Thapsigargin causes Ca2+ release and collapse of the membrane potential of Trypanosoma brucei mitochondria in situ and of isolated rat liver mitochondria

Thapsigargin, previously reported to release Ca2+ from non-mitochondrial stores of different cell types, as well as nigericin, were found, when used at high concentrations, to release Ca2+ and collapse the membrane potential of Trypanosoma brucei bloodstream and procyclic trypomastigotes mitochondria in situ. At similarly high concentrations (> 10 microM), thapsigargin was also found to release Ca2+ and collapse the membrane potential of isolated rat liver mitochondria. These results indicate that care should be taken when attributing the effects of thapsigargin in intact cells to the specific inhibition of the sarcoplasmic and endoplasmic reticulum Ca(2+)-ATPase family of calcium pumps. In addition, we have found no evidence for an increase in intracellular Ca2+ by release of the ion from intracellular stores by nigericin, measuring changes in cytosolic Ca2+ by dual wavelength spectrofluorometry in fura-2-loaded T. brucei bloodstream trypomastigotes or measuring Ca2+ transport in digitonin-permeabilized cells.     

Characterization of Ca2+ transport activity associated with a non-mitochondrial calcium pool in the rodent malaria parasite P. chabaudi

Non-mitochondrial calcium deposits were investigated in the intraerythrocytic malaria parasite Plasmodium chabaudi at the trophozoite stage by means of arsenazo III in the presence of ATP and the mitochondrial poisons, antimycin and oligomycin. Addition of vanadate and 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ), both known to interact with SERCA pump, induced calcium release by permeabilized parasites when the medium free calcium concentration was kept at 3.5 microM. The tumor promoter thapsigargin also caused elevation of the free calcium concentration in permeabilized parasites. Our results support the view that P. chabaudi sequesters calcium in an exchangeable form and maintains its calcium homeostasis by way of an endoplasmic reticulum Ca2+ pump.     

Direct effects of diazoxide on mitochondria in pancreatic B-cells and on isolated liver mitochondria

1. The direct effects of diazoxide on mitochondrial membrane potential, Ca2+ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B-cells and rat liver mitochondria. 2. Diazoxide, at concentrations commonly used to open adenosine 5'-triphosphate (ATP)-dependent K+-channels (K(ATP) channels) in pancreatic B-cells (100 to 1000 microM), decreased mitochondrial membrane potential in mouse intact perifused B-cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non-stimulating (5 mM) and stimulating (20 mM) glucose concentrations. 3. A decrease of mitochondrial membrane potential in perifused B-cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 microM each). 4. Measurements by a tetraphenylphosphonium-sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 microM) did not antagonize the effects of diazoxide. 5. In Fura 2-loaded B-cells perifused with the Ca2+ channel blocker, D 600, a moderate, reversible increase of intracellular Ca2+ concentration could be seen in response to 500 microM diazoxide. This intracellular Ca2+ mobilization may be due to mitochondrial Ca2+ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca2+ stored in the mitochondria. 6. In the presence of 500 microM diazoxide, ATP content of pancreatic islets incubated in 20 mM glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 mM K+ in the presence of 10 mM glucose was not inhibited by 500 microM diazoxide, suggesting that the energy-dependent processes of insulin secretion distal to Ca2+ influx were not affected by diazoxide at this concentration. 7. The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 mM succinate, 10 mM alpha-ketoisocaproic acid, 2 mM tetramethyl phenylenediamine plus 5 mM ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited alpha-ketoisocaproic acid-fuelled ATP production. 8. In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus-secretion coupling in pancreatic B-cells.     

