xestospongin-a and 2-aminoethoxydiphenyl-borate

Since its introduction to Ca2+ signaling in 1997, 2-aminoethoxydiphenyl borate (2-APB) has been used in many studies to probe for the involvement of inositol 1,4,5-trisphosphate receptors in the generation of Ca2+ signals. Due to reports of some nonspecific actions of 2-APB, and the fact that its principal antagonistic effect is on Ca2+ entry rather than Ca2+ release, this compound may not have the utility first suggested. However, 2-APB has thrown up some interesting results, particularly with respect to store-operated Ca2+ entry in nonexcitable cells. These data indicate that although it must be used with caution, 2-APB can be useful in probing certain aspects of Ca2+ signaling.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ion Transport; Macrocyclic Compounds; Mice; Oxazoles; Receptors, Cytoplasmic and Nuclear

Little is known about regulatory mechanisms of type 1 inositol-1,4,5-triphosphate receptor (IP(3)R-1) expression in conditioned place preference by methamphetamine (METH), though significant enhancement of IP(3)R-1 expression in the mouse frontal cortex and limbic forebrain by intermittent administration of cocaine is reported. The present study investigated the role and regulation of IP(3)R-1 in mice with METH-induced place preference. Injection of IP(3)R antagonists with different chemical structures, 2-aminophenoxyethane-borate and xestospongin C, into the mouse nucleus accumbens (NAcc) dose-dependently inhibited METH-induced place preference. The levels of IP(3)R-1 protein in the NAcc of METH-conditioned mice significantly increased, which was completely abolished by microinjection of SCH23390 and raclopride, selective dopamine D1-like and D2-like receptor (D1 and D2DR) antagonists respectively, into the mouse NAcc. Immunohistochemical assessment revealed co-localization of immunoreactivity for IP(3)R-1 and those for D1 and D2DRs in the NAcc. These findings suggest that IP(3)R-1 could be involved in the development of METH-induced place preference and that D1 and D2DRs in the NAcc of mice showing METH-induced place preference play possible regulatory roles in IP(3)R-1 expression.

Topics: Analysis of Variance; Animals; Boron Compounds; Central Nervous System Stimulants; Conditioning, Operant; Dopamine Agents; Dose-Response Relationship, Drug; Drug Interactions; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Methamphetamine; Mice; Microinjections; Nerve Tissue Proteins; Neurons; Nucleus Accumbens; Oxazoles; Receptors, Dopamine D1; Receptors, Dopamine D2; Up-Regulation

The norepinephrine transporter (NET) plays a crucial role in noradrenergic neurotransmission and is a target of many antidepressants and psychostimulants. Intracellular Ca2+ is reportedly involved in regulating NET activity, but the detailed mechanism is not clear. We employed a norepinephrine uptake assay using SH-SY5Y cells and found that the IP3 receptor inhibitors, 2-aminoethoxydiphenyl borate and xestospongin C, reduced the NET Vmax. These reductions were accompanied by the decreased cell surface expression of NET. Our findings suggest that intracellular Ca2+ mobilized by IP3 receptor is required for the maintenance of NET activity. This adds another pathway involving Ca2+ for the regulation of NET to other known mechanisms providing intracellular Ca2+.

Topics: Biological Transport; Boron Compounds; Calcium; Calcium Channels; Carbachol; Cell Line, Tumor; Humans; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Fluid; Macrocyclic Compounds; Norepinephrine; Norepinephrine Plasma Membrane Transport Proteins; Oxazoles; Receptors, Cytoplasmic and Nuclear

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Topics: Animals; Boron Compounds; Calcium; Calcium Channels; Carbachol; Dose-Response Relationship, Drug; Estrenes; Gastric Fundus; Gastrointestinal Motility; Guinea Pigs; Image Processing, Computer-Assisted; In Vitro Techniques; Inositol 1,4,5-Trisphosphate Receptors; Macrocyclic Compounds; Microscopy, Confocal; Myocytes, Smooth Muscle; Neomycin; Oxazoles; Phosphodiesterase Inhibitors; Pyrrolidinones; Receptors, Cytoplasmic and Nuclear; Ryanodine; Software; Tissue Distribution; Type C Phospholipases

