Thiol-reactive agents biphasically regulate inositol 1,4,5-trisphosphate binding and Ca(2+) release activities in bovine adrenal cortex microsomes.

Within all endocrine cells, the inositol 1,4,5-trisphosphate (InsP(3)) receptor plays an important role in regulation of the intracellular Ca(2+) 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(2+) release efficiency. The late dampening effect of thiol-reactive agents translated into a continuous reduction of the maximal binding capacity of the microsomes with a concomitant decrease in Ca(2+) release efficiency. Under these conditions, Western blot analyses demonstrated that the level of InsP(3) receptor protein was not modified. Sequential treatments with thimerosal and the reducing agent dithiothreitol showed that the InsP(3) receptor can readily oscillate between high and low affinity states that are related to its alkylation state. Our results suggest a common mode of action of thiol-reactive agents on the InsP(3) receptor. These results also support the contention that cellular mechanisms of thiol group modification could play important roles in regulation of the intracellular Ca(2+) concentration.

FK506 blocks intracellular Ca2+ oscillations in bovine adrenal glomerulosa cells.

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.

Different populations of inositol 1,4,5-trisphosphate receptors expressed in the bovine adrenal cortex.

The inositol 1,4,5-trisphosphate (InsP3) receptor forms a tetrameric channel responsible for the release of Ca2+ from intracellular stores. In the present study we showed that the experimental approach used to separate bound and free ligands may discriminate between two populations of InsP3 binding sites in bovine adrenal cortex microsomes. A large population of low affinity sites and a small population of high affinity sites were detected with centrifugation and filtration approaches, respectively. Both populations were found in the supernatant and the cytoskeleton fractions of Triton X-100 solubilized microsomes. After treatment of microsomes with thimerosal, an alkylating reagent known to increase InsP3 receptor affinity, the filtration and the centrifugation approaches yielded identical results. With selective anti-InsP3 receptor antibodies, we showed that types 1, 2 and 3 InsP3 receptors are present in intact microsomes and in the cytoskeleton fraction. Binding studies on immunoprecipitated receptors revealed that anti-type 1 antibody recognizes a large population of low affinity sites whereas anti-type 2 antibody recognizes a small population of high affinity sites. Our results indicate that the three types of InsP3 receptors are expressed at different levels in the bovine adrenal cortex. The presence of different types of InsP3 receptors with different ligand binding affinities and their association with the cytoskeleton offer a convenient way for the cell to simultaneously regulate its intracellular Ca2+ concentration and reorganize the spatial distribution of its Ca2+ stores.

Synthesis, acid-base behavior, and binding properties of 6-modified myo-inositol 1,4,5-tris(phosphate)s.

myo-Inositol 1,4,5-tris(phosphate) was modified at position 6. The analogues synthesized are reported in this publication are 6-deoxy-myo-inositol 1,4,5-tris(phosphate), 6-fluoro-6-deoxy-myo-inositol 1,4,5-tris(phosphate), epi-inositol 1, 4,5-tris(phosphate), and 6-amino-6-deoxy-myo-inositol 1,4, 5-tris(phosphate). These derivatives showed poor affinity for the Ins(1,4,5)P(3) receptors. The inframolecular acid-base behavior and the cooperative effects between the phosphate groups could help explain the loss of affinity of these 6-modified analogues.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production.

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 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.

Bidirectional activity of the endoplasmic reticulum Ca(2+)-ATPase of bovine adrenal cortex.

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 Ca(2+)-ATPase has been attributed an accessory role of refilling the intracellular Ca2+ store. In the present study, the activity of the microsomal Ca(2+)-ATPase of bovine adrenal cortex was investigated. We show that the Ca(2+)-pumping activity of the Ca(2+)-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 +/- 0.78 mM. ADP-induced Ca2+ release was not prevented by heparin, ruling out the participation of the inositol 1,4,5-trisphosphate receptor. ADP-induced Ca2+ release could not be attributed to the mere inhibition of the Ca(2+)-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 (2 microM). ADP-induced Ca2+ release experiments performed in the presence of [32P]PO4 revealed a concomitant production of [32P]ATP. ADP-induced [32P]ATP production was dose-dependent, with an EC50 of 5.50 +/- 0.70 mM. ADP-induced [32P]ATP production was prevented by ionomycin (10 microM) and by high concentrations of extramicrosomal Ca2+. These results demonstrate that the microsomal Ca(2+)-ATPase of adrenal cortex possesses a bidirectional activity that depends on ADP concentrations, the Ca2+ gradient across the microsomal membrane, and probably also ATP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)