Measurement of Ca2+ signaling dynamics in exocrine cells with total internal reflection microscopy

In nonexcitable cells, such as exocrine cells from the pancreas and salivary glands, agonist-stimulated Ca2+ signals consist of both Ca2+ release and Ca2+ influx. We have investigated the contribution of these processes to membrane-localized Ca2+ signals in pancreatic and parotid acinar cells using total internal reflection fluorescence (TIRF) microscopy (TIRFM). This technique allows imaging with unsurpassed resolution in a limited zone at the interface of the plasma membrane and the coverslip. In TIRFM mode, physiological agonist stimulation resulted in Ca2+ oscillations in both pancreas and parotid with qualitatively similar characteristics to those reported using conventional wide-field microscopy (WFM). Because local Ca2+ release in the TIRF zone would be expected to saturate the Ca2+ indicator (Fluo-4), these data suggest that Ca2+ release is occurring some distance from the area subjected to the measurement. When acini were stimulated with supermaximal concentrations of agonists, an initial peak, largely due to Ca2+ release, followed by a substantial, maintained plateau phase indicative of Ca2+ entry, was observed. The contribution of Ca2+ influx and Ca2+ release in isolation to these near-plasma membrane Ca2+ signals was investigated by using a Ca2+ readmission protocol. In the absence of extracellular Ca2+, the profile and magnitude of the initial Ca2+ release following stimulation with maximal concentrations of agonist or after SERCA pump inhibition were similar to those obtained with WFM in both pancreas and parotid acini. In contrast, when Ca2+ influx was isolated by subsequent Ca2+ readmission, the Ca2+ signals evoked were more robust than those measured with WFM. Furthermore, in parotid acinar cells, Ca2+ readdition often resulted in the apparent saturation of Fluo-4 but not of the low-affinity dye Fluo-4-FF. Interestingly, Ca2+ influx as measured by this protocol in parotid acinar cells was substantially greater than that initiated in pancreatic acinar cells. Indeed, robust Ca2+ influx was observed in parotid acinar cells even at low physiological concentrations of agonist. These data indicate that TIRFM is a useful tool to monitor agonist-stimulated near-membrane Ca2+ signals mediated by Ca2+ influx in exocrine acinar cells. In addition, TIRFM reveals that the extent of Ca2+ influx in parotid acinar cells is greater than pancreatic acinar cells when compared using identical methodologies.     

Novel kinetics of single cell Ca2+ transients in stimulated hepatocytes and A10 cells measured using fura-2 and fluorescent videomicroscopy

The kinetics of agonist-induced increases in cytosolic free Ca2+ have been measured in single A10 vascular smooth muscle cells and rat hepatocytes using fluorescent videomicroscopy with fura-2 as a Ca2+ indicator. At high agonist concentrations there was no difference in the kinetics of the Ca2+ transient measured in vasopressin-stimulated single A10 cells or in cell populations. However, stimulation of single A10 cells with concentrations of vasopressin below 0.5 nM produced characteristic Ca2+ transients composed of two distinct peaks. The two peaks appeared to represent a temporal separation between release of intracellular Ca2+ and influx of extracellular Ca2+. The double transient was not observed in single rat hepatocytes stimulated with low concentrations of vasopressin or phenylephrine. In both A10 cells and hepatocytes, the initial rate of increase in Ca2+ concentrations in response to submaximal agonist concentrations was faster in single cells than in cell populations. This difference was due to asynchrony of the cellular response, where there was a latent period of variable length before onset of a rapid increase in Ca2+ concentration. The duration of the latent period was dependent on the agonist concentration, higher concentrations of agonist giving a reduced latent period. The hormone-stimulated Ca2+ transient measured in single hepatocytes with fura-2 was different from the series of transient spikes as previously reported using aequorin as the Ca2+ indicator, suggesting that fura-2 and aequorin may report different aspects of the Ca2+ response in stimulated cells. Collectively, these results demonstrate that measurement of Ca2+ transients in single cells provide novel information concerning the nature of the Ca2+ transient that is not apparent from studies with cell populations.     

