A Mathematical Model of the Phosphoinositide Pathway

ABSTRACTPhosphoinositides are signaling lipids that constitute a complex network regulating many cellular processes. We propose a computational model that accounts for all known species of phosphoinositides in the plasma membrane of mammalian cells. The model replicates the steady-state of the phosphoinositide pathway and most known dynamic phenomena. Furthermore, sensitivity analysis demonstrates model robustness to moderate alterations in any of the parameters. Model analysis suggest that the greatest contributor to PI(4,5)P2production is a flux representing the direct transformation of PI into PI(4,5)P2and is also responsible for the maintenance of this pool when PI(4)P is decreased. PI(5)P is also shown to be a significant source for PI(4,5)P2production. The model was validated with data from siRNA screens that knocked down the expression of several enzymes in the pathway. The screen monitored the activity of the epithelium sodium channel, ENaC, which is activated by PI(4,5)P2. Moderating ENaC activity can have a therapeutic effect in Cystic Fibrosis (CF) patients. Our model suggests control strategies where the activities of the enzyme PIP5KI or the PI4K+PIP5KI+DVL protein complex are decreased and cause an efficacious reduction in PI(4,5)P2levels while avoiding undesirable alterations in other phosphoinositide pools.AbbreviationsAKTProtein Kinase B, a serine/threonine-specific protein kinaseASLAirway surface liquidBSTBiochemical systems theoryCFCystic fibrosisDAGDiacylglycerolENaCEpithelial Sodium ChannelEREndoplasmic ReticulumGMAGeneralized mass actionINPP5Inositol polyphosphate 5-phosphatasesIP3Inositol triphosphateLTPslipid transport proteinsMCSsmembrane contact sitesMDCK cellsMadin-Darby Canine Kidney Epithelial CellsMTMMyotubularinOCRLLowe Oculocerebrorenal Syndrome Protein; OCRL is an INPP5ODEOrdinary differential equationsPIPhosphatidylinositolPI(3)Pphosphatidylinositol 3-phosphatePI(3,4)P2Phosphatidylinositol 3,4-biphosphatePI(3,4,5)P3phosphatidylinositol 3,4,5-triphosphate, with phosphates in the third, fourth and fifth positionsPI(3,5)P2Phosphatidylinositol 3,5-biphosphatePI(4)Pphosphatidylinositol 4-phosphatePI(4,5)P2Phosphatidylinositol 4,5-biphosphate with phosphates in the fourth and fifth positions of the inositol ringPI(5)Pphosphatidylinositol 5-phosphatePI3KPhosphoinositide 3-kinasePI4KPhosphoinositide 4-kinasePIP5KPhosphoinositide 4-phosphate 5-kinasePIKfyveFYVE finger-containing phosphoinositide kinase.PLCPhospholipase CPLDPhospholipase DPLIPPTEN-like lipid phosphatasePTENPhosphatase and tensin homologPKCProtein kinase CSACSuppressor of actinSHIP1SH2 domain-containing phosphatidylinositol 5’-phosphataseSKIPSkeletal muscle and kidney enriched inositol polyphosphate phosphataseSYNJSynaptojaninsTPIPPTEN-Like Inositol Lipid PhosphataseWnt3aWingless-Type MMTV Integration Site Family, Member 3ADVLSegment Polarity Protein Dishevelled Homolog DVL

Vasopressin-induced nitric oxide production in rat inner medullary collecting duct is dependent on V2 receptor activation of the phosphoinositide pathway

We previously reported that arginine vasopressin (AVP) stimulates the production of nitric oxide (NO) in inner medullary collecting duct (IMCD) via activation of V2 receptors (V2R) and the mobilization of intracellular Ca2+. The aim of this study was to determine the pathway(s) through which this response is mediated. IMCDs were dissected from male Sprague-Dawley rats and intracellular Ca2+ concentration ([Ca2+]i) and NO production were measured using a fluorescence imaging system. AVP (100 nmol/l) produced a rapid increase [Ca2+]i of 381 ± 78 nmol/l that was followed by a significant increase of NO production (166 ± 61%). The specific nonpeptide V2R antagonist OPC31260 (1 μM), but not the V1R antagonist OPC21268 (1 μM), inhibited the increase in [Ca2+]i (up to 91 ± 5%) and abolished the NO response to AVP. Both the phospholipase C inhibitor U73112 (3 μM) and the inositol ( 1 , 4 , 5 ) tri-phosphate 3 receptor blocker 2-APB (75 μM) reduced the peak [Ca2+]i response to AVP (by 65 ± 9 and 59 ± 15%, respectively) and abolished the NO response. Although forskolin (100 μM; an activator of adenylyl cyclase) elicited a moderate increase in [Ca2+]i, neither preincubation with the adenylyl cyclase inhibitor 2′-5′-dideoxyadenosine (50 μM) nor the protein kinase A (PKA) inhibitor PKA14-22 (100 μM) significantly inhibited peak [Ca2+]i in response to AVP. IMCD [Ca2+]i responses to AVP were reduced by 72 ± 8% when incubated in Ca2+-free media and could be completely abolished by preincubation with the Ca2+-ATPase inhibitor thapsigargin. We conclude that AVP-induced NO production in IMCD is dependent on V2R activation of the phosphoinositide pathway and the mobilization of Ca2+ from both intracellular and extracellular pools.