Drosophila larvae lacking the bcl-2 gene, buffy, are sensitive to nutrient stress, maintain increased basal target of rapamycin (Tor) signaling and exhibit characteristics of altered basal energy metabolism

Background

B cell lymphoma 2 (Bcl-2) proteins are the central regulators of apoptosis. The two bcl-2 genes in Drosophila modulate the response to stress-induced cell death, but not developmental cell death. Because null mutants are viable, Drosophila provides an optimum model system to investigate alternate functions of Bcl-2 proteins. In this report, we explore the role of one bcl-2 gene in nutrient stress responses.

Results

We report that starvation of Drosophila larvae lacking the bcl-2 gene, buffy, decreases survival rate by more than twofold relative to wild-type larvae. The buffy null mutant reacted to starvation with the expected responses such as inhibition of target of rapamycin (Tor) signaling, autophagy initiation and mobilization of stored lipids. However, the autophagic response to starvation initiated faster in larvae lacking buffy and was inhibited by ectopic buffy. We demonstrate that unusually high basal Tor signaling, indicated by more phosphorylated S6K, was detected in the buffy mutant and that removal of a genomic copy of S6K, but not inactivation of Tor by rapamycin, reverted the precocious autophagy phenotype. Instead, Tor inactivation also required loss of a positive nutrient signal to trigger autophagy and loss of both was sufficient to activate autophagy in the buffy mutant even in the presence of enforced phosphoinositide 3-kinase (PI3K) signaling. Prior to starvation, the fed buffy mutant stored less lipid and glycogen, had high lactate levels and maintained a reduced pool of cellular ATP. These observations, together with the inability of buffy mutant larvae to adapt to nutrient restriction, indicate altered energy metabolism in the absence of buffy.

Conclusions

All animals in their natural habitats are faced with periods of reduced nutrient availability. This study demonstrates that buffy is required for adaptation to both starvation and nutrient restriction. Thus, Buffy is a Bcl-2 protein that plays an important non-apoptotic role to promote survival of the whole organism in a stressful situation.     

grim promotes programmed cell death of Drosophila microchaete glial cells

The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction with grim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim.     

Intracellular Ca2+ release through ryanodine receptors contributes to AMPA receptor-mediated mitochondrial dysfunction and ER stress in oligodendrocytes

Overactivation of ionotropic glutamate receptors in oligodendrocytes induces cytosolic Ca²⁺ overload and excitotoxic death, a process that contributes to demyelination and multiple sclerosis. Excitotoxic insults cause well-characterized mitochondrial alterations and endoplasmic reticulum (ER) dysfunction, which is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) to excitotoxicity in oligodendrocytes in vitro. First, we observed that oligodendrocytes express all previously characterized RyRs and IP₃Rs. Blockade of Ca²⁺-induced Ca²⁺ release by TMB-8 following α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor-mediated insults attenuated both oligodendrocyte death and cytosolic Ca²⁺ overload. In turn, RyR inhibition by ryanodine reduced as well the Ca²⁺ overload whereas IP₃R inhibition was ineffective. Furthermore, AMPA-triggered mitochondrial membrane depolarization, oxidative stress and activation of caspase-3, which in all instances was diminished by RyR inhibition. In addition, we observed that AMPA induced an ER stress response as revealed by α subunit of the eukaryotic initiation factor 2α phosphorylation, overexpression of GRP chaperones and RyR-dependent cleavage of caspase-12. Finally, attenuating ER stress with salubrinal protected oligodendrocytes from AMPA excitotoxicity. Together, these results show that Ca²⁺ release through RyRs contributes to cytosolic Ca²⁺ overload, mitochondrial dysfunction, ER stress and cell death following AMPA receptor-mediated excitotoxicity in oligodendrocytes.     

