The inositol 1,4,5-trisphosphate (InsP 3 ) receptor is a ligand-gated Ca 2+ channel playing an im... more The inositol 1,4,5-trisphosphate (InsP 3 ) receptor is a ligand-gated Ca 2+ channel playing an important role in the control of intracellular Ca 2+ . In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP 3 receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP 3 receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP 3 receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca 2+ induced by a submaximal dose of InsP 3 . We also showed that FK506 blocks intracellular Ca 2+ oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca 2+ concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP 3 receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP 3 receptor in the regulation of intracellular Ca 2+ oscillations and show that FK506, by maintaining the phosphorylated state of the InsP 3 receptor, causes important changes in the Ca 2+ oscillatory process.
European Journal of Pharmacology: Molecular Pharmacology, 1993
In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP 3) is an important second messenge... more In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP 3) is an important second messenger involved in the regulation of intracellular Ca 2÷ concentration. InsP 3 interacts with specific receptors and triggers the release of sequestered Ca 2+ from an internal store. We have synthesized a structural analogue of InsP 3 by phosphorylation of the free hydroxyl groups of 1,2,4-benzenetriol with dibenzylphosphorochloridate. The product benzene 1,2,4-trisphosphate (BzP 3) was shown to interact with InsP 3 receptor and InsP 3 metabolizing enzymes of bovine adrenal cortex. BzP 3 competitively blocked InsP 3 binding to adrenal cortex microsomes with a half-maximal efficiency at 34/zM. This affinity was about 10,000 times lower than that of InsP 3 for its receptor. The Ca 2÷ releasing activity of BzP 3 on the same microsomal preparation was monitored with the fluorescent indicator fura-2. BzP 3 had no agonistic effect on this activity but it was able to inhibit InsP3-induced Ca 2÷ release in a dose-dependent manner. The activity of InsP 3 phosphatase was also studied. BzP 3 inhibited the activity of the phosphatase with a half-maximal efficiency of 32/xM. BzP 3 was also able to inhibit the activity of the cytosolic InsP 3 kinase with a half-maximal efficiency of 6.1 ~M. These results show that BzP 3 is interacting with the three specific recognition sites for InsP 3 in the bovine adrenal cortex. The inhibitory effect of this compound is relatively more potent on the metabolizing enzymes than on the Ca2+-mobilizing receptor.
lnositol 1,4,&trisphosphate (InsPs) is a second messenger responsible for Ca*' release from a non... more lnositol 1,4,&trisphosphate (InsPs) is a second messenger responsible for Ca*' release from a non-mitochondrial intracellular store. An impoftant discrepancy has been observed between the affinity measured in binding studies (Kd) and the apparent afftnity obtained in Ca*+ mobilization studfes (EQo). It has been proposed that this dfscrepancy could be due to different experimental conditions used for Ca*+ mobilization studies and for InsP3 binding studies. With the fluorescent indicator Fura-2, we studied InsPs-induced Ca*+ release activp at 7-C and at 37*C, in bovine adrenal cortex microsomes. Under both conditions, the Ca ' releasing effect of lnsPs (1 m was completed within about 2 s, as a result of the quanta1 process of InsPs receptor action. The apparent afftnity (EC50) observed for InsPs-induced Ca*' rdease at 7% and at 37% were 0.64 f 0.2 w and 0.g f 0.2 jNl respectively. lnsP3 degradation studies, at 37X, indicated that less than 1096 of [SH]-InsPa was degraded within the first 10 s of incubation. InsP association rates were evaluated, at low temperature, with increasing concentrations of E HI&&%. These kinetic studies revealed a direct relationship between the initial rate of association (Vi) and InsPs concentration. From this relationship, we evaluated that the concentration of InsPs needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between Kd and EC50 is related to a kinetic con&mint dictated by the quanta1 process by which InsPa releases Ca*+.
Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor plays an importan... more Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor plays an important role in regulation of the intracellular Ca 2ϩ concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP 3 receptor activity) or thimerosal (known to increase InsP 3 receptor activity) caused time-dependent biphasic effects on the InsP 3 binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca 2ϩ release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding
It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adre... more It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125 I-[Tyr 8 ]BK revealed the presence of a relatively small amount (B max =180 55 fmol/mg of protein) of high affinity (K d =0·65 0·17 nM) binding sites. BK induced a timeand concentration-dependent increase of [ 3 H]inositol trisphosphate ([ 3 H]IP 3 ) in myo-[ 3 H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC 50 value was 1·0 0·5 nM. 125 I-[Tyr 8 ]BK binding and BK-induced IP 3 production were inhibited by the selective B 2 receptor antagonist Icatibant (1 µM) and unaffected by the selective B 1 receptor antagonist [DesArg 9 ,Leu 8 ]BK (1 µM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca 2+ response composed of a rapid and transient increase of intracellular Ca 2+ concentration [Ca 2+
It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate recepto... more It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca2+-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca2+-ATPase of bovine adrenal cortex was investigated. We show that the Ca2+-pumping activity of the Ca2+-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 f 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADPinduced Ca2+ release could not be attributed to the mere inhibition of the Ca2+-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin ). ADP-induced Ca2+ release experiments performed in the presence of [32P]P04 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 f 0.70 mM. ADP-induced [32P]ATP production was prevented by
The inositol 1,4,5-trisphosphate (InsP 3 ) receptor is a ligand-gated Ca 2+ channel playing an im... more The inositol 1,4,5-trisphosphate (InsP 3 ) receptor is a ligand-gated Ca 2+ channel playing an important role in the control of intracellular Ca 2+ . In the study presented here, we demonstrate that angiotensin (AngII), phorbol ester (PMA), and FK506 significantly increase the level of InsP 3 receptor phosphorylation in intact bovine adrenal glomerulosa cells. With a back-phosphorylation approach, we showed that the InsP 3 receptor is a good substrate for protein kinase C (PKC) and that FK506 increases the level of PKC-mediated InsP 3 receptor phosphorylation. With a microsomal preparation from bovine adrenal cortex, we showed that PKC enhances the release of Ca 2+ induced by a submaximal dose of InsP 3 . We also showed that FK506 blocks intracellular Ca 2+ oscillations in isolated adrenal glomerulosa cells by progressively increasing the intracellular Ca 2+ concentration to a high plateau level. This effect is consistent with an inhibitory role of FK506 on calcineurin dephosphorylation of the InsP 3 receptor, thus keeping the receptor in a phosphorylated, high-conductance state. Our results provide further evidence for the crucial role of the InsP 3 receptor in the regulation of intracellular Ca 2+ oscillations and show that FK506, by maintaining the phosphorylated state of the InsP 3 receptor, causes important changes in the Ca 2+ oscillatory process.
European Journal of Pharmacology: Molecular Pharmacology, 1993
In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP 3) is an important second messenge... more In a wide variety of cells, inositol 1,4,5-trisphosphate (InsP 3) is an important second messenger involved in the regulation of intracellular Ca 2÷ concentration. InsP 3 interacts with specific receptors and triggers the release of sequestered Ca 2+ from an internal store. We have synthesized a structural analogue of InsP 3 by phosphorylation of the free hydroxyl groups of 1,2,4-benzenetriol with dibenzylphosphorochloridate. The product benzene 1,2,4-trisphosphate (BzP 3) was shown to interact with InsP 3 receptor and InsP 3 metabolizing enzymes of bovine adrenal cortex. BzP 3 competitively blocked InsP 3 binding to adrenal cortex microsomes with a half-maximal efficiency at 34/zM. This affinity was about 10,000 times lower than that of InsP 3 for its receptor. The Ca 2÷ releasing activity of BzP 3 on the same microsomal preparation was monitored with the fluorescent indicator fura-2. BzP 3 had no agonistic effect on this activity but it was able to inhibit InsP3-induced Ca 2÷ release in a dose-dependent manner. The activity of InsP 3 phosphatase was also studied. BzP 3 inhibited the activity of the phosphatase with a half-maximal efficiency of 32/xM. BzP 3 was also able to inhibit the activity of the cytosolic InsP 3 kinase with a half-maximal efficiency of 6.1 ~M. These results show that BzP 3 is interacting with the three specific recognition sites for InsP 3 in the bovine adrenal cortex. The inhibitory effect of this compound is relatively more potent on the metabolizing enzymes than on the Ca2+-mobilizing receptor.
