Abstract
Digital imaging fluorescence microscopy of fura-2-loaded hepatocytes in primary culture has been used to examine the changes of cytosolic free Ca2+ ([Ca2+]i) in response to receptor activation by alpha 1-adrenergic agonists and vasopressin at the subcellular level. Agonist-induced Ca2+ oscillations did not occur synchronously within the cell but originated from a specific region adjacent to the cell membrane and then propagated throughout the rest of the cell, with each oscillation within a series originating from the same locus. Furthermore, hormones acting through different receptors produced Ca2+ waves with similar rates of progress (20-25 microns.s-1) which originated from the same subcellular locus. For a given cell, the rate of progress and amplitude of the Ca2+ waves were independent of applied agonist concentration and were unaffected by depletion of extracellular Ca2+. The kinetics of Ca2+ increase at different points within the cell indicated that the Ca2+ waves were not driven by diffusion but were characteristic of a self-propagating mechanism. Significantly, when cells were treated with A1F-4 to directly activate the G-protein which couples receptor occupancy to [Ca2+]i mobilization, the origin and kinetics of the Ca2+ waves were identical to those observed with hormonal stimulation. It is proposed that the spatial organization of the intracellular Ca2+ release mechanisms may have significance in the regulation of the asymmetric metabolic functions of hepatocytes and other functionally polarized cells.
Highlights
A number of mechanisms have been proposed to account for the [Ca2+]i oscillations induced by agonists which act through InsP3, including feed back inhibition of InsPa formation by protein kinase C, feed forward activation of phospholipase C by Ca’+, m. hibition of InsP, action by Ca*‘, and Ca’+-induced Ca*+ release (CICR) [1, 18,19,20,21]
Based on the finding that agonist dose affects only the frequency of oscillations, without altering the amplitude or rate of rise and fall of [Ca2+li,we have suggested that a mechanism based on the CICR model proposed by Berridge and Galione ((19) see Ref. 23) could be operating in hepatocytes [3]
InsPs causes a relatively limited and slow release of Ca*+ which is taken up by InsPa-insensitive Ca2+ storage pools. When the latter Ca” pools reach capacity, [Ca”“], begins to rise and a cycle of CICR is initiated followed by reuptake of the released Ca2+into both InsPs-sensitive and InsPs-insensitive Ca2+pools. The periodicity of this cycle will be set by the time taken for the CICR pool to refill to capacity, which is determined by the rate of Ca*+ transfer from the InsPs-sensitive Ca2+pool
Summary
Isolated hepatocytes were prepared by collagenase perfusion of livers obtained from male Sprague-Dawley rats (250-300 g) as described previously [3]. Prior to fura- loading, the hepatocytes were washed and incubated in insulin-free Williams. Fluorescence images were obtained as described previously [3]. [Caz+li was calibrated from the fluorescence measurements as previously described [3, 7]. Oscillatory Waves of Ca2+ in Single Hepatocytes loo- ’ ’ * b 1 ’ b n ’ ’ ’ ’ ’ ’ These data are taken from the hepatocyte indicated as cell I in Fig. Zy and represent the time courses of changes in [Ca’+li calculated from the digital images of fura- fluorescence.
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