ABSTRACT
The physiological functions and cellular signaling of Class II phosphoinositide 3-kinases (PI3Ks) remain largely unknown. Platelets express two Class II PI3Ks: PI3KC2α and PI3KC2ß. PI3KC2α deficiency was recently reported to cause disruption of the internal membrane reserve structure of platelets (open canalicular system, OCS) that results in dysregulated platelet adhesion and impaired arterial thrombosis in vivo. Notably, these effects on platelets occurred despite normal agonist-induced 3-phosphorylated phosphoinositide (3-PPI) production and cellular activation in PI3KC2α-deficient platelets. However, the potential compensatory actions of PI3KC2ß in platelets have not yet been investigated. Here, we report the first mice deficient in both PI3KC2α and PI3KC2ß (no Class II PI3Ks in platelets) and reveal a nonredundant role for PI3KC2α in mouse platelet structure and function. Specifically, we show that the disrupted OCS and impaired thrombus stability observed in PI3KC2α-deficient platelets does not occur in PI3KC2ß-deficient platelets and is not exaggerated in platelets taken from mice deficient in both enzymes. Furthermore, detailed examination of 3-PPI production in platelets from this series of mice revealed no changes in either unactivated or activated platelets, including those with a complete lack of Class II PI3Ks. These findings indicate a nonredundant role for PI3KC2α in regulating platelet structure and function, and suggest that Class II PI3Ks do not significantly contribute to the acute agonist-induced production of 3-PPIs in these cells.
Subject(s)
Blood Platelets/metabolism , Class II Phosphatidylinositol 3-Kinases/deficiency , Thrombosis/blood , Thrombosis/genetics , Animals , Blood Platelets/ultrastructure , Class II Phosphatidylinositol 3-Kinases/genetics , Class II Phosphatidylinositol 3-Kinases/metabolism , Class III Phosphatidylinositol 3-Kinases/genetics , Class III Phosphatidylinositol 3-Kinases/metabolism , Disease Models, Animal , Gene Expression Regulation , Mice , Mice, Knockout , Platelet Adhesiveness , Platelet Count , Platelet Function TestsABSTRACT
Thrombotic microangiopathy (TMA) commonly involves injury of kidney glomerular endothelial cells (ECs) and fibrin occlusion of the capillaries. The mechanisms underlying repair of the microvasculature and recovery of kidney function are poorly defined. In the developing vasculature, the phosphoinositide 3-kinase (PI3K) α isoform integrates many growth factor cues. However, the role of individual isoforms in repair of the established vasculature is unclear. We found that postnatal endothelial deletion of PI3Kß sensitizes mice to lethal acute kidney failure after TMA injury. In vitro, PI3Kß-deficient ECs show reduced angiogenic invasion of fibrin matrix with unaltered sensitivity to proapoptotic stress compared with wild-type ECs. This correlates with decreased expression of the EC tip cell markers apelin and Dll4 and is associated with a reduction in migration and proliferation. In vivo, PI3Kß-knockdown ECs are deficient in assembly of microvessel-like structures. These data identify a critical role for endothelial PI3Kß in microvascular repair following injury.
Subject(s)
Class II Phosphatidylinositol 3-Kinases/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Microvessels/metabolism , Microvessels/pathology , Thrombotic Microangiopathies/metabolism , Thrombotic Microangiopathies/pathology , Animals , Apoptosis/genetics , Biomarkers , Class II Phosphatidylinositol 3-Kinases/deficiency , Class II Phosphatidylinositol 3-Kinases/genetics , Disease Models, Animal , Endothelial Cells/metabolism , Enzyme Activation , Humans , Kidney Glomerulus/drug effects , Kidney Glomerulus/metabolism , Kidney Glomerulus/pathology , Mice , Mice, Knockout , PTEN Phosphohydrolase/metabolism , Phosphorylation/drug effects , Proto-Oncogene Proteins c-akt/metabolism , Thrombotic Microangiopathies/genetics , Thrombotic Microangiopathies/mortality , Vascular Endothelial Growth Factor A/pharmacologyABSTRACT
G protein-coupled opioid receptors undergo desensitization after prolonged agonist exposure. Recent in vitro studies of mu-opioid receptor (MOR) signaling revealed an involvement of phosphoinositide 3-kinases (PI3K) in agonist-induced MOR desensitization. Here we document a specific role of the G protein-coupled class IB isoform PI3Kgamma in MOR desensitization in mice and isolated sensory neurons. The tail-withdrawal nociception assay evidenced a compromised morphine-induced tolerance of PI3Kgamma-deficient mice compared to wild-type animals. Consistent with a role of PI3Kgamma in MOR signaling, PI3Kgamma was expressed in a subgroup of small-diameter dorsal root ganglia (DRG) along with MOR and the transient receptor potential vanilloid type 1 (TRPV1) receptor. In isolated DRG acute stimulation of MOR blocked voltage-gated calcium currents (VGCC) in both wild-type and PI3Kgamma-deficient DRG neurons. By contrast, following long-term opioid administration the attenuating effect of MOR was strongly compromised in wild-type DRG but not in PI3Kgamma-deficient DRG. Our results uncover PI3Kgamma as an essential modulator of long-term MOR desensitization and tolerance development induced by chronic opioid treatment in sensory neurons.
