scholarly journals Phospholipase C inhibitor, U73122, releases intracellular Ca2+, potentiates Ins(1,4,5)P3-mediated Ca2+ release and directly activates ion channels in mouse pancreatic acinar cells

1997 ◽  
Vol 324 (2) ◽  
pp. 645-651 ◽  
Author(s):  
Hideo MOGAMI ◽  
LLOYD MILLS Chris ◽  
David V. GALLACHER
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Phospholipase C ◽  
Single Cells ◽  
Specific Inhibitor ◽  
Acinar Cells ◽  
Cellular Systems ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Permeabilized Cells

It is recognized in many cellular systems that the receptor/G-protein activation of phospholipase C and Ins(1,4,5)P3 production is the transduction pathway regulating the release of Ca2+ from internal stores. Ca2+ signals can now be monitored at the level of single cells but the biochemical detection of Ins(1,4,5)P3 cannot match this resolution. It is often difficult or impossible to directly attribute responses evoked in single cells by putative phospholipase C-coupled agonists to changes in Ins(1,4,5)P3 levels. U73122 is an aminosteroid that is reported to act as a specific inhibitor of phospholipase C and it has become an important tool in establishing the link between phospholipase C activation and cellular Ca2+ signalling. In the present study we use both patch-clamp electrophysiology and the imaging of fluorescent Ca2+ indicators to investigate the effect of U73122 in mouse pancreatic acinar cells. The study reveals that U73122 has effects other than the inhibition of phospholipase C. U73122 can directly activate ion channels. It can itself promote the release of Ca2+ from intracellular stores in permeabilized cells and in intact cells it triggers a release of Ca2+ that is initiated specifically at the secretory pole of these morphologically and functionally polarized cells. We also present evidence that U73122 can potentiate the response to Ins(1,4,5)P3; this is seen both in permeabilized cells and in patch-clamp protocols in which cells are internally dialysed with submaximal concentrations of Ins(1,4,5)P3. The effects of U73122 are therefore multiple and not specific for the inhibition of phospholipase C. Importantly, all the effects described influence Ca2+ signalling yet in many experimental protocols some of these effects can go unnoticed and might in error be attributed simply to the inhibition of Ins(1,4,5)P3 production.

scholarly journals Effects of Ca2+ on phosphoinositide breakdown in exocrine pancreas

1986 ◽  
Vol 238 (3) ◽  
pp. 765-772 ◽  
Author(s):  
C W Taylor ◽  
J E Merritt ◽  
J W Putney ◽  
R P Rubin
Keyword(s):  
Arachidonic Acid ◽  
Phospholipase C ◽  
Acinar Cells ◽  
Major Effect ◽  
Arachidonic Acid Metabolism ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Receptor Agonists ◽  
Independent Effects

Recent studies have established that inositol 1,4,5-trisphosphate [I(1,4,5)P3] provides the link between receptor-regulated polyphosphoinositide hydrolysis and mobilization of intracellular Ca2+. Here, we report the effects of Ca2+ on inositol trisphosphate (IP3) formation from phosphatidylinositol bisphosphate (PIP2) catalysed by phospholipase C in intact and electrically permeabilized rat pancreatic acinar cells. In permeabilized cells, the Ca2+-mobilizing agonist caerulein stimulated [3H]IP3 formation when the free [Ca2+] was buffered at 140 nM, the cytosolic free [Ca2+] of unstimulated pancreatic acinar cells. When the free [Ca2+] was reduced to less than 10 nM, caerulein did not stimulate [3H]IP3 formation. Ca2+ in the physiological range stimulated [3H]IP3 formation and reduced the amount of [3H]PIP2 in permeabilized cells. The effects of Ca2+ and the receptor agonist caerulein were additive, but we have not established whether this reflects independent effects on the same or different enzymes. The effect of Ca2+ on [3H]IP3 formation by permeabilized cells was unaffected by inhibitors of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism; nor were the effects of Ca2+ mimicked by addition of arachidonic acid. These results suggest that the effects of Ca2+ on phospholipase C activity are not a secondary consequence of Ca2+ activation of phospholipase A2. Changes in free [Ca2+] (less than 10 nM-1.2 mM) did not affect the metabolism of exogenous [3H]I(1,4,5)P3 by permeabilized cells. In permeabilized cells, breakdown of exogenous [3H]IP3 to [3H]IP2 (inositol bisphosphate), and formation of [3H]IP3 in response to receptor agonists were equally inhibited by 2,3-bisphosphoglyceric acid. This suggests that the [3H]IP2 formed in response to receptor agonists is entirely derived from [3H]IP3. In intact cells, [3H]IP3 formation was stimulated when ionomycin was used to increase the cytosolic free [Ca2+]. However, a maximal concentration of caerulein elicited ten times as much IP3 formation as did the highest physiologically relevant [Ca2+]. We conclude that the major effect of receptor agonists on IP3 formation does not require an elevation of cytosolic free [Ca2+], although the increase in free [Ca2+] that normally follows IP3 formation may itself have a small stimulatory effect on phospholipase C.

