μM Thapsigargin Research Articles

Actions of endothelin-1 on calcium homeostasis in Madin—Darby canine kidney tubule cells

In both ischaemic and nephrotoxic models, renal failure is associated with increased endothelin-1 (ET-1) and cell calcium overload, and ET receptor antagonists are protective. Vascular and tubular actions of endothelins appear to be involved. This study examines the actions of ET-1 on intracellular Ca ([Ca2+]i) in the tubule model cell line MDCK (Madin-Darby canine kidney). Single-cell [Ca2+]i was measured using fura-2 and actions of ET-1 were compared with thapsigargin, which empties IP3-sensitive intracellular Ca stores. Mean resting [Ca2+]i was 84 nM (s.e.m. 6, n = 87). 1 microM thapsigargin and 100 nM ET-1 each caused a transient increase in [Ca2+]i by 696 nM (s.e.m. 160, n = 9) and 727 nM (s.e.m. 121, n = 5) respectively. After 1 microM thapsigargin, 100 nM ET-1 had no effect on [Ca2+]i. Oscillations in [Ca2+]i were frequently observed following 100 nM ET-1. In Ca(2+)-free extracellular solution, mean resting [Ca2+]i was reduced by 37 nM (s.e.m. 5, n = 11) and the mean transient increase in [Ca2+]i in response to ET-1 was 419 nM (s.e.m. 97, n = 5). Inhibition of the plasma membrane Ca-ATPase with La3+ halved the rate of [Ca2+]i removal from the cytoplasm following ET-1. The PKC inhibitor, chelerythrine (1 microM), reduced the ET-1 induced increase in [Ca2+]i to 349 nM (s.e.m. 97, n = 5) and also reduced the rate of removal of [Ca2+]i. Ligand binding studies demonstrated ETA receptor expression in MDCK cells sensitive to ET-1. ET-1 releases Ca2+ from IP3-sensitive stores in MDCK cells as well as stimulating extracellular Ca2+ entry leading to oscillations of [Ca2+]i. Ca2+ responses to ET-1 are potentiated by PKC; the plasma membrane Ca-ATPase contributes to removal of Ca2+ from the cytoplasm.

Evidence for the presence of two (Ca 2+-Mg 2+)ATPases with different sensitivities to thapsigargin and cyclopiazonic acid in the human flatworm Schistosoma mansoni

The subcellular localization of the (Ca 2+-Mg 2+)ATPase activities present in heterogeneous (P 1), nuclear (P 2), mitochondrial (P 3) and microsomal (P 4) fractions obtained by differential centrifugation of Schistosoma mansoni homogenate was investigated. In the microsomal fraction (P 4), the (Ca 2+-Mg 2+)ATPase activity was completely blocked by 3 μM thapsigargin, whereas in the more heterogeneous fraction (P 1), about 20–30% of this activity was resistant to the drug. The same pattern of inhibition was observed using 20 μM cyclopiazonic acid. The distribution pattern of (Ca 2+-Mg 2+)ATPase activity among the four subcellular fractions (P 1 > P 4 ⪢ P 3 > P 2) was completely different from that of [ 3H]-ouabain binding sites (P 1 ≥ P 4 = P 2 ≥ P 3). These results indicate that the (Ca 2+-Mg 2+)ATPase in S. mansoni is predominantly of the SERCA type (localized in the endoplasmic reticulum). However, there is another enzyme, present in lower proportion that could have a plasma membrane origin (PMCA type), because it is resistant to thapsigargin and cyclopiazonic acid and its inhibition by tamoxifen is antagonized by calmodulin.

Calcium store depletion potentiates a phosphodiesterase inhibitor- and dibutyryl cGMP-evoked calcium influx in rat pituitary GH 3 cells

A role for cGMP in the control of capacitative Ca 2+ influx was identified in rat pituitary GH 3 cells. Application of 50 μM-1 mM of the non-specific phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX), or the specific cGMP-phosphodiesterase inhibitor, zaprinast, induced a dose-dependent increase in the intracellular free Ca 2+ concentration [Ca 2+] i of the pituitary cell line, as assessed by video ratio imaging using fura-2. Response onset times were identical and response profiles were similar in all cells analysed. Application of 50 μM dibutyryl cGMP to GH 3 cells resulted in heterogeneous Ca 2+ responses, consisting of single or multiple transients with varying onset times. In all cases, increases in [Ca 2+] i were predominantly due to Ca 2+ influx, since no responses were detected in low Ca 2+ medium, or following pre-incubation of cells with 1 μM verapamil, or nicardipine. Depleting intracellular Ca 2+ stores by prior treatment of cells with 1 μM thapsigargin resulted in a dramatic potentiation in the Ca 2+ influx mediated by both phosphodiesterase inhibitors and dibutyryl cGMP, suggesting that cGMP modulates a dihydropyridine-sensitive Ca 2+ entry mechanism in GH 3 cells which is possibly regulated by the state of filling of Ca 2+ stores.