Evidence for mitochondrial Ca(2+)-induced Ca2+ release in permeabilised endothelial cells

Generally most intracellular Ca2+ is stored in the endoplasmic reticulum (ER) and mitochondria. Recently a mitochondrial Ca(2+)-induced Ca2+ release (mCICR) mechanism, unconnected with ryanodine receptors (RyR's), has been shown in tumour cells. The existence of a mitochondrial Ca2+ release mechanism in BAE cells was investigated using saponin-permeabilised BAE cells. When buffered intracellular solution were 'stepped' from 10 nM to 10 microM free Ca2+, the mitochondrial inhibitors CN (2 mM), FCCP (1 microM), and RR (20 microM) significantly reduced total CICR by approximately 25%. The ER Ca(2+)-ATPase inhibitor thapsigargin (100 nM) had no effect. Furthermore, cyclosporin A (200 nM), an inhibitor of the mitochondrial permeability transition pore (PTP), abolished total CICR. Therefore, the novel ryanodine-caffeine insensitive CICR mechanism previously reported in BAE cells involves mitochondrial Ca2 release. It is proposed that in BAE cells, mCICR occurs via the mitochondrial PTP and may be physiologically important in endothelial cell Ca2+ signalling.     

Rate of phosphate release after photoliberation of adenosine 5'-triphosphate in slow and fast skeletal muscle fibers

Inorganic phosphate (Pi) release was determined by means of a fluorescent Pi-probe in single permeabilized rabbit soleus and psoas muscle fibers. Measurements of Pi release followed photoliberation of approximately 1.5 mM ATP by flash photolysis of NPE-caged ATP in the absence and presence of Ca2+ at 15 degrees C. In the absence of Ca2+, Pi release occurred with a slow rate of 11 +/- 3 microM . s-1 (n = 3) in soleus fibers and 23 +/- 1 microM . s-1 (n = 10) in psoas fibers. At saturating Ca2+ concentrations (pCa 4.5), photoliberation of ATP was followed by rapid force development. The initial rate of Pi release was 0.57 +/- 0.05 mM . s-1 in soleus (n = 13) and 4.7 +/- 0.2 mM . s-1 in psoas (n = 23), corresponding to a rate of Pi release per myosin head of 3.8 s-1 in soleus and 31.5 s-1 in psoas. Pi release declined at a rate of 0.48 s-1 in soleus and of 5.2 s-1 in psoas. Pi release in soleus was slightly faster in the presence of an ATP regenerating system but slower when 0.5 mM ADP was added. The reduction in the rate of Pi release results from an initial redistribution of cross-bridges over different states and a subsequent ADP-sensitive slowing of cross-bridge detachment.     

Dynamic regulation of intracellular Ca2+ concentration in aortic endothelial cells

In non-excitable cells, a Ca2+ entry pathway is opened after the depletion of intracellular Ca2+ store sites. We have tried to estimate the sensitivity of this pathway to Ca2+ release using bovine aortic endothelial cells. Single application of a high concentration (30 microM) of ATP released almost all stored Ca2+ in Ca(2+)-free extracellular solution, whereas a low concentration of ATP (30 nM) produced a partial (57.3 +/- 3.0%) release of Ca2+. By 10 min of Ca2+ re-perfusion, the Ca2+ store site was reloaded to 97.1% of its initial filling state. When thapsigargin was applied to this cell in Mn2+ solution, Mn(2+)-induced quenching of fura-2 dye started when 19.3 +/- 5.3% of Ca2+ release, produced by 30 nM ATP, had occurred. Therefore, Ca2+ release required for Mn2+ entry was estimated as 11.1 +/- 3.0% of stored Ca2+. These results indicate that intracellular Ca2+ concentration is controlled dynamically by simultaneously occurring Ca2+ release and entry in bovine aortic endothelial cells.     

P2-purinoceptor-mediated formation of inositol phosphates and intracellular Ca2+ transients in human coronary artery smooth muscle cells