scholarly journals Collagen type IV stimulates an increase in intracellular Ca2+ in pancreatic acinar cells via activation of phospholipase C

1994 ◽  
Vol 299 (3) ◽  
pp. 603-611 ◽  
Author(s):  
L Somogyi ◽  
Z Lasić ◽  
S Vukičević ◽  
H Banfić
Keyword(s):  
Tyrosine Kinase ◽  
Phospholipase C ◽  
Collagen Type ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Collagen Type Iv ◽  
Type I ◽  
Rgd Sequence ◽  
Type Iv ◽  
Lipid Signalling

Intracellular Ca2+ responses to extracellular matrix molecules were studied in suspensions of pancreatic acinar cells loaded with Fura-2. Collagen type I, laminin, fibrinogen and fibronectin were unable to raise cytosolic free Ca2+ concentration ([Ca2+]i), whereas collagen type IV, at concentrations from 5 to 50 micrograms/ml, significantly increased it. The effect of collagen type IV was not due to possible contamination with type-I transforming growth factor beta or plasminogen, as neither of these agents was able to increase [Ca2+]i. Using highly specific mass assays, concentrations of inositol lipids, 1,2-diacylglycerol (DAG) and Ins(1,4,5) P3 were measured in pancreatic acinar cells stimulated with collagen type IV. A decrease in the concentrations of PtdIns(4,5) P2 and PtdIns4 P with a concomitant increase in the concentrations of DAG and InsP3 mass were observed, showing that collagen type IV increases [Ca2+]i by activation of phospholipase C. The observed [Ca2+]i signals had two components, the first resulting from Ca2+ release from the intracellular stores, and the second resulting from Ca2+ flux from the extracellular medium through the verapamil-insensitive channels. A tyrosine kinase inhibitor (tyrphostine) was able to block inositol lipid signalling caused by collagen type IV, which together with the insensitivity of this pathway to cholera toxin and pertussis toxin or to preactivation of protein kinase C, the longer duration of the increase in [Ca2+]i and a longer lag period needed for observation of increases in DAG and InsP3 concentration with collagen type IV than with carbachol (50 mM) suggest that activation of phospholipase C by collagen type IV is caused by tyrosine kinase activation. Inositol lipid signalling and increases in [Ca2+]i were also observed with Arg-Gly-Asp (RGD)-containing peptide but not with Arg-Asp-Gly (RDG)-containing peptide. Collagen type IV and RGD-containing peptide, but not carbachol, competed in increasing [Ca2+]i and DAG concentration, suggesting that the binding site of collagen type IV responsible for phospholipase C activation contains the RGD sequence. Together the present results suggest that, in pancreatic acinar cells, RGD sequence(s) within collagen type IV molecules cause activation of tyrosine kinase, probably through one of the integrin receptors, which then stimulates phospholipase C and increases [Ca2+]i.

scholarly journals Ca2+-sensitivity of inositol 1,4,5-trisphosphate-mediated Ca2+ release in permeabilized pancreatic acinar cells

1990 ◽  
Vol 265 (3) ◽  
pp. 681-687 ◽  
Author(s):  
P H G M Willems ◽  
M D De Jong ◽  
J J H H M De Pont ◽  
C H Van Os
Keyword(s):  
Acinar Cells ◽  
Inhibitory Effect ◽  
Feedback Mechanism ◽  
Pancreatic Acinar Cells ◽  
Release Mechanism ◽  
Concomitant Increase ◽  
Permeabilized Cells ◽  
Negative Feedback Mechanism ◽  
Inositol 1,4,5 Trisphosphate ◽  
Energy Dependent