Bombesin-induced cytosolic Ca2+ spiking in pancreatic acinar cells depends on cyclic ADP-ribose and ryanodine receptors

Different hormones and neurotransmitters, using Ca2+ as their intracellular messenger, can generate specific cytosolic Ca2+ signals in different parts of a cell. In mouse pancreatic acinar cells, cytosolic Ca2+ oscillations are triggered by activation of acetylcholine (ACh), cholecystokinin (CCK) and bombesin receptors. Low concentrations of these three agonists all induce local Ca(2+)spikes, but in the case of bombesin and CCK these spikes can also trigger global Ca2+ signals. Here we monitor cytosolic Ca2+ oscillations induced by low (2-5 pM) concentrations of bombesin and show that, like ACh- and CCK-induced oscillations, the bombesin-elicited responses are inhibited by ryanodine(50 microM). We then demonstrate that, like CCK- but unlike ACh-induced oscillations, the responses to bombesin are abolished by intracellular infusion of the cyclic ADP ribose (cADPr) antagonist 8-NH2-cADPr (20 microM). We conclude that in mouse pancreatic acinar cells, bombesin, CCK and ACh all produce local Ca2+ spikes by recruiting common oscillator units composed of ryanodine and inositol trisphosphate receptors. However, bombesin and CCK also recruit cADPr receptors, which may account for the global Ca2+ signals that can be evoked by these two agonists. Our new results indicate that each Ca2+ -mobilizing agonist, acting on mouse pancreatic acinar cells, recruits a unique combination of intracellular Ca2+ channels.     

A novel function of Noc2 in agonist-induced intracellular Ca2+ increase during zymogen-granule exocytosis in pancreatic acinar cells

Noc2, a putative Rab effector, contributes to secretory-granule exocytosis in neuroendocrine and exocrine cells. Here, using two-photon excitation live-cell imaging, we investigated its role in Ca(2+)-dependent zymogen granule (ZG) exocytosis in pancreatic acinar cells from wild-type (WT) and Noc2-knockout (KO) mice. Imaging of a KO acinar cell revealed an expanded granular area, indicating ZG accumulation. In our spatiotemporal analysis of the ZG exocytosis induced by agonist (cholecystokinin or acetylcholine) stimulation, the location and rate of progress of ZG exocytosis did not differ significantly between the two strains. ZG exocytosis from KO acinar cells was seldom observed at physiological concentrations of agonists, but was normal (vs. WT) at high concentrations. Flash photolysis of a caged calcium compound confirmed the integrity of the fusion step of ZG exocytosis in KO acinar cells. The decreased ZG exocytosis present at physiological concentrations of agonists raised the possibility of impaired elicitation of calcium spikes. When calcium spikes were evoked in KO acinar cells by a high agonist concentration: (a) they always started at the apical portion and traveled to the basal portion, and (b) calcium oscillations over the 10 μM level were observed, as in WT acinar cells. At physiological concentrations of agonists, however, sufficient calcium spikes were not observed, suggesting an impaired [Ca(2+)](i)-increase mechanism in KO acinar cells. We propose that in pancreatic acinar cells, Noc2 is not indispensable for the membrane fusion of ZG per se, but instead performs a novel function favoring agonist-induced physiological [Ca(2+)](i) increases.     

Different patterns of receptor-activated cytoplasmic Ca2+ oscillations in single pancreatic acinar cells: dependence on receptor type, agonist concentration and intracellular Ca2+ buffering.

Agonist-specific cytosolic Ca2+ oscillation patterns can be observed in individual cells and these have been explained by the co-existence of separate oscillatory mechanisms. In pancreatic acinar cells activation of muscarinic receptors typically evokes sinusoidal oscillations whereas stimulation of cholecystokinin (CCK) receptors evokes transient oscillations consisting of Ca2+ waves with long intervals between them. We have monitored changes in the cytosolic Ca2+ concentration ([Ca2+]i) by measuring Ca2(+)-activated Cl- currents in single internally perfused mouse pancreatic acinar cells. With minimal intracellular Ca2+ buffering we found that low concentrations of both ACh (50 nM) and CCK (10 pM) evoked repetitive short-lasting Ca2+ spikes of the same duration and frequency, but the probability of a spike being followed by a longer and larger Ca2+ wave was low for ACh and high for CCK. The probability that the receptor-evoked shortlasting Ca2+ spikes would initiate more substantial Ca2+ waves was dramatically increased by intracellular perfusion with solutions containing high concentrations of the mobile low affinity Ca2+ buffers citrate (10-40 mM) or ATP (10-20 mM). The different Ca2+ oscillation patterns normally induced by ACh and CCK would therefore appear not to be caused by separate mechanisms. We propose that specific receptor-controlled modulation of Ca2+ signal spreading, either by regulation of Ca2+ uptake into organelles and/or cellular Ca2+ extrusion, or by changing the sensitivity of the Ca2(+)-induced Ca2+ release mechanism, can be mimicked experimentally by different degrees of cytosolic Ca2+ buffering and can account for the various cytosolic Ca2+ spike patterns.     

