Alison M. Lawrie

Local and global cytosolic Ca2+ oscillations in exocrine cells evoked by agonists and inositol trisphosphate

Submaximal stimulation with agonists generating inositol 1,4,5-trisphosphate (IP3) evokes cytosolic Ca2+ oscillations in many different cell types. In general, each Ca2+ rise is initiated from a specific region near the plasma membrane and then spreads as a wave throughout the cell. We now demonstrate that low (physiological) agonist concentrations evoke local cytosolic Ca2+ spikes in the secretory pole of single mouse pancreatic acinar cells that are particularly sensitive to blockade by the IP3 receptor antagonist heparin. These spikes can occur alone or repetitively or can precede longer lasting Ca2+ signals that spread throughout the cell. Intracellular IP3 application mimics these agonist actions. The short-lasting local Ca2+ spikes provide an economical signaling mechanism and are of physiological significance since they activate Ca(2+)-dependent Cl- and cation currents important for control of fluid secretion.

Acetylcholine and cholecystokinin induce different patterns of oscillating calcium signals in pancreatic acinar cells

Receptor-activated cytoplasmic calcium (Ca2+) oscillations have been investigated in single pancreatic acinar cells by microfluorimetry (Fura-2 as indicator). At submaximal concentrations of the agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8), both give rise to oscillatory changes in the cytosolic free calcium concentration ([Ca2+]i). The patterns of oscillations are markedly and consistently different for each of these two agonists. The ACh induced oscillations are superimposed upon a median elevation in background [Ca2+]i. The CCK-8 induced oscillations are of longer duration with [Ca2+]i returning to prestimulus levels between the discrete spikes. The ACh induced oscillations are rapidly abolished upon removal of extracellular Ca2+ while the CCK-8 induced oscillations persist for many minutes in the absence of external Ca2+. The CCK-8, but not the ACh, induced oscillations are increased in duration by the protein kinase C (PKC) inhibitor staurosporine and abolished by the PKC activating phorbol ester PMA. It is clear that CCK-8 and ACh do not activate receptor transduction mechanisms in an identical manner to generate oscillating [Ca2+]i signals.

Ca2+ oscillations in pancreatic acinar cells : spatiotemporal relationships and functional implications

The pancreatic acinar cells are of particular interest for the study of cytosolic Ca2+ signals, since they are morphologically polarized and generate agonist-specific Ca2+ oscillation patterns. Recent data obtained by combining digital video imaging of Fura-2 fluorescence with patch-clamp whole-cell current recording have provided new information on the spatiotemporal relationships of the cytosolic Ca2+ signals and the Ca(2+)-activated ionic currents. Low agonist concentrations evoke repetitive short-lasting local Ca2+ spikes in the secretory pole region that activate shortlasting current spikes. In the case of acetylcholine stimulation the spikes are confined to this region. When cholecystokinin is used the shortlasting local spikes precede longer Ca2+ transients that spread to the whole of the cell. Infusion of non-metabolizable inositol trisphosphate analogues can mimic these responses. The shortlasting local Ca2+ spikes are particularly sensitive to blockade by the inositol trisphosphate receptor antagonist heparin. These results show that the secretory pole region has a particularly high sensitivity to inositol trisphosphate probably due to clustering of high affinity receptors.

Synchronous calcium oscillations in cerebellar granule cells in culture mediated by NMDA receptors

The concentration of cytosolic Ca2+ ([Ca2+]i) was monitored in cerebellar granule cell cultures by digital imaging of fura-2 loaded cells. In the presence of Mg2+, cells grown in low K+ cultures responded to N-methyl-D-aspartate (NMDA) with uniform increases in [Ca2+]i from a stable basal [Ca2+]i. In contrast, in Mg(2+)-free medium, low K+ cultures showed spontaneous, synchronized [Ca2+]i oscillations from 4 days in culture. The oscillations were rapidly blocked by Mg2+, D,L-2-amino-5-phosphonovalerate, or tetrodotoxin. The development of oscillatory behaviour depended on the culture conditions and was not observed in cultures grown in high K+. These data show a high degree of connectivity established within 4 days in culture by dissociated granule cells allowing synchronized activity mediated through synaptic mechanisms.

Two different spatiotemporal patterns for Ca2+ oscillations in pancreatic acinar cells : evidence of a role for protein kinase C in Ins(1,4,5)P3-mediated Ca2+ signalling

The oscillations in cytosolic Ca2+ evoked in pancreatic exocrine acinar cells by submaximal concentrations of the two phosphoinositidase-coupled agonists acetylcholine (ACh) and cholecystokinin octapeptide (CCK-8) have very different temporal patterns. In the present study we use digital video imaging of Fura-2 fluorescence to map the spatial distribution of Ca2+ during the oscillating responses to these two agonists. The spatial patterns induced are very different for each of these agonists. ACh oscillations are sinusoidal and initiated at the secretory pole of these morphologically and functionally polarized cells. As they spread across the cell, pronounced gradients in Ca2+ develop that persist throughout the oscillating response. CCK-8 induces a series of discrete Ca2+ transients of longer duration and lower frequency. These elevations in Ca2+ arise slowly, throughout the cells and without any detectable gradients in Ca2+. We consider that the different spatiotemporal patterns can be explained on the basis of a physiologically relevant interaction between Ins(1,4,5)P3 and protein kinase C in second messenger-mediated Ca2+ signalling.

Okadaic acid induces the release of Ca2+ from intracellular stores in ECV304 endothelial cells

The effects of serine/threonine phosphatase inhibition on endothelial cell cytosolic free Ca2+ ([Ca2+]c) were investigated using okadaic acid and Fura-2-loaded ECV304 endothelial cells. When added to confluent adherent cells, 500 nM okadaic acid induced a transient and oscillatory elevation of [Ca2+]c both in the presence and absence of extracellular Ca2+. In the absence of extracellular Ca2+, depletion of the intracellular Ca2+ stores with either ATP (1 microM) or thapsigargin (100 nM) prevented any further release of Ca2+ on the subsequent addition of okadaic acid. Likewise (in the absence of extracellular Ca2+), a prior release of Ca2+ induced by okadaic acid reduced the magnitude of the response to ATP (1 microM). Taken together these observations indicate that okadaic acid induces Ca2+ release from the agonist-sensitive pool. The okadaic acid-induced Ca2+ release was mimicked by another potent phosphatase inhibitor, calyculin A (10 nM), and also the less potent analogue of okadaic acid, 1-nor-okadone (500 nM). The response to okadaic acid was characterised by a series of asynchronous [Ca2+]c oscillations, which at their peak resulted in 40-100% cells, at any one time, having an elevated [Ca2+]c. The response appeared to propagate between adjacent cells and the elevation of [Ca2+]c appeared initially in the cell periphery. In adherent cells, the release of Ca2+ induced by okadaic acid was found to be dependent upon cell density, as the proportion of cells responding to okadaic acid increased as the cell density increased. The response to okadaic acid was not observed in ECV304 cell suspensions. The data suggest that a kinase activity stimulated either directly or indirectly by cell-cell interactions can lead to the release of Ca2+ from the agonist- and thapsigargin-sensitive intracellular stores.