lnositol 1,4,&trisphosphate (InsPs) is a second messenger responsible for Ca*' release from a non... more lnositol 1,4,&trisphosphate (InsPs) is a second messenger responsible for Ca*' release from a non-mitochondrial intracellular store. An impoftant discrepancy has been observed between the affinity measured in binding studies (Kd) and the apparent afftnity obtained in Ca*+ mobilization studfes (EQo). It has been proposed that this dfscrepancy could be due to different experimental conditions used for Ca*+ mobilization studies and for InsP3 binding studies. With the fluorescent indicator Fura-2, we studied InsPs-induced Ca*+ release activp at 7-C and at 37*C, in bovine adrenal cortex microsomes. Under both conditions, the Ca ' releasing effect of lnsPs (1 m was completed within about 2 s, as a result of the quanta1 process of InsPs receptor action. The apparent afftnity (EC50) observed for InsPs-induced Ca*' rdease at 7% and at 37% were 0.64 f 0.2 w and 0.g f 0.2 jNl respectively. lnsP3 degradation studies, at 37X, indicated that less than 1096 of [SH]-InsPa was degraded within the first 10 s of incubation. InsP association rates were evaluated, at low temperature, with increasing concentrations of E HI&&%. These kinetic studies revealed a direct relationship between the initial rate of association (Vi) and InsPs concentration. From this relationship, we evaluated that the concentration of InsPs needed to occupy half of the binding sites within the first second of incubation was 271 nM. We conclude that the discrepancy between Kd and EC50 is related to a kinetic con&mint dictated by the quanta1 process by which InsPa releases Ca*+.
Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor plays an importan... more Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP 3 ) receptor plays an important role in regulation of the intracellular Ca 2ϩ concentration. In the present study we showed that a single short-term treatment with either N-ethylmaleimide (known to decrease InsP 3 receptor activity) or thimerosal (known to increase InsP 3 receptor activity) caused time-dependent biphasic effects on the InsP 3 binding activity of bovine adrenal cortex microsomes. The early potentiating effect of thiol-reactive agents translated into a 2-fold increase in binding affinity and Ca 2ϩ release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding
It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adre... more It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125 I-[Tyr 8 ]BK revealed the presence of a relatively small amount (B max =180 55 fmol/mg of protein) of high affinity (K d =0·65 0·17 nM) binding sites. BK induced a timeand concentration-dependent increase of [ 3 H]inositol trisphosphate ([ 3 H]IP 3 ) in myo-[ 3 H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC 50 value was 1·0 0·5 nM. 125 I-[Tyr 8 ]BK binding and BK-induced IP 3 production were inhibited by the selective B 2 receptor antagonist Icatibant (1 µM) and unaffected by the selective B 1 receptor antagonist [DesArg 9 ,Leu 8 ]BK (1 µM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca 2+ response composed of a rapid and transient increase of intracellular Ca 2+ concentration [Ca 2+
It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate recepto... more It is generally accepted that the ryanodine receptor and the inositol 1,4,5-trisphosphate receptor play major roles in the complex mechanisms by which agonists increase intracellular Ca2+ concentration. In these mechanisms, the endoplasmic reticulum Ca2+-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca2+-ATPase of bovine adrenal cortex was investigated. We show that the Ca2+-pumping activity of the Ca2+-ATPase is related to the ADP/ATP ratio. Our results also show that a brisk increase of the ADP/ATP ratio upon addition of exogenous ADP triggered a rapid release of Ca2+ from preloaded microsomes. ADP released Ca2+ in a dose-dependent manner with an EC50 of 2.98 f 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADPinduced Ca2+ release could not be attributed to the mere inhibition of the Ca2+-ATPase, since the rate of ADP-induced Ca2+ release was 20 times faster than the rate of Ca2+ release induced by a maximal concentration of thapsigargin ). ADP-induced Ca2+ release experiments performed in the presence of [32P]P04 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 f 0.70 mM. ADP-induced [32P]ATP production was prevented by
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Papers by Marc Poitras