Kinetic control of multiple forms of Ca(2+) spikes by inositol trisphosphate in pancreatic acinar cells.

The mechanisms of agonist-induced Ca(2+) spikes have been investigated using a caged inositol 1,4,5-trisphosphate (IP(3)) and a low-affinity Ca(2+) indicator, BTC, in pancreatic acinar cells. Rapid photolysis of caged IP(3) was able to reproduce acetylcholine (ACh)-induced three forms of Ca(2+) spikes: local Ca(2+) spikes and submicromolar (<1 microM) and micromolar (1-15 microM) global Ca(2+) spikes (Ca(2+) waves). These observations indicate that subcellular gradients of IP(3) sensitivity underlie all forms of ACh-induced Ca(2+) spikes, and that the amplitude and extent of Ca(2+) spikes are determined by the concentration of IP(3). IP(3)-induced local Ca(2+) spikes exhibited similar time courses to those generated by ACh, supporting a role for Ca(2+)-induced Ca(2+) release in local Ca(2+) spikes. In contrast, IP(3)- induced global Ca(2+) spikes were consistently faster than those evoked with ACh at all concentrations of IP(3) and ACh, suggesting that production of IP(3) via phospholipase C was slow and limited the spread of the Ca(2+) spikes. Indeed, gradual photolysis of caged IP(3) reproduced ACh-induced slow Ca(2+) spikes. Thus, local and global Ca(2+) spikes involve distinct mechanisms, and the kinetics of global Ca(2+) spikes depends on that of IP(3) production particularly in those cells such as acinar cells where heterogeneity in IP(3) sensitivity plays critical role.     

Simultaneous detection of intracellular free calcium and zinc using fura-2FF and FluoZin-3

Elevation of intracellular free zinc ([Zn2+]i) probably contributes to cell death in injury paradigms involving calcium deregulation and oxidative stress such as glutamate excitotoxicity. However, it is difficult to monitor both ions simultaneously in live cells. Here we present a new method using fluorescence microscopy and the ion sensitive indicators fura-2FF and FluoZin-3 to monitor both [Ca2+]i and [Zn2+]i in primary cortical neurons. We show that the new single wavelength dye FluoZin-3 responds robustly to small zinc loads, is insensitive to high Ca2+ or Mg2+, and is relatively unaffected by low pH or oxidants. The ratiometric indicator fura-2FF is sensitive to both Ca2+ and Zn2+. However, in conditions analogous to excitotoxic glutamate exposure where [Ca2+]i is high relative to [Zn2+]i, we found that fura-2FF responds mostly to [Ca2+]i but is relatively unaffected by low [Zn2+]i. Moreover, fura-2FF ratio changes caused by high [Ca2+]i or high [Zn2+]i could be distinguished because each ion produces a different spectral response. Finally, dual dye experiments showed that FluoZin-3 and fura-2FF respond robustly to [Zn2+]i and [Ca2+]j, respectively, in the same neurons during intense glutamate exposure. These studies provide a novel method for the simultaneous detection of both calcium and zinc in cells.     

