Heterotrimeric G-protein subunit Gαi2 contributes to agonist-sensitive apoptosis and degranulation in murine platelets

Gαi2, a heterotrimeric G-protein subunit, regulates various cell functions including ion channel activity, cell differentiation, proliferation and apoptosis. Platelet-expressed Gαi2 is decisive for the extent of tissue injury following ischemia/reperfusion. However, it is not known whether Gαi2 plays a role in the regulation of platelet apoptosis, which is characterized by caspase activation, cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) translocation to the platelet surface. Stimulators of platelet apoptosis include thrombin and collagen-related peptide (CoRP), which are further known to enhance degranulation and activation of αII bβ3-integrin and caspases. Using FACS analysis, we examined the impact of agonist treatment on activation and apoptosis in platelets drawn from mice lacking Gαi2 and their wild-type (WT) littermates. As a result, treatment with either thrombin (0.01 U/mL) or CoRP (2 μg/mL or 5 μg/mL) significantly upregulated PS-exposure and significantly decreased forward scatter, reflecting cell size, in both genotypes. Exposure to CoRP triggered a significant increase in active caspase 3, ceramide formation, surface P-selectin, and αII bβ3-integrin activation. These molecular alterations were significantly less pronounced in Gαi2-deficient platelets as compared to WT platelets. In conclusion, our data highlight a previously unreported role of Gαi2 signaling in governing platelet activation and apoptosis.Fil: Cao, Hang. Universität Tübingen; AlemaniaFil: Qadri, Syed M.. Canadian Blood Services; Canadá. McMaster University; CanadáFil: Lang, Elisabeth. Heinrich-heine-universität Düsseldorf; AlemaniaFil: Pelzl, Lisann. Universität Tübingen; AlemaniaFil: Umbach, Anja T.. Universität Tübingen; AlemaniaFil: Leiss, Veronika. Universität Tübingen; AlemaniaFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Instituto de Investigaciones Biomédicas. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Biomédicas; ArgentinaFil: Nürnberg, Bernd. Universität Tübingen; AlemaniaFil: Pieske, Burkert. Berlin Institute of Health; Alemania. Universitätsmedizin Berlin; AlemaniaFil: Voelkl, Jakob. Berlin Institute of Health; Alemania. Universitätsmedizin Berlin; AlemaniaFil: Gawaz, Meinrad. Universität Tübingen; AlemaniaFil: Bissinger, Rosi. Universität Tübingen; AlemaniaFil: Lang, Florian. Universität Tübingen; Alemania. Heinrich-heine-universität Düsseldorf; Alemani

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death

Blunted apoptosis of erythrocytes in mice deficient in the heterotrimeric G-protein subunit Gαi2

Putative functions of the heterotrimeric G-protein subunit Gαi2-dependent signaling include ion channel regulation, cell differentiation, proliferation and apoptosis. Erythrocytes may, similar to apoptosis of nucleated cells, undergo eryptosis, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure. Eryptosis may be triggered by increased cytosolic Ca2+ activity and ceramide. In the present study, we show that Gαi2 is expressed in both murine and human erythrocytes and further examined the survival of erythrocytes drawn from Gαi2-deficient mice (Gαi2−/−) and corresponding wild-type mice (Gαi2+/+). Our data show that plasma erythropoietin levels, erythrocyte maturation markers, erythrocyte counts, hematocrit and hemoglobin concentration were similar in Gαi2−/− and Gαi2+/+ mice but the mean corpuscular volume was significantly larger in Gαi2−/− mice. Spontaneous PS exposure of circulating Gαi2−/− erythrocytes was significantly lower than that of circulating Gαi2+/+ erythrocytes. PS exposure was significantly lower in Gαi2−/− than in Gαi2+/+ erythrocytes following ex vivo exposure to hyperosmotic shock, bacterial sphingomyelinase or C6 ceramide. Erythrocyte Gαi2 deficiency further attenuated hyperosmotic shock-induced increase of cytosolic Ca2+ activity and cell shrinkage. Moreover, Gαi2−/− erythrocytes were more resistant to osmosensitive hemolysis as compared to Gαi2+/+ erythrocytes. In conclusion, Gαi2 deficiency in erythrocytes confers partial protection against suicidal cell death.Fil: Bissinger, Rosi. Eberhard Karls Universität Tübingen; AlemaniaFil: Lang, Elisabeth. Universitat Dusseldorf; AlemaniaFil: Ghashghaeinia, Mehrdad. Eberhard Karls Universität Tübingen; AlemaniaFil: Singh, Yogesh. Eberhard Karls Universität Tübingen; AlemaniaFil: Zelenak, Christine. Charité Medical University; AlemaniaFil: Fehrenbacher, Birgit. Eberhard Karls Universität Tübingen; AlemaniaFil: Honisch, Sabina. Eberhard Karls Universität Tübingen; AlemaniaFil: Chen, Hong. Eberhard Karls Universität Tübingen; AlemaniaFil: Fakhri, Hajar. Eberhard Karls Universität Tübingen; AlemaniaFil: Umbach, Anja T.. Eberhard Karls Universität Tübingen; AlemaniaFil: Liu, Guilai. Eberhard Karls Universität Tübingen; AlemaniaFil: Rexhepaj, Rexhep. Universitat Bonn; AlemaniaFil: Liu, Guoxing. Eberhard Karls Universität Tübingen; AlemaniaFil: Schaller, Martin. Eberhard Karls Universität Tübingen; AlemaniaFil: Mack, Andreas F.. Eberhard Karls Universität Tübingen; AlemaniaFil: Lupescu, Adrian. Eberhard Karls Universität Tübingen; AlemaniaFil: Birnbaumer, Lutz. National Institutes of Health; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; ArgentinaFil: Lang, Florian. Eberhard Karls Universität Tübingen; AlemaniaFil: Qadri, Syed M.. Eberhard Karls Universität Tübingen; Alemania. Pontificia Universidad Católica Argentina "Santa María de los Buenos Aires". Facultad de Ciencias Médicas. Instituto de Investigaciones Biomédicas; Argentin