Mechanism of action of the non-steroidal anti-inflammatory drug flufenamate on [Ca 2+], and Ca 2+-activated currents in neurons

We have shown previously that the non-steroidal anti-inflammatory drug flufenamate (FFA) causes a maintained increase in [Ca 2+] i and transient increases in a Ca 2+-activated nonselective cation current (I CAN) and a Ca 2+-activated slow, outward Cl − current (I o-slow) in molluscan neurons [Shaw T, Lee R.J., Partridge L.D. Action of diphenylamine carboxylate derivatives, a family of non-steroidal anti-inflammatory drugs, on [Ca 2+] i and Ca 2+-activated channels in neurons. Neurosci Lett 1995; 190: 121–124]. Here we demonstrate that pretreatment of neurons with 10 μM thapsigargin eliminates the FFA-induced increase in [Ca 2+] i and substantially reduces both I CAN and I o-slow supporting the hypothesis that the FFA-induced increase in [Ca 2+] i results primarily from Ca 2+ release from a thapsigargin-sensitive intracellular store. The [Ca 2+] i response appears to be sustained, not by influx of extracellular Ca 2+, but by inhibitory effects of FFA on Ca 2+ removal from the cytosol. Inhibition of Ca 2+ efflux may be an important component of the FFA-induced activation of both I CAN and I o-slow, as Ca 2+ release by thapsigargin alone is not sufficient to activate either current. Our data also demonstrate that the effects of FFA on [Ca 2+] i I CAN and I o-slow are reversible and suggest that protein phosphorylation as well as an increase in [Ca 2+] i are involved in the FFA-induced activation of I o-slow Effects on neuronal Ca 2+ handling as well as activation of I CAN or I o-slow, may partially explain the analgesic effects of FFA.

Ca 2+-induced Ca 2+ release activates K + currents by a cyclic GMP-dependent mechanism in single gastric smooth muscle cells

The participation of sarcoplasmic reticulum Ca 2+ release channels in the activation of Ca 2+-sensitive K + currents ( I K(Ca)) by cyclic dibutyryl GMP was investigated in smooth muscle cells from the circular layer of guinea-pig gastric fundus. All experiments were performed in the presence of 3 μM nicardipine into the bath and low Ca 2+ buffering capacity of the pipette-filling solution (pCa 7.4). Ruthenium red (10 μM) as well as its combination with 10 μM heparin abolished the cyclic GMP-induced activation of I K(Ca), while 10 μM heparin remained ineffective. Ryanodine (10 μM) and the subsequently added 1 μM thapsigargin induced a relatively small increase in I K(Ca) amplitudes. The addition of 10 μM ryanodine to 1 μM thapsigargin-containing bath solution caused a vast increase in I K(Ca). It is hypothesyzed that protein kinase G-induced vectorial Ca 2+ flux from the cell bulk and sarcoplasmic reticulum Ca 2+ stores toward the plasma membrane is realized by a spontaneous Ca 2+-induced Ca 2+ release from a superficially situated Ca 2+ store.

Dominant Role of Mitochondria in Clearance of Large Ca 2+ Loads from Rat Adrenal Chromaffin Cells

Cytosolic Ca 2+ (Ca 2+ c) clearance from adrenal chromaffin cells was studied by whole-cell patch clamp and indo-1 Ca 2+ photometry after influx of Ca 2+ through voltage-dependent Ca 2+ channels. We isolated the rates of Ca 2+ c clearance by several mechanisms using combinations of the following agents (with their expected targets): Li + or TEA substituted for Na + (Na +–Ca 2+ exchange), 1 mM La 3+ applied after the depolarization (Na +–Ca 2+ exchange and plasma membrane Ca 2+-ATPase), 1 μM thapsigargin (pumping into reticular stores), and 2 μM carbonyl cyanide m-chlorophenylhydrazone (uptake into mitochondria). Remarkably, whenever [Ca 2+] c rose above ∼500 nM, Ca 2+ c clearance by mitochondria exceeded clearance by either Na +–Ca 2+ exchange or the Ca 2+ pumps of the plasma and reticular membranes. As [Ca 2+] c fell again, Ca 2+ reemerged from mitochondria, prolonging the final return to basal levels.

