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First published online 15 February 2005
doi: 10.1242/jcs.01693

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Inositol (1,4,5)-trisphosphate receptor links to filamentous actin are important for generating local Ca²⁺ signals in pancreatic acinar cells

¹ Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 IPD, UK
² Biomedical Imaging Group, Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01650, USA

View larger version (28K): [in a new window]   Fig. 6. Latrunculin B inhibits Ca2+-dependent Cl– current spikes in single whole-cell patch-clamped acinar cells. Cells were held under voltage clamp at a membrane potential of –30 mV. Trains of current spikes (downward deflections in the current, i.e. outward movement of Cl–) were induced when 10 µM (2,4,5)IP3 was present in the whole-cell patch pipette (A). Bath application of 50-90 µM latrunculin B led to a dose-dependent cessation of spiking (Ba and C). Application of 100 µM ACh at the end of the recording (Ba) showed the cells were still capable of mobilizing a response even after (2,4,5)IP3-induced spikes were abolished. The ACh-induced response in the presence of latrunculin B (280±43 pA, mean±s.e.m.) was not different from control responses in the absence of latrunculin B (Bb) (397±57 pA, mean±s.e.m., Student's t-test P=0.79). DMSO, the drug vehicle, at the maximal concentration used in these experiments had no apparent effect on (2,4,5)IP3-induced current spikes (D). The horizontal line on the left of the current records is the zero current line. The current-voltage relationship of currents induced by an intracellular solution containing 600 nM Ca2+ was not affected by treatment with latrunculin B (E).

View larger version (58K): [in a new window]   Fig. 1. F-actin and IP3Rs are both contained within the apical subplasmalemmal compartment of pancreatic acini. Isolated pancreatic acinar cell clusters were fixed and stained with (A) phalloidin to highlight F-actin, or with (B) antibodies to IP3R2 (pAb) and IP3R3 (mAb). The image in A is a confocal section taken through the middle of the cell cluster. The images in B are 10 confocal serial sections separated by approximately 1 µm in the z-axis projected onto a single plane. Arrows denote the apical membrane and arrowheads denote the basolateral membrane. Bar, 10 µm.

View larger version (65K): [in a new window]   Fig. 2. Actin and IP3Rs are contained within the same region of the apical subplasamlemmal compartment of pancreatic acini. Isolated pancreatic cell clusters were fixed and stained with (A) antibodies to actin (mAb, green) and IP3R2 (pAb, red), or (B) antibodies to actin (pAb, green) and IP3R3 (mAb, red). Images are of confocal sections taken through the middle of the cell clusters. Arrows denote the apical membrane and arrowheads denote the basolateral membrane. Lower panels represent enlarged images of the regions covered by squares. Bar, 10 µm for image of cell cluster and 2 µm for enlarged image.

View larger version (39K): [in a new window]   Fig. 3. After treatment with latrunculin B, IP3Rs remain localized in the same region as the residual actin in the apical subplasmalemmal compartment of pancreatic acini. Isolated pancreatic acinar cell clusters were treated with 100 µM latrunculin B for 30 minutes to 1 hour and then fixed and stained with (A) phalloidin, (B) antibodies to actin (mAb, green) and IP3R2 (pAb, red), or (C) antibodies to actin (pAb, green) and IP3R3 (mAb, red). Images are of confocal sections taken through the middle of the cell clusters. Arrows denote the apical membrane and arrowheads denote the basolateral membrane. Lower panels represent enlarged images of the regions covered by squares. Bar, 10 µm for images of cell clusters and 2 µm for magnified image.

View larger version (25K): [in a new window]   Fig. 4. Gelsolin effects on the actin cytoskeleton in clusters of pancreatic acinar cells. (A) Permeabilized cells were treated for 5 minutes with a BRB80 solution containing 1.3 µM recombinant gelsolin either in the presence of EGTA (left) or in the presence of 1 mM Ca2+ (right). Cells were then fixed in paraformaldehyde and stained with phalloidin. We observed gelsolin effects on the F-actin network only in the presence of Ca2+, with a reduction in apical F-actin (arrows) and a substantial loss in the basal pole (arrowheads). Bars, 10 µm.

View larger version (11K): [in a new window]   Fig. 5. Western blot analysis indicates that IP3Rs are resistant to detergent extraction. Pancreatic acini were homogenized and used to prepare a Triton X-100-soluble supernatant (S) and a Triton X-100-insoluble pellet (P). Proteins contained in the supernatant and pellet fractions were separated by SDS-PAGE and probed with antibodies recognising (A) actin, (B) myosin IIa and (C) IP3R3.

View larger version (35K): [in a new window]   Fig. 7. Inhibitory effects of latrunculin B on Ca2+-dependent Cl– current spikes are mediated by an inhibition of the underlying local Ca2+ signal. Latrunculin B inhibits Ca2+-dependent Cl– current spikes induced by (2,4,5)IP3 in whole-cell patch-clamped pancreatic acinar cells (A). At time points i-v shown in the patch-clamp trace the average Ca2+ signal, denoted by an asterisk and measured within the white box shown in the phase image, was measured (B). The pseudocolour fluorescence ratio image at these times is also shown.