase. FBS (1%) initiated oscillations characterized by return of [Ca²⁺]_i after each peak to the baseline level, occurring prior to stimulation, and by maintenance of roughly constant amplitude and frequency (of the order of 1 min⁻¹). Oscillations persisted longer (more than 15 min in 87% of cells) than with ACh. Repeated stimulation of cells by FBS revealed a strongly reduced sensitivity after 1 h of rest, whereas responses to ACh partially restored within 3 min. Investigation of the involvement of IP₃R and RyR in FBS-induced oscillations gave completely inverse results relative to ACh and demonstrated a leading role of IP₃R without a considerable contribution of RyR and of its activation pathways. With both stimuli, Ca²⁺ entry through the plasma membrane was necessary only as a support of oscillations. The results show that in adipocytes different agonists can engage distinct subsystems of Ca²⁺ signaling, each of them generating oscillations with a specific temporal pattern.