Role of calcium messenger systems in ACTH-induced cortisol production in bovine adrenal fasciculo-reticularis cells.
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We investigated the regulation of each intracellular signal transduction system including cyclic AMP (cAMP)-dependent and calcium (Ca2+) messenger systems in bovine adrenal fasciculo-reticularis cells to clarify the exact mode of action of ACTH. Pretreatment with primaquine and quinacrine, which are phospholipase A2 inhibitors, significantly inhibited cortisol production activated by both low and high concentrations of ACTH. Therefore, it seems that metabolites induced by phospholipase A2 are quite essential for cortisol synthesis induced by ACTH, either at low or high concentrations. At low concentrations of ACTH (10(-13)-10(-12) M), significant increases of cytosolic calcium ([Ca2+]i), but not of cAMP, were observed. Calphostin C, a specific protein kinase C inhibitor, apparently suppressed cortisol production activated by low concentrations of ACTH, while H-89, a specific inhibitor of cAMP-dependent protein kinase, did not. These findings suggest that, at physiologically low concentrations, ACTH activates [Ca2+]i and phospholipase A2 without affecting cAMP formation, resulting in an increased biosynthesis of cortisol, partly via protein kinase C-dependent processes. At high concentrations, ACTH (10(-9)-10(-7) M) induced an increase of cAMP and [Ca2+]i. The cortisol production induced by high concentrations of ACTH was significantly inhibited by pretreatment with calphostin C, H-89 and H-7, suggesting the participation of cAMP-dependent protein kinase and protein kinase C systems in the regulation of cortisol production in the presence of high concentrations of ACTH. In conclusion, cytosolic calcium is biphasically enhanced by ACTH, although cAMP accumulation is increased only by high concentrations of ACTH. A phospholipase A2-dependent process may partly play a crucial role in the regulation of cortisol biosynthesis, when stimulated by low and high concentrations of ACTH. (+info)
Inhibition of the Na+/dicarboxylate cotransporter by anthranilic acid derivatives.
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The Na(+)/dicarboxylate cotransporter NaDC1 absorbs citric acid cycle intermediates from the lumen of the small intestine and kidney proximal tubule. No effective inhibitor has been identified yet, although previous studies showed that the nonsteroidal anti-inflammatory drug, flufenamate, inhibits the human (h) NaDC1 with an IC(50) value of 2 mM. In the present study, we have tested compounds related in structure to flufenamate, all anthranilic acid derivatives, as potential inhibitors of hNaDC1. We found that N-(p-amylcinnamoyl) anthranilic acid (ACA) and 2-(p-amylcinnamoyl) amino-4-chloro benzoic acid (ONO-RS-082) are the most potent inhibitors with IC(50) values lower than 15 microM, followed by N-(9-fluorenylmethoxycarbonyl)-anthranilic acid (Fmoc-anthranilic acid) with an IC(50) value of approximately 80 microM. The effects of ACA on NaDC1 are not mediated through a change in transporter protein abundance on the plasma membrane and seem to be independent of its effect on phospholipase A(2) activity. ACA acts as a slow inhibitor of NaDC1, with slow onset and slow reversibility. Both uptake activity and efflux are inhibited by ACA. Other Na(+)/dicarboxylate transporters from the SLC13 family, including hNaDC3 and rbNaDC1, were also inhibited by ACA, ONO-RS-082, and Fmoc-anthranilic acid, whereas the Na(+)/citrate transporter (hNaCT) is much less sensitive to these compounds. The endogenous sodium-dependent succinate transport in Caco-2 cells is also inhibited by ACA. In conclusion, ACA and ONO-RS-082 represent promising lead compounds for the development of specific inhibitors of the Na(+)/dicarboxylate cotransporters. (+info)
Role of phospholipase A2 (group I secreted) in the genesis of basal tone in the internal anal sphincter smooth muscle.
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The role of phospholipase A(2) (PLA(2)) in the genesis of basal tone in the internal anal sphincter (IAS) is not known. We determined the effects of PLA(2) and inhibitors on the basal tone and intraluminal pressures (IASP) in the rat IAS vs. rectal smooth muscles (RSM). In addition, we determined the correlations between the IAS tone, PLA(2) levels, and the actual enzymatic activity. Inhibition of PLA(2) by 4-bromophenacyl bromide (universal inhibitor of PLA(2)) and MJ33 [selective inhibitor of secreted isoform of PLA(2) (sPLA(2))] caused concentration-dependent decrease in the IAS tone and in the IASP. Maximal decreases in the IAS tone and IASP by 4-bromophenacyl bromide and MJ33 were 58.8 +/- 6.9 and 51.5 +/- 6.3%, and 66.7 +/- 5.1 and 79.8 +/- 8.2%, respectively. The sPLA(2) inhibitors were approximately 100 times more potent in decreasing the IASP than the mean blood pressure. Conversely, the selective inhibitors of the cytosolic and calcium-independent PLA(2) arachidonyl trifluoromethyl ketone and bromoenol lactone, respectively, produced no significant effect. The IAS had characteristically higher levels of sPLA(2) activity (26.5 +/- 4.9 micromol.min(-1).ml(-1)) vs. the RSM (3.2 +/- 0.4 micromol.min(-1).ml(-1)), and higher levels of sPLA(2) as shown by Western blot and RT-PCR. Interestingly, administration of sPLA(2) transformed RSM into the tonic smooth muscle like that of the IAS: it developed basal tone and relaxed in response to the electrical field stimulation. From the present data, we conclude that sPLA(2) plays a critical role in the genesis of tone in the IAS. PLA(2) inhibitors may provide potential therapeutic target for treating anorectal motility disorders. (+info)
Inhibition of cytosolic phospholipase A(2) suppresses production of cholesteryl ester through the reesterification of free cholesterol but not formation of foam cells in oxidized LDL-stimulated macrophages.
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Macrophage-derived foam cells are formed as a result of the accumulation of cholesteryl ester (CE) not only in cytoplasm where CE is produced by the reesterification of free cholesterol derived from oxidized low density lipoprotein (OxLDL) undergoing hydrolysis, but also in lysosomes where the remaining CE of OxLDL is deposited. We examined the possible involvement of cytosolic phospholipase A(2)s (cPLA(2)s) in the production of CE through the reesterification and in the formation of foam cells. In [(3)H]oleic acid-labeled human acute monocytic leukemia (THP-1) cell-derived macrophages (THP-M) and mouse peritoneal macrophages (MPM), which possessed at least cPLA(2)alpha and cPLA(2)gamma, stimulation with OxLDL induced the production of [(3)H]cholesteryl oleate ([(3)H]CE).The production was suppressed by an inhibitor of cPLA(2)s. However, the inhibitor tended to slightly decrease total intracellular levels of CE, and did not affect the formation of foam cells, as estimated by staining with Oil Red O. In cPLA(2)alpha-knockout MPM, OxLDL-induced increases in [(3)H]CE and total CE did not differ from those in wild-type MPM. Our results suggest that cPLA(2)s other than cPLA(2)alpha contribute to the supply of fatty acids, which are utilized for the production of CE through the reesterification, in OxLDL-stimulated macrophages. However, the formation of foam cells could not be inhibited only by the suppression of cPLA(2)-mediated CE production. (+info)
Lipidomic analysis reveals differential defense responses of Taxus cuspidata cells to two elicitors, methyl jasmonate and cerium (Ce4+).
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