thapsigargin and 1-oleoyl-2-acetoyl-sn-glycerol

Perturbation of calcium signaling that occurs during cell injury and disease, promotes cell death. In mouse lung fibroblasts A23187 triggered mitochondrial permeability transition pore (MPTP) formation, lactate dehydrogenase (LDH) release, and necrotic cell death that were blocked by cyclosporin A (CsA) and EGTA. LDH release temporally correlated with arachidonic acid release but did not involve cytosolic phospholipase A2α (cPLA2α) or calcium-independent PLA2. Surprisingly, release of arachidonic acid and LDH from cPLA2α-deficient fibroblasts was inhibited by the cPLA2α inhibitor pyrrophenone, and another serine hydrolase inhibitor KT195, by preventing mitochondrial calcium uptake. Inhibitors of calcium/calmodulin-dependent protein kinase II, a mitochondrial Ca(2+) uniporter (MCU) regulator, also prevented MPTP formation and arachidonic acid release induced by A23187 and H2O2. Pyrrophenone blocked MCU-mediated mitochondrial calcium uptake in permeabilized fibroblasts but not in isolated mitochondria. Unlike pyrrophenone, the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol and CsA blocked cell death and arachidonic acid release not by preventing mitochondrial calcium uptake but by inhibiting MPTP formation. In fibroblasts stimulated with thapsigargin, which induces MPTP formation by a direct effect on mitochondria, LDH and arachidonic acid release were blocked by CsA and 1-oleoyl-2-acetyl-sn-glycerol but not by pyrrophenone or EGTA. Therefore serine hydrolase inhibitors prevent necrotic cell death by blocking mitochondrial calcium uptake but not the enzyme releasing fatty acids that occurs by a novel pathway during MPTP formation. This work reveals the potential for development of small molecule cell-permeable serine hydrolase inhibitors that block MCU-mediated mitochondrial calcium overload, MPTP formation, and necrotic cell death.

Topics: Animals; Arachidonic Acid; Calcium; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Cell Line, Transformed; Chelating Agents; Cyclosporine; Diglycerides; Egtazic Acid; Fibroblasts; Group IV Phospholipases A2; Isoenzymes; L-Lactate Dehydrogenase; Mice; Mice, Knockout; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Necrosis; Phospholipase A2 Inhibitors; Pyrrolidines; Thapsigargin

TRPC (canonical transient receptor potential) channel subunits have been shown to assemble into homo- or hetero-meric channel complexes, including different Ca2+-handling proteins, required for the activation of CCE (capacitative Ca2+ entry) or NCCE (non-CCE) pathways. In the present study we found evidence for the dynamic interaction between endogenously expressed hTRPC6 (human TRPC6) with either both Orai1 and STIM1 (stromal interaction molecule 1) or hTRPC3 to participate in CCE or NCCE. Electrotransjection of cells with an anti-hTRPC6 antibody, directed towards the C-terminal region, reduces CCE induced by TPEN [N,N,N',N'-tetrakis-(2-pyridylmethyl)-ethylenediamine], which reduces the intraluminal free Ca2+ concentration. Cell stimulation with thrombin or extensive Ca2+-store depletion by TG (thapsigargin)+ionomycin enhanced the interaction between hTRPC6 and the CCE proteins Orai1 and STIM1. In contrast, stimulation with the diacylglycerol analogue OAG (1-oleoyl-2-acetyl-sn-glycerol) displaces hTRPC6 from Orai1 and STIM1 and enhances the association between hTRPC6 and hTRPC3. The interaction between hTRPC6 and hTRPC3 was abolished by dimethyl-BAPTA [1,2-bis-(o-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid] loading, which indicates that this phenomenon is Ca2+-dependent. These findings support the hypothesis that hTRPC6 participates both in CCE and NCCE through its interaction with the Orai1-STIM1 complex or hTRPC3 respectively.

Topics: Blood Platelets; Blotting, Western; Calcium; Calcium Channels; Cell Survival; Chelating Agents; Diglycerides; Egtazic Acid; Ethylenediamines; Humans; Immunoprecipitation; Ionomycin; Membrane Proteins; Neoplasm Proteins; ORAI1 Protein; Protein Binding; Signal Transduction; Stromal Interaction Molecule 1; Thapsigargin; TRPC Cation Channels; TRPC6 Cation Channel

