thapsigargin and 2-5-di-tert-butylbenzoquinone

2,5-Di-(tert-butyl)-1,4-benzohydroquinone (TBQ) is a reversible inhibitor of SERCA, potentially making it a useful tool to study the effects of SERCA inhibition in cardiac cells. However, it is unknown if TBQ also has effects on other components of ventricular Ca handling. The aim of these experiments was to characterise the effects of TBQ on Ca handling in rat ventricular myocytes and assess its suitability as a specific inhibitor of SERCA. This was achieved by voltage clamp via perforated patch and [Ca(2+)]i measurement using Fluo-3 AM. TBQ produced a fully reversible, concentration dependent decrease in the rate of systolic Ca decay. 10μM TBQ decreased the amplitude of the systolic Ca transient by 48±5% and the rate of decay by 54±6%. SR Ca content was also reduced by 62±4%. However, 10μM TBQ also decreased the peak L-type Ca current by 23±7%. At higher concentrations (100μM), TBQ also activated an outward current with a current-voltage relationship consistent with a potassium current. This outward current was abolished by Glibenclamide (100μM). These data show that TBQ can be used to reversibly inhibit SERCA. However, at concentrations that decrease SERCA activity, TBQ also decreases the L-type Ca current and (at higher concentrations) activates an outward current which appears to be an ATP dependent potassium current. We conclude that TBQ cannot be used as a specific inhibitor of SERCA in rat ventricular myocytes.

Topics: Animals; Benzoquinones; Calcium; Calcium Channels, L-Type; Cells, Cultured; Heart Ventricles; Male; Membrane Potentials; Myocytes, Cardiac; Patch-Clamp Techniques; Rats; Rats, Wistar; Sarcoplasmic Reticulum; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Thapsigargin

Mutational analysis of trans-membrane helices M3, M4, M5 and M7 of the Ca2+-ATPase revealed a novel phenotypic variant, M4 [Y295A (the one-letter symbols are used for amino acid residues throughout)], displaying an increased affinity for Pi and decreased affinity for MgATP, while retaining the ability to translocate Ca2+ ions across the endoplasmic reticulum membrane. The properties of this mutant suggest that the E1-E2 equilibrium is shifted towards E2, and indicate a key role for this aromatic residue (Y295) at the end of trans-membrane helix M4. A mutant containing three amino acid residue substitutions at the end of the seventh trans-membrane helix, M7 (F834A, F835A, T837F), showed a complete loss of ATPase activity and a reduced ability to phosphorylate with Pi, although MgATP-initiated phosphorylation was unaffected. The observation that single mutations in this cluster of residues had no effect on Ca2+ transport suggests that correct anchoring of the helix at the lipid-water interface by these aromatic residues is important in the functioning of the ATPase. Mutation of polar residues in helix M3 did not affect inhibition of the ATPase by thapsigargin, thapsivillosin A or t-butyl hydroquinone, suggesting that hydrogen-bonding partners for the essential -OH groups on these inhibitors lie elsewhere in the ATPase.

Topics: Adenosine Triphosphatases; Animals; Benzoquinones; Calcium-Transporting ATPases; COS Cells; DNA Mutational Analysis; Enzyme Inhibitors; Genes, Synthetic; Phosphorylation; Protein Conformation; Protein Folding; Protein Structure, Secondary; Thapsigargin

The human lung small cell adenocarcinoma cell line, A549, demonstrates a concentration-dependent rise in [Ca2+]i in response to extracellular nucleotides. The cells show Ca2+ mobilization on addition of various nucleotides, with an order of agonist potency: UTP > or = ATP > ADP > ADP beta S > AMP; adenosine is ineffective. The EC50 values for UTP and ATP are 12.5 +/- 0.4 microM and 18.9 +/- 0.5 microM, respectively. Together, these results are strongly indicative of the P2U subclass being the major nucleotide receptor expressed in these cells. The Ca2+ response was typically biphasic consisting of an initial spike, representing release of Ca2+ from internal stores, and a subsequent plateau representing Ca2+ influx. The majority of cells showed an agonist-induced Ca2+ increase that was unaffected by pretreatment with the Ca(2+)-ATPase inhibitors 2,5-di(tert-butyl)1,4-benzohydroquinone or thapsigargin. Caffeine did not raise [Ca2+]i above basal levels and applied in conjunction with nucleotide did not attenuate the agonist-mediated response. The Ca2+ influx was sensitive to protein kinase C, and agonist addition in the presence of a protein kinase C inhibitor, D-erythrosphingosine, produced a significantly potentiated Ca2+ influx. Furthermore, agonist-mediated Ca2+ influx was abolished in the presence of a protein kinase C activator, phorbol 12,13-dibutyrate. It is concluded that these cells posses a functional P2U receptor that, upon activation, causes Ca2+ mobilization from TBQ and thapsigargin insensitive stores followed by protein kinase C regulated Ca2+ influx.

