Effect of Src Kinase Inhibition on Cytochrome c, Smac/DIABLO and Apoptosis Inducing Factor (AIF) Following Cerebral Hypoxia-Ischemia in Newborn Piglets.

We have previously shown that cerebral Hypoxia-ischemia (HI) results in activation of Src kinase in the newborn piglet brain. We investigated the regulatory mechanism by which the pre-apoptotic proteins translocate from mitochondria to the cytosol during HI through the Src kinase. Newborn piglets were divided into 3 groups (n = 5/group): normoxic (Nx), HI and HI pre-treated with Src kinase inhibitor PP2 (PP2 + HI). Brain tissue HI was verified by neuropathological analysis and by Adenosine Triphosphate (ATP) and Phosphocreatine (PCr) levels. We used western blots, immunohistochemistry, H&E and biochemical enzyme assays to determine the role of Src kinase on mitochondrial membrane apoptotic protein trafficking. HI resulted in decreased ATP and PCr levels, neuropathological changes and increased levels of cytochrome c, Smac/DIABLO and AIF in the cytosol while their levels were decreased in mitochondria compared to Nx. PP2 decreased the cytosolic levels of pre-apoptotic proteins, attenuated the neuropathological changes and apoptosis and decreased the HI-induced increased activity of caspase-3. Our data suggest that Src kinase may represent a potential target that could interrupt the enzymatic activation of the caspase dependent cell death pathway

Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

Background. Protein tyrosine phosphatases (PTPs) in conjunction with protein tyrosine kinases (PTKs) regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B) is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks. Methods. Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO 2 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO 2 for 1 hour and PTP-1B expression was measured via immunoblotting. Results. PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression. Conclusions. Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression

The Role of Src Kinase in the Caspase-1 Pathway After Hypoxia in the Brain of Newborn Piglets

Hypoxia induces a cerebral inflammatory response, which contributes to brain injury. Inflammasomes are complex intracellular molecular structures that initiate the inflammatory cascade. Caspase-1 and interleukin 1-β (IL-1β), have been established as markers of inflammasome activation. Src kinase, a cytosolic non-receptor protein tyrosine kinase, is linked to cell proliferation and differentiation and is up regulated during hypoxia. The role of Src kinase in the above pathway is not fully understood. The present study tests the hypothesis that inhibition of Src kinase, by a selective inhibitor, PP2, will prevent the activation of caspase-1 and production of IL-1β acutely, as well as at 1 and 15 days after hypoxia in the cerebral cortex of the newborn piglet. Piglets were divided into: Normoxia (Nx), Hypoxia acute (Hx), Hypoxia-day 1 (Hx-day 1), and Hypoxia day 15 (Hx-day 15). Piglets pretreated with Src kinase inhibitor, PP2, 1 mg/kg IV, 30 min prior to hypoxia were divided into: Hypoxia acute (Hx + PP2), 1 day (Hx + PP2-day 1), and day 15 (Hx + PP2-day 15). Hypoxia was induced by exposing the piglets to an FiO2 of 0.07 for 1 hour. Caspase-1 activity and expression were determined with spectrophotometry and Western blot respectively, while IL-1β levels were measured by solid phase ELISA. Caspase-1 activation was achieved immediately (within 1 h) after hypoxia and persisted for 15 days. IL-1β level was also increased after hypoxia reaching a maximum level at 24 h following hypoxia and returned to baseline by 15 days. Administration of PP2 attenuated the activity acutely, but not the expression of the caspase-1. IL-1β level at 24 h after hypoxia returned to baseline in piglets that were pretreated with PP2. We provide evidence that inhibition of Src kinase in the acute phase after hypoxia involves changes in the production or processing of caspase-1 subunits. Our data suggest that Src kinase mediates hypoxia-induced caspase-1 activation in the cerebral cortex of newborn piglets. Inhibition of Src kinase may attenuate the neuroinflammatory response and could represent a potential target for neuroprotection after hypoxic injury

Temporal Changes in Caspase-1 and Caspase-8 Activities Following Brain Hypoxia With and Without Src kinase Inhibition in a Piglet Animal Model

The Src family kinases are a family of intracellular, non-receptor tyrosine kinases that are involved in a variety of cellular functions including the regulation of inflammation and apoptosis after brain hypoxia. Caspase-1 (C1) activates IL-1β through the formation of complex structures, the inflammasomes, while caspase-8 (C8) is part of the extrinsic apoptotic pathway. C8 has been found to directly activate the production of IL-1β. Previously, we observed that C1 and IL-1β are increased in the acute phase after hypoxia in the brain of piglets, but they follow a different pattern long term, with C1 remaining activated throughout the period of observation, while IL-1β returning to baseline at 15 days. Src kinase inhibition ameliorated the activation of C1 and IL-1β early, but did not appear to have any effect long term. Prompted by these findings, we assessed the changes that occur over time (1 h and 15 days) in C1 and C8 activities after brain hypoxia as well as the effect of pretreatment with a Src kinase inhibitor, PP2 on these biochemical markers. Enzymatic activities were determined by spectrophotometry with measurements of C1 and C8 in each cytosolic brain sample (N = 4 in each group). We found that C1 and C8 activities increase in the acute phase following hypoxia in the brain of newborn piglets, with C8 relatively more than C1 (C8/C1 ratio increased from 2:1 as baseline to 3:1 in hypoxia). Fifteen days after hypoxia C8/C1 ratio decreased to about 1:1. In piglets that were pretreated with a Src kinase selective inhibitor (PP2) and then subjected to hypoxia, the C8/C1 ratio early increase was not observed. Immediately after hypoxia C8 and C1 follow a similar pattern of increase while long term this appears to dissociate. We propose that following this experimental methodology, the previously observed IL-1β production after hypoxia might be associated with C8 rather than C1 and that Src kinase is involved in the above process