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A V Tepikin

Publications and source records attributed to A V Tepikin.

At least 19 recordsLinked to original sources
PubMed1998

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells.

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet techniques we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca+]i but induced vigorous extracellular Ca+ concentration ([Ca2+]o) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]o indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]o, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]o spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mm. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.

Acetylcholine↗
PubMed1998

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells.

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.

Acetylcholine↗
PubMed1998

Termination of cytosolic Ca2+ signals: Ca2+ reuptake into intracellular stores is regulated by the free Ca2+ concentration in the store lumen.

The mechanism by which agonist-evoked cytosolic Ca2+ signals are terminated has been investigated. We measured the Ca2+ concentration inside the endoplasmic reticulum store of pancreatic acinar cells and monitored the cytoplasmic Ca2+ concentration by whole-cell patch-clamp recording of the Ca2+-sensitive currents. When the cytosolic Ca2+ concentration was clamped at the resting level by a high concentration of a selective Ca2+ buffer, acetylcholine evoked the usual depletion of intracellular Ca2+ stores, but without increasing the Ca2+-sensitive currents. Removal of acetylcholine allowed thapsigargin-sensitive Ca2+ reuptake into the stores, and this process stopped when the stores had been loaded to the pre-stimulation level. The apparent rate of Ca2+ reuptake decreased steeply with an increase in the Ca2+ concentration in the store lumen and it is this negative feedback on the Ca2+ pump that controls the Ca2+ store content. In the absence of a cytoplasmic Ca2+ clamp, acetylcholine removal resulted in a rapid return of the elevated cytoplasmic Ca2+ concentration to the pre-stimulation resting level, which was attained long before the endoplasmic reticulum Ca2+ store had been completely refilled. We conclude that control of Ca2+ reuptake by the Ca2+ concentration inside the intracellular store allows precise Ca2+ signal termination without interfering with store refilling.

Acetylcholine↗
PubMed1997

Ca2+ flow via tunnels in polarized cells: recharging of apical Ca2+ stores by focal Ca2+ entry through basal membrane patch.

Intracellular Ca2+ store depletion induces Ca2+ entry across the plasma membrane, allowing the store to recharge. In our experiments, Ca2+ stores in pancreatic acinar cells were depleted by acetylcholine (ACh) stimulation in Ca2+-free solution. Thereafter, Ca2+ entry was only allowed through a CaCl2-containing pipette attached to the basal membrane. Recharging intracellular Ca2+ stores via a patch pipette occurred without a rise in the cytosolic Ca2+ concentration and depended on the operation of a thapsigargin-sensitive Ca2+ pump. After a period of focal Ca2+ entry, ACh could again evoke a rise in the cytosolic Ca2+ concentration, and this rise always started in the apical secretory pole. Recharging the apical Ca2+ store therefore depends on Ca2+ flow through a tunnel from the basal to the secretory pole, and the endoplasmic reticulum Ca2+ pump is essential for this process.

Acetylcholine↗
PubMed1997

Distribution of Ca2+ extrusion sites on the mouse pancreatic acinar cell surface.

The localizations of Ca2+ extrusion sites in mouse pancreatic acinar cells during elevation of the intracellular free calcium concentration ([Ca2+]i) have been studied. During an agonist stimulated calcium elevation as well as when intracellular calcium is released from a 'caged compound', Ca2+ is primarily extruded from the apical secretory pole of the cells in spite of different spatial patterns of [Ca2+]i different sources of Ca2+, and the presence or absence of agonist. This is most likely due to a relatively high density of calcium pumps in the secretory granule region, although it could be explained by calcium pumps in this part of the cell having different characteristics from those in the basal membrane. The intensity of Ca2+ extrusion in the apical secretory pole is such that substantial (several millimoles per litre) changes of the free calcium concentration in the lumen of the acinus can occur during agonist stimulation.

Acetylcholine↗
PubMed1996

Localization of Ca2+ extrusion sites in pancreatic acinar cells.