1. The effects of extracellular adenosine 5'-triphosphate (ATP) on smooth muscles are mediated by a variety of purinoceptors. In this study we addressed the identity of the purinoceptors on smooth muscle cells (SMC) cultured from human large coronary arteries. Purinoceptor-mediated increases in [Ca2+]i were measured in single fura-2 loaded cells by applying a digital imaging technique, and the formation of inositol phosphate compounds was quantified after separation on an anion exchange column. 2. Stimulation of the human coronary artery SMC (HCASMC) with extracellular ATP at concentrations of 0.1-100 microM induced a transient increase in [Ca2+]i from a resting level of 49 +/- 21 nM to a maximum of 436 +/- 19 nM. The effect was dose-dependent with an EC50 value for ATP of 2.2 microM. 3. The rise in [Ca2+]i was independent of the presence of external Ca2+, but was abolished after depletion of intracellular stores by incubation with 100 nM thapsigargin. 4. [Ca2+]i was measured upon stimulation of the cells with 0.1-100 microM of the more specific P2-purinoceptor agonists alpha, beta-methyleneadenosine 5'-triphosphate (alpha,beta-MeATP), 2-methylthioadenosine 5'-triphosphate (2MeSATP) and uridine 5'-triphosphate (UTP). alpha, beta-MeATP was without effect, whereas 2MeSATP and UTP induced release of Ca2+ from internal stores with 2MeSATP being the most potent agonist (EC50 = 0.17 microM), and UTP having a potency similar to ATP. The P1 purinoceptor agonist adenosine (100 microM) did not induce any changes in [Ca2+]i. 5. Stimulation with a submaximal concentration of UTP (10 microM) abolished a subsequent ATP-induced increase in [Ca2+]i, whereas an increase was induced by ATP after stimulation with 10 microM 2MeSATP. 6. The phospholipase C (PLC) inhibitor U73122 (5 microM) abolished the purinoceptor-activated rise in [Ca2+]i, whereas pretreatment with the Gi protein inhibitor pertussis toxin (PTX, 500 ng ml-1) was without effect on ATP-evoked [Ca2+]i increases. 7. Receptor activation with UTP and ATP resulted in formation of inositol phosphates with peak levels of inositol 1, 4, 5-trisphosphate (Ins(1, 4, 5)P3) observed 5-20 s after stimulation. 8. These findings show, that cultured HCASMC express G protein-coupled purinoceptors, which upon stimulation activate PLC to induce enhanced Ins(1, 4, 5)P3 production causing release of Ca2+ from internal stores. Since a release of Ca2+ was induced by 2MeSATP as well as by UTP, the data indicate that P2y- as well as P2U-purinoceptors are expressed by the HCASMC.     

Cloning and tissue distribution of two new potassium channel alpha-subunits from rat brain

The release of excitatory amino acids from Schwann cell cultures in the rat was monitored using high-performance liquid chromatography. The basal concentration of glutamate and aspartate was 33 +/- 4 nM (mean +/- S.E.M., n = 12) and 8 +/- 1 nM (mean +/- S.E.M., n = 12), respectively. ATP (100 microM) caused a receptor-mediated increase in release of glutamate and aspartate from Schwann cell cultures. Bath application of adenosine (100 microM) was without effect on release of excitatory amino acids suggesting involvement of P2 receptors. Suramin, a competitive antagonist at P2 receptors, prevented the response to ATP. The release of excitatory amino acids evoked by ATP was not abolished in calcium-depleted saline. Pretreatment of the Schwann cultures with 50 microM 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetracetic acid-acetoxymethyl ester (BAPTA-AM) abolished the effect of ATP. ATP-evoked release of glutamate from cultured Schwann cells was significantly reduced by thapsigargin (1 microM), an inhibitor of Ca(2+)-ATPase of the Ca2+ pump of internal stores. U73122, a selective inhibitor of receptor-coupled phospholipase C-dependent processes, abolished stimulatory effect of ATP suggesting that ATP's action is mediated through an inositol 1,4,5-triphosphate-sensitive calcium store. The action of ATP was not blocked by L-trans-pyrrolidine-2,4-dicarboxylate, an inhibitor of the electrogenic glutamate transporter, nor was it blocked in Na(+)-free medium, and glutamate release was not stimulated by a depolarizing stimulus, suggesting that ATP-evoked release of glutamate from Schwann cells is not due to the reversal of the glutamate uptake. An anion transport blocker, furosemide, reduced ATP-induced glutamate release. These results suggest that ATP-stimulated glutamate and aspartate release from Schwann cells may be through a calcium-dependent furosemide-sensitive mechanism.     