Hormonal and phorbol ester pretreatment of pancreatic acinar cells markedly decreases the Ins(1,4,5)P3-induced release of actively stored Ca2+ [Willems, Van Den Broek, Van Os & De Pont (1989) J. Biol. Chem. 264, 9762-9767]. Inhibition occurred at an ambient free Ca2+ concentration of 0.1 microM, suggesting a receptor-mediated increase in Ca2(+)-sensitivity of the Ins(1,4,5)P3-operated Ca2+ channel. To test this hypothesis, the Ca2(+)-dependence of Ins(1,4,5)P3-induced Ca2+ release was investigated. In the presence of 0.2 microM free Ca2+, permeabilized cells accumulated 0.9 nmol of Ca2+/mg of acinar protein in an energy-dependent pool. Uptake into this pool increased 2.2- and 3.3-fold with 1.0 and 2.0 microM free Ca2+ respectively. At 0.2, 1.0 and 2.0 microM free Ca2+, Ins(1,4,5)P3 maximally released 0.53 (56%), 0.90 (44%) and 0.62 (20%) nmol of Ca2+/mg of acinar protein respectively. Corresponding half-maximal stimulatory Ins(1,4,5)P3 concentrations were calculated to be 0.5, 0.6 and 1.4 microM, suggesting that the affinity of Ins(1,4,5)P3 for its receptor decreases beyond 1.0 microM free Ca2+. The possibility that an inhibitory effect of sub-micromolar Ca2+ is being masked by the concomitant increase in size of the releasable store is excluded, since Ca2+ release from cells loaded in the presence of 0.1 or 0.2 microM free Ca2+ and stimulated at higher ambient free Ca2+ was not inhibited below 1.0 microM free Ca2+. At 2.0 and 10.0 microM free Ca2+, Ca2+, Ca2+ release was inhibited by approx. 30% and 75% respectively. The results presented show that hormonal pretreatment does not lead to an increase in Ca2(+)-sensitivity of the release mechanism. Such an increase in Ca2(+)-sensitivity to sub-micromolar Ca2+ is required to explain sub-micromolar oscillatory changes in cytosolic free Ca2+ by a Ca2(+)-dependent negative-feedback mechanism.

Patch-clamp study of single-channel and whole-cell K+ currents in guinea pig pancreatic acinar cells

1988 ◽  
Vol 255 (3) ◽  
pp. G275-G285 ◽  
Author(s):  
K. Suzuki ◽  
O. H. Petersen
Keyword(s):  
Patch Clamp ◽  
Guinea Pig ◽  
Single Channel ◽  
Pancreatic Acinar Cells ◽  
Intact Cells ◽  
Membrane Area ◽  
Pancreatic Acini ◽  
Whole Cell ◽  
Single Channel Currents ◽  
K Currents

K+ channels in the plasma membrane of isolated guinea pig pancreatic acini were studied by patch-clamp single-channel and whole-cell current recording techniques. Three types of K+-permeable pores were found in excised patch experiments: Ca2+-activated nonselective cation channels with a unit conductance of approximately 25 pS that could be inhibited by ATP acting on the membrane inside, and two kinds of Ca2+- and voltage-activated K+-selective channels with unit conductances (in symmetrical K+-rich solutions) of about 200 and 30 pS, respectively. In intact cells, pentagastrin activation of currents through the 30 pS K+-selective pores was demonstrated. In these experiments pentagastrin was added to the bath solution and had no direct contact with the electrically isolated membrane area from which the single-channel currents were recorded, suggesting that the activation is mediated via an intracellular messenger system. Pentagastrin stimulation of voltage-gated K+ currents was also observed in whole-cell recording experiments. Results from these experiments suggest that in the stimulated condition the membrane electrical properties were dominated by the 30 pS K+-selective channels.

