Bosch R R, Harris A B, van Emst-de Vries S E, De Pont J J, Willems P H
Department of Biochemistry, University Medical Centre Nijmegen, The Netherlands.
Pflugers Arch. 2001 Sep;442(6):910-9. doi: 10.1007/s004240100611.
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.
胰腺腺泡细胞中存在受调控的磷脂酶D（PLD）活性的证据存在矛盾。这些知识很重要，因为信号激活的PLD除其他外还与受调控的胞吐作用有关。在本研究中，使用新鲜分离的大鼠胰腺腺泡通过RT-PCR鉴定PLD转录本，通过蛋白质印迹评估PLD蛋白的存在和亚细胞定位，并通过PLD催化的转磷脂酰化反应评估促分泌素调节的PLD活性的存在。腺泡细胞中存在PLD1b和PLD2的转录本，但不存在PLD1a的转录本。此外，一种特异性抗人PLD1抗体证明了大量PLD1蛋白的表达。然而，有趣的是，亚细胞分级分离后观察到的腺泡PLD1的分布模式明显不典型，因为免疫反应主要发生在腺泡细胞质中。用佛波酯（4β-佛波醇12-肉豆蔻酸13-乙酸酯，PMA）预处理完整腺泡以依赖性激活PLD1蛋白激酶C（PKC），并没有改变PLD1的亚细胞分布。同样，用鸟苷5'-O-（3-硫代三磷酸）（GTPγS）预处理腺泡破碎细胞制剂以通过小GTP酶激活PLD和PMA，也没有影响这种分布。在乙醇存在下，胆囊收缩素-（26-33）-肽酰胺（CCK8）不会增加用o-[32P]磷酸盐或[3H]肉豆蔻酸预标记的完整腺泡中放射性标记的磷脂酰乙醇（PtdEth）的量。同样，内质网Ca2 + -ATP酶的特异性抑制剂毒胡萝卜素引起的细胞质Ca2 +浓度升高不会刺激腺泡PLD活性，而用PMA进行的高水平PKC激活会引起轻微刺激。相比之下，已知所有这三种刺激物在表达大鼠胰腺腺泡细胞CCKA受体的中国仓鼠卵巢（CHO）细胞中很容易增加PLD活性。最后，PMA和GTPγS的组合在腺泡细胞质和膜的同源重组后不会增加PLD活性，而相同的操作在CHO细胞中导致PLD活性的显著刺激。异源重组实验表明，在腺泡细胞质存在下，CHO膜中的PLD活性很容易受到刺激，这表明腺泡细胞质中含有PMA / GTPγS诱导的膜PLD活性刺激所需的因子。相比之下，CHO细胞细胞质不会赋予腺泡膜PMA / GTPγS对PLD活性的刺激，这与腺泡PLD主要位于细胞质中的定位一致。目前的研究结果表明，大鼠胰腺腺泡细胞表达一种细胞质PLD1同工型，其不受生理上重要的促分泌素CCK的调节。
