We investigated spatiotemporal changes in cytoplasmic free Ca2 concentration ([Ca2 Jj) in norepinephrine (NE)-stimulated and fura-2-loaded individual H-35 rat hepatoma cells, using digital imaging microscopy and high time-resolution microspectrofluorometry. Application of NE (5 x 10 M) resulted in an initial transient increase in [Ca2 jj, followed by a small sustained [Ca2 Ji plateau above the pre-stimulaiion level. The initial peak and the small sustained plateau originated from intracellular stores and the extracellular space, respectively. The initial transient evoked by NE was totally blocked by phentolamine, an a-adrenergic antagonist, but was not blocked by either pre-incubation with nominally Ca2-free medium or by pre-treatment ofcells with La3. On the other hand, the sustained plateau was eliminated by act synergistically (2,7,29).

ABSTRACT. Spatiotemporal change of the cytosolic free Ca2+ concentration ([Ca2+L) in response to a variety of secretagogues was examined in rat pancreatoma AR-42J and AR-IP cells by microspectroflurometry and digital imaging microscopy after loading with fura-2. In the presence of external Ca2+, carbachol, CCK-OP (cholecystokinin-octapeptide), gastrin, norepinephrine or high K+ evoked a large transient increase in [Ca2+]i in AR-42J cells which declined to a sustained level before slowly declining towards the resting level. In the absence of external Ca2+, a transient increase in [Ca2+L were evoked by all the ligands except for high K+ stimulation, which declined rapidly towards the resting level. The [Ca2+]i increase caused by carbachol and high K+ treatment was inhibited by muscarinic receptor antagonist, atropin, and by L-type Ca2+ channel blocker, nifedipin, respectively. The transient [Ca2+]i increase induced by gastrin stimulation was not blocked by Ca2+ channel blocker, lanthanum. In the AR-IP cells, which are non-differentiated pancreatoma cell line, all stimulations including high K+ treatment have failed to evoke [Ca2+]i response. These intracelluar Ca2+ mobilizations in response to ligands in AR-42J cells were displayed by digital imaging microscopy. From these results we conclude that AR-42Jcells has an a-adrenergic receptor, in addition to muscarnic acetylcholine receptor, CCK-OP receptor, gastrin receptor and voltage dependent Ca2+ channel. In marked contrast, AR-IP cells have neither any hormonereceptor for the above ligands nor voltage dependent Ca2+channel.