Recent studies have defined roles for STIM1 and Orai1 as calcium sensor and calcium channel, respectively, for Ca(2+)-release activated Ca(2+) (CRAC) channels, channels underlying store-operated Ca(2+) entry (SOCE). In addition, these proteins have been suggested to function in signalling and constructing other channels with biophysical properties distinct from the CRAC channels. Using the human kidney cell line, HEK293, we examined the hypothesis that STIM1 can interact with and regulate members of a family of non-selective cation channels (TRPC) which have been suggested to also function in SOCE pathways under certain conditions. Our data reveal no role for either STIM1 or Orai1 in signalling of TRPC channels. Specifically, Ca(2+) entry seen after carbachol treatment in cells transiently expressing TRPC1, TRPC3, TRPC5 or TRPC6 was not enhanced by the co-expression of STIM1. Further, knockdown of STIM1 in cells expressing TRPC5 did not reduce TRPC5 activity, in contrast to one published report. We previously reported in stable TRPC7 cells a Ca(2+) entry which was dependent on TRPC7 and appeared store-operated. However, we show here that this TRPC7-mediated entry was also not dependent on either STIM1 or Orai1, as determined by RNA interference (RNAi) and expression of a constitutively active mutant of STIM1. Further, we determined that this entry was not actually store-operated, but instead TRPC7 activity which appears to be regulated by SERCA. Importantly, endogenous TRPC activity was also not regulated by STIM1. In vascular smooth muscle cells, arginine-vasopressin (AVP) activated non-selective cation currents associated with TRPC6 activity were not affected by RNAi knockdown of STIM1, while SOCE was largely inhibited. Finally, disruption of lipid rafts significantly attenuated TRPC3 activity, while having no effect on STIM1 localization or the development of I(CRAC). Also, STIM1 punctae were found to localize in regions distinct from lipid rafts. This suggests that TRPC signalling and STIM1/Orai1 signalling occur in distinct plasma membrane domains. Thus, TRPC channels appear to be activated by mechanisms dependent on phospholipase C which do not involve the Ca(2+) sensor, STIM1.

Topics: Arginine Vasopressin; Barium; beta-Cyclodextrins; Calcium Channels; Calcium Signaling; Carbachol; Cell Adhesion Molecules; Cell Line; Cell Membrane; Chelating Agents; Diglycerides; Electrophysiological Phenomena; Gadolinium; Humans; Inositol 1,4,5-Trisphosphate; Membrane Microdomains; Membrane Proteins; Myocytes, Smooth Muscle; Neoplasm Proteins; ORAI1 Protein; RNA, Small Interfering; Stromal Interaction Molecule 1; Stromal Interaction Molecule 2; Thapsigargin; Transfection; TRPC Cation Channels; TRPC6 Cation Channel

The mammalian canonical transient receptor channels (TRPCs) are considered to be candidates for store-operated calcium channels (SOCCs). Many studies have addressed how TRPC3 channels are affected by depletion of intracellular calcium stores. Conflicting results have been shown for TRPC3 regarding its function, and this has been linked to its level of expression in various systems. In the present study, we have investigated how overexpression of TRPC3 interferes with the regulation of intracellular calcium stores. We demonstrate that overexpression of TRPC3 reduces the mobilization of calcium in response to stimulation of the cells with thapsigargin (TG) or the G-protein coupled receptor agonist sphingosine-1-phosphate (S1P). Our results indicate that this is the result of the expression of TRPC3 channels in the endoplasmic reticulum (ER), thus depleting ER calcium stores. OAG evoked calcium entry in cells overexpressing TRPC3, indicating that functional TRPC3 channels were also expressed in the plasma membrane. Taken together, our results show that overexpression of the putative SOCC, TRPC3, actually reduces the calcium content of intracellular stores, but does not enhance agonist-evoked or store-dependent calcium entry. Our results may, in part, explain the conflicting results obtained in previous studies on the actions of TRPC3 channels.

Topics: Animals; Calcium; Calcium Channels; Calnexin; Cell Line; Diglycerides; Endoplasmic Reticulum; Humans; Ion Channel Gating; Lysophospholipids; Patch-Clamp Techniques; Recombinant Fusion Proteins; Sphingosine; Thapsigargin; TRPC Cation Channels

The role of PKC in the regulation of store-operated Ca2+ entry (SOCE) is rather controversial. Here, we used Ca2+-imaging, biochemical, pharmacological, and molecular techniques to test if Ca2+-independent PLA2beta (iPLA2beta), one of the transducers of the signal from depleted stores to plasma membrane channels, may be a target for the complex regulation of SOCE by PKC and diacylglycerol (DAG) in rabbit aortic smooth muscle cells (SMCs). We found that the inhibition of PKC with chelerythrine resulted in significant inhibition of thapsigargin (TG)-induced SOCE in proliferating SMCs. Activation of PKC by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (OAG) caused a significant depletion of intracellular Ca2+ stores and triggered Ca2+ influx that was similar to TG-induced SOCE. OAG and TG both produced a PKC-dependent activation of iPLA2beta and Ca2+ entry that were absent in SMCs in which iPLA2beta was inhibited by a specific chiral enantiomer of bromoenol lactone (S-BEL). Moreover, we found that PKC regulates TG- and OAG-induced Ca2+ entry only in proliferating SMCs, which correlates with the expression of the specific PKC-epsilon isoform. Molecular downregulation of PKC-epsilon impaired TG- and OAG-induced Ca2+ influx in proliferating SMCs but had no effect in confluent SMCs. Our results demonstrate that DAG (or OAG) can affect SOCE via multiple mechanisms, which may involve the depletion of Ca2+ stores as well as direct PKC-epsilon-dependent activation of iPLA2beta, resulting in a complex regulation of SOCE in proliferating and confluent SMCs.

Topics: Animals; Benzophenanthridines; Calcium; Calcium Signaling; Cell Proliferation; Cells, Cultured; Diglycerides; Enzyme Activation; Enzyme Inhibitors; Group VI Phospholipases A2; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Naphthalenes; Protein Kinase C-epsilon; Pyrones; Rabbits; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin; Time Factors; Transfection