The Bcl-2 Protein Family Member Bok Binds to the Coupling Domain of Inositol 1,4,5-Trisphosphate Receptors and Protects Them from Proteolytic Cleavage

Bok is a member of the Bcl-2 protein family that controls intrinsic apoptosis. Bok is most closely related to the pro-apoptotic proteins Bak and Bax, but in contrast to Bak and Bax, very little is known about its cellular role. Here we report that Bok binds strongly and constitutively to inositol 1,4,5-trisphosphate receptors (IP₃Rs), proteins that form tetrameric calcium channels in the endoplasmic reticulum (ER) membrane and govern the release of ER calcium stores. Bok binds most strongly to IP₃R1 and IP₃R2, and barely to IP₃R3, and essentially all cellular Bok is IP₃R bound in cells that express substantial amounts of IP₃Rs. Binding to IP₃Rs appears to be mediated by the putative BH4 domain of Bok and the docking site localizes to a small region within the coupling domain of IP₃Rs (amino acids 1895–1903 of IP₃R1) that is adjacent to numerous regulatory sites, including sites for proteolysis. With regard to the possible role of Bok-IP₃R binding, the following was observed: (i) Bok does not appear to control the ability of IP₃Rs to release ER calcium stores, (ii) Bok regulates IP₃R expression, (iii) persistent activation of inositol 1,4,5-trisphosphate-dependent cell signaling causes Bok degradation by the ubiquitin-proteasome pathway, in a manner that parallels IP₃R degradation, and (iv) Bok protects IP₃Rs from proteolysis, either by chymotrypsin in vitro or by caspase-3 in vivo during apoptosis. Overall, these data show that Bok binds strongly and constitutively to IP₃Rs and that the most significant consequence of this binding appears to be protection of IP₃Rs from proteolysis. Thus, Bok may govern IP₃R cleavage and activity during apoptosis.     

Mitochondrial Calcium Signaling Driven by the IP3 Receptor

Many agonists bring about their effects on cellular functions through a rise incytosolic [Ca²⁺]([Ca²⁺]_c) mediated by the second messenger inositol 1,4,5-trisphosphate (IP₃). Imaging studiesof single cells have demonstrated that [Ca²⁺]_c signals display cell specific spatiotemporalorganization that is established by coordinated activation of IP₃ receptor Ca²⁺ channels.Evidence emerges that cytosolic calcium signals elicited by activation of the IP₃ receptors areefficiently transmitted to the mitochondria. An important function of mitochondrial calciumsignals is to activate the Ca²⁺-sensitive mitochondrial dehydrogenases, and thereby to meetdemands for increased energy in stimulated cells. Activation of the permeability transitionpore (PTP) by mitochondrial calcium signals may also be involved in the control of cell death.Furthermore, mitochondrial Ca²⁺ transport appears to modulate the spatiotemporal organizationof [Ca²⁺]_c responses evoked by IP₃ and so mitochondria may be important in cytosolic calciumsignaling as well. This paper summarizes recent research to elucidate the mechanisms andsignificance of IP₃-dependent mitochondrial calcium signaling.     

Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism

Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP₆) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP₆ increased rhythm amplitude and shortened period in NIH3T3 cells. The IP₆ effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP₆ did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP₆ involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP₆, decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP₆ in NIH3T3 cells or neurons. Pharmacological inhibition of the IP₆ kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP₆. Overall, we conclude that inositol phosphates, in particular IP₆ have effects on circadian rhythms. Manipulations affecting IP₆ and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated.     

Endoplasmic reticulum Ca release through ryanodine and IP3 receptors contributes to neuronal excitotoxicity

Overactivation of ionotropic glutamate receptors induces a Ca²⁺ overload into the cytoplasm that leads neurons to excitotoxic death, a process that has been linked to several neurodegenerative disorders. While the role of mitochondria and its involvement in excitotoxicity have been widely studied, the contribution of endoplasmic reticulum (ER), another crucial intracellular store in maintaining Ca²⁺ homeostasis, is not fully understood. In this study, we analyzed the contribution of ER-Ca²⁺ release through ryanodine (RyR) and IP₃ (IP₃R) receptors to a neuronal in vitro model of excitotoxicity. NMDA induced a dose-dependent neuronal death, which was significantly decreased by ER-Ca²⁺ release inhibitors in cortical neurons as well as in organotypic slices. Furthermore, ryanodine and 2APB, RyR and IP₃R inhibitors respectively, attenuated NMDA-triggered intracellular Ca²⁺ increase and oxidative stress, whereas 2APB reduced mitochondrial membrane depolarization and caspase-3 cleavage. Consistent with ER-Ca²⁺ homeostasis disruption, we observed that NMDA-induced ER stress, characterized here by eIF2α phosphorylation and over-expression of GRP chaperones which were regulated by ER-Ca²⁺ release inhibitors. These results demonstrate that Ca²⁺ release from ER contributes to neuronal death by both promoting mitochondrial dysfunction and inducing specific stress and apoptosis pathways during excitotoxicity.     