The micromeres, the first cells to be specified in sea urchin embryos, are generated by unequal cleavage at the fourth cell division. The micromeres differentiate autonomously to form spicules and dispatch signals to induce endomesoderm in the neighbouring macromeres cells in the embryo. Using a calcium indicator Fura-2/AM and a mixture of dextran conjugated Oregon green-BAPTA 488 and Rhodamine red, the intracellular calcium ion concentration ([Ca2+]i) was studied in embryos at the 16-cell stage. [Ca2+]i was characteristically elevated in the micromeres during furrowing at the 4th cleavage. Subsequently, Ca2+ oscillated for about 10 min in the micromeres, resulting in episodic high levels of [Ca2+]i. High [Ca2+]i regions were associated with regional localizations of the endoplasmic reticulum (ER), though not with ER accumulated at the vegetal pole of the micromeres during the 4th division. Pharmacological studies, using a blocker of IP3-mediated Ca2+ release (Xestospongin), a store-operated Ca2+ entry inhibitor (2 aminoethoxydiphenyl borate (2-APB)) and an inhibitor of stretch-dependent ion channels (gadolinium), suggest that the high [Ca2+]i and oscillations in the micromeres are triggered by calcium influx caused by the activation of stretch-dependent calcium channels, followed by the release of calcium ions from the endoplasmic reticulum. On the basis of these new findings, a possible mechanism for autonomous formation of the micromeres is discussed.

Topics: Animals; Boron Compounds; Calcium; Calcium Signaling; Embryo, Nonmammalian; Endoplasmic Reticulum; Gadolinium; Imidazoles; Inositol 1,4,5-Trisphosphate; Macrocyclic Compounds; Microtubules; Oxazoles; Sea Urchins

Intracellular Ca(2+) ([Ca(2+)](i)) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca(2+) release from intracellular Ca(2+) stores produces [Ca(2+)](i) oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca(2+)](i) oscillations in ICCs are preserved in the presence of dihydropyridine Ca(2+) antagonists, which suppress Ca(2+) activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca(2+)](i) oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca(2+)](i) oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP(3)Rs. The reconstituted [Ca(2+)](i) oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.

Topics: Anesthetics, Local; Animals; Biological Clocks; Boron Compounds; Caffeine; Calcium; Calcium Channel Blockers; Cell Line; Enzyme Inhibitors; Fluorescent Antibody Technique, Indirect; Fluorescent Dyes; Fura-2; Humans; Ileum; Immunohistochemistry; Kinetics; Macrocyclic Compounds; Mice; Microscopy, Fluorescence; Muscle, Smooth; Nifedipine; Oxazoles; Proto-Oncogene Proteins c-kit; RNA, Messenger; Ryanodine; Ryanodine Receptor Calcium Release Channel; Tacrolimus; Tetracaine

Exogenous phosphatidic acid (PA) was observed to produce a concentration-dependent increase in [Ca(2+)](i) in cultured A10 vascular smooth muscle cells. Preincubation of cells with sarcoplasmic reticulum Ca(2+)-ATPase inhibitors (cyclopiazonic acid and thapsigargin), a phospholipase C inhibitor (2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate), inositol 1,4,5-trisphosphate receptor antagonists (2-aminoethoxydiphenyl borate and xestospongin), and an activator of protein kinase C (PKC) (phorbol 12-myristate 13-acetate) depressed the PA-evoked increase in [Ca(2+)](i). Although EGTA, an extracellular Ca(2+) chelator, decreased the PA-induced increase in [Ca(2+)](i), sarcolemmal Ca(2+)-channel blockers (verapamil or diltiazem) did not alter the action of PA. On the other hand, inhibitors of PKC (bisindolylmaleimide I) and G(i)-protein (pertussis toxin) potentiated the increase in [Ca(2+)](i) evoked by PA significantly. These results suggest that the PA-induced increase in [Ca(2+)](i) in vascular smooth muscle cells may occur upon the activation of phospholipase C and the subsequent release of Ca(2+) from the inositol 1,4,5-trisphosphate-sensitive Ca(2+) pool in the sarcoplasmic reticulum. This action of PA may be mediated through the involvement of PKC.

Topics: Animals; Boron Compounds; Calcium; Carbamates; Cells, Cultured; Diltiazem; Drug Interactions; Egtazic Acid; Indoles; Macrocyclic Compounds; Maleimides; Muscle, Smooth, Vascular; Oxazoles; Pertussis Toxin; Phenylcarbamates; Phosphatidic Acids; Rats; Tetradecanoylphorbol Acetate; Thapsigargin; Verapamil

Beta-amyloid (Abeta) peptide has been suggested to play important roles in the pathogenesis of Alzheimer's disease (AD). Abeta peptide neurotoxicity was shown to induce disturbance of cellular calcium homeostasis. However, whether modulation of calcium release from the endoplasmic reticulum (ER) can protect neurons from Abeta toxicity is not clearly defined. In the present study, Abeta peptide-triggered ER calcium release in primary cortical neurons in culture is modulated by Xestospongin C, 2-aminoethoxydiphenyl borate or FK506. Our results showed that reduction of ER calcium release can partially attenuate Abeta peptide neurotoxicity evaluated by LDH release, caspase-3 activity and quantification of apoptotic cells. While stress signals associated with perturbations of ER functions such as up-regulation of GRP78 was significantly attenuated, other signaling machinery such as activation of caspase-7 transmitting death signals from ER to other organelles could not be altered. We further provide evidence that molecular signaling in mitochondria play also a significant role in determining neuronal apoptosis because Abeta peptide-triggered activation of caspase-9 was not significantly reduced by attenuating ER calcium release. Our results suggest that neuroprotective strategies aiming at reducing Abeta toxicity should include molecular targets linked to ER perturbations associated with ER calcium release as well as mitochondrial stress.

