Search PubMed⌕ Search

Biomedical subjects

I M Bird

Publications and source records attributed to I M Bird.

At least 55 records · Page 3Linked to original sources
PubMed1997

Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression.

Pregnancy is characterized by elevations in uterine but not omental artery nitric oxide synthase (NOS)-specific activity. We hypothesized that increases in NO production during pregnancy are associated with elevations in protein expression of the constitutive isoform, endothelial cell NOS (ecNOS), in uterine but not systemic arteries. Arterial NOS-specific activity and guanosine 3',5'-cyclic monophosphate (cGMP) production were tested in pregnant sheep in the presence or absence [+5 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] of Ca2+. With the use of Western analysis, ecNOS and neuronal NOS (nNOS) constitutive isoform expressions were evaluated in intact and denuded [vascular smooth muscle (VSM)] uterine and systemic (omental and renal) arteries as well as in isolated endothelium-derived proteins from nonpregnant and pregnant sheep. Uterine and omental artery NOS activity and cGMP production were inhibited 75-85% by Ca2+ removal. ecNOS was localized only in uterine and systemic artery endothelium (not VSM) by immunohistochemistry and Western analysis; nNOS was not detected. Compared with nonpregnant ewes, pregnancy increased expression of ecNOS in uterine [2.1- to 4.2-fold (P < 0.0001)] and omental [1.3- to 2.2-fold (P = 0.032)] but not renal (P = 0.1367) artery endothelium. Increases in uterine were greater than in omental artery endothelium. Levels of plasma and urinary cGMP were elevated (P < 0.01) proportionally (1.8- to 2.0-fold) in pregnant versus nonpregnant ewes. During pregnancy, expression of uterine artery endothelium-derived (not VSM) ecNOS constitutive isoform is increased, whereas expression in systemic vessels shows little or no change.

Animals↗
PubMed1997

Pregnancy induces an increase in angiotensin II type-1 receptor expression in uterine but not systemic artery endothelium.

During pregnancy, the uterine artery demonstrates refractoriness to vasoconstriction by infused angiotensin II (AII). AII increases prostacyclin (PGI2) production by uterine artery endothelium from pregnant ewes, and this response is mediated via the AT1 receptor (AT1-R). This response is also unique to pregnancy because AII does not stimulate PGI2 production by uterine artery endothelium from nonpregnant ewes. We therefore hypothesize that the increase in uterine artery PGI2 production in response to AII in pregnancy is associated in part with a concomitant increase in AT1-R expression in uterine artery endothelium. Endothelium-derived protein was directly removed from the lumenal surface of freshly isolated uterine and systemic (omental) arteries from nonpregnant and pregnant ewes. AT1-R expression was then measured in both the endothelium-derived fraction and endothelium-denuded vascular smooth muscle (VSM) fraction by Western analysis. AT1-R was detected as 54- and 65-kDa proteins in all samples, as well as adrenal cortex control. AT1-R expression increased more than 8-fold in uterine artery endothelium of pregnant ewes over that in nonpregnant ewes at each of four gestational ages (P < 0.05 at 110, 120, 130, 142 days, n = 4 each vs. n = 6 nonpregnant). No significant differences were seen, however, from 110 to 142 days of gestation. In contrast, whereas the level of AT1-R staining in omental artery endothelium in nonpregnant ewes was higher than in uterine artery, AT1-R increased less in pregnant ewes (2-fold) and only reached significance over nonpregnant values at 110 and 120 days, or when data was combined irrespective of gestational age (P < 0.05). Although AT1-R was also detected in uterine and omental artery VSM, little or no change in expression was observed in pregnancy. Results were confirmed by immunohistochemical staining of arterial cross sections, and the increase in AT1-R expression in uterine artery endothelium was confirmed by RT/PCR amplification of AT1-R messenger RNA from collagenase dispersed cells (n = 4 pregnant vs. n = 4 nonpregnant, mean 20-fold increase, P < 0.028). We conclude that increased uterine artery endothelial PGI2 responsiveness to AII during pregnancy is indeed associated with a correspondingly marked and localized increase in expression of the endothelial AT1-R receptor. We believe our findings allow a more detailed understanding of the molecular mechanisms that underlie increased uterine blood flow that is so central to the normal development of the growing fetus, and on dysfunction may lead to conditions such as preeclampsia.