Inositol 1,4,5-trisphosphate induces calcium release from a non-mitochondrial pool in amoebae of Dictyostelium

Evidence is presented for two distinct Ca²⁺ pools in amoebae of Dictyostelium discoideum. One pool, presumably mitochondrial, was sensitive to the mitochondrial inhibitors oligomycin and dinitrophenol and showed an affinity for Ca²⁺ in the μM concentration range. The other Ca²⁺ pool, which was insensitive to these inhibitors, was of lower capacity but had higher affinity (in the nM range). Inositol 1,4,5-trisphosphate (5 μM) added to saponin-permeabilized amoebae induced a rapid release of Ca²⁺ from the latter pool but had no effect on the presumed mitochondrial pool. Controls using addition of inositol 1,4-bisphosphate (the hydrolytic product of IP₃) induced no such Ca²⁺ release. The results provide strong support for the involvement of IP₃ in signal transmission during chemotaxis of D. discoideum.     

Selective down-regulation of IP3receptor subtypes by caspases and calpain during TNF α -induced apoptosis of human T-lymphoma cells

There are at least three types of inositol 1,4,5-trisphosphate receptor (IP₃R) [IP₃-gated Ca²⁺channels], which are expressed in different cell types and mammalian tissues. In this study, we have identified three IP₃R subtypes in human Jurkat T-lymphoma cells. All three subtypes have a molecular mass of about 260 kDa, and display Ca²⁺channel properties in an IP₃-dependent manner. We have also demonstrated that TNFα promotes the activity of different proteases (e.g. caspase-8, caspase-3 and calpain), alters the TCR-mediated Ca²⁺response and subsequently induces apoptosis in Jurkat cells. During the first 6 h of incubation with TNFα, several IP₃R subtype-related changes occur (e.g. proteolysis of IP₃R subtypes, inhibition of IP₃binding and impairment of IP₃-mediated Ca²⁺flux) concomitantly with an elevation of protease (caspase-8, caspase-3 and calpain) activity. Furthermore, the caspase inhibitor, Z-VAD-fmk, significantly reduces TNFα-mediated perturbation of IP₃R1 and IP₃R2 (but not IP₃R3) function; whereas the calpain inhibitor I, ALLN, is capable of blocking the inhibitory effect of TNFα on IP₃R3 function. These findings suggest that IP₃R1 and IP₃R2 serve as cellular substrates for caspases, and IP₃R3 is a substrate for calpain. We propose that the selective down-regulation of IP₃R subtype-mediated Ca²⁺function by caspase-dependent and calpain-sensitive mechanisms may be responsible for the early onset of the apoptotic signal by TNFα in human T-cells.     

Decreased expression of the Augmenter of Liver Regeneration results in increased apoptosis and oxidative damage in human-derived glioma cells

