Peter Thorn

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

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

Cyclic ADP-ribose regulation of ryanodine receptors involved in agonist evoked cytosolic Ca2+ oscillations in pancreatic acinar cells.

We have investigated the role of the ryanodine‐sensitive intracellular Ca2+ release channel (ryanodine receptor) in the cytosolic Ca2+ oscillations evoked in pancreatic acinar cells by acetylcholine (ACh) or cholecystokinin (CCK). Ryanodine abolished or markedly inhibited the agonist evoked Ca2+ spiking, but enhanced the frequency of spikes evoked by direct internal inositol trisphosphate (InsP3) application. We have also investigated the possibility that cyclic ADP‐ribose (cADP‐ribose), the putative second messenger controlling the ryanodine receptor, plays a role in Ca2+ oscillations. We found that cADP‐ribose could itself induce repetitive Ca2+ spikes localized in the secretory pole and that these spikes were blocked by ryanodine, but also by the InsP3 receptor antagonist heparin. Our results indicate that both the ryanodine and the InsP3 receptors are involved in Ca2+ spike generation.

Hyaluronic acid modified mesoporous silica nanoparticles for targeted drug delivery to CD44-overexpressing cancer cells

In this paper, a targeted drug delivery system has been developed based on hyaluronic acid (HA) modified mesoporous silica nanoparticles (MSNs). HA-MSNs possess a specific affinity to CD44 over-expressed on the surface of a specific cancer cell line, HCT-116 (human colon cancer cells). The cellular uptake performance of fluorescently labelled MSNs with and without HA modification has been evaluated by confocal microscopy and fluorescence-activated cell sorter (FACS) analysis. Compared to bare MSNs, HA-MSNs exhibit a higher cellular uptake via HA receptor mediated endocytosis. An anticancer drug, doxorubicin hydrochloride (Dox), has been loaded into MSNs and HA-MSNs as drug delivery vehicles. Dox loaded HA-MSNs show greater cytotoxicity to HCT-116 cells than free Dox and Dox-MSNs due to the enhanced cell internalization behavior of HA-MSNs. It is expected that HA-MSNs have a great potential in targeted delivery of anticancer drugs to CD44 over-expressing tumors.

Cytosolic Ca2+ spikes evoked by the thiol reagent thimerosal in both intact and internally perfused single pancreatic acinar cells

Cytosolic calcium signals evoked by the sulphydryl-group-oxidising agent, thimerosal, have been investigated in acutely isolated pancreatic acinar cells. Two techniques were employed for the assessment of the cytosolic free-calcium concentration ([Ca2+]i): measurement of calcium-dependent chloride and non-specific cation currents (whole-cell patch-clamp recording) and microfluorimetry (fura-2). Thimerosal (0.5–100 μM) evoked repetitive spikes in both chloride and cation currents as seen by patch-clamp recording, and in [Ca2+]i as seen by microfluorimetry, with a latency of 1–3 min. The response increased in magnitude over time and was not reversed on removal of thimerosal. The thimerosal-induced spikes were reversibly blocked by 2 mM dithiothreitol and by 20 mM caffeine. Inclusion of heparin (200 μg/ml) in the pipette solution blocked the thimerosal-induced spikes. The calcium spikes continued after the removal of extracellular calcium; however, low concentrations of thimerosal (0.5–5 μM) were unable to initiate a current response in the absence of external calcium. High concentrations of thimerosal (50–100 μM) could initiate spikes without extracellular calcium. Thimerosal, at concentrations that failed to produce an independent effect, potentiated the acetylcholine-evoked oscillations in [Ca2+]i. We conclude that thimerosal is able to mobilise calcium from an intracellular store; the blockade by heparin may indicate that thimerosal exerts an action on the inositol trisphosphate pathway. The dependence on extracellular calcium for initiation, but not for continuation of the thimerosal-induced calcium spikes suggests that thimerosal may have the additional effect of inhibiting the plasma membrane calcium ATPase.