Topics: Adenocarcinoma; Adenosine Diphosphate; Adenosine Monophosphate; Adenosine Triphosphate; Benzoquinones; Caffeine; Calcium; Down-Regulation; Enzyme Inhibitors; Humans; Lung Neoplasms; Phosphodiesterase Inhibitors; Protein Kinase C; Purinergic P2 Receptor Agonists; Receptors, Purinergic P2; Thapsigargin; Thionucleotides; Tumor Cells, Cultured; Uridine Triphosphate

Depletion of intracellular calcium stores induces transmembrane Ca2+ influx. We studied Ca(2+)- and Ba(2+)-permeable ion channels in A431 cells after store depletion by dialysis of the cytosol with 10 mM BAPTA solution. Cell-attached patches of cells held at low (0.5 microM) external Ca2+ exhibited transient channel activity, lasting for 1-2 min. The channel had a slope conductance of 2 pS with 200 mM CaCl2 and 16 pS with 160 mM BaCl2 in the pipette. Channel activity quickly ran down in excised inside-out patches and was not restored by InsP3 and/or InsP4. Thapsigargin induced activation in cells kept in 1 mM external Ca2+ after BAPTA dialysis. These channels represent one Ca2+ entry pathway activated by depletion of internal calcium stores and are clearly distinct from previously identified calcium repletion currents.

Topics: Barium Compounds; Benzoquinones; Calcium; Calcium Channels; Calcium Chloride; Calcium-Transporting ATPases; Carcinoma, Squamous Cell; Cell Adhesion; Cell Line; Chlorides; Egtazic Acid; Electric Conductivity; Humans; Inositol Phosphates; Ion Channel Gating; Kinetics; Magnesium Chloride; Membrane Potentials; Terpenes; Thapsigargin; Time Factors; Tumor Cells, Cultured

In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL/IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL/IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.

Topics: Antibodies, Monoclonal; Benzoquinones; Biological Transport; Blood Platelets; Calcium; Calcium-Transporting ATPases; Cell Compartmentation; Endoplasmic Reticulum; Humans; Indoles; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Isoenzymes; Signal Transduction; Terpenes; Thapsigargin

Specific inhibitors of the endoplasmic-reticulum Ca2+ pump will deplete intracellular stores and are therefore useful to study the role of store depletion on plasma-membrane Ca2+ permeability. We now report that the Ca(2+)-pump inhibitor 2,5-di-(tert-butyl)-1,4-benzohydroquinone (tBuBHQ) reduces the passive Ca2+ leak from the internal stores in permeabilized A7r5 vascular smooth-muscle cells. This aspecific effect occurred at concentrations that are normally used to empty the stores in intact cells. Cyclopiazonic acid exerted a similar, although less pronounced effect, while thapsigargin did not affect the passive Ca2+ leak. The inositol 1,4,5-trisphosphate-mediated Ca2+ release was not affected. tBuBHQ and cyclopiazonic acid cannot therefore be used as specific tools to probe the mechanism of receptor-mediated Ca2+ entry.

Topics: Benzoquinones; Calcium; Calcium-Transporting ATPases; Carcinogens; Cell Membrane Permeability; Cells, Cultured; Endoplasmic Reticulum; Indoles; Muscle, Smooth, Vascular; Terpenes; Thapsigargin