Angiotensin II↗
PubMed1997

Influence of dietary sodium restriction on angiotensin II receptors in rat adrenals.

We studied the distribution of angiotensin II (AII) receptors type 1 (AT1) and type 2 (AT2) and the effects of a low sodium intake on these two subtypes of receptors in male rat adrenals. Binding studies on adrenal slices, on cell membranes and on cell suspensions were performed using [125I]AII and specific analogs for AT1 (Losartan) and AT2 (PD 123319) receptors. The distribution of AT1 was also studied by immunofluorescence. Complementary approaches were necessary to reach our goal. Indeed, by autoradiography on adrenal slices, [125I]AII was shown to bind to the zona glomerulosa (ZG) and to the medulla (M). When coincubated with [125I]AII, PD 123319 displaced [125I]AII from the medulla and from the ZG, indicating the presence of AT2 receptors in both zones. Losartan partially displaced [125I]AII from the ZG, indicating the presence of AT1 receptors in that zone. Furthermore, the labeling intensity of the medulla (AT2 receptors) was much stronger in adrenal sections from rats kept on a low sodium regimen than from controls. Immunofluorescence microscopy revealed that AT1 receptors were located mainly in the ZG of control rats. After sodium restriction, AT1 receptors appeared to be uniformly distributed within an enlarged ZG; furthermore AT1 receptor-positive cells were found to a limited degree in the zona fasciculata and possibly in the zona reticularis, and a greater number of these positive cells appeared in these zones under sodium restriction. Cell suspensions from rats fed a low sodium diet showed a 2.7- and 2.1-fold increase in total AII receptors in adrenal ZG and ZFR + M cells when compared with controls. Based on Losartan displacement, we calculated that [125I]AII bound to AT1 and to AT2 receptors was increased in both ZG and ZFR + M cell preparations under sodium restriction. Results of binding studies on cell membranes were also indicative of an increasing effect of sodium restriction on AT1 and AT2 receptors binding capacity. Furthermore, Northern blotting analysis revealed 3.0- and 2.5-fold increases in the level of AT1 receptor mRNA in the ZG and the ZFR + M of rats fed a low sodium diet as compared with those fed a normal diet. The low sodium intake resulted in a weaker increase (1.5-fold) in the level of AT2 receptor messenger RNA in the ZG, with no changes in the ZFR + M preparations. In conclusion, in this study complementary approaches were needed to determine the localization of AT1 and AT2 receptors in the rat adrenal, and to show the increasing effects of a low sodium regimen on the adrenal level of these receptors. Immunofluorescence studies revealed AT1 receptors mainly in the ZG and also in some cells of the inner adrenal cortex zones; in adrenals of rats kept on a low sodium diet the ZG was markedly enlarged, and an increased number of immunoreactive cells with AT1 receptors were observed throughout that zone; also more immunoreactive cells were present in the inner zones of the adrenal cortex. Furthermore in the adrenals of rats kept on a low sodium diet, we observed: 1) an increased number of AT1 and AT2 receptors in cell suspensions from the ZG, and in cell suspensions of the ZFR + M; 2) an increased level of AT1 and AT2 receptor mRNAs in the ZG; 3) an increased level of AT1 receptor mRNA, with no changes in the AT2 mRNA level in the ZFR + M. These results suggest a role for AT1 as well as for AT2 receptors in controlling adrenal function and differentiation under normal as well as under physiological stimulation of AII production following sodium restriction.

Adrenal Glands↗
PubMed1996

Differential regulation of 11 beta-hydroxylase and aldosterone synthase in human adrenocortical H295R cells.