The phospholipase C inhibitor U73122 increases cytosolic calcium in MDCK cells by activating calcium influx and releasing stored calcium

The effects of the phospholipase C (PLC) inhibitor U73122 on intracellular calcium levels ([Ca2+]i) were studied in MDCK cells. U73122 elevated [Ca2+]i dose-dependently. Ca2+ influx contributed to 75% of 20 microM U73122-induced Ca2+ signals. U73122 pretreatment abolished the [Ca2+]i transients evoked by ATP and bradykinin, suggesting that U73122 inhibited PLC. The Ca2+ signals among individual cells varied considerably. The internal Ca2+ source for the U73122 response was the endoplasmic reticulum (ER) since the response was abolished by thapsigargin. The depletion of the ER Ca2+ store triggered a La3+-sensitive capacitative Ca2+ entry. Independently of the internal release and capacitative Ca2 entry, U73122 directly evoked Ca2+ influx through a La3+-insensitive pathway. The U73122 response was augmented by pretreatment of carbonylcyanide m-chlorophynylhydrozone (CCCP), but not by Na+ removal, implicating that mitochondria contributed significantly in buffering the Ca2+ signal, and that efflux via Na+/Ca2+ exchange was insignificant.     

Transport of calcium ions by Ehrlich ascites-tumour cells

Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.     

Long-term activation of capacitative Ca2+ entry in mouse microglial cells

The cytoplasmic free calcium concentration ([Ca2+]i) was measured in cultured microglial cells with the Ca2+-sensitive fluorescent dye Fura-2 using a digital imaging system. Stimulation of P2 purinergic receptors by ATP or UTP always evoked a [Ca2+]i elevation. The ATP-induced Ca2+ response involved both Ca2+ influx through ionotropic receptors and Ca2+ release from intracellular pools, whereas UTP selectively stimulated intracellular Ca2+ release. When intracellular Ca2+ release was stimulated in the absence of extracellular Ca2+, the readmission of extracellular Ca2+ caused a large rebound [Ca2+]i increase. Following this rebound, [Ca2+]i did not return to the initial resting level, but remained for long periods of time (up to 20 min), at a new, higher steady-state level. Both the amplitude of the rebound Ca2+ transient and the new plateau level strongly correlated with the degree of intracellular Ca2+ depletion, indicating the activation of a store-operated Ca2+ entry pathway. The elevated steady-state [Ca2+]i level was associated with a significant increase in the plasma membrane permeability to Ca2+, as changes in extracellular Ca2+ were reflected in almost immediate changes of [Ca2+]i. Similarly, blocking plasma-lemmal Ca2+ channels with the non-specific agonist La3+ (50 microM) caused a decrease in [Ca2+]i, despite the continuous presence of Ca2+ ions in the extracellular medium. After the establishment of the new, elevated steady-state [Ca2+]i level, stimulation of P2U metabotropic purinoreceptors did not induce a [Ca2+]i response. In addition, application of either thapsigargin (1 microM) or carbonyl cyanide chlorophenyl hydrazone (10 microM) failed to affect [Ca2+]i. We conclude that the maximal depletion of intracellular Ca2+ stores in mouse brain microglia determines the long-term activation of a plasma membrane Ca2+ entry pathway. This activation appears to be associated with a significant decrease in the capability of the intracellular Ca2+ stores to take up cytosolic Ca2+ once they have been maximally depleted.     

Neomycin and spermine have similar effects on secretion and phosphoinositide metabolism in ATP-depleted permeabilized adrenal chromaffin cells

To evaluate the requirement of bovine adrenal chromaffin cells for inositol phospholipids in secretion, the effects of neomycin and spermine on secretion and phosphoinositide metabolism were compared. Spermine and neomycin had virtually identical effects on secretion and phosphoinositide levels. Both polyamines 1) partially maintained secretion when added to permeabilized cells in the absence of ATP, 2) had no effect when added to cells in the presence of ATP and 3) inhibited secretion when present with the Ca2+ stimulus. In the absence of ATP the enhancements to secretion due to incubation with either polyamine were associated with sustained levels of the polyphosphoinositides. The ability of spermine and neomycin to maintain secretion and the polyphosphoinositides in the absence of ATP supports the hypothesis that the maintenance of the polyphosphoinositides is necessary for secretion. Neomycin was found to inhibit Ca2+ stimulated production of both inositol bis- and tris-phosphates while spermine was found to selectively inhibit inositol bis-phosphate production and had no effect on Ca(2+)-stimulated inositol tris-phosphate production in permeabilized cells.     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

Intracellular calcium stores modulate miniature GABA-mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.     