Continuous superfusion of rat glioma cells with medium containing bradykinin (from 0.2 nM) induced a transient hyperpolarization followed by regular hyperpolarizing oscillations of the membrane potential. Similar repetitive hyperpolarizing oscillations were caused by extracellularly applied bradykinin or muscarine or by intracellularly injected GTP-gamma-S. The frequency of the oscillations was 1 per minute at bradykinin concentrations ranging from 0.2 nM to 2 microM, but the amplitude and duration increased with rising peptide concentration. The muscarine-induced oscillations were blocked by atropine. In the presence of extracellular Ca2+, the substances thapsigargin, 2,5-di(tert-butyl)-1,4-benzohydroquinone (tBuBHQ), and ionomycin reversibly suppressed the bradykinin-induced oscillations. Thapsigargin and tBuBHA, which are known to block the Ca2+ ATPase of endoplasmic reticulum, caused a transient rise in cytosolic Ca2+ activity, monitored with Fura-2, in suspensions of rat glioma cells or of mouse neuroblastoma-rat glioma hybrid cells. After a transient Ca2+ rise caused by thapsigargin, tBuBHQ, or ionomycin, the Ca2+ response to bradykinin which is known to be due to release of Ca2+ from internal stores was suppressed. This indicates that thapsigargin and tBuBHQ deplete internal Ca2+ stores as already seen previously for ionomycin. Thus, the inhibition of the membrane potential oscillations by thapsigargin, tBuBHQ, and ionomycin indicates that the oscillations are associated with activation of InsP3-sensitive Ca2+ stores. In some cells composite oscillation patterns which consisted of two independent oscillations with different amplitudes that overlapped additively were seen. We discuss that this pattern and the concentration dependency of the oscillations could be due to "quantal" Ca2+ release from stores with different inositol 1,4,5-triphosphate sensitivities. Subsidence of the oscillations after omission of extracellular Ca2+ seems to be due to a lack of replenishment of the intracellular stores with Ca2+, which comes from the extracellular compartment.

Topics: Animals; Benzoquinones; Bradykinin; Calcium; Cytosol; Glioma; Hybrid Cells; Inositol 1,4,5-Trisphosphate; Membrane Potentials; Mice; Muscarine; Neuroblastoma; Rats; Terpenes; Thapsigargin; Tumor Cells, Cultured

We have investigated the role of the intracellular Ca2+ pool in regulating Ca2+ entry into vascular endothelial cells. The intracellular Ca2+ pool was mobilized using either thapsigargin (TG) or 2',5'-di(tert-butyl)-1,4-benzohydroquinone (BHQ), inhibitors of the endoplasmic reticulum Ca(2+)-adenosinetriphosphatase (ATPase). Mobilization of intracellular Ca2+ stores with either inhibitor depleted intracellular Ca2+ and greatly reduced subsequent mobilization of the inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ pool by bradykinin. However, bradykinin-induced mobilization of the IP3-sensitive intracellular Ca2+ pool only partially reduced the subsequent response of cells to TG and BHQ. Mobilization of the intracellular Ca2+ pool by either TG or BHQ led to a concentration-dependent elevation of cytosolic Ca2+ concentrations ([Ca2+]i) without initiating inositol polyphosphate formation. In contrast to the rapidly developing, transient rise in Ca2+ concentration initiated by bradykinin, maximal concentrations of TG and BHQ stimulated a slowly developing, prolonged elevation of [Ca2+]i that required extracellular Ca2+ and could be blocked by extracellular Ni2+. Extracellular Ca2+ entered the cell through an activated cation entry pathway, since bradykinin, TG, and BHQ stimulated Mn2+ and 45Ca2+ entry. Bradykinin-stimulated 45Ca2+ uptake reached a peak within 2 min, whereas 45Ca2+ influx initiated by TG or BHQ continued for at least 8 min. Importantly, the [Ca2+]i response after low concentrations of BHQ was more transient than that seen after TG. The return of [Ca2+]i to basal values after low concentrations of BHQ was associated with reversal of Ca(2+)-ATPase inhibition and refilling of the IP3-sensitive Ca2+ pool. The continued elevation of [Ca2+]i and prolonged Ca2+ entry seen with TG was associated with continued Ca(2+)-ATPase inhibition and an empty IP3-sensitive Ca2+ pool. We conclude that mobilization of intracellular Ca2+ stores induces Ca2+ entry in endothelial cells which continues until the intracellular Ca2+ pool is refilled.

Topics: Animals; Benzoquinones; Bradykinin; Calcium; Calcium-Transporting ATPases; Endothelium, Vascular; Extracellular Space; Intracellular Membranes; Osmolar Concentration; Terpenes; Thapsigargin

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.

Topics: Animals; Benzoquinones; Calcium; Calcium-Transporting ATPases; Carbachol; Cell Membrane Permeability; Fura-2; In Vitro Techniques; Indoles; Inositol 1,4,5-Trisphosphate; Kinetics; Male; Microsomes; Parotid Gland; Rats; Rats, Inbred Strains; Terpenes; Thapsigargin