Arachidonic Acid Modulates the Spatiotemporal Characteristics of Agonist-evoked Ca2+ Waves in Mouse Pancreatic Acinar Cells

Abstract

MAPK and p42/44MAPK could also be observed in the presence of cholecystokinin (CCK), which also causes activation of cPLA 2 . ACh-and CCK-induced Ca 2؉ waves were slowed down when p38 MAPK (4,5). The speed of Ca 2ϩ waves depends on the type of agonist used for stimulation and to some extent also on the agonist concentration (6). The propagation rate can be modified by application of arachidonic acid (AA) or by activation of protein kinase C (PKC) with phorbol esters (6, 7). Both application of AA and activation of PKC leads to inhibition of CICR and thereby slows down spreading of Ca 2ϩ signals. Analysis of the propagation rate of secretagogueevoked Ca 2ϩ waves in pancreatic acinar cells can therefore be used to investigate coupling of hormone receptors to intracellular signal cascades that lead to endogenous production of AA and/or to activation of PKC. In previous studies we could demonstrate that in mouse pancreatic acinar cells bombesin receptors couple to phospholipase D (7), which produces diacylglycerol and thereby activates PKC, whereas high affinity CCK receptors couple to cytosolic phospholipase A 2 (cPLA 2 ) (6), an enzyme that leads to release of AA from membrane phospholipids. Bombesin-induced activation of PKC via phospholipase D-dependent diacylglycerol production, as well as CCK-induced formation of AA by cPLA 2 , takes place within the very first seconds after hormone application, before or during development of the initial inositol 1,4,5-trisphosphate-induced Ca 2ϩ signal in the luminal cell pole.In the present study we investigated the role of cPLA 2 in ACh-and bombesin-evoked Ca 2ϩ signaling. Our data indicate that in pancreatic acinar cells activation of cPLA 2 is an early event in ACh-but not in bombesin-evoked intracellular signaling. ACh-and CCK-induced activation of cPLA 2 involves MAP kinase activation. Furthermore, we demonstrate that secretagogue-induced Ca 2ϩ signaling in pancreatic acinar cells is modified by AA itself and not by metabolites of AA. MATERIALS AND METHODSCell Preparation-Adult male CD-1 mice (35-40 g) were sacrificed by cervical dislocation. The pancreas was removed and transferred into a "preparation buffer" consisting of (in mM): 130 NaCl, 4.7 KCl, 1.3 CaCl 2 , 1 MgCl 2 , 1.2 KH 2 PO 4 , 10 glucose, 0.2% (w/v) albumin, 0.01% (w/v) trypsin inhibitor, 10 HEPES, pH 7.4. Single acinar cells were isolated by enzymatic digestion with collagenase type V (30 units/ml, 10 min, 37°C; Sigma) as described previously (8). Enzymatic digestion was followed by mechanical dissociation of cells by gentle pipetting. The resulting cell suspension was centrifuged, and the cells were washed twice in preparation buffer without collagenase.Confocal Microscopy-Freshly prepared acinar cells were loaded with 4 M fluo-3/AM for 30 min at room temperature and then stored at 4°C. Experiments were performed within 4 h after cell isolation. For measurement of cytosolic Ca 2ϩ signals, cells were transferred to a perfusion chamber and were allowed to adhere to the glass coverslip for several minut...

Molecular Localization of the Inhibitory Arachidonic Acid Binding Site to the Pore of hIK1

“…Arachidonic acid is an ubiquitous second messenger released in response to both Ca 2ϩ -and cAMP-mediated agonists, including acetylcholine (40,41), UTP (42)(43)(44)(45), bile acids (17), adenosine (16), and vasoactive intestinal polypeptide (14). Inflammatory responses are also characterized by significant increases in AA production, both via direct release from inflammatory cells (20) as well as through Ca 2ϩ -dependent, kinin-mediated generation (18).…”

Cholecystokinin and Gastrin Receptors

“…The pattern of calcium oscillations is also regulated by the phosphorylation of IP 3 receptors in response to physiological doses of CCK through a mechanism dependent on the protein kinase A (PKA) pathway (271,502). In addition, the high-affinity binding site of CCK1R is coupled to the phospholipase A 2 /arachidonic acid cascade that inhibits IP 3 and ryanodine receptors (180,469). Activation of the low-affinity binding sites by high concentrations of CCK generates a very different pattern of calcium mobilization, which consists of a rapid global elevation of intracellular calcium that decreases to a sustained plateau (394).…”

“…In addition to ␤-and ␥-PLC isoforms, two other phospholipases are activated by the CCK receptors: cytosolic phospholipase A 2 (cPLA 2 ), an enzyme that produces arachidonic acid from membrane phospholipids (166, 180,266,469,474,531), and phospholipase D (PLD), which induces a sustained diacylglycerol (DAG) production and activates PKCs (7,54,180,414). Whereas the high-affinity binding site of CCK1R activates cPLA2, the low-affinity binding site is coupled to PLD (180,454).…”

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“…AA also activates leukocyte NADPH oxidase [17], activates PPAR receptors [18], affects H + channel [19], K + channel [20] and Ca 2+ channel activities [21]. It elevates heat shock gene expression [22], induces apoptosis [23] and inhibits Ca 2+ induced Ca 2+ release [24]. In human embryonic kidney cells it down-regulates sterol regulatory element-binding proteins (SREBPs), especially the SREBP-1a isoform [25].…”