Chromaffin granules release calcium on contact with annexin VI: implications for exocytosis

Chromaffin granules represent a substantial and exchangeable intracellular calcium pool which is thought to be regulated by a sodium/calcium exchange protein and also by a putative inositol trisphosphate-activated calcium channel. A family of calcium-binding proteins, called the annexins, has been shown to bind to chromaffin granules. We have therefore investigated the possible involvement of these proteins in the regulation of chromaffin granule sequestered calcium. Annexin VI (A-VI) produced a concentration-dependent release of 45Ca2+ from chromaffin granules; half-maximal release occurred at 1 microM A-VI, with near-maximum release being at 20 microM A-VI. The A-VI-induced release of 45Ca2+ was rapid, being essentially complete by our first time point of 7 s, and corresponded to 40% of the total sequestered 45Ca2+. A-VI-induced release occurred at extravesicular Ca2+ concentrations ranging from a pCa2+ of 4.12 to 6.86 and also appeared specific to this protein since neither annexin I nor annexin II (tetramer) could evoke any 45Ca2+ release. Given the predominant localization of A-VI to the apical plasmalemma, these results suggest that this protein could participate in the secretory event by mediating the localized release of Ca2+ at sites of contact between the chromaffin granule and plasma membrane.     

Calcium pools in Ehrlich carcinoma cells. A major, high affinity Ca2+ pool is sensitive to both inositol 1,4,5-trisphosphate and thapsigargin

To investigate the presence and the size of different non-mitochondrial Ca2+ pools of Ehrlich ascites tumor cells (EATCs), digitonin-permeabilized cells were allowed to accumulate Ca2+ in the presence of mitochondrial inhibitors and treated with the reticular Ca(2+)-ATPase inhibitor thapsigargin, IP3 and the Ca2+ ionophore A23187. Emptying of thapsigargin-sensitive Ca2+ stores prevented any Ca2+ release by IP3, and, after IP3 addition, little or no Ca2+ was released by thapsigargin. In both instances, a further Ca2+ release was accomplished by A23187. The IP3-thapsigargin-sensitive pool and the residual A23187-sensitive one corresponded to approximately 60 and 37% of non-mitochondrial stored Ca2+, respectively. In intact EATCs, IP3-dependent agonists and thapsigargin discharged Ca2+ pools almost completely overlapping, and A32187 released a minor residual Ca2+ pool. The IP3-insensitive pool appeared to have a relatively low affinity for Ca2+ (below 600 nM). The high affinity, IP3-sensitive Ca2+ pool was discharged in a 'quantal' manner following step additions of sub maximal [IP3], and the IP3-induced fractional Ca2+ release was more marked at higher concentrations of stored (luminal) Ca2+, The IP3-sensitive Ca2+ pool appeared to be devoid of the Ca(2+)-activated Ca2+ release channel since caffeine did not released any Ca2+ in intact and permeabilized EATCs, and Western blot analyses of EATC microsomal membranes failed to detect any known ryanodine receptor isoform.     

Endoplasmic reticulum Ca(2+)-ATPase in microsomal vesicles isolated from bovine retinae

Active Ca2+ transport was measured in microsomal vesicles prepared from bovine retinae and was compared with that in disk membranes of the photoreceptor cells of the same retina. The 45Ca uptake was dependent on the presence of Mg(2+)-ATP and was inhibited by vanadate or when GTP substituted for ATP. The dependence of calcium uptake on the external free Ca2+ concentration gave a KM = 13 microM or a KM = 0.1 microM for disks and microsomal vesicles, respectively. A phosphorylated intermediate (E-P) of Ca(2+)-ATPase of about 100 kDa was isolated in microsomal vesicles. The E-P formation was strongly inhibited by thapsigargin and partially by 2,5-di-(-butyl)benzohydroquinone. Digestion of disks or microsomes with calpain had no effect on the phosphorylated intermediate, while digestion with trypsin produced two fragments of approximately 55 kDa and 35 kDa. These results suggest that bovine retinal microsomes contain a calcium pump belonging to the SERCA family.     

