Pardaxin Stimulation of Phospholipases A 2 and Their Involvement in Exocytosis in PC-12 Cells

ABSTRACT Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A 2 inhibitors we show that PX stimulation of phospholipase A 2 (PLA 2 ) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA 2 activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E 2 (PGE 2 ), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3 ) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE 2 , and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2Ј-amino-3Ј-methoxyflavone (PD98059) reduced by 100 and 82%, respectively, the release of AA and PGE 2 induced by PX. In the absence of extracellular calcium, the calcium-independent PLA 2 (iPLA 2 ) inhibitors methyl arachidonyl fluorophosphonate, AACOCF 3 , and bromoenol lactone (BEL) inhibited by 80 to 90% PX stimulation of AA release, by 65 to 85% PX stimulation of PGE 2 release, and by 80 to 90% PX-induced dopamine release. Using vesicle fusion-based enzyme-linked immunosorbent assay we found similar levels of inhibition of PX-induced exocytosis by these inhibitors. Also, PX induced the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor complexes, an effect that was augmented by N-methylmaleimide. This complex formation was completely inhibited by BEL. Botulinum toxins type C1 and F significantly inhibited the release of AA, PGE 2 , and dopamine induced by PX. Our data suggest that PX stimulates exocytosis by activating cystolic PLA 2 and iPLA 2 , leading to the generation of AA and eicosanoids, which, in turn, stimulate vesicle competence for fusion and neurotransmitter release. Hormones and neurotransmitters are usually released from cells by exocytosis, when a rise in cytosolic calcium triggers fusion of the secretory vesicle membrane with the plasma membrane SNAREs are targets for the botulinum and tetanus toxins Aside from toxins that inhibit neurotransmitter release, there are others that cause a massive release of neurotrans-E.B.-S. and S.A.-R. contributed equally to this work

The role and dynamics of β-catenin in precondition induced neuroprotection after traumatic brain injury.

Preconditioning via heat acclimation (34°C 30 d) results in neuroprotection from traumatic brain injury due to constitutive as well as dynamic changes triggered by the trauma. Among these changes is Akt phosphorylation, which decreases apoptosis and induces HIF1α. In the present study we investigated the Akt downstream GSK3β/β-catenin pathway and focused on post injury alternations of β catenin and its impact on the cellular response in preconditioned heat acclimated mice. We found that the reduction in motor disability is accompanied with attenuation of depressive like behavior in heat acclimated mice that correlates with the GSK3β phosphorylation state. Concomitantly, a robust β catenin phosphorylation is not followed by its degradation, or by reduced nuclear accumulation. Enhanced tyrosine phosphorylation of β catenin in the injured area weakens the β catenin-N cadherin complex. Membrane β catenin is transiently reduced in heat acclimated mice and its recovery 7 days post TBI is accompanied by induction of the synaptic marker synaptophysin. We suggest a set of cellular events following traumatic brain injury in heat acclimated mice that causes β catenin to participate in cell-cell adhesion alternations rather than in Wnt signaling. These events may contribute to synaptogenesis and the improved motor and cognitive abilities seen heat acclimated mice after traumatic brain injury

Dynamic changes in the recovery after traumatic brain injury in mice: effect of injury severity on T2-weighted MRI abnormalities, and motor and cognitive functions

Memory and neurobehavioral dysfunctions are among the sequelae of traumatic brain injury (TBI). The Neurological Severity Score (NSS) includes 10 tasks and was previously designed to assess the functional status of mice after TBI. The object recognition task (ORT) measures specific episodic memory and is expressed by the percent time spent by an animal at a novel, unfamiliar object (Discrimination Index [DI]). It is an ideal tool for evaluating cognitive function after TBI. The present study sought to validate the use of the NSS and ORT in severe and mild focal TBI in mice, and to confirm that the spontaneous recovery and the radiological abnormalities, shown by T2-weighted magnetic resonance imaging (MRI), are dependent upon injury severity. Mice were subjected to severe and mild closed head injury (NSS at 1 h 7.52 ± 0.34 and 4.62 ± 0.14, respectively). NSS was evaluated for 25 days and showed a decrease by 3.86 ± 0.26 and 2.54 ± 0.35 units in the severely and mildly injured mice, respectively. ORT revealed DI in severely injured group of 51.7 ± 6.15%, (vs ∼75–80% in uninjured animal) on day 3 and 66.2 ± 6.81% on day 21. In contrast, the mildly injured mice did not show cognitive impairment throughout the same period. The damage seen by MRI at 24 h after injury, strongly correlated with NSS(1h) (R = 0.87, p < 0.001). We conclude that NSS is a reliable tool for evaluation of neurological damage in head-injured mice, NSS(1h) predicts the motor dysfunction, cognitive damage, and brain-damage characteristics as depicted by T2-weighted MRI. The combined assessment of neurobehavioral and cognitive function along with MRI is most useful in evaluating recovery from injury, especially when testing effectiveness of novel treatments or genetic manipulations

Ischemic preconditioning increases antioxidants in the brain and peripheral organs after cerebral ischemia

