Biomedical subjects

O H Petersen

Publications and source records attributed to O H Petersen.

At least 19 recordsLinked to original sources

PubMed1999

Edible mushroom (Agaricus bisporus) lectin, which reversibly inhibits epithelial cell proliferation, blocks nuclear localization sequence-dependent nuclear protein import.

The Galbeta1-3GalNAcalpha (TF antigen)-binding lectin (ABL) from the common edible mushroom (Agaricus bisporus) has a potent anti-proliferative effect without any apparent cytotoxicity. This unusual combination of properties prompted investigation of its mechanism of action. In contrast to soluble lectin, agarose-immobilized, and hence noninternalizable ABL had no effect on proliferation of HT29 colon cancer cells. Electron microscopy of HT29 cells incubated with fluorescein- and gold-conjugated ABL showed internalization of the lectin into endocytotic vesicles and multivesicular bodies. Confocal microscopy showed perinuclear accumulation of fluorescein isothiocyanate-conjugated lectin, which also inhibits HT29 cell proliferation, raising the possibility that the lectin might interfere with nuclear pore function. Transport of heat shock protein 70 into the nucleus in response to heat shock was blocked by preincubation of HT29 cells for 6 h with 40 micrograms/ml ABL. In digitonin-permeabilized cells, nuclear uptake of bovine albumin conjugated to a nuclear localization sequence (NLS)-containing peptide was also inhibited by a 15-min preincubation with 40-100 micrograms/ml ABL. In contrast, serum-stimulated nuclear translocation of mitogen-activated protein kinase, which is NLS-independent, was not affected by pretreatment of cells with the lectin. These results suggest that the anti-proliferative effect of ABL is likely to be a consequence of the lectin trafficking to the nuclear periphery, where it blocks NLS-dependent protein uptake into the nucleus.

Adult↗

PubMed1998

Calcium uptake via endocytosis with rapid release from acidifying endosomes.

A number of specific cellular Ca2+ uptake pathways have been described in many different cell types [1] [2] [3]. The possibility that substantial quantities of Ca2+ could be imported via endocytosis has essentially been ignored, although it has been recognized that endosomes can store Ca2+ [4] [5]. Exocrine cells can release significant amounts of Ca2+ via exocytosis [6], so we have investigated the fate of Ca2+ taken up via endocytosis into endosomes. Ca2+-sensitive and H+-sensitive fluorescent probes were placed in the extracellular solution and subsequently taken up into fibroblasts by endocytosis. Confocal microscopy was used to assess the distribution of fluorescence intensity. Ca2+ taken up by endocytosis was lost from the endosomes within a few minutes, over the same period as endosomal acidification took place. The acidification was inhibited by reducing the extracellular Ca2+ concentration, and Ca2+ loss from the endosomes was blocked by bafilomycin (100 nM), a specific inhibitor of the vacuolar proton ATPase. Quantitative evaluation indicated that endocytosis causes substantial import of Ca2+ because of rapid loss from early endosomes.

3T3 Cells↗

PubMed1998

Intracellular glucose switches between cyclic ADP-ribose and inositol trisphosphate triggering of cytosolic Ca2+ spiking.

Cyclic ADP-ribose (cADPR) is a potentially important intracellular Ca2+ releasing messenger [1-5]. In pancreatic acinar cells where intracellular infusion of both inositol trisphosphate (IP3) and cADPR evoke repetitive Ca2+ spiking [6], the cADPR antagonist 8-NH2-cADPR [7], which blocks cADPR-evoked but not IP3-evoked Ca2+ spiking, can abolish Ca2+ spiking induced by physiological levels of the peptide hormone cholecystokinin (CCK) [8]. We have tested the effect of intracellular glucose on the ability of IP3, cADPR and CCK to induce cytosolic Ca2+ spikes in pancreatic acinar cells. In order to gain access to the intracellular cytosol, we used the whole-cell configuration of the patch-clamp technique [9] and monitored cytosolic Ca2+ concentration changes by measuring the Ca(2+)-dependent ionic current [10-13]. Glucose (300 microM to 10 mM) in the patch pipette/intracellular solution prevented cADPR from evoking Ca2+ spiking. The same effect was observed with 2-deoxy-glucose, but not L-glucose. In contrast, glucose potentiated IP3-evoked Ca2+ spiking. CCK evoked Ca2+ spiking irrespective of the presence or absence of intracellular glucose, but the cADPR antagonist 8-NH2-cADPR blocked CCK-evoked Ca2+ spiking only in the absence of intracellular glucose. This suggests that the hormone can evoke Ca2+ spiking via either the IP3 or the cADPR pathway. The intracellular glucose level may control a switch between these two pathways.

Adenosine Diphosphate Ribose↗

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↗

PubMed1998

The cyclic ADP ribose antagonist 8-NH2-cADP-ribose blocks cholecystokinin-evoked cytosolic Ca2+ spiking in pancreatic acinar cells.

