We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor th... more We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca 2+ ] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca 2+ ] around 40 M. Cell stimulation with either ATP, caffeine or high-K + depolarization increased cytosolic [Ca 2+ ] and decreased secretory granule [Ca 2+ ] ([Ca 2+ ] SG ). Inositol-(1,4,5)trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca 2+ from the granules. Changes in cytosolic [Na + ] (0-140 mM) or [Ca 2+ ] (0-10 M) did not modify either ([Ca 2+ ] SG ). Instead, [Ca 2+ ] SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca 2+ ] SG , while cytosolic alcalinization reversibly increased [Ca 2+ ] SG . These results are consistent with the operation of a H + /Ca 2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K + on [Ca 2+ ] SG , because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca 2+ ] SG was reversible in 10-15 min even in the absence of cytosolic Ca 2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca 2+ buffer. This large store buffers [Ca 2+ ] SG changes in the long-term but allows highly dynamic free [Ca 2+ ] SG changes to occur in seconds or minutes.
1Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2... more 1Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2+-dependent phenomena. It has been known for many years that this Ca2+ uptake is mediated by the mitochondrial Ca2+ uniporter, a specific Ca2+ channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter.2The specific α-ER agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca2+ uptake in permeabilized HeLa cells by 10-fold at 2 μM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca2+] peak and reduced the cytosolic one.3Diethylstilbestrol and 17-β-estradiol (but not 17-α-estradiol) were active at pharmacological concentrations while the β-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective.4The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca2+ uptake (IC50 2.5±1.5 and 2.5±1.4 μM, mean±s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect.5Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum.6Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca2+ uptake.7These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca2+ uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2+-dependent phenomena. It has been known for many years that this Ca2+ uptake is mediated by the mitochondrial Ca2+ uniporter, a specific Ca2+ channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter.The specific α-ER agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca2+ uptake in permeabilized HeLa cells by 10-fold at 2 μM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca2+] peak and reduced the cytosolic one.Diethylstilbestrol and 17-β-estradiol (but not 17-α-estradiol) were active at pharmacological concentrations while the β-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective.The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca2+ uptake (IC50 2.5±1.5 and 2.5±1.4 μM, mean±s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect.Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum.Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca2+ uptake.These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca2+ uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.British Journal of Pharmacology (2005) 145, 862–871. doi:10.1038/sj.bjp.0706265
Background and purpose:The thiourea derivative KB-R7943, originally developed as inhibitor of the... more Background and purpose:The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes.The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes.Experimental approach.[Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells.[Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells.Key results:KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU.KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU.Conclusions and Implications:Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260
During cell activation, mitochondria play an important role in Ca 2+ homoeostasis due to the pres... more During cell activation, mitochondria play an important role in Ca 2+ homoeostasis due to the presence of a fast and specific Ca 2+ channel in its inner membrane, the mitochondrial Ca 2+ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca 2+ ] changes and controls the intramitochondrial Ca 2+ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca 2+ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca 2+ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca 2+ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca 2+ uptake by 85 + − 15 % (mean + − S.E.M., n = 4) and the histamine-induced mitochondrial Ca 2+ peak by 139 + − 19 % (mean + − S.E.M., n = 5) at a concentration of 1 µM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.
Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, ... more Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.British Journal of Pharmacology (2004) 141, 263–268. doi:10.1038/sj.bjp.0705609
There is increasing evidence that mitochondria play an important role in the control of cytosolic... more There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca 2+ signaling. We show here that the main mitochondrial Ca 2+ -exit pathway, the mitochondrial Na + /Ca 2+ exchanger, controls the pattern of cytosolic Ca 2+ oscillations in nonexcitable cells. In HeLa cells, the inhibitor of the mitochondrial Na + /Ca 2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca 2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca 2+ ] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca 2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca 2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na + /Ca 2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca 2+ release and in that way controls cytosolic Ca 2+ oscillations.