Measurement of cytosolic free Ca2+ in individual small cells using fluorescence microscopy with dual excitation wavelengths

Free Ca2+ concentrations in the cytosol of individual small cells can be recorded with a new fluorescent Ca2+ indicator, "fura-2", and a fluorescence microscope modified to chop rapidly between two wavelengths of excitation. Both fura-2 and its Ca2+ complex fluoresce strongly, but their excitation peaks differ in wavelength. Alternation between the two preferred wavelengths allows assessment of the ratio of Ca2+-bound dye to free dye and hence cytosolic free Ca2+. This ratio measurement largely cancels out the effects of cell thickness, dye content, or instrumental efficiency, uncertainties that can jeopardize measurements at single wavelengths. We describe instrumentation that supplies rapidly alternating excitation wavelengths to either a standard cuvet or a fluorescence microscope. Its use is illustrated by experiments showing changes in cytosolic [Ca2+] accompanying activation of human platelets in suspension or single mouse thymocytes on the microscope.     

Two functionally distinct cholecystokinin receptors show different modes of action on Ca2+ mobilization and phospholipid hydrolysis in isolated rat pancreatic acini. Studies using a new cholecystokinin analog, JMV-180.

A new hepatapeptide cholecystokinin (CCK) analog, JMV-180 (Boc-Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-2-phenylethylester), acts as an agonist at high affinity CCK receptors on rat pancreatic acini to stimulate amylase release but unlike cholecystokinin octapeptide (CCK8) does not act on low affinity CCK receptors to inhibit amylase release (Galas, M. D., Lignon, M. F., Rodriguez, M., Mendre, C., Fulcrand, P., Laur, J., and Martinez, J. (1988) Am. J. Physiol. 254, G176-G188). To investigate the biochemical mechanisms initiated by CCK acting on each class of CCK receptor, the effects of JMV-180 and CCK8 on amylase release, Ca2+ mobilization, and phospholipid hydrolysis were studied in isolated rat pancreatic acini. When acini were loaded with the intracellular Ca2+ chelator BAPTA, amylase release stimulated by both JMV-180 and CCK8 was reduced. Measurement of 45Ca2+ efflux and cytosolic free calcium concentration ([Ca2+]i) by the fluorescence of fura-2-loaded acini in a stirred cuvette showed that JMV-180 induced a concentration-dependent increase but with a maximal response only two-thirds that induced by CCK8. When [Ca2+]i of individual fura-2-loaded acinar cells was measured by microspectrofluorometry, all concentrations of JMV-180 (1 nM-10 microM) induced repetitive transient [Ca2+]i spikes (Ca2+ oscillations). By contrast, stimulation with a high concentration of CCK8 (1 nM) caused a large increase in [CA2+]i followed by a small sustained elevation of [Ca2+]i. The measurement of inositol trisphosphate (IP3) production by both [3H]inositol labeling and 1,4,5-IP3 radioreceptor assay showed that JMV-180 had only minimal effects at 10 microM in contrast to the large increase induced by high concentrations of CCK8 (more than 1 nM). JMV-180 blocked the effect of a high concentration of CCK8 on both [Ca2+]i and 1,4,5-IP3 productions but did not affect the response to carbamylcholine. JMV-180 caused a delayed monophasic stimulation of 1,2-diacylglycerol (DAG) sustained to 60 min without the early increase in DAG observed in response to CCK8. Furthermore, JMV-180 stimulated the release of [3H]choline metabolites, primarily phosphorylated choline, from [3H]choline-labeled acini at low concentrations and to the same extent as CCK8. Since JMV-180 interacts not only with high affinity CCK receptors as an agonist but also with low affinity CCK receptors as a functional antagonist, the present results indicate that the occupancy of high affinity state receptors by CCK induces Ca2+ oscillations, DAG formation from phosphatidylcholine hydrolysis, and amylase release with minimal phosphatidylinositol 4,5-bisphosphate hydrolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