In order to investigate the possible involvement of cyclic ADP ribose as an intracellular messenger for hormone-evoked cytosolic Ca2+ signalling, we performed experiments on intracellularly perfused mouse pancreatic acinar cells. Both a stable inositol 1,4,5 trisphosphate analogue (IP3) and cyclic ADP ribose (cADPR) evoked regular spikes of Ca2+ dependent ion current, reflecting cytosolic Ca2+ spiking. The effect of cADPR, but not IP3, was abolished by the presence intracellularly of the cADPR antagonist 8-NH2-cADPR. External application of cholecystokinin (CCK) in a physiological concentration (2.5-5 pM) evoked a mixture of short-lasting and longer-lasting spikes of Ca2+-dependent ion current. These effects were abolished by the presence intracellularly of 8-NH2-cADPR (18 muM). Increasing the CCK concentration to 15 pM could overcome the inhibition by 18 muM of the antagonist. These experiments provide fresh evidence for the involvement of cADPR receptors in the hormone-evoked cytosolic Ca2+ signalling process in pancreatic acinar cells.

Action Potentials↗

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

Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules.

In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca2+ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP3) is believed to release Ca2+ from the endoplasmic reticulum, but we have now tested whether the Ca(2+)-releasing messengers IP3 and cyclic ADP-ribose (cADPr) can liberate Ca2+ from AGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP3 and cADPr evoke a marked decrease in the free intragranular Ca2+ concentration. Using a novel high resolution method, we have measured changes in the Ca2+ concentration in the vicinity of an isolated AG and show that IP3 and cADPr cause rapid Ca2+ release from the granule, explaining the agonist-evoked cytosolic Ca2+ rise in the secretory pole.

Adenosine Diphosphate Ribose↗

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

Simultaneous presence of cAMP and cGMP exert a co-ordinated inhibitory effect on the agonist-evoked Ca2+ signal in pancreatic acinar cells.

The stimulation of the pancreatic acinar cells by physiological secretagogues, such as acetycholine (ACh), activates a well-established intracellular signalling pathway, which involves the generation of Inositol 1,4,5-trisphosphate (InsP3) and the release of Ca2+ from intracellular stores. Caffeine, which inhibits this agonist-evoked Ca2+ response reversibly and competitively also blocks the Ca2+ signal generated by the non-specific activation of the membrane guanine nucleotide-binding proteins (G-proteins). Removal of caffeine is associated with an increase of intracellular [Ca2+] ([Ca2+]i) and the spatial and temporal characteristics of this Ca2+ signal are identical to those of the signal generated by the initial agonist stimulation. Caffeine is also a potent non-specific inhibitor of various cellular phosphodiesterases (PDE) and its inhibitory effect can be reproduced by other PDE inhibitors, chemically related (theophylline) or not (papaverine). Various protocols designed to increase the concentration of either of the major intracellular cyclic nucleotides [adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP)] failed to reproduce the full extent of the caffeine inhibition: at maximal agonist concentration (1 microM ACh) increases of either cAMP or cGMP did not affect the Ca2+ signal, whereas at submaximal doses of agonist (0.1-0.3 microM ACh) they induced partial inhibition. Here we show that only the simultaneous increase of the cellular concentrations of both cyclic nucleotides (either simultaneous or sequential) are effective in mimicking the blocking effect of caffeine and other non-specific PDE inhibitors. These data indicate, thus, that, in addition to other independent intracellular effects, cAMP and cGMP can exert a co-ordinated inhibitory effect of the agonist-evoked Ca2+ signal in pancreatic acinar cells.

Acetylcholine↗

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↗

PubMed1996

Progressive disruption of acinar cell calcium signaling is an early feature of cerulein-induced pancreatitis in mice.

BACKGROUND & AIMS: Disruption of pancreatic exocrine secretion is an important feature of acute pancreatitis. Because cytosolic calcium is a key intracellular messenger controlling pancreatic secretion, this study examined patterns of calcium signaling during the early stages of cerulein-induced pancreatitis. METHODS: Mice were administered hourly intraperitoneal injections of cerulein (50 micrograms/kg), and paired controls were administered saline. Acini were isolated by collagenase from pancreatic tissue harvested after injections 1, 3, 5, and 7 and were loaded with Fura-2. Individual cellular calcium responses to acetylcholine and cholecystokinin were studied using digital imaging. RESULTS: The proportion of cells maintaining a normal oscillatory calcium response to physiological secretagogue stimulation diminished progressively after increasing cerulein injections. Also, the normal polarized spatial pattern of calcium Increase within individual acinar cells was progressively lost. A sustained response to high-dose stimulation was maintained but with diminishing amplitude. The characteristic calcium response to the Ca(2+)-adenosine triphosphatase inhibitor thapsigargin was maintained, implying that calcium reuptake and extrusion were not impaired. CONCLUSIONS: Progressive disruption of physiological patterns of pancreatic acinar cell calcium signaling, notably in the secretory pole of the cell, is an early feature of pancreatitis induced by cerulein hyperstimulation. These changes may be important in contributing to the disruption of exocrine secretion in acute pancreatitis.

Acetylcholine↗

PubMed1996

Can Ca2+ be released from secretory granules or synaptic vesicles?

Recent studies on single isolated secretory granules, as well as secretory granules in intact cells, show that Ca2+ is released from the granule store into the cytosol via a messenger-mediated pathway following agonist binding. This is important for generation of localized cytosolic Ca2+ signals which can initiate exocytosis.

Calcium↗