We have investigated the localization of Ca2+ extrusion sites in mouse pancreatic acinar cells. Employing a new technique, in which high resolution localization of cellular Ca2+ exit is achieved by confocal microscopy and a Ca2+-sensitive fluorescent probe coupled to heavy dextran to slow down diffusion of extracellular Ca2+, it is shown directly that the secretory pole (secretory granule area) is the major site for Ca2+ extrusion following agonist stimulation. This Ca2+ extrusion appears not to be a consequence of exocytosis, as assessment of secretion under our experimental conditions (low external Ca2+ concentration, room temperature) using the technique of monitoring quinacrine fluorescence shows little loss of secretory granules in spite of sustained Ca2+ exit. We conclude that Ca2+ is primarily extruded by Ca2+ pumps from the secretory pole and propose that this process is useful for maintaining a high Ca2+ concentration in the acinar lumen, which is necessary for promotion of endocytosis.

Acetylcholine↗
PubMed1996

Calcium dependence of calcium extrusion and calcium uptake in mouse pancreatic acinar cells.

1. The droplet technique was used to investigate the calcium dependence of calcium extrusion from pancreatic acinar cells with preserved intracellular environments. The calcium dependence of calcium extrusion indicated a strong co-operativity (Hill coefficient, 3). The half-maximal rate of calcium extrusion occurred at an intracellular free calcium concentration ([Ca2+]i) of approximately 200 nM. At [Ca2+]i levels higher than 400 nM the calcium extrusion mechanism was almost completely saturated. 2. The rate of [Ca2+]i recovery was measured with the same cells under conditions where both calcium extrusion and calcium reuptake occurred simultaneously and under conditions when calcium reuptake was prevented and recovery depended entirely upon calcium extrusion. The rate of [Ca2+]i recovery due to calcium reuptake displayed a very sharp dependence on [Ca2+]i. The rate of [Ca2+]i recovery due to reuptake increased approximately 10 times (from 4.3 to 44.1 nM s-1) for an increase of [Ca2+]i of only 100 nM (from 120 to 220 nM). 3. With a decrease of [Ca2+]i the ratio of rate of calcium extrusion to rate of calcium uptake into internal stores increased, indicating that extrusion plays a more important role at low [Ca2+]i levels. Data for [Ca2+]i recovery rates due to extrusion and due to reuptake allowed us to evaluate the absolute rate of calcium translocation into the internal stores during the recovery process. When [Ca2+]i = 350 nM the total (i.e. bound and free) calcium concentration in the cytosol decreased by approximately 100 microM s-1 due to calcium uptake into internal stores. The rate of uptake was approximately 20 times slower when [Ca2+]i = 120 nM.

Animals↗
PubMed1996

Calcium transport pathways in the nucleus.

Due to the availability of new biophysical and biochemical techniques, there has recently been considerable progress in our understanding of Ca2+ transport inside, as well as into and out of, the nucleus. A number of Ca2+ transport pathways have been localized specifically in the outer or inner nuclear membrane and the Ca2+ permeability through the nuclear pore complex has been assessed. The nuclear envelope has characteristics similar to those of a leaky epithelium. The leak is through the nuclear pore complex. The outer nuclear membrane contains the Ca2+ ATPase whereas the functionally important inositol trisphosphate (IP3)-activated Ca2+ release channels are specifically localized in the inner nuclear membrane.

Animals↗
PubMed1996

Decrease of acidity inside zymogen granules inhibits acetylcholine- or inositol trisphosphate-evoked cytosolic Ca2+ spiking in pancreatic acinar cells.

In isolated pancreatic acinar cells application of the proton-potassium ionophore nigericin or the proton-sodium ionophore monensin led to a reduction of acidity inside the zymogen granules which could be visualized in an imaging system by a rapid reduction in the intragranular quinacrine fluorescence. Cytosolic Ca2+ spikes in response to acetylcholine stimulation or intracellular inositol trisphosphate application were assessed by recording Ca2+ -sensitive ionic currents in the patch clamp whole-cell recording configuration. Both nigericin and monensin evoked marked reductions in frequency and amplitude of spikes and in many experiments abolished spiking altogether. The Ca2+ -sensitive membrane currents could still be activated after nigericin or monensin treatment since subsequent application of the Ca2+ ionophore ionomycin evoked a large current response. The decrease in intragranular acidity would appear to inhibit intracellular Ca2+ release perhaps due to a reduction in the free intragranular Ca2+ concentration.