In humans the last steps in the synthesis of aldosterone and cortisol rely on the activity of two cytochrome P450 genes termed CYP11B2 (aldosterone synthase; P450aldo) and CYP11B1 (11 beta hydroxylase; P450cl1). The mechanisms which lead to differential expression of these two genes within the adrenal cortex are not well-defined. The human adrenocortical cell line. H295R, was utilized in this study to examine the intracellular second messenger pathways regulating expression of P450aldo and P450c11. using specific ribonuclease protection assays. Treatment of H295R cells with angiotensin II or potassium (K+) caused a time-dependent induction in the level of P450aldo transcripts. While K+ treatment was more specific for the induction of P450aldo mRNA, treatment with angiotensin II increased levels of both P450aldo and P450c11 transcripts. To define the second messenger systems which influence transcript levels for these enzymes, the effects of agonists of the protein kinase A, protein kinase C, and calcium pathways were tested on the expression of P450aldo and P450c11. Activation of the protein kinase A pathway by the agonists, dibutyryl cAMP or forskolin, preferentially increased the P450c11 transcript to a greater degree than P450aldo. Interestingly, activation of the protein kinase C pathway by tetradecanoylphorbol acetate (TPA) did not alter transcripts for either P450aldo or P450c11. The calcium channel agonist BAYK 8644 mimicked the effects of K+ by increasing the transcript for P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo. However, the calcium channel blocker nifedipine attenuated the stimulatory effects of angiotensin II and K+ on the levels of P450aldo transcripts without affecting the stimulatory effect of dbcAMP. This study demonstrates that the protein kinase A pathway preferentially induces P450c11 mRNA over that of P450aldo. In addition, pharmacologic agents that affect calcium levels provide evidence for an additional regulatory mechanism in modulating the expression of P450aldo. This is of importance since the major physiologic regulators of aldosterone secretion, angiotensin II and K+ are able to increase intracellular calcium but have little effect on intracellular cAMP levels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Immunohistochemical analysis of AT1 receptor versus P450c17 and 3 beta HSD expression in ovine adrenals.

Previous studies in ovine adrenocortical cells in vitro have shown angiotensin II (AII) receptors are expressed on the zona fasciculata (ZF) cells and are functionally coupled to phosphoinositidase C and increased [Ca2+]i, but AII stimulation does not cause an acute change in cortisol biosynthesis. AII can, however, chronically regulate differential expression of P450c17 and 3 beta HSD in ovine adrenocortical cells in vitro. We have stained ovine adrenal sections with specific antisera to the angiotensin II Type-1 receptor (AT1-R), as well as P450c17 and 3 beta HSD in order to further test the hypothesis that changes in AT1-R expression underlie changes in zonal expression of P450c17 and 3 beta HSD in vivo. AT1-R expression was found to be highest in the outermost layer of cells (zona glomerulosa, ZG) which stained negatively for P450c17 and only faintly positive for 3 beta HSD, as expected. The adjacent layer of cells (ZF) stained much less strongly for AT1-R but stronger for P450c17 and 3 beta HSD. These findings are consistent with our previously reported in vitro expression data, and suggest that the transition from ZG to ZF phenotype, i.e. increased P450c17 and 3 beta HSD expression, may require reduced expression of AT1-R, but maintenance of reduced levels of AT1-R expression in the ovine ZF still allows for differential control of the P450c17: 3 beta HSD ratio. Thus, even though there is no acute cortisol response to AII alone in these cells, AII stimulation can oppose C19 steroid production in the face of cortisol biosynthesis by the ZF in response to agonists such as ACTH.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Human ovarian tumor cells: a potential model for thecal cell steroidogenesis.