Background and purpose. Low molecular weight antioxidants (LMWA), which reflect tissue reducing power, are among the endogenous mechanisms for neutralizing reactive oxygen species (ROS). Ischemic preconditioning (IPC) was associated with decreased oxidative stress. We examined the effect of focal ischemia on LMWA and on prostaglandin E 2 (PGE 2, a product of arachidonic acid oxidation) in the brain, heart, liver, and lungs of rats subjected to 90 min of ischemia and in IPC rats subjected to similar insult. Methods. Transient right middle cerebral artery occlusion (MCAO) was performed for 90 min and at 0, 5, 30, 60, or 240 min of reperfusion, LMWA and PGE 2 were evaluated by cyclic voltametry (CV) and radioimmunoassay, respectively. IPC was induced by 2 min of MCAO, 24 h prior to the major ischemic episode. Results. LMWA decreased at 5 min of reperfusion in the brain, heart, liver, and lung and rose 4 h later only in the brain. PGE 2 levels increased three to fivefold in all tissues examined. Surprisingly, in IPC rats a dramatic increase of LMWA occurred at 5 min of reperfusion in the brain and in the peripheral organs. Uric acid, but not ascorbic, is the major LMWA increased. Conclusions. We propose that after ischemia, ROS rapidly consume the antioxidants reserves in the brain and also in peripheral organs, suggesting that the whole body is under oxidative stress. Moreover, part of the neuroprotection afforded by IPC is mediated by the brain's ability to mobilize antioxidants, especially uric acid, that attenuate the massive ROS-mediated oxidative stress

Heat acclimation provides sustained improvement in functional recovery and attenuates apoptosis after traumatic brain injury

Heat acclimation (HA) offers functional neuroprotection in mice after traumatic brain injury (TBI). This study further characterizes endogenous neuroprotection acquired by HA (34±1°C, 30 d) after TBI. We establish here the ability of HA to induce sustained functional benefits and to reduce activation of apoptotic pathways. Neurobehavioral recovery, assessed by the Neurological Severity Score, was greater in HA mice up to 8 days after injury as compared with normothermic controls (P<0.05) and lesion volume was also smaller in the HA group (P<0.05). Reduced apoptotic cell death in HA mice was confirmed using caspase-3 activity measurements and immunohistochemistry. To investigate the underlying molecular pathways, expression levels of intrinsic apoptotic pathway-related proteins were examined. HA mice displayed higher mitochondrial levels of antiapoptotic Bcl-xL, accompanied by lower proapoptotic Bad levels and decreased cytochrome c release, suggesting a higher apoptotic threshold. Taken together with our previous reports, indicating increased Akt phosphorylation and antioxidative capacity, alongside with reduced tumor necrosis α levels after TBI in HA animals, the current results support the involvement of an antiapoptotic effect in HA-induced neuroprotection. Current results warrant further study as TBI-induced apoptosis may persist over weeks after injury, possibly providing a target for belated therapeutic intervention

Pardaxin Stimulation of Phospholipases A 2 and Their Involvement in Exocytosis in PC-12 Cells

Pardaxin (PX) is a voltage-dependent ionophore that stimulates catecholamine exocytosis from PC-12 pheochromocytoma cells both in the presence and absence of extracellular calcium. Using a battery of phospholipase A 2 inhibitors we show that PX stimulation of phospholipase A 2 (PLA 2) enzymes is coupled with induction of exocytosis. We investigated the relationship between PX-induced PLA 2 activity and neurotransmitter release by measuring the levels of arachidonic acid (AA), prostaglandin E 2 (PGE 2), and dopamine release. In the presence of extracellular calcium, the cytosolic PLA 2 inhibitor arachidonyl trifluoromethyl ketone (AACOCF 3) inhibited by 100, 70, and 73%, respectively, the release of AA, PGE 2, and dopamine induced by PX. The mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor 2�-amino-3�-methoxyflavon

Heat acclimation induces transient reduction in membrane β-catenin and synaptophysin.

<p>Membrane cellular fraction extracts were separated on SDS-PAGE gels and analyzed using western blotting. After the injury a significant reduction in membrane bound β-catenin were seen at 72 h post injury in heat acclimated (HA) mice and after 7 days in normothermic (NT) mice. Membrane bound β-catenin levels were recovered by 7 days after injury (<b><i>A</i></b>) Blots of membrane extracts show significant reduction in synaptophysin levels in both groups at 72 h after injury with a recovery seen only in HA mice by 7 days post injury (<b><i>B</i></b>). The absence of synaptophysin in the cytosol verifies membrane protein enrichment. Values represent the mean ± SEM. n = 5–6 per group. *<i>p</i><0.05 vs. NT mice, **<i>p</i><0.05 vs. sham mice,within the same group.</p

HA induces inhibition of glycogen synthase kinase 3β (GSK3β) and c-Jun N terminal kinase (JNK).

<p>Mice were subjected to CHI or sham operation, and injured cortexes were removed at 6, 12 or 24β showing significant inhibition of GSK3β by 24 h post injury in heat acclimated mice (HA) as compared with normothermic mice (NT) (<b><i>A</i></b>). Representative western blots of JNK phosphorylation, that is related to enhanced kinase activity, showing reduced JNK activity in HA mice after CHI (<b><i>B</i></b>). Values represent the mean ± SEM. n = 5–6 per group. *<i>p</i><0.05 vs. NT mice, **<i>p</i><0.05 vs. sham HA mice.</p

Heat acclimation induces tyrosine 654 phosphorylation of β catenin.

<p>Mice were subjected to CHI or sham operation, and injured cortexes were removed at 6 or 24β catenin, and Y654 β catenin was detected using western blots. Total β catenin was used as loading control (<b><i>A</i></b>). Immunostaining of the injured brain for Y654 β catenin (green) and DAPI (blue) shows induced Y654 β catenin phosphorylation restricted to injury area (<b><i>B</i></b>). Values represent the mean ± SEM. n = 5–6 per group. *<i>p</i><0.05 vs. NT mice, **<i>p</i><0.05 vs. sham mice, within the same group.</p