Topics: Amyloid beta-Peptides; Analysis of Variance; Animals; Blotting, Western; Boron Compounds; Calcium; Carrier Proteins; Caspase 3; Caspases; Cell Death; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Enzyme Inhibitors; Heat-Shock Proteins; Immunosuppressive Agents; Indoles; Intracellular Space; L-Lactate Dehydrogenase; Macrocyclic Compounds; Molecular Chaperones; Neurons; Oxazoles; Peptide Fragments; Rats; Rats, Sprague-Dawley; Tacrolimus; Time Factors

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Estrenes; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Macrocyclic Compounds; Male; Muscle Cells; Oxazoles; Phosphodiesterase Inhibitors; Portal Vein; Pyrrolidinones; Rabbits; Receptors, Cytoplasmic and Nuclear; Ryanodine Receptor Calcium Release Channel; Type C Phospholipases

1. Agonists increase endothelial cell intracellular Ca(2+), in part, by capacitative entry, which is triggered by the filling state of intracellular Ca(2+) stores. It has been suggested that depletion of endoplasmic reticulum (ER) Ca(2+) stores either leads to a physical coupling between the ER and a plasma membrane channel, or results in production of an intracellular messenger which affects the gating of membrane channels. As an axis involving the IP(3) receptor has been implicated in a physical coupling mechanism the aim of this study was to examine the effects of the putative IP(3) receptor antagonists/modulators, 2 aminoethoxydiphenyl borate (2APB) and xestospongin C, on endothelial cell Ca(2+) entry. 2. Studies were conducted in fura 2 loaded cultured bovine aortic endothelial cells and endothelial cells isolated from rat heart. 3. 2APB (30 - 300 microM) inhibited Ca(2+) entry induced by both agonists (ATP 1 microM, bradykinin 0.1 microM) and receptor-independent mechanisms (thapsigargin 1 microM, ionomycin 0.5 and 5 microM). 2APB did not diminish endothelial cell ATP-induced production of IP(3) nor effect in vitro binding of [(3)H]-IP(3) to an adrenal cortex binding protein. Capacitative Ca(2+) entry was also blocked by disruption of the actin cytoskeleton with cytochalasin (100 nM) while the initial Ca(2+) release phase was unaffected. 4. Similarly to 2APB, xestospongin C (3 - 10 microM) inhibited ATP-induced Ca(2+) release and capacitative Ca(2+) entry. Further, xestospongin C inhibited capacitative Ca(2+) entry induced by thapsigargin (1 microM) and ionomycin (0.5 microM). 5. The data are consistent with a mechanism of capacitative Ca(2+) entry in vascular endothelial cells which requires (a) IP(3) receptor binding and/or an event distal to the activation of the ER receptor and (b) a spatial relationship, dictated by the cytoskeleton, between Ca(2+) release and entry pathways.

Topics: Actins; Adenosine Triphosphate; Animals; Aorta; Boron Compounds; Calcium; Calcium Channels; Cattle; Cell Movement; Cells, Cultured; Cytoskeleton; Endothelium, Vascular; Enzyme Inhibitors; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Ionomycin; Ionophores; Macrocyclic Compounds; Oxazoles; Rats; Receptors, Cytoplasmic and Nuclear; Stereoisomerism; Thapsigargin

1. The role of internal Ca(2+) stores in the generation of the rhythmic electrical and contractile activity in the guinea-pig proximal renal pelvis was examined using intracellular microelectrode and muscle tension recording techniques. 2. Ryanodine (30 microM) transiently increased contraction amplitude, while caffeine (0.5 - 3 mM) reduced contraction amplitude and frequency. Contractility was also reduced by 2-aminoethoxy-diphenylborate (2-APB 60 microM), xestospongin C (1 microM), U73122 (5 microM) and neomycin (4 mM), blockers of IP(3)-dependent release from Ca(2+) stores. 3. 60 mM K(+) saline-evoked contractions were reduced by caffeine (1 mM), U73122 (5 microM) and neomycin (4 mM), but little affected by ryanodine or 2-APB (60 microM). 4. Spontaneous action potentials consisting of an initial spike followed by a long plateau were recorded (frequency 8.6+/-1.0 min(-1)) in small urothelium-denuded strips of proximal renal pelvis. 5. Action potential discharge was blocked in 75 and 35% of cells by 2-APB (60 microM) and caffeine (1 mM), respectively. In the remaining cells, only a truncation of the plateau phase was observed. 6. Cyclopiazonic acid (CPA 10 microM for 10 - 180 min), blocker of CaATPase, transiently increased contraction frequency and amplitude. Action potential durations were increased 3.6 fold. Contraction amplitude and frequency slowly declined during a prolonged (>60 min) CPA exposure. 7. We conclude that the action potential in caffeine-sensitive cells and the shoulder component of caffeine-insensitive action potential arise from the entry of Ca(2+) through Ca(2+) channels. The inhibitory actions of modulators of internal Ca(2+) release were partially explained by a blockade of Ca(2+) entry.

