Evidence for the presence of a regulated phospholipase D (PLD) activity in pancreatic acinar cells is conflicting. Such knowledge is important because signal-activated PLD has been implicated in, amongst other things, regulated exocytosis. In this study, freshly isolated rat pancreatic acini were used to identify PLD transcripts by RT-PCR, to assess the presence and subcellular localization of PLD protein by Western blotting and to evaluate the presence of secretagogue-regulated PLD activity by means of the PLD-catalysed transphosphatidylation reaction. Transcripts of PLD1b and PLD2, but not PLD1a, were present in acinar cells. Moreover, a specific anti-human PLD1 antibody demonstrated the expression of substantial amounts of PLD1 protein. Intriguingly, however, the distribution pattern of acinar PLD1 seen following subcellular fractionation was clearly atypical in that immunoreactivity occurred predominantly in the acinar cytosol. Pretreatment of intact acini with a phorbol ester (4beta-phorbol 12-myristate 13-acetate, PMA) to activate PLD1 protein kinase C (PKC) dependently did not change the subcellular distribution of PLD1. Similarly, pretreatment of a broken cell preparation of acini with guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) to activate PLD via small GTPases and PMA also did not influence this distribution. In the presence of ethanol, cholecystokinin-(26-33)-peptide amide (CCK8) did not increase the amount of radiolabelled phosphatidylethanol (PtdEth) in intact acini prelabelled with either o-[32P]phosphate or [3H]myristic acid. Similarly, an increased cytosolic Ca2+ concentration evoked by the specific inhibitor of the endoplasmic reticulum Ca2+-ATPase, thapsigargin, did not stimulate acinar PLD activity whereas high-level PKC activation with PMA elicited slight stimulation. In contrast, all three stimuli are known to increase PLD activity readily in Chinese hamster ovary (CHO) cells expressing the rat pancreatic acinar cell CCKA receptor. Finally, the combination of PMA and GTPgammaS did not increase PLD activity following homologous reconstitution of acinar cytosol and membranes, whereas the same manoeuvre resulted in marked stimulation of PLD activity in CHO cells. Heterologous reconstitution experiments revealed that PLD activity in CHO membranes was stimulated readily in the presence of acinar cytosol, indicating that the acinar cytosol contains the necessary factors for PMA/GTPgammaS-induced stimulation of membrane PLD activity. In contrast, CHO cell cytosol did not confer PMA/GTPgammaS-stimulation of PLD activity on acinar membranes, in agreement with the predominantly cytosolic localization of acinar PLD. The present findings show that rat pancreatic acinar cells express a cytosolic PLD1 isoform that is not regulated by the physiologically important secretagogue CCK.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.