Vip Modulates Intracellular Calcium Oscillations in Human Lymphoblasts

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Abstract
Vasoactive intestinal polypeptide (VIP) has been shown to stimulate adenylate cyclase in a human lymphoblast cell line (MOLT 4). In the present study, we monitored fluorescence in cell suspensions and in single fura-2 loaded MOLT 4 lymphoblasts to determine if VIP modulates intracellular calcium concentrations ([Ca2+]i), and if this modulation is mediated by adenylate cyclase. The distribution of [Ca2+]i in resting and stimulated cells was non-homogeneous, with gradients of high [Ca2+]i present in the subplasmalemmal space. In a subset of cells (10-30% of all cells studied), [Ca2+]i showed La3+-sensitive, temporal changes in the form of [Ca2+]i oscillations with a baseline [Ca2+]i value of 115±10 nM, an oscillation amplitude of 150±18 nM and a mean period of 9.2±2s. The remaining non-oscillating cells showed a constant [Ca2+]i level of 75±5 nM (n=65 cells from 4 experiments). In the subset of cells with spontaneous [Ca2+]i oscillations, VIP dose-dependendy (10-12 to 10-8M) increased the amplitude of oscillations but did not stimulate their frequency. The stimulatory effect of VIP was correlated with baseline [Ca2+]i in these cells, was attenuated in the presence of La3+ (25 μM), but was unaffected by cell depolarization (126 mM KC1). Dibutyryl cyclic AMP (10-4 to 10-3 M) and forskolin (10-4M) had no effect on [Ca2+]i oscillations, or on [Ca2+]i in cells without oscillations. In cell suspensions, baseline [Ca2+]i was found to be 55. 1±11.2 nM (meanS.E.M., n=11); VIP, cyclic AMP analogues or forskolin had no significant effect on [Ca2+]i. These findings suggest that: a) VIP modulates the amplitude of [Ca2+]i oscillations generated by a cytosolic [Ca2+] oscillator in a subset of cells at a concentration of 10-12M, a thousand-fold below the KD for the VIP receptor; b) baseline [Ca2+] values may be related to both the ability of cells to generate spontaneous [Ca2+] oscillations and of oscillating cells to respond to VIP; c) due to the small number of responding cells, VIP-induced [Ca2+]i changes are not detectable when studied in cell suspensions.