• Disruption-induced mucus secretion: repair and protection.

      Miyake, Katsuya; Tanaka, Tomoaki; McNeil, Paul L.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2006-08-28)
      When a cell suffers a plasma membrane disruption, extracellular Ca(2+) rapidly diffuses into its cytosol, triggering there local homotypic and exocytotic membrane fusion events. One role of this emergency exocytotic response is to promote cell survival: the internal membrane thus added to the plasma membrane acts as a reparative "patch." Another, unexplored consequence of disruption-induced exocytosis is secretion. Many of the cells lining the gastrointestinal tract secrete mucus via a compound exocytotic mechanism, and these and other epithelial cell types lining the digestive tract are normally subject to plasma membrane disruption injury in vivo. Here we show that plasma membrane disruption triggers a potent mucus secretory response from stomach mucous cells wounded in vitro by shear stress or by laser irradiation. This disruption-induced secretory response is Ca(2+) dependent, and coupled to cell resealing: disruption in the absence of Ca(2+) does not trigger mucus release, but results instead in cell death due to failure to reseal. Ca(2+)-dependent, disruption-induced mucus secretion and resealing were also demonstrable in segments of intact rat large intestine. We propose that, in addition to promoting cell survival of membrane disruptions, disruption-induced exocytosis serves also the important protective function of liberating lubricating mucus at sites of mechanical wear and tear. This mode of mechanotransduction can, we propose, explain how lubrication in the gastrointestinal tract is rapidly and precisely adjusted to widely fluctuating, diet-dependent levels of mechanical stress.
    • Extranuclear estrogen receptors mediate the neuroprotective effects of estrogen in the rat hippocampus.

      Yang, Li-cai; Zhang, Quan-Guang; Zhou, Cai-feng; Yang, Fang; Zhang, Yi-dong; Wang, Rui-min; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2010-05-18)
      BACKGROUND: 17beta-estradiol (E2) has been implicated to exert neuroprotective effects in the brain following cerebral ischemia. Classically, E2 is thought to exert its effects via genomic signaling mediated by interaction with nuclear estrogen receptors. However, the role and contribution of extranuclear estrogen receptors (ER) is unclear and was the subject of the current study. METHODOLOGY/PRINCIPAL FINDINGS: To accomplish this goal, we employed two E2 conjugates (E2 dendrimer, EDC, and E2-BSA) that can interact with extranuclear ER and exert rapid nongenomic signaling, but lack the ability to interact with nuclear ER due to their inability to enter the nucleus. EDC or E2-BSA (10 microM) was injected icv 60 min prior to global cerebral ischemia (GCI). FITC-tagged EDC or E2-BSA revealed high uptake in the hippocampal CA1 region after icv injection, with a membrane (extranuclear) localization pattern in cells. Both EDC and E2-BSA exerted robust neuroprotection in the CA1 against GCI, and the effect was blocked by the ER antagonist, ICI182,780. EDC and E2-BSA both rapidly enhanced activation of the prosurvival kinases, ERK and Akt, while attenuating activation of the proapoptotic kinase, JNK following GCI, effects that were blocked by ICI182,780. Administration of an MEK or PI3K inhibitor blocked the neuroprotective effects of EDC and E2-BSA. Further studies showed that EDC increased p-CREB and BDNF in the CA1 region in an ERK- and Akt-dependent manner, and that cognitive outcome after GCI was preserved by EDC in an ER-dependent manner. CONCLUSIONS/SIGNIFICANCE: In conclusion, the current study demonstrates that activation of extranuclear ER results in induction of ERK-Akt-CREB-BDNF signaling in the hippocampal CA1 region, which significantly reduces ischemic neuronal injury and preserves cognitive function following GCI. The study adds to a growing literature that suggests that extranuclear ER can have important actions in the brain.
    • Lectin-based food poisoning: a new mechanism of protein toxicity.

      Miyake, Katsuya; Tanaka, Toru; McNeil, Paul L.; Institute of Molecular Medicine and Genetics; Department of Cellular Biology and Anatomy (2007-08-01)
      BACKGROUND: Ingestion of the lectins present in certain improperly cooked vegetables can result in acute GI tract distress, but the mechanism of toxicity is unknown. In vivo, gut epithelial cells are constantly exposed to mechanical and other stresses and consequently individual cells frequently experience plasma membrane disruptions. Repair of these cell surface disruptions allows the wounded cell to survive: failure results in necrotic cell death. Plasma membrane repair is mediated, in part, by an exocytotic event that adds a patch of internal membrane to the defect site. Lectins are known to inhibit exocytosis. We therefore tested the novel hypothesis that lectin toxicity is due to an inhibitory effect on plasma membrane repair. METHODS AND FINDINGS: Repair of plasma membrane disruptions and exocytosis of mucus was assessed after treatment of cultured cell models and excised segments of the GI tract with lectins. Plasma membrane disruptions were produced by focal irradiation of individual cells, using a microscope-based laser, or by mechanical abrasion of multiple cells, using a syringe needle. Repair was then assessed by monitoring the cytosolic penetration of dyes incapable of crossing the intact plasma membrane. We found that cell surface-bound lectins potently inhibited plasma membrane repair, and the exocytosis of mucus that normally accompanies the repair response. CONCLUSIONS: Lectins potently inhibit plasma membrane repair, and hence are toxic to wounded cells. This represents a novel form of protein-based toxicity, one that, we propose, is the basis of plant lectin food poisoning.
    • Mitochondrial BNIP3 upregulation precedes endonuclease G translocation in hippocampal neuronal death following oxygen-glucose deprivation.

      Zhao, Shen-Ting; Chen, Ming; Li, Shu-Ji; Zhang, Ming-Hai; Li, Bo-Xing; Das, Manas; Bean, Jonathan C; Kong, Ji-Ming; Zhu, Xin-Hong; Gao, Tian-Ming; et al. (2009-09-23)
      BACKGROUND: Caspase-independent apoptotic pathways are suggested as a mechanism for the delayed neuronal death following ischemic insult. However, the underlying signalling mechanisms are largely unknown. Recent studies imply the involvement of several mitochondrial proteins, including endonuclease G (EndoG) and Bcl-2/adenovirus E1B 19 kDa-interacting protein (BNIP3), in the pathway of non-neuronal cells. RESULTS: In this report, using western blot analysis and immunocytochemistry, we found that EndoG upregulates and translocates from mitochondria to nucleus in a time-dependent manner in cultured hippocampal neurons following oxygen-glucose deprivation (OGD). Moreover, the translocation of EndoG occurs hours before the observable nuclear pyknosis. Importantly, the mitochondrial upregulation of BNIP3 precedes the translocation of EndoG. Forced expression of BNIP3 increases the nuclear translocation of EndoG and neuronal death while knockdown of BNIP3 decreases the OGD-induced nuclear translocation of EndoG and neuronal death. CONCLUSION: These results suggest that BNIP3 and EndoG play important roles in hippocampal neuronal apoptosis following ischemia, and mitochondrial BNIP3 is a signal protein upstream of EndoG that can induce neuronal death.
    • Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

      Raz, Limor; Zhang, Quan-Guang; Zhou, Cai-feng; Han, Dong; Gulati, Priya; Yang, Li-cai; Yang, Fang; Wang, Rui-min; Brann, Darrell W; Institute of Molecular Medicine and Genetics (2010-09-7)
      Background: Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.