The acrosomal vesicle of mouse sperm is a calcium store

Subsequent to binding to the zona pellucida, mammalian sperm undergo a regulated sequence of events that ultimately lead to acrosomal exocytosis. Like most regulated exocytotic processes, a rise in intracellular calcium is sufficient to trigger this event although the precise mechanism of how this is achieved is still unclear. Numerous studies on mouse sperm have indicated that a voltage-operated Ca2+ channel plays some immediate role following sperm binding to the zona pellucida glycoprotein ZP3. However, there is also evidence that the mammalian sperm acrosome contains a high density of IP3 receptors, suggesting that the exocytotic event involves the release of Ca2+ from the acrosome. The release of Ca2+ from the acrosome may directly trigger exocytosis or may activate store-operated Ca2+ channels in the plasma membrane. To test the hypothesis that the acrosome is an intracellular store we loaded mammalian sperm with the membrane permeant forms of three Ca2+-sensitive fluorescent indicator dyes: fura-2, indo-1, and Calcium Green-5N. Fluorescence microscopy revealed that the sperm were labeled in all intracellular compartments. When fura-2 labeled sperm were treated with 150 microM MnCl2 to quench all fluorescence in the cytosol, or when the sperm were labeled with the low affinity dye Calcium Green-5N, there was a large Ca2+ signal in the acrosome. Consistent with the acrosome serving as an intracellular Ca2+ reservoir, the addition of 20 microM thapsigargin, a potent inhibitor of the smooth endoplasmic reticular Ca2+-ATPase (SERCA), to populations of capacitated sperm resulted in nearly 100% acrosomal exocytosis within 60 min (tau1/2 approximately 10 min), in the absence of extracellular Ca2+. Additionally, treatment of sperm with 100 microM thimerosal, an IP3 receptor agonist, also resulted in acrosomal exocytosis. Taken together, these data suggest that the mouse sperm acrosome is a Ca2+ store that regulates its own exocytosis through an IP3 Ca2+ mobilization pathway.     

Identification of cyclic ADP-ribose-dependent mechanisms in pancreatic muscarinic Ca(2+) signaling using CD38 knockout mice

We showed that muscarinic acetylcholine (ACh)-stimulation increased the cellular content of cADPR in the pancreatic acinar cells from normal mice but not in those from CD38 knockout mice. By monitoring ACh-evoked increases in the cytosolic Ca(2+) concentration ([Ca(2+)](i)) using fura-2 microfluorimetry, we distinguished and characterized the Ca(2+) release mechanisms responsive to cADPR. The Ca(2+) response from the cells of the knockout mice (KO cells) lacked two components of the muscarinic Ca(2+) release present in wild mice. The first component inducible by the low concentration of ACh contributed to regenerative Ca(2+) spikes. This component was abolished by ryanodine treatment in the normal cells and was severely impaired in KO cells, indicating that the low ACh-induced regenerative spike responses were caused by cADPR-dependent Ca(2+) release from a pool regulated by a class of ryanodine receptors. The second component inducible by the high concentration of ACh was involved in the phasic Ca(2+) response, and it was not abolished by ryanodine treatment. Overall, we conclude that muscarinic Ca(2+) signaling in pancreatic acinar cells involves a CD38-dependent pathway responsible for two cADPR-dependent Ca(2+) release mechanisms in which the one sensitive to ryanodine plays a crucial role for the generation of repetitive Ca(2+) spikes.     

Fluorescence measurements of free Ca2+ concentration in human erythrocytes using the Ca2+-indicator fura-2

We report here the use of the fluorescent Ca2+-chelator fura-2 to directly measure free Ca2+ concentration within intact human erythrocytes and the influence of viscosity on the fluorescence of this probe. The bright fluorescence of fura-2 has permitted the use of low concentrations of indicator and cells, thus minimizing the screening effect and the intrinsic fluorescence of haemoglobin. Erythrocytes (10(8) cells/ml) were loaded with 0.5 microM fura-2AM then diluted at 10(7) cells per ml for measurements. The extracellular signal was suppressed by addition of manganese ions just before recording spectra. Under these conditions, a blood sample of 100 microliter was sufficient for analysis. To study the influence of viscosity on fura-2 fluorescence, gelatin and polyvinylpyrrolidone at various concentrations were added to a physiological buffer to perform fura-2-Ca fluorescence standard curves. Fluorescence intensities and the apparent affinity constant for Ca2+ were modified by viscosity. When intra-erythrocytic viscosity was simulated with 21 g/l polyvinylpyrrolidone to obtain a mean viscosity of 14 mPa.s similar to that observed in human erythrocytes, the mean value of free Ca2+ concentration measured in erythrocytes from healthy subjects was 78 +/- 16 nM (mean +/- S.D., n = 29).     