Uridine triphosphate-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum of rat skeletal muscle fibers

The pyrimidine nucleotide, uridine triphosphate (UTP), was tested with skinned skeletal muscle fibers in order to investigate the UTP-sensitive pathway of Ca2+ release from the sarcoplasmic reticulum. The presence of ryanodine (200 microM), ruthenium red (10 microM) or heparin (2.5 mg/ml) did not affect the tension elicited in the presence of UTP, demonstrating that the UTP-induced Ca2+ release involved neither ryanodine nor inositol triphosphate-sensitive channels. Drugs such as compound 48/80 or cyclopiazonic acid used to inhibit Ca2+-ATPase in its reverse function appeared to be, respectively, non-specific or without any inhibitory effect on the tension induced by UTP. Finally, the UTP-induced tension as well as the trifluoperazine-induced tension were abolished in the presence of spermidine (50 mM), supporting the hypothesis that the UTP-sensitive pathway of the SR Ca2+ release might occur through the uncoupled calcium ATPase.     

Dendritic cell development: multiple pathways to nature''s adjuvants

The endothelin (ET) isoforms ET-1, ET-2 and ET-3 applied at 100 nM triggered a transient increase in [Ca2+]i in Bergmann glial cells in cerebellar slices acutely isolated from 20-25 day-old mice. The intracellular calcium concentration ([Ca2+]i) was monitored using Fura-2-based [Ca2+]i microfluorimetry. The ET-triggered [Ca2+]i transients were mimicked by ETB receptor agonist BQ-3020 and were inhibited by ETB receptor antagonist BQ-788. ET elevated [Ca2+]i in Ca(2+)-free extracellular solution and the ET-triggered [Ca2+]i elevation was blocked by 500 nM thapsigargin indicating that the [Ca2+]i was released from InsP3-sensitive intracellular pools. The ET-triggered [Ca2+]i increase in Ca(2+)-free solution was shorter in duration. Restoration of normal extracellular [Ca2+] briefly after the ET application induced a second [Ca2+]i increase indicating the presence of a secondary Ca2+ influx which prolongs the Ca2+ signal. Pre-application of 100 microM ATP or 10 microM noradrenaline blocked the ET response suggesting the involvement of a common Ca2+ depot. The expression of ETB receptor mRNAs in Bergmann glial cells was revealed by single-cell RT-PCR. The mRNA was also found in Purkinje neurones, but no Ca2+ signalling was triggered by ET. We conclude that Bergmann glial cells are endowed with functional ETB receptors which induce the generation of intracellular [Ca2+]i signals by activation of Ca2+ release from InsP3-sensitive intracellular stores followed by a secondary Ca2+ influx.     

Zinc inhibits Ca2+ transport by rat brain NA+/Ca2+ exchanger

Calcium transport by the Na+/Ca2+ exchanger was measured in plasma membranes vesicles purified from rat brain and in primary rat cortical cell culture. Sodium-loaded vesicles rapidly accumulate Ca2+ via Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ uptake). Extravesicular zinc inhibited Na+/Ca2+ exchange as evidenced by a reduction of the initial velocity of Ca2+ uptake. Significant inhibition of Ca2+ uptake was seen at concentrations of zinc as low as 3 microM. Lineweaver-Burk analysis of the data was consistent with noncompetitive inhibition with respect to extravesicular Ca2+ concentration. The Ki for zinc inhibition of Ca2+ uptake determined from a Dixon plot was 14.5 microM. This is within the range of zinc concentrations thought to be obtained extracellularly after excitation. When vesicles were preloaded with Ca2+, extravesicular zinc also inhibited reversal of Na+/Ca2+ exchange (Na+(i)-dependent Ca2+ release) although its potency was much less: concentrations of > or = 30 microM zinc were required. Zinc inhibition of Ca2+ release was not Na+ dependent. Na+(i)-dependent calcium uptake by rat cortical cells in primary culture also was inhibited by zinc. The extent of inhibition was similar to that seen for inhibition of Na+(i)-dependent Ca2+ uptake in membrane vesicles, but the potency was less. The results suggest that Ca2+ transport by the Na+/Ca2+ exchanger is inhibited by concentrations of zinc thought to be attained extracellularly after excitation.