Polarized Ca(2+) signals that originate at and spread from the apical pole have been shown to occur in acinar cells from lacrimal, parotid, and pancreatic glands. However, "local" Ca(2+) signals, that are restricted to the apical pole of the cell, have been previously demonstrated only in pancreatic acinar cells in which the primary function of the Ca(2+) signal is to regulate exocytosis. We show that submandibular acinar cells, in which the primary function of the Ca(2+) signal is to drive fluid and electrolyte secretion, are capable of both Ca(2+) waves and local Ca(2+) signals. The generally accepted model for fluid and electrolyte secretion requires simultaneous Ca(2+)-activation of basally located K(+) channels and apically located Cl(-) channels. Whereas a propagated cell-wide Ca(2+) signal is clearly consistent with this model, a local Ca(2+) signal is not, because there is no increase in intracellular Ca(2+) concentration at the basal pole of the cell. Our data provide the first direct demonstration, in submandibular acinar cells, of the apical and basal location of the Cl(-) and K(+) channels, respectively, and confirm that local Ca(2+) signals do not Ca(2+)-activate K(+) channels. We reevaluate the model for fluid and electrolyte secretion and demonstrate that Ca(2+)-activation of the Cl(-) channels is sufficient to voltage-activate the K(+) channels and thus demonstrate that local Ca(2+) signals are sufficient to support fluid secretion.

Ca(2+) signalling in exocrine acinar cells has been shown to be both polarized and pulsatile in all cell types tested, except acutely isolated mouse lacrimal acinar cells. Lacrimal cells are also unusual in that they display a very low sensitivity to Ins(1,4,5) P (3) (Ins P (3)) that may be enhanced by placing the cells in primary culture for 12-72 h or by intracellular infusion of a low concentration of Ins(1,3,4,5) P (4) (Ins P (4)). We have proposed previously that this atypical behaviour stemmed from vesiculation of the endoplasmic reticulum (ER) incurred during isolation of the cells and, furthermore, that time in culture or Ins P (4) increased sensitivity to Ins P (3) by increasing ER integrity [Smith, Harmer, Letcher and Irvine (2000) Biochem. J. 347, 77-82]. We have measured the half time for fluorescence recovery after photobleaching (FRAP) of a fluorescent marker (Mag-fluo 4) loaded into the ER lumen in order to determine directly the functional integrity of the ER in lacrimal cells. The half-time for FRAP was increased (indicating a reduction in the functional integrity of the ER) following exposure to anti-microtubule agents (taxol and nocodazole) known to perturb ER structure and decreased (indicating an increase in the functional integrity of the ER) by time in culture and exposure to Ins P (4). The action of Ins P (4) was particularly pronounced because it occurred under patch-clamp whole-cell conditions that were themselves found to reduce ER functional integrity. These data show that ER remodelling could be a physiological regulator of Ca(2+) signalling and indicate a role for Ins P (4) in control of this process.

1. The acidic interior of neuroendocrine secretory vesicles provides both an energy gradient for amine-proton exchangers (VMATs) to concentrate small transmitter molecules, for example catecholamines, and an optimal pH for the prohormone convertases which cleave hormone precursors. There is evidence that VMAT activity modulates prohormone cleavage, but in the absence of measurements of pH in secretory vesicles in intact cells, it has not been possible to establish whether these effects are attributable to raised intravesicular pH due to proton transport through VMATs. 2. Clones were generated of the hamster insulinoma cell line HIT-T15 expressing a pH-sensitive form of green fluorescent protein (GFP-F64L/S65T) targeted to secretory vesicles, with and without co-expression of VMAT2. In order to study prohormone cleavage, further clones were generated that expressed preprogastrin with and without co-expression of VMAT2. 3. Confocal microscopy of GFP fluorescence indicated that the pH in the secretory vesicles was 5.6 in control cells, compared with 6.6 in cells expressing VMAT2; the latter was reduced to 5.8 by the VMAT inhibitor reserpine. 4. Using a pulse-chase labelling protocol, cleavage of 34-residue gastrin (G34) was found to be inhibited by co-expression with VMAT2, and this was reversed by reserpine. Similar effects on vesicle pH and G34 cleavage were produced by ammonium chloride. 5. We conclude that VMAT expression confers the linked abilities to store biogenic amines and modulate secretory vesicle pH over a range influencing prohormone cleavage and therefore determining the identity of regulatory peptide secretory products.

cADP-ribose (cADPr) and nicotinic acid–adenine dinucleotide phosphate (NAADP) are two putative second messengers; they were first shown to stimulate Ca2+ mobilization in sea urchin eggs. We have used the patch-clamp whole-cell technique to determine the role of cADPr and NAADP in relation to that of Ins(1,4,5)P3 in mouse submandibular acinar cells by measuring agonist-evoked and second-messenger-evoked changes in Ca2+-dependent K+ and Cl- currents. Both Ins(1,4,5)P3 and cADPr were capable of reproducing the full range of responses normally seen in response to stimulation with acetylcholine (ACh). Low concentrations of agonist (10–20nM ACh) or second messenger [1–10µM Ins(1,4,5)P3 or cADPr] elicited a sporadic transient activation of the Ca2+-dependent currents; mid-range concentrations [50–500nM ACh, 50µM Ins(1,4,5)P3 or 50–100µM cADPr] elicited high-frequency (approx. 2Hz) trains of current spikes; and high concentrations [more than 500nM ACh, more than 50µM Ins(1,4,5)P3 or more than 100µM cADPr] gave rise to a sustained current response. The response to ACh was inhibited by antagonists of both the Ins(1,4,5)P3 receptor [Ins(1,4,5)P3R] and the ryanodine receptor (RyR) but could be completely blocked only by an Ins(1,4,5)P3R antagonist (heparin). NAADP (50nM to 100µM) did not itself activate the Ca2+-dependent ion currents, nor did it inhibit the activation of these currents by ACh. These results show that, in these cells, both Ins(1,4,5)P3R and RyR are involved in the propagation of the Ca2+ signal stimulated by ACh and that cADPr can function as an endogenous regulator of RyR. Furthermore, although NAADP might have a role in hormone-stimulated secretion in pancreatic acinar cells, it does not contribute to ACh-evoked secretion in submandibular acinar cells.