Ca2+ oscillations induced by hormonal stimulation of individual fura-2-loaded hepatocytes

Isolated rat hepatocytes were loaded with the Ca2+ indicator fura-2 to measure cytosolic free Ca2+ concentrations ([Ca2+]i) in individual cells by digital ratio imaging microscopy. Stimulation with 0.1 nM vasopressin, 0.5 microM phenylephrine, or 0.5 microM ATP caused repetitive spikes of high [Ca2+]i in a high percentage of cells, in agreement with Woods et al. (Woods, N. M., Cuthbertson, K. S. R., and Cobbold, P. H. (1986) Nature 319, 600-602), but unlike the results of Monck et al. (Monck, J. R., Reynolds, E. E., Thomas, A. P., and Williamson, J. R. (1988) J. Biol. Chem. 263, 4569-4575). Reduction in extracellular [Ca2+] decreased the frequency but not the amplitude of the spikes, suggesting that the spikes result from dumping of intracellular stores and that the entry of extracellular Ca2+ affects only the rate of replenishment of those stores. Membrane depolarization failed to elevate [Ca2+]i and had an effect similar to removal of extracellular Ca2+ in decreasing the frequency of agonist-evoked [Ca2+]i oscillations or inhibiting them altogether, arguing against any significant role for voltage-operated Ca2+ channels.     

Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells

The fluorescent indicator fura-2 has been applied to a variety of cell types in order to set up appropriate conditions for measurements of the cytosolic concentration of free ionized Ca2+ [( Ca2+]i) in both cell suspensions and single cells analyzed in a conventional fluorimeter or in a fluorescence microscope equipped for quantitative analyses (with or without computerized image analyses), respectively. When the usual procedure for fluorescence dye loading (i.e., incubation at 37 degrees C with fura-2 acetoxy-methyl ester) was used, cells often exhibited a nonhomogeneous distribution of the dye, with marked concentration in multiple small spots located preferentially in the perinuclear area. These spots (studied in detail in human skin fibroblasts), were much more frequent in attached than in suspended cells, and were due to the accumulation (most probably by endocytosis) of the dye within acidic organelles after hydrolysis by lysosomal enzyme(s). When loading with fura-2 was performed at low (15 degrees C) temperature, no spots appeared, and cells remained diffusely labeled even after subsequent incubation at 32-37 degrees C for up to 2 h. Homogeneous distribution of the dye is a prerequisite for appropriate [Ca2+]i measurement. In fact, comparison of the results obtained in human skin fibroblasts labeled at either 37 or 15 degrees C demonstrated in spotty cells a marked apparent blunting of Ca2+ transients evoked by application of bradykinin. Additional problems were encountered when using fura-2. Leakage of the dye from loaded cells to the extracellular medium markedly affected the measurements in cell suspensions. This phenomenon was found to depend on the cell type, and to markedly decrease when temperature was lowered, suggesting the involvement of a facilitated transport. Calibration of fluorescence signals in terms of absolute [Ca2+]i was complicated by the increased fluorescence of fura-2 in the intracellular environment. To solve this problem we propose an in situ calibration procedure based on measurements carried out on cells in which [Ca2+]i was massively lowered (by loading the probe in a Ca2+-free medium) or increased (by treatment with the Ca2+ ionophore ionomycin, applied in a medium containing 3 mM Ca2+). These results provide explanations and, at least partial, solutions to the major problems encountered when using fura-2, and should thus be of help in clarifying the proper usage of the dye in [Ca2+]i measurements.     

Calcium-magnesium interactions in pancreatic acinar cells.