Ovarian thecal cell production of C19 steroids (i.e. dehydroepiandrosterone, androstenedione, and testosterone) is necessary to provide substrate for granulosa cell biosynthesis of estrogen; however, excessive production of C19 steroids can lead to disorders associated with androgen excess. Because of difficulties in obtaining adequate numbers of thecal cells, the biomolecular regulation of C19 steroid production and expression of steroidogenic enzymes is not well defined. We have overcome this obstacle by developing a highly dependable and unique human ovarian thecal-like tumor (HOTT) cell culture model system from an ovarian tumor found to produce excessive amounts of C19 steroids. Aliquots of freshly dispersed tumor cells were frozen for future use. Once placed in monolayer culture, HOTT cells proliferated and could be maintained for extended periods. Acutely, cultured HOTT cells increased progesterone and cAMP production in response to 2 h of forskolin treatment. These cells were, however, unresponsive to treatment with LH. Steroid hormone production continued in cells that were maintained in culture for up to 2 months. Analysis of the steroids produced by HOTT cells was accomplished using RIA and high performance liquid chromatography. Under basal conditions, HOTT cells produced mainly 17 alpha-hydroxyprogesterone and progesterone. Treatment with forskolin or dibutyryl cAMP (dbcAMP) increased the production of progesterone and 17 alpha-hydroxyprogesterone as well as C19 steroids. Treatment of monolayer cultures of HOTT cells with forskolin (0.01 to 20 mumol/L) or dbcAMP (0.01 to 1 mmol/L) for 48 h increased the production of androstenedione (8- to 15-fold) and progesterone (2- to 5-fold). In HOTT cells chronically treated with forskolin or dbcAMP (up to 72 h), progesterone production was observed to plateau, although the amount of androstenedione continued to increase. The enzymatic activities of both 3 beta-hydroxysteroid dehydrogenase (6-fold), and 17 alpha-hydroxylase P450 (P450c17; 9-fold) were also increased by activation of the protein kinase A messenger pathway. Treatment of HOTT cells with forskolin caused a time-dependent induction of the messenger RNAs for cholesterol side-chain cleavage P450, 3 beta-hydroxysteroid dehydrogenase, and P450c17. No changes in steroidogenic enzyme expression were observed following treatment with LH. In conclusion, these data demonstrate that certain ovarian tumor cells may serve well as appropriate models to study the molecular mechanisms regulating human ovarian thecal cell C19 steroidogenesis and the expression of steroid-metabolizing enzymes.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Differential control of 17 alpha-hydroxylase and 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of human adrenocortical cells have given inconsistent findings concerning the effects of angiotensin II (AII) alone or in combination with activators of the protein kinase A-signaling pathway on expression of cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450c17), and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD), as well as the corresponding effects on adrenocortical cell steroid secretory products. We have used the human adrenocortical carcinoma H295R cell to evaluate further this question and determine the role of protein kinase C in each of these responses to AII. Treatment with AII alone (10 nmol/L, 48 h) resulted in a significant increase in cortisol production (1.8-fold), as well as a much greater effect on aldosterone production. This increased formation of 17 alpha-hydroxysteroids was accompanied by increased expression of P450c17 as determined at the level of messenger RNA (mRNA) and enzyme activity. Similar increases in expression of P450scc were observed at the level of mRNA. Increases in 3 beta-HSD expression were also seen at the level of mRNA and, to a lesser extent, at the level of enzyme activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activity of H295R cells, however, the overall effect of AII treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. Whereas treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of AII on 3 beta-HSD expression, TPA failed to reproduce the effects of AII on P450c17 and P450scc and even resulted in a marked decrease in expression of P450c17. Thus, the stimulatory effect of AII alone on P450c17 expression was not mediated via protein kinase C but, like the action of K+, was probably mediated via the Ca(2+)-signaling pathway. Treatment with forskolin (10 mumol/L, 48 h) resulted in a dramatic increase in both cortisol and dehydroepiandrosterone production together with increases in expression of P450c17, P450scc, and 3 beta-HSD as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on 17 alpha-hydroxylase expression was greater than that on 3 beta-HSD at the level of enzyme activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and AII, however, resulted in a dose-dependent reduction in cortisol and DHEA production concomitant with a marked attenuation of P450scc and P450c17 expression. Although forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with AII, additivity was observed at the level of changes in enzyme activity. Thus, AII cotreatment resulted in a marked reduction of the forskolin-induced increase in 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so, 17 alpha-hydroxysteroid synthesis was attenuated. These effects of AII cotreatment on expression of P450c17 and P450scc were reproduced by cotreatment with TPA (10 nmol/L), suggesting the involvement of protein kinase C in these attenuative responses. Furthermore, the effect of AII cotreatment on changes in forskolin-induced 17 alpha-hydroxylase and 3 beta-HSD activities were blocked by the AII Type 1 (AT1) receptor antagonist DuP753 (Losartan), confirming the involvement of an AT1 receptor-linked phospholipase C in activating protein kinase C.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Inhibin and activin differentially regulate androgen production and 17 alpha-hydroxylase expression in human ovarian thecal-like tumor cells.

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-beta superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) and 17 alpha-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 microM) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3 beta HSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3 beta HSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1.5-fold) and activity (twofold) but with minimal effects on the mRNA for 3 beta HSD and P450scc. HOTT cell responses to activin (0.05-50 ng/ml) and inhibin (0.05-50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome.