Nitric oxide induces intracellular Ca2+ mobilization and increases secretion of incorporated 5-hydroxytryptamine in rat pancreatic β-cells

This study is the first to demonstrate that low concentrations of aqueous NO induce intracellular Ca2+ mobilization and an increase in secretory activity of rat pancreatic β-cells. Application of NO solution (2 μM) resulted in a transient increase in the free intracellular Ca2+ concentration ([Ca2+]i) of isolated cells, as assessed by video ratio imaging and single wavelength microfluorimetry. Amperometry revealed a simultaneous increase in the release of preloaded 5-hydroxytryptamine from the isolated cells. The NO-induced Ca2+ response primarily involves mobilization of endoplasmic reticulum Ca2+ stores, since the response was retained when cells were transferred to low Ca2+ medium, and completely inhibited when cells were pretreated with 10 μM thapsigargin. The Ca2+ response was also inhibited when cells were incubated with a high concentration of ryanodine (200 μM), suggesting that Ca2+ mobilization is via a ryanodine-sensitive store.

Slow kinetics of InsP 3-induced Ca 2+release: differences between uni- and bi-directional 45Ca 2+ fluxes

The effects of a long-lasting stimulation with inositol 1,4,5-trisphosphate (InsP 3) have been studied in monolayers of permeabilized A7r5 cells. When measured under unidirectional 45Ca 2+ efflux conditions, i.e. in the presence of 2 μM thapsigargin, an initial fast release was observed which then progressively slowed down into a slow phase which persisted for up to 20 min. When measured under bidirectional 45Ca 2+ flux conditions with functional Ca 2+ pumps, a transient phase of re-uptake occurred between the initial fast and the subsequent slow release phase. These kinetics are compatible with intrinsic inactivation of the InsP 3 receptor. However, this inactivation did not prevent the slow release component. The slow component was not due to the accumulation of an InsP 3 metabolite nor to a GTP-dependent translocation of Ca 2+ between stores. The slow release phase was more pronounced when the Ca 2+ pumps were active than when they were inhibited. This observation is compatible with other findings indicating that the InsP 3 receptor is controlled by luminal Ca 2+. The decreasing effectiveness of a 20 min lasting InsP 3 challenge in mobilizing Ca 2+ from less filled stores is most likely due to a progressive depletion of the store and cannot be considered as an experimental artifact caused by a preferential emptying of InsP 3-sensitive Ca 2+ stores. We conclude that the InsP 3 receptor can intrinsically inactivate but that this inactivation is unable to prevent the slow release, which is especially pronounced when Ca 2+ pumps are active.

Isradipine affects histamine-induced cytosolic Ca 2+ movements in human endothelial cells

Although endothelial actions of dihydropyridines remain controversial, isradipine has been observed to exert anti-atherosclerotic actions in which endothelium could be involved. This study was designed to investigate the direct effects of isradipine on cytosolic Ca 2+ concentration in cultured human umbilical vein endothelial cells. Isradipine (from 10 nM to 1 μM) had no effect on unstimulated cells but dose-dependently decreased both the transient [Ca 2+] i peak and the sustained increase induced by histamine. Its maximal effects were reached at 0.1 μM. In the absence of Ca 2+ influx or in depolarized cells, 1 μM isradipine still significantly decreased the transient [Ca 2+]i peak (by 23 ± 8% and 42 ± 11%). Casu2+ influx induced by re-establishment of transmembrane Ca 2+ gradient was also inhibited by isradipine, as was that induced by 1 μM thapsigargin. These results demonstrate that isradipine is able to reduce both Ca 2+ release from internal stores and the consequent Ca 2+ entry in stimulated human endothelial cells.

