Abstract
Chronic stimulation (24 h) with vasopressin leads to hypertrophy in H9c2 cardiomyoblasts and this is accompanied by continuous activation of phospholipase C. Consequently, vasopressin stimulation leads to a depletion of phosphatidylinositol levels. The substrate for phospholipase C is phosphatidylinositol (4, 5) bisphosphate (PIP2) and resynthesis of phosphatidylinositol and its subsequent phosphorylation maintains the supply of PIP2. The resynthesis of PI requires the conversion of phosphatidic acid to CDP-diacylglycerol catalysed by CDP-diacylglycerol synthase (CDS) enzymes. To examine whether the resynthesis of PI is regulated by vasopressin stimulation, we focussed on the CDS enzymes. Three CDS enzymes are present in mammalian cells: CDS1 and CDS2 are integral membrane proteins localised at the endoplasmic reticulum and TAMM41 is a peripheral protein localised in the mitochondria. Vasopressin selectively stimulates an increase CDS1 mRNA that is dependent on protein kinase C, and can be inhibited by the AP-1 inhibitor, T-5224. Vasopressin also stimulates an increase in cFos protein which is inhibited by a protein kinase C inhibitor. We conclude that vasopressin stimulates CDS1 mRNA through phospholipase C, protein kinase C and cFos and provides a potential mechanism for maintenance of phosphatidylinositol levels during long-term phospholipase C signalling.
Highlights
Several agonists including norepinephrine, angiotensin II and vasopressin (VP) acting on Gq-protein coupled receptors induce hypertrophy of cardiac myocytes [1,2,3,4,5]
We identify protein kinase C and cFos to be responsible for the increase in CDS1 mRNA
Addition of VP for 20 min results in the robust activation of phospholipase C (PLC) monitored by measuring the accumulation of [3H]-inositol phosphates (IPs) in lithium chloride-treated cells. (Li+ inhibits the enzyme, inositol monophosphatase). (Confluent H9c2 cells were prelabelled by maintaining the cells in the presence of [3H]-inositol for 72 h)
Summary
Angiotensin II and vasopressin (VP) acting on Gq-protein coupled receptors induce hypertrophy of cardiac myocytes [1,2,3,4,5]. VP stimulates protein synthesis without an increase in cell number [1] Cardiomyocyte cell lines such as H9c2 cells are established models for studying hypertrophy and show similar hypertrophic responses to primary. PI species are unusual amongst the major class of lipids in that they are often found to have a highly restricted range of acyl chains with a C18:0 (stearoyl) chain at the sn-1 position and C20:4 (arachidonoyl) chain at the sn-2 position predominantly [22,23] This is the case for mammalian tissue as opposed to cultured cell-lines [22]. We confirm that H9c2 cells show a hypertrophic response when exposed to VP for 24 h and demonstrate that this is accompanied by continuous stimulation of PLC activity. We identify protein kinase C and cFos to be responsible for the increase in CDS1 mRNA
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