3-Hydroxysteroid Dehydrogenases↗
PubMed1996

Regulation of 3 beta-hydroxysteroid dehydrogenase expression in human adrenocortical H295R cells.

Previous studies of the effects of angiotensin II (All), alone or in combination with activators of the protein kinase. A signalling pathway, have yielded inconsistent findings on the expression of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD and 17 alpha-hydroxylase cytochrome P450 (P450c17) as well as the corresponding responses on steroid secretory products in human adrenocortical cells. We have used the human adrenocortical carcinoma H295R cell further to evaluate this question, as well as to determine the role of protein kinase C in each of these responses to All. Treatment with All alone resulted in a marked increase in aldosterone secretion and a significant increase in cortisol secretion (1-8-fold). The increased formation of 17-hydroxysteroids was accompanied by an increased level of P450c17 mRNA and activity. Increases in 3 beta-HSD expression were also seen at the level of mRNA and to a lesser extent, at the level of activity. Because of the comparatively low basal 17 alpha-hydroxylase and high basal 3 beta-HSD activities of H295R cells, however, the overall effect of All treatment was actually a rise in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, so resulting in increased formation of 17 alpha-hydroxysteroids such as cortisol. While treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) reproduced the effect of All on 3 beta-HSD expression, TPA failed to reproduce the effects of All on P450c17 because it caused a marked decrease in P450c17 expression. Thus the stimulatory effect of All on P450c17 expression, unlike that on 3 beta-HSD expression, was not mediated by protein kinase C but, like the action of K, was probably mediated via the Ca2+ signalling pathway. Treatment with forskolin resulted in a dramatic increase in both cortisol and dehydroepiandrosterone (DHEA) secretion together with increases in expression of 3 beta-HSD and P450c17 as measured at the level of mRNA and activity. Consistent with the increase in 17 alpha-hydroxysteroid formation, the effect on P450c17 expression was greater than that on 3 beta-HSD at the level of activity, so a larger 17 alpha-hydroxylase/3 beta-HSD activity ratio was achieved. Cotreatment with forskolin and All, however, resulted in a dose-dependent reduction in cortisol and DHEA secretion concomitant with a marked attenuation of 3 beta-HSD and P450c17 expression. While forskolin-induced expression of 3 beta-HSD was not further increased at the level of mRNA by cotreatment with All, additivity was observed as the level of activity changed. Thus All cotreatment resulted in a marked reduction in the forskolin-induced increase in the 17 alpha-hydroxylase/3 beta-HSD activity ratio, and so 17 alpha-hydroxysteroid synthesis was attenuated. The effect of All cotreatment on changes in forskolin-induced 3 beta-HSD activity was blocked by the All type 1 (AT1) antagonist DuP753 (Losartan), confirming the involvement of the AT1 receptor-linked phospholipase C in activating protein kinase C.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Adrenal androgen biosynthesis with special attention to P450c17.

In vitro studies of human adrenal androgen synthesis are limited because of the difficulties in obtaining adrenals. We describe the use of the human adrenocortical tumor H295 cell line as a model to evaluate mechanisms controlling C19-steroid production. The cells were characterized with regard to responsiveness to a variety of agents as measured by steroid secretion and induction of 17 alpha-hydroxylase cytochrome P450 (P450c17) expression, a key enzyme in C19-steroid production. Forskolin and dibutyryl cAMP, which were more effective than ACTH, enhanced the production of DHEA and androstenedione over a 48-hour treatment period. Agents that act by increasing intracellular calcium (angiotensin II and K+ ions) as well as protein kinase A pathway activators (ACTH, forskolin, and dibutyryl cAMP) individually increased the mRNA levels and activity of P450c17. In addition, angiotensin II but not K+ ions attenuated the increased expression promoted by the kinase A agonists. Thus, the complexity of human adrenal P450c17 expression through multiple signaling pathways may contribute importantly to the diverse patterns of human adrenocortical steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Potassium negatively regulates angiotensin II type 1 receptor expression in human adrenocortical H295R cells.

