calyculin-a and 2-aminoethoxydiphenyl-borate

Using a combination of fluorescence measurements of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) and membrane potential we have investigated the sensitivity to serine/threonine phosphatase inhibition of Ca(2+) entry stimulated by activation of the Ca(2+) release-activated Ca(2+) (CRAC) entry pathway in rat basophilic leukemia cells. In both suspension and adherent cells, addition of the type 1/2A phosphatase inhibitor calyculin A, during activation of CRAC uptake, resulted in a fall in [Ca(2+)](i) to near preactivation levels. Pre-treatment with calyculin A abolished the component of the Ca(2+) rise associated with activation of CRAC uptake and inhibited Mn(2+) entry, consistent with a requirement of phosphatase activity for activation of the pathway. Depletion of intracellular Ca(2+) stores is accompanied by a large depolarisation which is absolutely dependent upon Ca(2+) entry via the CRAC uptake pathway. Application of calyculin A or okadaic acid, a structurally unrelated phosphatase antagonist inhibits this depolarisation. Taken in concert, these data demonstrate a marked sensitivity of the CRAC entry pathway to inhibition by calyculin A and okadaic acid.

Topics: Animals; Boron Compounds; Calcium; Calcium Signaling; Cations, Divalent; Cells, Cultured; Enzyme Inhibitors; Ion Transport; Leukemia, Basophilic, Acute; Manganese; Marine Toxins; Membrane Potentials; Okadaic Acid; Oxazoles; Phosphoprotein Phosphatases; Protein Phosphatase 1; Rats; Thapsigargin

We showed that 5-amino-3-(3,4-dichlorophenyl)1,2,3,4-oxatriazolium (GEA3162), a lipophilic nitric oxide (NO)-releasing agent, induced Ca(2+) entry into rat neutrophils in a concentration-dependent manner, whereas the guanylyl cyclase inhibitors, 6-anilino-5,8-quinolinequinone (LY83583) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), had no effect on GEA3162-induced response. The GEA3162-induced Ca(2+) entry was not observed in a Ca(2+)-free medium. GEA3162 did not potentiate but reduced the store-emptying activated Ca(2+) entry caused by cyclopiazonic acid. Stimulation of cells with GEA3162 in the absence of extracellular Ca(2+) followed by addition of cations showed that only Ca(2+) but not Ba(2+) and Sr(2+) entry occurs. Store-operated Ca(2+) entry was sensitive to La(3+) and Ni(2+) inhibition, whereas the GEA3162-induced Ca(2+) entry was sensitive to La(3+) but resistant to Ni(2+). cis-N-(2-Phenylcyclopentyl)azacyclotridec-1-en-2-amine (MDL-12,330A) and calyculin A diminished the Ca(2+) entry activated by cyclopiazonic acid as well as by GEA3162. In contrast, 2-aminoethyldiphenyl borate (2-APB) diminished cyclopiazonic acid-but enhanced GEA3162-induced [Ca(2+)](i) change. Genistein effectively attenuated the cyclopiazonic acid-but slightly inhibited GEA3162-induced [Ca(2+)](i) change. Application of neomycin and high extracellular Ca(2+) concentration did not induce [Ca(2+)](i) rise. These data suggest that GEA3162 induced Ca(2+) entry and regulated Ca(2+) signal, through direct protein thiol oxidation. The action of GEA3162 demonstrates characteristics that distinguish it from the store-operated mechanism in neutrophils and therefore is likely to represent an entirely distinct pathway. Extracellular Ca(2+)-sensing receptor is not existing in neutrophils.

Topics: Adenylyl Cyclase Inhibitors; Animals; Barium; Boron Compounds; Calcium; Calcium Signaling; Calcium-Transporting ATPases; Cell Membrane Permeability; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genistein; Imines; Indoles; Manganese; Marine Toxins; Neutrophils; Nitric Oxide Donors; Oxazoles; Phosphoprotein Phosphatases; Rats; Rats, Sprague-Dawley; S-Nitrosothiols; Strontium; Triazoles

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca(2+) signals and replenishment of Ca(2+) stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (InsP(3)R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca(2+) entry between 1-10 microm. 2-APB activated authentic SOCs because the entry was totally selective for Ca(2+) (no detectable entry of Ba(2+) or Sr(2+) ions), and highly sensitive to La(3+) ions (IC(50) 30-100 nm). To assess the role of InsP(3)Rs in this response, we used the DT40 triple InsP(3)R-knockout (ko) cell line, DT40InsP(3)R-ko, in which the absence of full-length InsP(3)Rs or InsP(3)R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP(3)R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP(3)R-ko, cells indicating InsP(3)Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca(2+) entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca(2+) release from stores in intact DT40wt cells but not in DT40InsP(3)R-ko cells, indicating an InsP(3)R-mediated effect. In contrast, 2-APB blocked InsP(3)Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP(3)Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP(3)R activation but did not alter InsP(3)R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP(3)Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca(2+) entry.