Acetylcholine↗
PubMed1996

Short pulses of acetylcholine stimulation induce cytosolic Ca2+ signals that are excluded from the nuclear region in pancreatic acinar cells.

We have investigated the spreading of cytosolic Ca2+ signals generated by acetylcholine stimulation (using either microionophoresis or pressure application) of isolated pancreatic acinar cells (or small cell clusters) using confocal microscopy of Ca2+-sensitive fluorescence (fura red). We have been particularly interested in the effects of short vigorous pulses of acetylcholine (ACh) stimulation since, in the pancreas, ACh secreted from nerve endings is quickly eliminated by the action of ACh esterase. We focused on three regions: the secretory pole (secretory granule area), the nucleus and the basal area outside the nucleus. The nuclei were visualized by using the specific nuclear stain Hoechst 33342. With ionophoretic application, a long-lasting stimulation with ACh (10 s and longer) induces large Ca2+ transients of similar amplitude in all the three selected regions of the cell. Short applications (about 3 s) of ACh result in a Ca2+ rise in the secretory pole, whereas no changes in cytoplasmic Ca2+ were detected in the basal, nonnuclear region or in the nucleus. We found that at the peak of such localised Ca2+ responses, evoked either by ACh ionophoresis or pressure application, significant Ca2+ concentration gradients (up to 400 nM/microm) can be established along the line connecting the secretory pole with the nucleus. In some experiments slightly longer applications (about 5 s) of ACh produce Ca2+ transients in both the secretory region and in the basal, nonnuclear regions of the cells, whereas the nuclear [Ca2+] remained largely unaffected. Estimation of the ACh concentration in the vicinity of the cell under investigation indicated that values of about 1 microM were attained in the pressure application experiments. These results show directly that the nucleus of pancreatic acinar cells can be effectively protected from relatively large Ca2+ transients generated in the secretory pole of pancreatic acinar cells by short pulses of near-maximal ACh concentrations. This indicates that calcium-dependent secretion (both fluid and digestive enzymes) can occur without changes of the intranuclear [Ca2+] and consequently without activation of numerous calcium dependent nuclear processes.

Acetylcholine↗
PubMed1995

ATP-dependent accumulation and inositol trisphosphate- or cyclic ADP-ribose-mediated release of Ca2+ from the nuclear envelope.

Uptake and release of Ca2+ from isolated liver nuclei were studied with fluorescent probes. We show with the help of digital imaging and confocal microscopy that the Ca(2+)-sensitive fluorescent probe Fura 2 is concentrated in or around the nuclear envelope and that the distribution of Fura 2 fluorescence is similar to that of an endoplasmic reticulum marker. The previously demonstrated ATP-dependent uptake of Ca2+ into isolated nuclei and release of the accumulated Ca2+ by inositol 1,4,5-trisphosphate (IP3) are therefore due to transport of Ca2+ into and out of the nuclear envelope and not the nucleoplasm. Dextrans labeled with fluorescent Ca2+ indicators (calcium-Green 1 and Fura 2) are distributed uniformly in the nucleoplasm and can be used to show that changes in the external Ca2+ concentration produce rapid changes in the nucleoplasmic Ca2+ concentration. Nevertheless, IP3 and cyclic ADP-ribose evoke transient intranuclear Ca2+ elevations. The release from the Ca2+ stores in or around the nuclear envelope appears to be directed into the nucleoplasm from where it can diffuse out through the permeable nuclear pore complexes.

Adenosine Diphosphate Ribose↗
PubMed1994

The droplet technique: measurement of calcium extrusion from single isolated mammalian cells.

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.

Calcium↗
PubMed1993

Calcium clamp in single nerve cells.

Free calcium concentration in isolated single neurons was clamped using a new technical approach based on a feed-back connection between the Fura-2 fluorescence signal measuring the intracellular Ca2+ concentration ([Ca2+]i) and iontophoretic current injecting Ca2+ into the cell. Beginning of [Ca2+]i clamping at a level above the basal one triggered fast (few seconds) current transients equal to injection of 36 +/- 20 microM Ca2+ (for a 0.1 microM change of [Ca2+]i), representing the filling of a fast cytosolic buffer. Continuation of clamping required very small clamping currents (corresponding to injection of 0.39 +/- 0.20 microM.s-1 Ca2+). This value increased proportionally to the magnitude of the change of [Ca2+]i above basal level, indicating the activation of calcium-dependent mechanisms for Ca2+ removal from the cytosol. The described approach allowed measurement, under physiological conditions, of the capacitative and kinetic properties of different Ca-regulating systems functioning in a single nerve cell as well as other types of cells.