Temsirolimus Sensitive Stimulation of Platelet Activity, Apoptosis and Aggregation by Collagen Related Peptide

Background/Aims: The mammalian target of rapamycin (mTOR) inhibitor temsirolimus stimulates apoptosis of tumor cells and is thus therapeutically used for the treatment of diverse malignancies. On the other hand, temsirolimus has been shown to protect against apoptosis of hippocampal neurons. Similar to nucleated cells, blood platelets may enter suicidal death characterized by cell shrinkage and cell membrane scrambling. Platelet apoptosis is frequently preceded by Ca2+ entry, degranulation, integrin activation and stimulation of caspases. Those events could be triggered by collagen related peptide (CRP). The present study explored whether treatment of platelets with temsirolimus modifies platelet activation, caspase activity, platelet shrinkage, and phosphatidylserine abundance. Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to temsirolimus (40 µg/ml) without or with additional CRP (2 µg/ ml or 5 µg/ml) treatment. Flow cytometry was employed to estimate cytosolic Ca2+-activity ([Ca2+]i) from Fluo-3 fuorescence, platelet degranulation from P-selectin abundance, integrin activation from αIIbβ3 integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding and relative platelet volume from forward scatter. Results: In the absence of CRP, the administration of temsirolimus (40 µg/ml) significantly decreased [Ca2+]i, but did not significantly modify P-selectin abundance, activated αIIbβ3 integrin, annexin-V-binding, cell volume, caspase activity and aggregation. Exposure of platelets to CRP was followed by significant increase of [Ca2+]i, P-selectin abundance, αIIbβ3 integrin activity, annexin-V-binding, ROS, caspase activity and aggregation, effects significantly blunted in the presence of temsirolimus. CRP further decreased forward scatter, an effect again significantly blunted by temsirolimus. Conclusions: Temsirolimus is a powerful inhibitor of platelet activation and suicidal platelet death

Intestinal Na+ Loss and Volume Depletion in JAK3-Deficient Mice

Background/Aims: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na+ channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na+ exretion, blood pressure and extracellular fluid volume regulation. Methods: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3-/-) and in wild type mice (jak3+/+). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na+ concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results: The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na+ excretion significantly higher in jak3-/- mice than in jak3+/+ mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3-/- mice than in jak3+/+ mice. Both, absolute and fractional renal Na+ excretion were significantly lower in jak3-/- mice than in jak3+/+ mice. Conclusions: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na+ loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na+ reabsorption

Sgk1 Sensitive Pendrin Expression in Murine Platelets

Background: The anion exchanger pendrin (SLC26A4) is required for proper development of the inner ear, and contributes to iodide organification in thyroid glands as well as anion transport in various epithelia, such as airways and renal tubules. SLC26A4 deficiency leads to Pendred syndrome, which is characterized by hearing loss with enlarged vestibular aqueducts and variable hypothyroidism and goiter. Pendrin expression in kidney, heart, lung and thyroid is up-regulated by the mineralocorticoid deoxycorticosterone (DOCA). Platelets express anion exchangers but virtually nothing is known about the molecular identity and regulation of those carriers. Other carriers such as the Na+/H+ exchanger are regulated by the mineralocorticoid-sensitive serum and glucocorticoid inducible kinase SGK1. Methods: The present study utilized i) quantitative reverse transcription polymerase chain reaction (RT-qPCR) to quantify the transcript levels of Slc26a4 as compared to Gapdh and ii) western blotting to assess Slc26a4 protein abundance in murine platelets from gene-targeted mice lacking Sgk1 (sgk1-/-) and respective wild type animals (sgk1+/+) treated without or with a subcutaneous injection of 2.5 mg DOCA for 3 h, or in sgk1+/+ platelets with or without in vitro treatment for 1 h with 10 µg/ml DOCA. Results: Slc26a4 was expressed in platelets, and in vitro DOCA treatment increased Slc26a4 mRNA levels in platelets isolated from sgk1+/+ mice. Moreover, in vivo DOCA treatment significantly up-regulated Slc26a4 mRNA levels in platelets isolated from sgk1+/+ but not sgk1-/- mice. An increase in Sgk1 mRNA levels paralleled that of Slc26a4 mRNA levels in platelets of sgk1+/+ mice. In addition, DOCA treatment further increased Slc26a4 protein abundance in platelets isolated from sgk1+/+ mice. Conclusions: Pendrin is expressed in platelets and is presumably regulated by SGK1 and mineralocorticoids