The mammalian growth factor erv1-like (GFER) gene encodes a sulfhydryl oxidase enzyme, named Augmenter of Liver Regeneration (ALR). Recently it has been demonstrated that ALR supports cell proliferation acting as an anti-apoptotic factor. This effect is determined by ALR ability to support the anti-apoptotic gene expression and to preserve cellular normoxic conditions. We recently demonstrated that the addition of recombinant ALR (rALR) in the culture medium of H₂O₂-treated neuroblastoma cells reduces the lethal effects induced by the hydrogen peroxide. Similar data have been reported in the regenerating liver tissue from partially hepatectomized rats treated with rALR. The purpose of the present study was to evaluate the effect of the GFER inhibition, via the degradation of the complementary mRNA by the specific siRNA, on the behaviour of the apoptosis (apoptotic gene and caspase expression and apoptotic cell number) and of the oxidative stress-induced parameters (reactive oxygen species (ROS), clusterin expression and mitochondrial integrity) in T98G glioma cells. The results revealed a reduction of (i) ALR, (ii) clusterin and (iii) bcl-2 and an increase of (iv) caspase-9, activated caspase-3, ROS, apoptotic cell number and mitochondrial degeneration. These data confirm the anti-apoptotic role of ALR and its anti-oxidative properties, and shed some light on the molecular pathways through which ALR modulates its biological effects.     

Control of protein translation by IP3R-mediated Ca2+ release in Drosophila neuroendocrine cells

The inositol 1,4,5-trisphosphate receptor (IP₃R) is one of two Ca²⁺ channels that gates Ca²⁺ release from ER-stores. The ligand IP₃, generated upon specific G-protein coupled receptor activation, binds to IP₃R to release Ca²⁺ into the cytosol. IP₃R also mediates ER-store Ca²⁺ release into the mitochondria, under basal as well as stimulatory conditions; an activity that influences cellular bioenergetics and thus, cellular growth and proliferation. In Drosophila neuroendocrine cells expressing a hypomorphic mutant of IP₃R, we observed reduced protein translation levels. Here, we discuss the possible molecular mechanism for this observation. We hypothesize that the cellular energy sensor, AMPK connects IP₃R mediated Ca²⁺ release into the mitochondria, to protein translation, via the TOR pathway.     

A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H₂O₂ and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H₂O₂ levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism.     

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP₃) stimulates an efflux of ⁴⁵Ca²⁺ from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP₃ might serve as a second messenger for the mobilization of intracellular Ca²⁺ in higher plant cells. To determine whether or not IP₃ and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-³H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [³H]inositol metabolites coeluted with inositol bisphosphate (IP₂) and IP₃ when separated by anion exchange chromatography. However, we could not detect IP₂ or IP₃ when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP₂ and IP₃, were present in these cells. Thus, [³H] inositol metabolites other than IP₂ and IP₃ had coeluted on the anion exchange columns. The data indicate that either IP₃ is rapidly metabolized or that it is not present at a detectable level in the carrot cells.     

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study

The inositol 1,4,5-trisphosphate receptor (IP₃R) is an intracellular Ca²⁺ release channel responsible for mobilizing stored Ca²⁺. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP₃R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP₃R2 and IP₃R3, respectively). We studied the expression of the IP₃R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP₃R1, the levels of expression of IP₃R2 and IP₃R3 mRNAs were low in all of the tissues tested. IP₃R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP₃R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP₃R2 or IP₃R3 mRNA are known to have a secretory function in which IP₃/Ca²⁺ signalling has been shown to be involved, and thus either IP₃R2 or IP₃R3 may be a prerequisite to secretion in these cells.     

The role of endoplasmic reticulum in cadmium-induced mesangial cell apoptosis

Cd is an industrial and environmental pollutant that affects many organs in humans and other mammals. However, the molecular mechanisms of Cd-induced nephrotoxicity are unclear. In this study, we show that endoplasmic reticula (ER) played a pivotal role in Cd-induced apoptosis in mesangial cells. Using Fluo-3 AM, the intracellular concentration of calcium ([Ca²⁺]_i) was detected as being elevated as time elapsed after Cd treatment. Co-treatment with BAPTA-AM, a calcium chelator, was able to significantly suppress Cd-induced apoptosis. Calcineurin is a cytosolic phosphatase, which was able to dephosphorylate the inositol-1,4,5-triphosphate receptor (IP₃R) calcium channel to prevent the release of calcium from ER. Cyclosporine A, a calcineurin inhibitor, increased both [Ca²⁺]_i and the percentage of Cd-induced apoptosis. However, EGTA and the IP₃R inhibitor, 2-APB, were able to partially modulate Cd cytotoxicity. These results led us to suggest that the extracellular and ER-released calcium plays a crucial role in Cd-induced apoptosis in mesangial cells. Following this line, we further detected the ER stress after Cd treatment since ER is one of the major calcium storage organelles. After Cd exposure, GADD153, a hallmark of ER stress, was upregulated (at 4 h of exposure), followed by activation of ER-specific caspase-12 and its downstream molecule caspase-3 (at 16 h of exposure). The pan caspase inhibitor, Z-VAD, and BAPTA-AM were able to reverse the Cd-induced cell death and ER stress, respectively. Furthermore, the mitochondrial membrane potential (ΔΨ_m) was depolarized significantly and cytochrome c was released after 24 h of exposure to Cd and followed by mild activation of caspase-9 at the 36-h time point, indicating that mitochondria stress is a late event. Therefore, we concluded that ER is the major killer organelle in Cd-induced mesangial cell apoptosis and that calcium oscillation plays a pivotal role.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