Differential release of histamine and prostaglandin D 2 in rat peritoneal mast cells: roles of cytosolic calcium and protein tyrosine kinases

We studied how the release of histamine and prostaglandin D 2 (PGD 2) were connected in stimulated rat peritoneal mast cells, and to what extent these processes were controlled by the cytosolic Ca 2+ concentration, [Ca 2+] i, and protein tyrosine kinases. In the presence of 1 mM CaCl 2, the G-protein activating compound 48/80 (10 μg/ml) evoked a transient rise in [Ca 2+] i and a relatively high secretion of histamine, but only a low release of PGD 2. In contrast, 5 μM thapsigargin (an inhibitor of endomembrane Ca 2+-ATPases) and 5 μM ionomycin evoked high and prolonged rises in [Ca 2+] i, and stimulated the cells to release relatively small amounts of histamine and high amounts of PGD 2. Stimulation of the cells with CaCl 2 and 10 μM ATP 4− gave only minor quantities of histamine and PGD 2, despite of the micromolar level of [Ca +] i reached. When CaCl 2 was replaced by EGTA, rises in [Ca 2+] i as well as release of histamine and PGD 2 were reduced with each agonist, but the preference of agonists to release more histamine or PGD 2 remained unchanged. In mast cells with depleted Ca 2+ stores, the addition of CaCl 2 stimulated the store-regulated Ca 2+ entry resulting in a prolonged rise in [Ca 2+] i. However, simultaneous addition of compound 48/80 and CaCl 2 was required for release of histamine and PGD 2. In cells with full stores, PGD 2 release evoked by compound 48/80 was greatly reduced by genistein and methyl-2,5-dihydroxycinnamate, two structurally unrelated inhibitors of protein tyrosine kinases, whereas histamine secretion was not influenced by these inhibitors. Similarly, with thapsigargin or ionomycin as agonist, PGD 2 release was more sensitive to the tyrosine kinase inhibitors than histamine secretion. We conclude that in activated rat peritoneal mast cells: (i) the influx of extracellular Ca 2+ potentiates agonist-evoked rises in [Ca 2+] i as well as histamine secretion and PGD 2 release; (ii) the amplitude of the [Ca 2+] i rise does not determine the preferential effect of agonists to release more histamine or more PGD 2; (iii) the relatively high PGD 2 release evoked by thapsigargin and ionomycin is probably due to their potency to evoke a prolonged rise in [Ca 2+] i and to activate protein tyrosine kinases.

The influence of thapsigargin on Na,K-ATPase activity in cultured nonpigmented ciliary epithelial cells.

Experiments were conducted to test the influence of thapsigargin on the NaK-ATPase activity of cultured cells (ODM2) derived from human nonpigmented ciliary epithelium. The rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was diminished in cells that had been pretreated with thapsigargin then permeabilized. Following 20 min exposure of intact cells to thapsigargin, the cells were permeabilized with digitonin and the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was measured immediately in a calcium-free buffer. In permeabilized cells that had been pretreated with 1 microM thapsigargin for 20 min, the rate of ouabain-sensitive ATP hydrolysis (Na,K-ATPase activity) was reduced by 38%. Pretreatment with lesser concentrations of thapsigargin caused smaller changes of Na,K-ATPase activity. The decrease of Na,K-ATPase activity was the same whether or not calmodulin antagonists W7 or trifluoperazine were present during the thapsigargin pretreatment period. This inhibitory effect upon the Na,K-ATPase may serve to limit the extent of sodium pump activation that takes place in intact cells when thapsigargin causes sodium pump stimulation by a mechanism that appears to involve changes in cytoplasmic ion levels when potassium channels open.

Inhibition of capacitative Ca 2+ entry by a Cl − channel blocker in human endothelial cells

We have used the patch clamp technique in combination with intracellular calcium measurements to measure simultaneously Ca 2+ entry and ionic currents activated by emptying of intracellular Ca 2+ stores (capacitative Ca 2+ entry and Ca 2+ release-activated Ca 2+ currents, CRAC) in human endothelial cells from umbilical veins. Intracellular stores were depleted of Ca 2+ by preincubating endothelial cells for 20 minutes with 2 μM thapsigargin in Ca 2+-free solution. Reapplication of 10 mM [Ca 2+] e evoked an increase in [Ca 2+] i indicating Ca 2+ influx after the thapsigargin-induced store depletion (capacitative Ca 2+ entry), however no measurable CRAC could be detected. The increase in [Ca 2+] i after [Ca 2+] e resubmission was substantially reduced in the presence of 50 μM NPPB (5-nitro-2-(3-phenylpropylamino)-benzoic acid) from 0.77 ± 0.25 μM to 0.2 ± 0.06 μM (n = 6) at a holding potential of −40 mV. Estimates of the capacitative Ca 2+ entry at various membrane potentials from the first time derivative of the Ca 2+ transients showed a highly inwardly rectifying I–V curve with a Ca 2+ inward current amplitude of 1.0 ± 0.3 pA (membrane capacitance 59 ± 9 pF, n = 8) at −80 mV. This current amplitude was decreased to 0.32 ± 0.12 pA( n = 6) in the presence of 50 μM NPPB. This corresponds to a decrease in the Ca 2+ permeability of the endothelial cell membrane from 0.15·10 −8 cm/s (control) to 0.06·10 −8 cm/s (50 μM NPPB).