Actividad 2. El Contexto socioeconómico en mi etapa de educación secundaria. FECHA DE ELABORACIÓN... more Actividad 2. El Contexto socioeconómico en mi etapa de educación secundaria. FECHA DE ELABORACIÓN: 21 de septiembre del 2014.
We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor th... more We have used an aequorin chimera targeted to the membrane of the secretory granules to monitor the free [Ca 2+ ] inside them in neurosecretory PC12 cells. More than 95% of the probe was located in a compartment with an homogeneous [Ca 2+ ] around 40 M. Cell stimulation with either ATP, caffeine or high-K + depolarization increased cytosolic [Ca 2+ ] and decreased secretory granule [Ca 2+ ] ([Ca 2+ ] SG ). Inositol-(1,4,5)trisphosphate, cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate were all ineffective to release Ca 2+ from the granules. Changes in cytosolic [Na + ] (0-140 mM) or [Ca 2+ ] (0-10 M) did not modify either ([Ca 2+ ] SG ). Instead, [Ca 2+ ] SG was highly sensitive to changes in the pH gradient between the cytosol and the granules. Both carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and nigericin, as well as cytosolic acidification, reversibly decreased [Ca 2+ ] SG , while cytosolic alcalinization reversibly increased [Ca 2+ ] SG . These results are consistent with the operation of a H + /Ca 2+ antiporter in the vesicular membrane. This antiporter could also mediate the effects of ATP, caffeine and high-K + on [Ca 2+ ] SG , because all of them induced a transient cytosolic acidification. The FCCP-induced decrease in [Ca 2+ ] SG was reversible in 10-15 min even in the absence of cytosolic Ca 2+ or ATP, suggesting that most of the calcium content of the vesicles is bound to a slowly exchanging Ca 2+ buffer. This large store buffers [Ca 2+ ] SG changes in the long-term but allows highly dynamic free [Ca 2+ ] SG changes to occur in seconds or minutes.
1Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2... more 1Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2+-dependent phenomena. It has been known for many years that this Ca2+ uptake is mediated by the mitochondrial Ca2+ uniporter, a specific Ca2+ channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter.2The specific α-ER agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca2+ uptake in permeabilized HeLa cells by 10-fold at 2 μM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca2+] peak and reduced the cytosolic one.3Diethylstilbestrol and 17-β-estradiol (but not 17-α-estradiol) were active at pharmacological concentrations while the β-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective.4The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca2+ uptake (IC50 2.5±1.5 and 2.5±1.4 μM, mean±s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect.5Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum.6Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca2+ uptake.7These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca2+ uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.Ca2+ uptake by mitochondria is a key element in the control of cellular Ca2+ homeostasis and Ca2+-dependent phenomena. It has been known for many years that this Ca2+ uptake is mediated by the mitochondrial Ca2+ uniporter, a specific Ca2+ channel of the inner mitochondrial membrane. We have shown previously that this channel is strongly activated by a series of natural phytoestrogenic flavonoids. We show here that several agonists and antagonists of estrogen receptors (ERs) also modulate the activity of the uniporter.The specific α-ER agonist 4,4′,4″-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT) was the strongest activator, increasing the rate of mitochondrial Ca2+ uptake in permeabilized HeLa cells by 10-fold at 2 μM. Consistently, PPT largely increased the histamine-induced mitochondrial [Ca2+] peak and reduced the cytosolic one.Diethylstilbestrol and 17-β-estradiol (but not 17-α-estradiol) were active at pharmacological concentrations while the β-estrogen-receptor agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) was little effective.The ER modulators tamoxifen and 4-hydroxy-tamoxifen inhibited mitochondrial Ca2+ uptake (IC50 2.5±1.5 and 2.5±1.4 μM, mean±s.d., respectively) both in the presence and in the absence of PPT, but raloxifene and the pure estrogen antagonist ICI 182,780 produced no effect.Activation by PPT was immediate and inhibition by tamoxifen or 4-hydroxy-tamoxifen required only 5 min to reach maximum.Tamoxifen did not modify mitochondrial membrane potential and PPT induced a slow mitochondrial depolarization at higher concentrations than those required to activate mitochondrial Ca2+ uptake.These results suggest that some kind of ER or related protein located in mitochondria controls the activity of the Ca2+ uniporter by a nongenomic mechanism. This novel mechanism of action of estrogen agonists and antagonists can provide a new interpretation for several previously reported effects of these compounds.British Journal of Pharmacology (2005) 145, 862–871. doi:10.1038/sj.bjp.0706265