Regulation of adenylyl cyclase by membrane potential

Mammalian adenylyl cyclases possess 12 transmembrane-spanning domains and bear a superficial resemblance to certain classes of ion channels. Some evidence suggests that bacterial and sea urchin sperm adenylyl cyclases can be regulated by membrane depolarization. In the present study, we explored the effect of altering membrane potential on the adenylyl cyclase activity of cerebellar granule cells with acute potassium depolarization. A biphasic stimulatory and then inhibitory response is evoked by progressive increases in the extracellular [K]:[Na] ratio in the absence of extracellular Ca2+. This effect does not mimic the linear increase in membrane potential elicited under the same conditions. Instead it appears as though membrane depolarization opens L-type (nimodipine-sensitive) Ca2+ channels, allowing the entry of Na+, which directly stimulates adenylyl cyclase activity. Gramicidin, which generates pores that are permeable to monovalent cations, and concurrently eliminates the membrane potential, permits a similar stimulation by extracellularly applied Na+. Although the results indicate no direct sensitivity of cerebellar granule cell adenylyl cyclase to membrane potential, they do demonstrate that, as a result of membrane depolarization, the influx of Na+, as well as Ca2+, will elevate cAMP levels.

Vesicle-associated membrane protein 8 (VAMP8) is a SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) selectively required for sequential granule-to-granule fusion.

Compound exocytosis is found in many cell types and is the major form of regulated secretion in acinar and mast cells. Its key characteristic is the homotypic fusion of secretory granules. These then secrete their combined output through a single fusion pore to the outside. The control of compound exocytosis remains poorly understood. Although soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as syntaxin 2, SNAP23 (synaptosome-associated protein of 23 kDa), and SNAP25 have been suggested to play a role, none has been proven. Vesicle-associated membrane protein 8 (VAMP8) is a SNARE first associated with endocytic processes but more recently has been suggested as an R-SNARE in regulated exocytosis. Secretion in acinar cells is reduced when VAMP8 function is inhibited and is less in VAMP8 knock-out mice. Based on electron microscopy experiments, it was suggested that VAMP8 may be involved in compound exocytosis. Here we have tested the hypothesis that VAMP8 controls homotypic granule-to-granule fusion during sequential compound exocytosis. We use a new assay to distinguish primary fusion events (fusion with the cell membrane) from secondary fusion events (granule-granule fusion). Our data show the pancreatic acinar cells from VAMP8 knock-out animals have a specific reduction in secondary granule fusion but that primary granule fusion is unaffected. Furthermore, immunoprecipitation experiments show syntaxin 2 association with VAMP2, whereas syntaxin 3 associates with VAMP8. Taken together our data indicate that granule-to-granule fusion is regulated by VAMP8 containing SNARE complexes distinct from those that regulate primary granule fusion.

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

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

A novel large-conductance Ca(2+)-activated potassium channel and current in nerve terminals of the rat neurohypophysis.

