07-1210 Sigma-AldrichAnti-IP3 Receptor Antibody

The endoplasmic reticulum (ER) is not only a home for folding and posttranslational modifications of secretory proteins but also a reservoir for intracellular Ca(2+). Perturbation of ER homeostasis contributes to the pathogenesis of various neurodegenerative diseases, such as Alzheimer's and Parkinson diseases. One key regulator that underlies cell survival and Ca(2+) homeostasis during ER stress responses is inositol-requiring enzyme 1α (IRE1α). Despite extensive studies on this ER membrane-associated protein, little is known about the molecular mechanisms by which excessive ER stress triggers cell death and Ca(2+) dysregulation via the IRE1α-dependent signaling pathway. In this study, we show that inactivation of IRE1α by RNA interference increases cytosolic Ca(2+) concentration in SH-SY5Y cells, leading to cell death. This dysregulation is caused by an accelerated ER-to-cytosolic efflux of Ca(2+) through the InsP3 receptor (InsP3R). The Ca(2+) efflux in IRE1α-deficient cells correlates with dissociation of the Ca(2+)-binding InsP3R inhibitor CIB1 and increased complex formation of CIB1 with the pro-apoptotic kinase ASK1, which otherwise remains inactivated in the IRE1α-TRAF2-ASK1 complex. The increased cytosolic concentration of Ca(2+) induces mitochondrial production of reactive oxygen species (ROS), in particular superoxide, resulting in severe mitochondrial abnormalities, such as fragmentation and depolarization of membrane potential. These Ca(2+) dysregulation-induced mitochondrial abnormalities and cell death in IRE1α-deficient cells can be blocked by depleting ROS or inhibiting Ca(2+) influx into the mitochondria. These results demonstrate the importance of IRE1α in Ca(2+) homeostasis and cell survival during ER stress and reveal a previously unknown Ca(2+)-mediated cell death signaling between the IRE1α-InsP3R pathway in the ER and the redox-dependent apoptotic pathway in the mitochondrion.

Holes within the internal elastic lamina (IEL) of blood vessels are sites of fenestration allowing for passage of diffusible vasoactive substances and interface of endothelial cell membrane projections with underlying vascular smooth muscle. Endothelial projections are sites of dynamic Ca(2+) events leading to endothelium dependent hyperpolarization (EDH)-mediated relaxations and the activity of these events increase as vessel diameter decreases. We tested the hypothesis that IEL fenestration is greater in distal vs. proximal arteries in skeletal muscle, and is unlike other vascular beds (mesentery). We also determined ion channel protein composition within the endothelium of intramuscular and non-intramuscular skeletal muscle arteries. Popliteal arteries, subsequent gastrocnemius feed arteries, and first and second order intramuscular arterioles from rat hindlimb were isolated, cut longitudinally, fixed, and imaged using confocal microscopy. Quantitative analysis revealed a significantly larger total fenestration area in second and first order arterioles vs. feed and popliteal arteries (58% and 16% vs. 5% and 3%; N = 10 images/artery), due to a noticeably greater average size of holes (9.5 and 3.9 µm(2) vs 1.5 and 1.9 µm(2)). Next, we investigated via immunolabeling procedures whether proteins involved in EDH often embedded in endothelial cell projections were disparate between arterial segments. Specific proteins involved in EDH, such as inositol trisphosphate receptors, small and intermediate conductance Ca(2+)-activated K(+) channels, and the canonical (C) transient receptor potential (TRP) channel TRPC3 were present in both popliteal and first order intramuscular arterioles. However due to larger IEL fenestration in first order arterioles, a larger spanning area of EDH proteins is observed proximal to the smooth muscle cell plasma membrane. These observations highlight the robust area of fenestration within intramuscular arterioles and indicate that the anatomical architecture and endothelial cell hyperpolarizing apparatus for distinct vasodilatory signaling is potentially present.

In this study we have used saponin to permeabilize bovine endothelial cell membranes in order to directly test the involvement of IP3 in regulating internal Ca2+ release. Our results indicate that the release of internal Ca2+ occurs as early as 1-3 seconds after IP3 addition. This IP3-induced internal Ca2+ release can be inhibited by heparin (an IP3 receptor antagonist). Further binding of [3H]IP3 to saponin-permeabilized bovine endothelial cells reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of approximately 0.50 (+/- 0.03) nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the bovine endothelial cell IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Immunofluorescence data indicates that the IP3 receptor is preferentially located at the perinuclear region of the cells. In addition, PCR analysis of first-strand cDNAs from both bovine endothelial cells and rat brain tissues reveals that the IP3 receptor transcript in bovine endothelial cells belongs to the short non-neuronal form and not the long neuronal form detected in rat brain tissue. These findings suggest that the IP3 receptor in endothelial cells is both structurally and functionally analogous to that reported in non-neuronal cell systems and probably plays an important role in agonist-induced endothelial cell activation.

Mouse T-lymphoma cells contain a unique type of internal vesicle which bands at the relatively light density of 1.07 g/cc. These vesicles do not contain any detectable Golgi, endoplasmic reticulum, plasma membrane, or lysosomal marker protein activities. Binding of [3H]inositol 1,4,5-trisphosphate (IP3) to these internal vesicles reveals the presence of a single, high affinity class of IP3 receptor with a dissociation constant (Kd) of 1.6 +/- 0.3 nM. Using a panel of monoclonal and polyclonal antibodies against IP3 receptor, we have established that the IP3 receptor (approximately 260 kDa) displays immunological cross-reactivity with the rat brain IP3 receptor. Polymerase chain reaction analysis of first-strand cDNAs from both mouse T-lymphoma cells and rat brain tissues reveals that the IP3 receptor transcript in mouse T-lymphoma cells belongs to the short form (non-neuronal form) and not the long form (neuronal form) detected in rat brain tissue. Scatchard plot analysis shows that high affinity binding occurs between ankyrin and the IP3 receptor with a Kd of 0.2 nM. Most importantly, the binding of ankyrin to the light density vesicles significantly inhibits IP3 binding and IP3-induced internal Ca2+ release. These findings suggest that the cytoskeleton plays a pivotal role in the regulation of IP3 receptor-mediated internal Ca2+ release during lymphocyte activation.

In this study we have used saponin to permeabilize platelet membranes in order to test directly the involvement of IP3 in regulating internal Ca2+ release, and to measure IP3 binding to its receptor. Our results indicate that platelet vesicles release Ca2+ as early as 3 seconds after IP3 addition. Using [3H]IP3, we have found that platelets contain a single class of high affinity IP3 binding sites with a Kd of approximately 0.20 (+/- 0.01) nM. Immuno-blotting shows that platelets contain a 260 kDa polypeptide which shares immunological cross reactivity with brain IP3 receptor. Immunofluorescence staining data indicate that the IP3 receptor is preferentially located at the periphery of the platelet plasma membrane. Most importantly, both IP3 binding and IP3-induced Ca2+ release activities are significantly inhibited by cytochalasin D (a microfilament inhibitor) and colchicine (a microtubule inhibitor). These findings suggest that the cytoskeleton is involved in the regulation of IP3 binding and IP3 receptor-mediated Ca2+ release during platelet activation.