Background and purpose:The thiourea derivative KB-R7943, originally developed as inhibitor of the... more Background and purpose:The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes.The thiourea derivative KB-R7943, originally developed as inhibitor of the plasma membrane Na+/Ca2+ exchanger, has been shown to protect against myocardial ischemia-reperfusion injury. We have studied here its effects on mitochondrial Ca2+ fluxes.Experimental approach.[Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells.[Ca2+] in cytosol, mitochondria and endoplasmic reticulum (ER), and mitochondrial membrane potential were monitored using both luminescent (targeted aequorins) and fluorescent (fura-2, tetramethylrhodamine ethyl ester) probes in HeLa cells.Key results:KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU.KB-R7943 was also a potent inhibitor of the mitochondrial Ca2+ uniporter (MCU). In permeabilized HeLa cells, KB-R7943 inhibited mitochondrial Ca2+ uptake with a Ki of 5.5±1.3 μM (mean±S.D.). In intact cells, 10μM KB-R7943 reduced by 80% the mitochondrial [Ca2+] peak induced by histamine. KB-R7943 did not modify the mitochondrial membrane potential and had no effect on the mitochondrial Na+/Ca2+ exchanger. KB-R7943 inhibited histamine-induced ER-Ca2+ release in intact cells, but not in cells loaded with a Ca2+-chelator to damp cytosolic [Ca2+] changes. Therefore, inhibition of ER-Ca2+-release by KB-R7943 was probably due to the increased feedback Ca2+-inhibition of inositol 1,4,5-trisphosphate receptors after MCU block. This mechanism also explains why KB-R7943 reversibly blocked histamine-induced cytosolic [Ca2+] oscillations in the same range of concentrations required to inhibit MCU.Conclusions and Implications:Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260Inhibition of MCU by KB-R7943 may contribute to its cardioprotective activity by preventing mitochondrial Ca2+-overload during ischemia-reperfusion. In addition, the effects of KB-R7943 on Ca2+ homeostasis provide new evidence for the role of mitochondria modulating Ca2+-release and regenerative Ca2+-oscillations. Search for permeable and selective MCU inhibitors may yield useful pharmacological tools in the future.British Journal of Pharmacology (2007) 151, 647–654; doi:10.1038/sj.bjp.0707260
During cell activation, mitochondria play an important role in Ca 2+ homoeostasis due to the pres... more During cell activation, mitochondria play an important role in Ca 2+ homoeostasis due to the presence of a fast and specific Ca 2+ channel in its inner membrane, the mitochondrial Ca 2+ uniporter. This channel allows mitochondria to buffer local cytosolic [Ca 2+ ] changes and controls the intramitochondrial Ca 2+ levels, thus modulating a variety of phenomena from respiratory rate to apoptosis. We have described recently that SB202190, an inhibitor of p38 MAPK (mitogen-activated protein kinase), strongly activated the uniporter. We show in the present study that a series of natural plant flavonoids, widely distributed in foods, produced also a strong stimulation of the mitochondrial Ca 2+ uniporter. This effect was of the same magnitude as that induced by SB202190 (an approx. 20-fold increase in the mitochondrial Ca 2+ uptake rate), developed without measurable delay and was rapidly reversible. In intact cells, the mitochondrial Ca 2+ peak induced by histamine was also largely increased by the flavonoids. Stimulation of the uniporter by either flavonoids or SB202190 did not require ATP, suggesting a direct effect on the uniporter or an associated protein which is not mediated by protein phosphorylation. The most active compound, kaempferol, increased the rate of mitochondrial Ca 2+ uptake by 85 + − 15 % (mean + − S.E.M., n = 4) and the histamine-induced mitochondrial Ca 2+ peak by 139 + − 19 % (mean + − S.E.M., n = 5) at a concentration of 1 µM. Given that flavonoids can reach this concentration range in plasma after ingestion of flavonoid-rich food, these compounds could be modulating the uniporter under physiological conditions.
Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, ... more Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.Cyclosporin A (CsA) is a widely used compound because of its potent immunosupressive properties, derived mainly from the inhibition of calcineurin, and also because of its ability to block the mitochondrial permeability transition pore (PTP). This second effect has been involved in the protection against apoptosis mediated by release of mitochondrial factors. We show here that CsA (1–10μM) has an additional effect on Ca2+ homeostasis in mitochondria that cannot be attributed to inhibition of PTP.By measuring specifically mitochondrial [Ca2+] with targeted aequorin, we show that CsA inhibited Ca2+ entry into mitochondria both in intact and in permeabilized cells, and this effect was stronger when Ca2+ entry was triggered by low cytosolic [Ca2+], below 5 μM.Inhibition of mitochondrial Ca2+ uptake required micromolar concentrations of CsA and was not mimicked by other inhibitors of calcineurin such as FK-506 or cypermethrin, nor by a different inhibitor of the PTP, bongkrekic acid.CsA blocked the increase in mitochondrial Ca2+ uptake rate induced by the mitochondrial Ca2+ uniporter activator SB202190.Our results suggest that CsA inhibits Ca2+ entry through the Ca2+ uniporter by a mechanism independent of the inhibition of PTP or calcineurin. This effect may contribute to reduce depolarization and Ca2+ overloading in mitochondria after cell stimulation, and thus cooperate with the direct inhibition of PTP to prevent apoptosis.British Journal of Pharmacology (2004) 141, 263–268. doi:10.1038/sj.bjp.0705609
There is increasing evidence that mitochondria play an important role in the control of cytosolic... more There is increasing evidence that mitochondria play an important role in the control of cytosolic Ca 2+ signaling. We show here that the main mitochondrial Ca 2+ -exit pathway, the mitochondrial Na + /Ca 2+ exchanger, controls the pattern of cytosolic Ca 2+ oscillations in nonexcitable cells. In HeLa cells, the inhibitor of the mitochondrial Na + /Ca 2+ exchanger CGP37157 changed the pattern of the oscillations induced by histamine from a high-frequency irregular one to a lower frequency baseline spike type, surprisingly with little changes in the average Ca 2+ values of a large cell population. In human fibroblasts, CGP37157 increased the frequency of the baseline oscillations in cells having spontaneous activity and induced the generation of oscillations in cells without spontaneous activity. This effect was dose-dependent, disappeared when the inhibitor was washed out and was not mimicked by mitochondrial depolarization. CGP37157 increased mitochondrial [Ca 2+ ] and ATP production in histamine-stimulated HeLa cells, but the effect on ATP production was only transient. CGP37157 also activated histamine-induced Ca 2+ release from the endoplasmic reticulum and increased the size of the cytosolic Ca 2+ peak induced by histamine in HeLa cells. Our results suggest that the mitochondrial Na + /Ca 2+ exchanger directly modulates inositol 1,4,5-trisphosphate-induced Ca 2+ release and in that way controls cytosolic Ca 2+ oscillations.
Actividad 2. El Contexto socioeconómico en mi etapa de educación secundaria. FECHA DE ELABORACIÓN... more Actividad 2. El Contexto socioeconómico en mi etapa de educación secundaria. FECHA DE ELABORACIÓN: 21 de septiembre del 2014.
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