Topics: Animals; B-Lymphocytes; Blotting, Western; Boron Compounds; Calcium; Cell Line; Chickens; CHO Cells; Cricetinae; Cytoskeleton; Enzyme Inhibitors; Inositol 1,4,5-Trisphosphate; Marine Toxins; Muscle, Smooth; Oxazoles; Protein Binding; Protein Structure, Tertiary; Time Factors; Type C Phospholipases

Notwithstanding extensive efforts, the mechanism of capacitative calcium entry (CCE) remains unclear. Two seemingly opposed theories have been proposed: secretion-like coupling (Patterson, R. L., van Rossum, D. B., and Gill, D. L. (1999) Cell 98, 487-499) and the calcium influx factor (CIF) (Randriamampita, C., and Tsien, R. Y. (1993) Nature 364, 809-814). In the current study, a combinatorial approach was taken to investigate the mechanism of CCE in corneal endothelial cells. Induction of cytochrome P-450s by beta-naphthoflavone (BN) enhanced CCE measured by Sr(2+) entry after store depletion. 5,6-Epoxyeicosatrienoic acid (5,6-EET), a proposed CIF generated by cytochrome P-450s (Rzigalinski, B. A., Willoughby, K. A., Hoffman, S. W., Falck, J. R., and Ellis, E. F. (1999) J. Biol. Chem. 274, 175-182), induced Ca(2+) entry. Both BN-enhanced CCE and the 5,6-EET-induced Ca(2+) entry were inhibited by the CCE blocker 2-aminoethoxydiphenyl borate, indicating a role for cytochrome P-450s in CCE. Treatment with calyculin A (CalyA), which causes condensation of cortical cytoskeleton, inhibited CCE. The actin polymerization inhibitor cytochalasin D partially reversed the inhibition of CCE by CalyA, suggesting a secretion-like coupling mechanism for CCE. However, CalyA could not inhibit CCE in BN-treated cells, and 5,6-EET caused a partial activation of CCE in CalyA-treated cells. These results further support the notion that cytochrome P-450 metabolites may be CIFs. The vesicular transport inhibitor brefeldin A inhibited CCE in both vehicle- and BN-treated cells. Surprisingly, Sr(2+) entry in the absence of store depletion was enhanced in BN-treated cells, which was also inhibited by 2-aminoethoxydiphenyl borate. An integrative model suggests that both CIF from cytochrome P-450 metabolism and secretion-like coupling mechanisms play roles in CCE in corneal endothelial cells.

Topics: 8,11,14-Eicosatrienoic Acid; Adenosine Triphosphate; Animals; beta-Naphthoflavone; Boron Compounds; Brefeldin A; Calcium; Cattle; Cornea; Cytochalasin D; Cytochrome P-450 Enzyme System; Cytoskeleton; Endothelium, Corneal; Enzyme Inhibitors; Marine Toxins; Models, Biological; Oxazoles; Protein Conformation; Strontium; Time Factors

One popular model for the activation of store-operated Ca2+ influx is the secretion-like coupling mechanism, in which peripheral endoplasmic reticulum moves to the plasma membrane upon store depletion thereby enabling inositol 1,4,5-trisphosphate (InsP3) receptors on the stores to bind to, and thus activate, store-operated Ca2+ channels. This movement is regulated by the underlying cytoskeleton. We have examined the validity of this mechanism for the activation of I(CRAC), the most widely distributed and best characterised store-operated Ca2+ current, in a model system, the RBL-1 rat basophilic cell line. Stabilisation of the peripheral cytoskeleton, disassembly of actin microfilaments and disaggregation of microtubules all consistently failed to alter the rate or extent of activation of I(CRAC). Rhodamine-phalloidin labelling was used wherever possible, and revealed that the cytoskeleton had been significantly modified by drug treatment. Interference with the cytoskeleton also failed to affect the intracellular calcium signal that occurred when external calcium was re-admitted to cells in which the calcium stores had been previously depleted by exposure to thapsigargin/ionomycin in calcium-free external solution. Application of positive pressure through the patch pipette separated the plasma membrane from underlying structures (cell ballooning). However, I(CRAC) was unaffected irrespective of whether cell ballooning occurred before or after depletion of stores. Pre-treatment with the membrane-permeable InsP3 receptor antagonist 2-APB blocked the activation of I(CRAC). However, intracellular dialysis with 2-APB failed to prevent I(CRAC) from activating, even at higher concentrations than those used extracellularly to achieve full block. Local application of 2-APB, once I(CRAC) had been activated, resulted in a rapid loss of the current at a rate similar to that seen with the rapid channel blocker La3+. Studies with the more conventional InsP3 receptor antagonist heparin revealed that occupation of the intracellular InsP3-sensitive receptors was not necessary for the activation or maintenance of I(CRAC). Similarly, the InsP3 receptor inhibitor caffeine failed to alter the rate or extent of activation of I(CRAC). Exposure to Li+, which reduces InsP3 levels by interfering with inositol monophosphatase, also failed to alter I(CRAC). Caffeine and Li+ did not affect the size of the intracellular Ca2+ signal that arose when external Ca2+ was re-admitted to cells

Topics: Animals; Basophils; Boron Compounds; Caffeine; Calcium; Calcium Channels; Calcium Signaling; Cell Line; Cell Size; Cytochalasin D; Cytoskeleton; Depsipeptides; Enzyme Inhibitors; Heparin; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lithium; Marine Toxins; Microscopy, Fluorescence; Nocodazole; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Rats; Receptors, Cytoplasmic and Nuclear; Thapsigargin; Time Factors

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