Although the role of calcium (Ca2+) in the signal transduction and pathobiology of the exocrine pancreas is firmly established, the role of magnesium (Mg2+) remains unclear. We have characterized the intracellular distribution of Mg2+ in response to hormone stimulation in isolated mouse pancreatic acinar cells and studied the role of Mg2+ in modulating Ca2+ signaling using microspectrofluorometry and digital imaging of Ca2+- or Mg2+-sensitive fluorescent dyes as well as Mg2+-sensitive intracellular microelectrodes. Our results indicate that an increase in intracellular Mg2+ concentrations reduced the cholecystokinin (CCK) -induced Ca2+ oscillations by inhibiting the capacitive Ca2+ influx. An intracellular Ca2+ mobilization, on the other hand, was paralleled by a decrease in [Mg2+]i, which was reversible upon hormone withdrawal independent of the electrochemical gradients for Mg2+, Ca2+, Na+, and K+, and not caused by Mg2+ efflux from acinar cells. In an attempt to characterize possible Mg2+ stores that would explain the reversible, hormone-induced intracellular Mg2+ movements, we ruled out mitochondria or ATP as potential Mg2+ buffers and found that the CCK-induced [Mg2+]i decrease was initiated at the basolateral part of the acinar cells, where most of the endoplasmic reticulum (ER) is located, and progressed from there toward the apical pole of the acinar cells in an antiparallel fashion to Ca2+ waves. These experiments represent the first characterization of intracellular Mg2+ movements in the exocrine pancreas, provide evidence for possible Mg2+ stores in the ER, and indicate that the spatial and temporal distribution of intracellular Mg concentrations profoundly affects acinar cell Ca2+ signaling.     

Myoplasmic binding of fura-2 investigated by steady-state fluorescence and absorbance measurements.

Binding of the fluorescent Ca2+ indicator dye fura-2 by intracellular constituents has been investigated by steady-state optical measurements. Fura-2's (a) fluorescence intensity, (b) fluorescence emission anisotropy, (c) fluorescence emission spectrum, and (d) absorbance spectra were measured in glass capillary tubes containing solutions of purified myoplasmic proteins; properties b and c were also measured in frog skeletal muscle fibers microinjected with fura-2. The results indicate that more than half, and possibly as much as 85%, of fura-2 molecules in myoplasm are in a protein-bound form, and that the binding changes many properties of the dye. For example, in vitro characterization of the Ca2+-dye reaction indicates that when fura-2 is bound to aldolase (a large and abundant myoplasmic protein), the dissociation constant of the dye for Ca2+ is three- to fourfold larger than that measured in the absence of protein. The problems raised by intracellular binding of fura-2 to cytoplasmic proteins may well apply to cells other than skeletal muscle fibers.     

NPC-14686 (Fmoc-l-homophenylalanine)-induced CaCa2+ increases and death in human prostate cancer cells

The effect of NPC-14686, a potential anti-inflammatory drug, on cytosolic free Ca2+ levels ([Ca2+]i) and growth in PC3 human prostate cancer cells was examined by using fura-2 as a fluorescent Ca2+ indicator and WST-1 as a fluorescent growth dye. NPC-14686 at concentrations above 10 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 100 microM. NPC-14686-induced Ca2+ influx was confirmed by Mn2+ quench of fura-2 fluorescence. The Ca2+ signal was also reduced by removing extracellular Ca2+. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ nearly abolished 200 microM NPC-14686-induced Ca2+ release; and conversely pretreatment with NPC-14686 completely inhibited thapsigargin-induced Ca2+ release. The Ca2+ release induced by 200 microM NPC-14686 was not affected by inhibiting phospholipase C with 2 microM U73122. Overnight treatment with 1-500 microM NPC-14686 decreased cell viability in a concentration-dependent manner. These findings suggest that in human PC3 prostate cancer cells, NPC-14686 increases [Ca2+]i by evoking extracellular Ca2+ influx and releasing intracellular Ca2+ from the endoplasmic reticulum via a phospholiase C-independent manner. NPC-14686 may be cytotoxic to prostate cancer cells.     

Cytoplasmic Ca2+ oscillations evoked by receptor stimulation, G-protein activation, internal application of inositol trisphosphate or Ca2+: simultaneous microfluorimetry and Ca2+ dependent Cl- current recording in single pancreatic acinar cells.