Animals↗
PubMed1992

Pulsatile Ca2+ extrusion from single pancreatic acinar cells during receptor-activated cytosolic Ca2+ spiking.

Ca2+ extrusion was measured simultaneously with the free intracellular Ca2+ concentration ([Ca2+]i) from single pancreatic acinar cells placed in microdroplets of extracellular solution (Tepikin, A. V., Voronina, S. G., Gallacher, D. V., and Petersen, O. H. (1992) J. Biol. Chem. 267, 3569-3572). Submaximal stimulation with cholecystokinin usually evoked discrete cytosolic Ca2+ spikes and each of these spikes was associated with a discrete and virtually synchronous pulse of Ca2+ extrusion into the extracellular microdroplet solution. When ACh evoked repetitive discrete [Ca2+]i spikes, each spike was also accompanied by a discrete pulse of Ca2+ extrusion. The velocity of Ca2+ extrusion oscillated with a time course similar to that of [Ca2+]i. The extracellular solution in our experiments had a low total calcium concentration (15-35 microM) and only a limited number of [Ca2+]i spikes (2-8) could be evoked. The magnitudes of the [Ca2+]i spikes and the amounts of Ca2+ extruded during each spike gradually decreased in each experiment. During the first cholecystokinin-evoked cytosolic Ca2+ spike the Ca2+ extrusion corresponded to a loss of 15-70% (mean value 39% +/- 12) of the mobilizable cellular calcium pool. The substantial pulsatile Ca2+ extrusion occurring synchronously with the receptor-activated cytosolic Ca2+ spikes is therefore an important element in repetitively bringing back [Ca2+]i to the resting level.

Acetylcholine↗
PubMed1992

Acetylcholine-evoked increase in the cytoplasmic Ca2+ concentration and Ca2+ extrusion measured simultaneously in single mouse pancreatic acinar cells.

The intracellular free Ca2+ concentration ([free Ca2+]i) was measured simultaneously with the Ca2+ extrusion from single isolated mouse pancreatic acinar cells placed in a microdroplet of extracellular solution using the fluorescent probes fura-2 and fluo-3. The extracellular solution had a low total calcium concentration (15-35 microM), and acetylcholine (ACh), applied by microionophoresis, therefore only evoked a transient elevation of [free Ca2+]i lasting about 2-5 min. The initial sharp rise in [free Ca2+]i from about 100 nM toward 0.5-1 microM was followed within seconds by an increase in the total calcium concentration in the microdroplet solution ([Ca]o). The rate of this rise of [Ca]o was dependent on the [free Ca2+]i elevation, and as [free Ca2+]i gradually decreased Ca2+ extrusion declined with the same time course. Ca2+ extrusion following ACh stimulation was not influenced by removal of all Na+ in the microdroplet solution indicating that the Ca2+ extrusion is not mediated by Na(+)-Ca2+ exchange but by the Ca2+ pump. The amount of Ca2+ extruded during the ACh-evoked transient rise in [free Ca2+]i corresponded to a decrease in the total intracellular Ca concentration of about 0.7 mM which is close to previously reported values (0.5-1 mM) for the total concentration of mobilizable calcium in these cells. Our results therefore demonstrate directly the ability of the Ca2+ pump to rapidly remove the large amount of Ca2+ released from the intracellular pools during receptor activation.

Acetylcholine↗
PubMed1991

Free calcium transients and oscillations in nerve cells.

Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xanthine-induced release. IP3 and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Extrusion of calcium from a single isolated neuron of the snail Helix pomatia.

Simultaneous optical measurements of extra- and intracellular Ca2+ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca2+. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the micro-chamber containing the cell. The velocity of Ca2+ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2-0.5 microM, the velocity of Ca2+ extrusion from the neuron was approximately 0.3-4.6 microM/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca2+ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 microM/sec per cell volume.

Animals↗