A novel fluorescent method employing the FRET-based biosensor “LIBRA” for the identification of ligands of the inositol 1,4,5-trisphosphate receptors

LIBRA is a fluorescent biosensor of inositol 1,4,5-trisphosphate (IP₃) and is composed of the ligand-binding domain of the rat type 3 IP₃ receptor and cyan and yellow fluorescent proteins. We examined the responses of LIBRA and its IP₃-insensitive mutant LIBRA-N to compounds known to inhibit IP₃-induced Ca²⁺ release. Heparin, a competitive antagonist of IP₃ receptors, increased the emission ratio of LIBRA but not that of LIBRA-N. In contrast, 2-aminoethoxydiphenyl borate, a known non-competitive inhibitor of IP₃ receptor, decreased the emission ratios of both LIBRA and LIBRA-N. Thus, the concurrent use of LIBRA-N with LIBRA identifies nonspecific responses. These results indicate that LIBRA and its mutant control can be used to detect specific agonists and antagonists of IP₃ receptors. We also demonstrate the utility of LIBRA and LIBRA-N in discriminating between specific and nonspecific responses in intact cells.     

Tetrahydropalmatine protects rat pulmonary endothelial cells from irradiation-induced apoptosis by inhibiting oxidative stress and the calcium sensing receptor/phospholipase C-γ1 pathway

The aim of this study was to confirm the protective effect of tetrahydropalmatine (THP) against irradiation-induced rat pulmonary endothelial cell apoptosis and to explore the underlying mechanism, with a focus on the calcium-sensing receptor (CaSR)/phospholipase C-γ1 (PLC-γ1) pathway. We established a model of irradiation-induced primary rat pulmonary endothelial cell injury. Cell apoptosis and mitochondrial membrane potential (Δψm) were measured by flow cytometry. The expression of CaSR, cytochrome c, PLC-γ1, reactive oxygen species (ROS) and [Ca²⁺]_i was also determined. Caspase-3 and caspase-9 activities were measured using commercial kits. Inositol triphosphate (IP₃) and the production of inflammatory cytokines were detected by enzyme-linked immunosorbent assay. The results showed that THP significantly inhibited irradiation-induced cell apoptosis and intracellular accumulation of ROS. Pretreatment with THP significantly decreased the expression of CaSR, inhibited the CaSR/PLC-γ1 pathway and subsequent [Ca²⁺]_i overload stimulated by irradiation. THP, NPS2390 (inhibitor of CaSR), U73122 (inhibitor of PLC-γ1) and 2-APB (inhibitor of IP₃) further decreased cell apoptosis, along with down-regulation of cytochrome c, caspase-3 and caspase-9 activation, disruption of Δψm and the production of inflammatory cytokines. These findings suggest that THP protects primary rat pulmonary endothelial cells against irradiation-induced apoptosis by inhibiting oxidative stress and the CaSR/PLC-γ1 pathway.     

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5‐trisphosphate (IP₃) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP₃ signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up‐regulating IP₃ signalling decreases sensitivity. Tissue‐specific rescue experiments suggest IP₃ functions in the intestine. We also exploit IP₃ signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.