The effects of acetylcholine (ACh), cholecystokinin (CCK), internally applied GTP-gamma-S, inositol trisphosphate [Ins (1,4,5) P3] or Ca2+ on the cytoplasmic free Ca2+ concentration [( Ca2+]i) were assessed by simultaneous microfluorimetry (fura-2) and measurement of the Ca2(+)-dependent Cl- current (patch-clamp whole-cell recording) in single internally perfused mouse pancreatic acinar cells. ACh (0.1-0.2 microM) evoked an oscillating increase in [Ca2+]i measured in the cell as a whole (microfluorimetry) which was synchronous with oscillations in the Ca2(+)-dependent Cl- current reporting [Ca2+]i close to the cell membrane. In the same cells a lower ACh concentration (0.05 microM) evoked shorter repetitive Cl- current pulses that were not accompanied by similar spikes in the microfluorimetric recording. When cells did not respond to 0.1 microM ACh, caffeine (1 mM) added on top of the sustained ACh stimulus resulted in [Ca2+]i oscillations seen synchronously in both types of recording. CCK (10 nM) also evoked [Ca2+]i oscillations, but with much longer intervals between slightly broader Ca2+ pulses. Internal perfusion with 100 microM GTP-gamma-S evoked [Ca2+]i oscillations with a similar pattern. Ins (1,4,5) P3 (10 microM) evoked repetitive shortlasting spikes in [Ca2+]i that were only seen in the Cl- current traces, except in one small cell where these spikes were also observed synchronously in the microfluorimetric recording. Caffeine (1 mM) broadened these Ca2+ pulses. [Ca2+]i was also directly changed, bypassing the normal signalling process, by infusion of a low or high Ca2+ solution into the pipette.(ABSTRACT TRUNCATED AT 250 WORDS)     

Amine weak bases disrupt vesicular storage and promote exocytosis in chromaffin cells

The vesicular contents in bovine chromaffin cells are maintained at high levels owing to the strong association of its contents, which is promoted by the low vesicular pH. The association is among the catecholamines, Ca2+, ATP, and vesicular proteins. It was found that transient application of a weak base, methylamine (30 mM), amphetamine (10 microM), or tyramine (10 microM), induced exocytotic release. Exposure to these agents was also found to increase both cytosolic catecholamine and intracellular Ca2+ concentration, as measured by amperometry and fura-2 fluorescence. Amphetamine, the most potent amine with respect to evoking exocytosis, was found to be effective even in buffer without external Ca2+; however, the occurrence of spikes was suppressed when BAPTA-acetoxymethyl ester was used to complex intracellular Ca2+. Amphetamine-induced spikes in Ca2+-free medium were not suppressed by thapsigargin or ruthenium red, inhibitors of the sarco(endo)plasmic reticulum Ca2+-ATPase and mitochondrial Ca2+ stores. Atomic absorption measurements of amphetamine- and methylamine-treated vesicles reveal that intravesicular Ca2+ stores are decreased after a 15-min incubation. Taken together, these data indicate that amphetamine and methylamine can disrupt vesicular stores to a sufficient degree that Ca2+ can escape and trigger exocytosis.     

Simultaneous recording of calcium transients in skeletal muscle using high- and low-affinity calcium indicators.

To monitor cytosolic [Ca2+] over a wide range of concentrations in functioning skeletal muscle cells, we have used simultaneously the rapid but relatively low affinity calcium indicator antipyrylazo III (AP III) and the slower but higher affinity indicator fura-2 in single frog twitch fibers cut at both ends and voltage clamped with a double vaseline gap system. When both dyes were added to the end pool solution the cytosolic fura-2 concentration reached a steady level equal to the end pool concentration within approximately 2.5 h, a time when the AP III concentration was still increasing. For depolarizing pulses of increasing amplitude, the fura-2 fluorescence signal approached saturation when the simultaneously recorded AP III absorbance change was far from saturation. Comparison of simultaneously recorded fura-2 and AP III signals indicated that the mean values of the on and off rate constants for calcium binding to fura-2 in 18 muscle fibers were 1.49 x 10(8) M-1 s-1 and 11.9 s-1, respectively (mean KD = 89 nM), if all AP III in the fiber is assumed to behave as in calibrating solution and to be in instantaneous equilibrium with [Ca2+]. [Ca2+] transients calculated from the fura-2 signals using these rate constants were consistent with the [Ca2+] transients calculated from the AP III signals. Resting [Ca2+] or small changes in [Ca2+] which could not be reliably monitored with AP III could be monitored with fura-2 with little or no interference from changes in [Mg2+] or from intrinsic signals. The fura-2 signal was also less sensitive to movement artifacts than the AP III signal. After a [Ca2+] transient the fura-2 signal demonstrated a relatively small elevation of [Ca2+] that was maintained for many seconds.