Ca 2+ entry pathways activated by the tumor promoter thapsigargin in human platelets

Thapsigargin-activated Ca 2+ entry into platelets was examined in the presence of S-145, a thromboxane A 2 receptor antagonist, to inhibit indirect effects by endogenously formed prostaglandin H 2/thromboxane A 2. With external Ca 2+ present, 0.2 μM thapsigargin caused a prompt increase in intracellular Ca 2+ concentration ([Ca 2+] 1) followed by a gradual increase. Pretreatment with 6 μM wortmannin, a specific inhibitor of myosin light chain kinase, partly inhibited the increase in [Ca 2+] i. In Ca 2+-free EGTA buffer, thapsigargin induced a smaller increase in [Ca 2+] i, and subsequent addition of Ca 2+ to the buffer caused a further prompt increase in [Ca 2+] i, demonstrating external Ca 2+ entry. Wortmannin only partly inhibited this entry of external Ca 2+. The wortmannin-insensitive Ca 2+ entry pathway remained open for more than 6 min in Ca 2+-free buffer. On the other hand, when receptor agonists such as thrombin and U46619 were substituted for thapsigargin, activation of the wortmannin-insensitive Ca 2+ entry was transient (Hashimoto et al., J. Biol. Chem (1992) 267, 17078–17081). In the presence of S-145 and wortmannin, thapsigargin stimulated phosphorylation of neither the 20-kDa myosin light chain nor the 47-kDa protein, a substrate of protein kinase C. These results suggest that thapsigargin induces external Ca 2+ entry by two mechnisms: (1) a mechanism involving myosin light chain kinase; (2) a mechanism, not activated by receptor agonists, that is independent of the major protein kinases of platelets.

Muscarinic m3 receptors and dynamics of intracellular Ca 2+ cerebellar granule neurons

Activation of muscarinic receptors with carbachol has no effect on intracellular Ca 2+ concentration in cerebellar granule cell cultures. Only after elevating intracellular Ca 2+ concentrations using either 40 mM KCl or activating glutamatergic receptors was carbachol able to increase intracellular Ca 2+. The response lasted about 10 s, and the median increase in intracellular Ca 2+ with either 100 μM or 300 μM carbachol was about 85 nM. Carbachol at 30 μM elicited an increase in intracellular calcium that was half maximal. After a 16 min or 32 min delay following cell depolarization, responses to carbachol were only found in about 20% of the cells studied and the median increase in intracellular Ca 2+ was about 14 nM. (−)-Quinuclidinylxanthene-9-carboxylate hemioxalate (QNX) at 10 nM (a muscarinic m3 antagonist), but not methoctramine at 5 μM (a muscarinic m2 antagonist), attenuated the action of 100 μM carbachol. This carbachol-mediated response was elicited in the absence of extracellular Ca 2+ and in the presence of 10 μM dantrolene, but was blocked by 1 μM thapsigargin. Lastly, treatments of cerebellar granule cell cultures with carbachol (100 μM) for up to 12 h results in a desensitization of the carbachol-mediated release of stored Ca 2+. Thus, carbachol acts on muscarinic m3 receptors to induce the release of Ca 2+ from IP 3-sensitive Ca 2+ stores that must be filled by a prior period of high intracellular Ca 2+.

Thapsigargin blocks the mobilisation of intracellular calcium caused by activation of human NK1 (long) receptors expressed in chinese hamster ovary cells

Neurokinin receptors can couple to several second messenger systems including phospholipase C and intracellular calcium mobilisation. Using FURA-2 microspectrofluorimetry, this study examines the mobilisation of calcium in CHO cells which had been stably transfected with the long isoform of the human NK1 receptor. Substance P caused a concentration-dependent rise in inositol phosphate production which was correlated with a reversible increase in intracellular calcium levels. The mobilisation of calcium was reduced by the removal of extracellular calcium from the bathing solution, and almost totally abolished by depletion of intracellular calcium pools with 1 μM thapsigargin. These data suggest that the transduction mechanism associated with NK1 (long) receptor activation can utilise both intracellular and extracellular calcium pools in this expression system.