Calcium release by cholecystokinin analogue OPE is IP3 dependent in single rat pancreatic acinar cells

1994 ◽  
Vol 267 (1) ◽  
pp. C220-C228 ◽  
Author(s):  
H. Y. Gaisano ◽  
D. Wong ◽  
L. Sheu ◽  
J. K. Foskett
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Calcium Release ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Low Molecular Weight ◽  
Ip3 Receptor ◽  
Intact Cells ◽  
Inositol 1,4,5 Trisphosphate ◽  
Low Levels

Cholecystokinin (CCK) and carbachol raise intracellular Ca2+ concentration ([Ca2+]i) in pancreatic acinar cells by elevating inositol 1,4,5-trisphosphate (IP3). CCK analogues JMV-180 and OPE stimulate fully efficacious enzyme secretion and [Ca2+]i oscillations but release Ca2+ from intracellular stores by apparently IP3-independent mechanisms in permeabilized acinar cells. In the present study, we investigated whether OPE mobilizes Ca2+ from IP3-sensitive Ca2+ stores and whether IP3 mediates such responses in single intact cells. OPE and JMV-180 similarly elevated IP3 to low levels compared with those elicited by 10 nM CCK. Depletion of IP3-sensitive stores by elevation of intracellular IP3 using carbachol, microinjection of a nonmetabolizable IP3 analogue, or exposure to thapsigargin, in the absence of extracellular Ca2+, depleted the same Ca2+ stores that were sensitive to OPE. In converse experiments, OPE depleted carbachol- or thapsigargin-sensitive stores, indicating that carbachol-, thapsigargin-, IP3-, and OPE-sensitive Ca2+ stores overlap completely and that stores mobilized by OPE are IP3 sensitive. To determine whether IP3 mediates responses to OPE, cells were microinjected with low-molecular-weight heparin, a competitive inhibited the rise of [Ca2+]i in response to carbachol, OPE, or JMV-180, whereas de-N-sulfated heparin, an inactive heparin, was without effect. These results indicate that CCK analogues release Ca2+ from IP3-sensitive Ca2+ stores by mechanisms involving the IP3 receptor.

scholarly journals Relationship between hormonal, GTP and Ins(1,4,5)P3-stimulated Ca2+ uptake and release in pancreatic acinar cells

1989 ◽  
Vol 263 (2) ◽  
pp. 333-339 ◽  
Author(s):  
S Muallem ◽  
T G Beeker
Keyword(s):  
Protein Kinase C ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini ◽  
Intracellular Pool ◽  
Permeabilized Cells ◽  
Kinase C ◽  
Uptake Medium ◽  
Uptake And Release ◽  
Stimulation Of

Electrically permeabilized rat pancreatic acini were used to evaluate the contributions of GTP and Ins(1,4,5) P3 to hormone-stimulated Ca2+ uptake and release from intracellular pools. Treatment of permeabilized acini with Ca2+-mobilizing hormones, GTP or GTP[S] resulted in stimulation of an ATP-dependent, VO4(2-)-sensitive Ca2+ uptake into a non-mitochondrial intracellular pool. GTP and GTP[S] also augmented the hormone-mediated stimulation of Ca2+ uptake. Including oxalate in the uptake medium increased Ca2+ uptake into this pool but did not modify the stimulation of Ca2+ uptake induced by hormones or GTP. Ins(1,4,5)P3 released all the extra Ca2+ accumulated as a result of hormone, GTP or GTP[S] stimulation. Hence, these stimuli activated the Ca2+ pump localized in the membrane of the hormone and Ins(1,4,5)P3-sensitive Ca2+ pool. Including 2,3-diphosphoglyceric acid (PGA) [an inhibitor of Ins(1,4,5)P3 hydrolysis] in the incubation medium blunted the GTP and GTP[S]-stimulated Ca2+ uptake. In the presence of PGA, the hormones inhibited Ca2+ accumulation, and GTP and GTP[S] augmented this effect. Accordingly, PGA stabilized the Ins(1,4,5)P3-evoked Ca2+ release from intracellular pools. Only in the presence of PGA was it possible to demonstrate hormonally-evoked Ca2+ release from permeabilized cells. GTP, and more importantly GTP[S], augmented the hormone-evoked Ca2+ release. Hormones and Ins(1,4,5)P3 in the presence or absence of GTP or GTP[S] released Ca2+ from the same intracellular pool. The extent of Ca2+ release caused by the combination of hormones and GTP or GTP[S] was similar to that evoked by Ins(1,4,5)P3 alone. Taken together, these results suggest that GTP or GTP[S] facilitates stimulation of phospholipase C by hormones. Such stimulation results in stimulation of protein kinase C and increased levels of Ins(1,4,5)P3 and is sufficient to explain the effects of GTP and GTP[S] on Ca2+ uptake and release from pancreatic acinar cells.

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