Topics: Animals; Boron Compounds; Caffeine; Calcium; Electric Conductivity; Electrophysiology; Estrenes; Guinea Pigs; Indoles; Kidney Pelvis; Macrocyclic Compounds; Muscle Contraction; Muscle, Smooth; Neomycin; Oxazoles; Pyrrolidinones; Ryanodine

We examined the single channel properties and regulation of store-operated calcium channels (SOCC). In human submandibular gland cells, carbachol (CCh) induced flickery channel activity while thapsigargin (Tg) induced burst-like activity, with relatively lower open probability (NP(o)) and longer mean open time. Tg- and CCh-activated channels were permeable to Na(+) and Ba(2+), but not to NMDG, in the absence of Ca(2+). The channels exhibited similar Ca(2+), Na(+), and Ba(2+) conductances and were inhibited by 2-aminoethoxydiphenylborate, xestospongin C, Gd(3+), and La(3+). CCh stimulated flickery activity changed to burst-like activity by (i) addition of Tg, (ii) using Na(+) instead of Ca(2+), (iii) using Ca(2+)-free bath solution, or (iv) buffering [Ca(2+)](i) with BAPTA-AM. Buffering [Ca(2+)](i) induced a 2-fold increase in NP(o) of Tg-stimulated SOCC. Reducing free [Ca(2+)] in the endoplasmic reticulum with the divalent cation chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), induced burst-like channel activity similar to that seen with CCh + Tg. Thus, SOCC is activated by stimulation of muscarinic receptors, inhibition of the sarcoendoplasmic Ca(2+) pump, and lowering [Ca(2+)] in the internal store. Importantly, SOCC activity depends on [Ca(2+)](i) and the free [Ca(2+)] in the internal store. These novel findings reveal that SERCA plays a major role in the gating of SOCC by (i) refilling the internal Ca(2+) store(s) and (ii) decreasing the [Ca(2+)](i)-dependent inhibition.

Topics: Barium; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium-Transporting ATPases; Carbachol; Cations; Cell Line; Cholinergic Agonists; Electrophysiology; Endoplasmic Reticulum; Enzyme Inhibitors; Ethylenediamines; Gadolinium; Glycerol; Humans; Kinetics; Macrocyclic Compounds; Oxazoles; Sarcoplasmic Reticulum; Sodium; Thapsigargin

The coupling mechanism between endoplasmic reticulum (ER) calcium ion (Ca2+) stores and plasma membrane (PM) store-operated channels (SOCs) is crucial to Ca2+ signaling but has eluded detection. SOCs may be functionally related to the TRP family of receptor-operated channels. Direct comparison of endogenous SOCs with stably expressed TRP3 channels in human embryonic kidney (HEK293) cells revealed that TRP3 channels differ in being store independent. However, condensed cortical F-actin prevented activation of both SOC and TRP3 channels, which suggests that ER-PM interactions underlie coupling of both channels. A cell-permeant inhibitor of inositol trisphosphate receptor (InsP3R) function, 2-aminoethoxydiphenyl borate, prevented both receptor-induced TRP3 activation and store-induced SOC activation. It is concluded that InsP3Rs mediate both SOC and TRP channel opening and that the InsP3R is essential for maintaining coupling between store emptying and physiological activation of SOCs.

Topics: Actins; Boron Compounds; Calcium; Calcium Channels; Calcium Signaling; Carbachol; Cell Line; Cell Membrane; Diglycerides; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate Receptors; Ionomycin; Macrocyclic Compounds; Marine Toxins; Oxazoles; Phosphoprotein Phosphatases; Receptors, Cytoplasmic and Nuclear; Strontium; Thapsigargin; Transfection; TRPC Cation Channels; Type C Phospholipases

Topics: Animals; Boron Compounds; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Signaling; Cell Line; Cell Membrane; Endoplasmic Reticulum; Humans; Inositol 1,4,5-Trisphosphate Receptors; Intracellular Membranes; Ion Channels; Macrocyclic Compounds; Oxazoles; Protein Conformation; Receptors, Cytoplasmic and Nuclear; TRPC Cation Channels