1. Nerve terminals of the rat posterior pituitary were acutely dissociated and identified using a combination of morphological and immunohistochemical techniques. Terminal membrane currents were studied using the ‘whole‐cell’ patch clamp technique and channels were studied using inside‐out and outside‐out patches. 2. In physiological solutions, but with 7 mM 4‐aminopyridine (4‐AP), depolarizing voltage clamp steps from different holding potentials (‐90 or ‐50 mV) elicited a fast, inward current followed by a slow, sustained, outward current. This outward current did not appear to show any steady‐state inactivation. 3. The threshold for activation of the outward current was ‐30 mV and the current‐voltage relation was ‘bell‐shaped’. The amplitude increased with increasingly depolarized potential steps. The outward current reversal potential was measured using tail current analysis and was consistent with that of a potassium current. 4. The sustained potassium current was determined to be dependent on the concentration of intracellular calcium. Extracellular Cd2+ (80 microM), a calcium channel blocker, also reversibly abolished the outward current. 5. The current was delayed in onset and was sustained over the length of a 150 ms‐duration depolarizing pulse. The outward current reached a peak plateau and then decayed slowly. The decay was fitted by a single exponential with a time constant of 9.0 +/‐ 2.2 s. The decay constants did not show a dependence on voltage but rather on intracellular Ca2+. The time course of recovery from this decay was complex with full recovery taking > 190 s. 6. 4‐AP (7 mM), dendrotoxin (100 nM), apamin (40‐80 nM), and charybdotoxin (10‐100 nM) had no effect on the sustained outward current. In contrast Ba2+ (200 microM) and tetraethylammonium inhibited the current, the latter in a dose‐dependent manner (apparent concentration giving 50% of maximal inhibition (IC50) = 0.51 mM). 7. The neurohypophysial terminal outward current recorded here corresponds most closely to a Ca(2+)‐activated K+ current (IK(Ca)) and not to a delayed rectifier or IA‐like current. It also has properties different from that of the Ca(2+)‐dependent outward current described in the magnocellular neuronal cell bodies of the hypothalamus. 8. A large conductance channel is often observed in isolated rat neurohypophysial nerve terminals. The channel had a unit conductance of 231 pS in symmetrical 150 mM K+.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple, coordinated Ca2+ -release events underlie the inositol trisphosphate-induced local Ca2+ spikes in mouse pancreatic acinar cells.

Ca2+ wave initiation and non‐propagating Ca2+ spikes occur as a result of localized Ca2+ release from the more sensitive intracellular Ca2+ stores. Using high spatial and temporal Ca2+ ‐imaging techniques we have investigated inositol 1,4,5 triphosphate (InsP3)‐induced local Ca2+ spiking, which occurs at the site of Ca2+ wave initiation in pancreatic acinar cells. The spatial and temporal organization of a single spike suggested discrete hot spots of Ca2+ release. Further analysis of long trains of Ca2+ spikes demonstrated that these hot spots showed regenerative Ca2+ ‐release events which were consistently active from spike to spike. Regions adjacent to these hot spots also showed regenerative Ca2+ ‐release events of similar amplitude but with a much lower frequency of occurrence. We conclude that the InsP3‐induced non‐propagating Ca2+ spikes can be devolved into smaller components of release. Our results are consistent with a model of coordinated activity of pacemaker hot spots of Ca2+ release that recruit and entrain active Ca2+ ‐release events from surrounding regions.

The Plasma Membrane Q-SNARE Syntaxin 2 Enters the Zymogen Granule Membrane during Exocytosis in the Pancreatic Acinar Cell

During exocytosis in the pancreatic acinar cell, zymogen granules fuse directly with the apical plasma membrane and also with granules that have themselves fused with the plasma membrane. Together, these primary and secondary fusion events constitute the process of compound exocytosis. It has been suggested that the sequential nature of primary and secondary fusion is a consequence of the requirement for plasma membrane soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors, such as syntaxin 2, to enter the membrane of the primary fused granule. We have tested this possibility by determining the location of syntaxin 2 in unstimulated and stimulated pancreatic acini. Syntaxin 2 was imaged by confocal immunofluorescence microscopy. Fused granules were detected both through their filling with the aqueous dye lysine-fixable Texas Red-dextran and through the decoration of their cytoplasmic surfaces with filamentous actin. In unstimulated cells, syntaxin 2 was exclusively present on the apical plasma membrane. In contrast, after stimulation, syntaxin 2 had moved into the membranes of fused granules, as judged by its location around dye-filled structures of 1-μm diameter that were coated with filamentous actin. At long times of stimulation (5 min), the majority (85%) of dye-filled granules were also positive for syntaxin 2. In contrast, at shorter times (1 min), more dye-filled granules (29%) were syntaxin 2-negative. We conclude that syntaxin 2 enters the membrane of a fused zymogen granule after the opening of the fusion pore, and we suggest that this movement might permit the onset of secondary fusion.