The tumour-promoting agent thapsigargin has been shown to inhibit the microsomal Ca(2+)-ATPase and cause Ca2+ mobilization in a variety of cell types including exocrine acinar cells [Bird, Obie and Putney (1992) J. Biol. Chem. 267, 18382-18386]. When applied to acutely isolated lacrimal acinar cells, thapsigargin caused a slow biphasic activation of both the Ca(2+)-dependent K+ and Cl- currents measured using the whole-cell patch-clamp technique. If the only action of thapsigargin is to inhibit sequestration into Ca2+ pools, then Ca2+ mobilization following exposure to thapsigargin indicates that there is a significant ‘leak’ of Ca2+ into the cytoplasm, which is normally countered by Ca(2+)-ATPase activity. In the present study, we introduced the Ins(1,4,5)P3 receptor antagonist heparin (200 micrograms/ml) into lacrimal acinar cells via the patch-clamp pipette. Following a 5 min preincubation in the presence of heparin, neither acetylcholine (1 microM) nor thapsigargin (1 microM) caused any significant increase in either Ca(2+)-dependent current. Caffeine has been shown to suppress basal Ins(1,4,5)P3 levels in exocrine acinar cells [Toescu, O'Neill, Petersen and Eisner (1992) J. Biol. Chem. 267, 23467-23470]. Preincubation with caffeine (10 mM) also inhibited the response to subsequent exposure to thapsigargin. These data suggest that, in acutely isolated lacrimal cells, the source of the Ca2+ leak which gives rise to Ca2+ mobilization following inhibition of Ca2+ re-uptake by thapsigargin is Ca2+ release, from Ins(1,4,5)P3-dependent Ca2+ pools, caused by resting Ins(1,4,5)P3 levels.

This paper contains a description of the modified droplet technique that is designed to monitor Ca2+ extrusion from single isolated pancreatic acinar cells. A cell loaded with calcium indicator is maintained in a small droplet of solution containing another calcium indicator. Differences in the optical properties of the intracellular and extracellular indicators allows one to monitor simultaneously intracellular and extracellular calcium concentrations. The paper contains a description of the calibration procedure that is used to calculate intracellular and extracellular calcium concentrations. The advantages and disadvantages of different pairs of extracellular and intracellular indicators are discussed.

The initial increase of intracellular free Ca2+ concentration ([Ca2+]i) following agonist stimulation is spatially restricted to one pole of the cell, from where a wave of [Ca2+]i spreads across the cytosol. In the present study we have investigated the dynamic properties of the agonist-activated Ca(2+)-release mechanisms in different regions of the acinar cell and show that, during maximal agonist stimulation, the rate of [Ca2+]i increase at the secretory pole is identical with that recorded at the basal pole. Furthermore, the relationship between [Ca2+]i and the apparent rate of [Ca2+]i increase is similar in both regions of the cell. The data show that whereas the sensitivity to the Ca(2+)-releasing agent is different in different regions of the cell, the process of [Ca2+]i increase, once triggered, will proceed in an identical fashion, irrespective of the area of the cell.

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 Ca2+-dependent Cl- and cation currents important for control of fluid secretion.

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.

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 Ca2+-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 mimick 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.

The agonists acetylcholine (ACh) and cholecystokinin (CCK) have been shown to evoke markedly different patterns of cytosolic Ca2+ oscillations in the same isolated pancreatic acinar cells. ACh induces high frequency sinusoidal oscillations (spiking) associated with activation of Ca2+ influx. CCK evokes longer lasting discrete transients separated by long intervals, and these low frequency transients persist for many minutes in the absence of extracellular Ca2+. Using digital imaging of fura-2 fluorescence, we have now monitored the free cytoplasmic Ca2+ concentration ([Ca2+]i) simultaneously in many individual cells from the same population. In the resting condition [Ca2+]i ranged from about 50 to 300 nM. When the resting [Ca2+]i was below 150 nM, ACh (50-100 nM) invariably evoked typical high frequency spiking. In the majority of cells which had a resting [Ca2+]i higher than 150 nM, ACh also evoked low frequency transients. Although initiated by ACh, these transients displayed the temporal and functional characteristics of the CCK-evoked transients. Removal of extracellular Ca2+ for a few minutes had no effect on this type of oscillation, whereas such a procedure reversibly abolished the ACh-evoked high frequency response. For the response evoked by 10-30 pM Ca2+ signal amplitude and the resting [Ca2+]i. Because the Ca2+ signal amplitude and the resting [Ca2+]i. Because CCK could never induce high frequency spiking there is some receptor specificity in dictating the time course of Ca2+ oscillations, but the resting [Ca2+]i is a major determinant of the Ca2+ signal pattern.