We have previously shown that the human adrenocortical H295R cell line expresses the type 1 angiotensin II receptor (AT1-R) and that expression of this receptor is downregulated at the level of mRNA by forskolin or dibutyryl-cAMP as well as by angiotensin II (Ang II). In this study we examine the effects of K+ on both AT1-R mRNA and receptors, as monitored through 125I-Ang II binding in the presence of PD 123319. After treatment with a maximal stimulatory steroidogenic dose of K+ (14 mmol/L), H295R cells showed an increase in cytosolic free Ca2+ from 113 to 212 nmol/L. Unlike the effects of Ang II, this increase could be abolished by pretreatment with the Ca2+ channel antagonist nifedipine (1 mumol/L). AT1-R mRNA levels also fell in response to elevated extracellular K+ in a dose-dependent (Kd, 9 mmol/L; maximal fall in message at 12 mmol/L) and time-dependent (maximum 50% at 12 hours) manner. The change in AT1-R mRNA level was less rapid than that in response to activation of phosphoinositidase C by Ang II or adenylyl cyclase by forskolin or by dibutyryl-cAMP. Unlike the action of Ang II but similar to the action of forskolin or dibutyryl-cAMP, the action of K+ was sustained. Changes in mRNA level in response to treatment with K+, Ang II, or dibutyryl-cAMP were also paralleled by changes in 125I-Ang II binding in each case.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex↗
PubMed1995

Ca(2+)-regulated expression of steroid hydroxylases in H295R human adrenocortical cells.

Although changes in the expression of key steroidogenic enzymes such as cytochrome P450 cholesterol side-chain cleavage, 17 alpha-hydroxylase (P450c17), aldosterone synthase, and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the human adrenal cortex are known to be controlled by factors activating the protein kinase A or protein kinase C signaling pathways, little is known concerning the effects of increased intracellular Ca2+. In this study we describe the effects of K+, an agent known to increase intracellular Ca2+ through the opening of voltage-sensitive Ca2+ channels, on steroidogenesis in H295R human adrenocortical cells and corresponding changes in expression of these vital steroidogenic enzymes. Treatment of cells for 48 h with K+ (14 mM) resulted in an increase in aldosterone (3.5-fold) as well as the 17 alpha-hydroxylated steroids cortisol (2.9-fold) and dehydroepiandrosterone (DHEA; 3.7-fold). This action of K+ was accompanied by a dose-dependent (P < 0.05 at 6 mM K+ or above) and time-dependent (P < 0.05 at 24 h and beyond) increase in expression of P450c17 and, to a lesser extent, cytochrome P450 cholesterol side-chain cleavage messenger RNA (mRNA). Treatment with K+ also caused a time-dependent increase in aldosterone synthase mRNA levels, which were detectable by 12 h. Treatment with K+, however, was without effect on 3 beta HSD expression. These effects contrast with those of (Bu)2cAMP, which stimulated a greater increase in cortisol and DHEA secretion as well as P450c17 expression. The effects of K+ treatment also differ from those of AII, which promoted a greater aldosterone secretory response (5.7-fold), but a lesser effect on DHEA secretion (2.2-fold) and P450c17 expression. Although AII and TPA (known activators of protein kinase C) as well as forskolin and (Bu)2cAMP (known activators of protein kinase A) increased the expression of 3 beta HSD mRNA, K+ treatment was without effect, suggesting that elevation of [Ca2+]i in response to K+ did not activate the protein kinase C or protein kinase A signaling pathways. Furthermore, the effects of K+ on steroid secretion and 17 alpha-hydroxylase activity were reproduced by the voltage-sensitive Ca2+ channel activator BAYK 8644, and increases in P450c17 mRNA in response to K+ were reversed by the Ca2+ channel antagonist, nifedipine. We conclude that K+ can modulate the expression of key steroidogenic enzymes in H295R cells through the Ca2+ signaling pathway without involvement of the protein kinase A or protein kinase C pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines.

Corticotropin (ACTH) binds to specific receptors in the adrenal cortex and thereby regulates glucocorticoid and mineralocorticoid production. The number of ACTH binding sites on adrenocortical cells is increased by exposure of cells to activators of the cAMP pathway. The mechanism responsible for the increase in ACTH binding sites is not known. We therefore studied the levels of ACTH-R mRNA in mouse Y-1 and human NCI-H295 (H295) adrenocortical carcinoma cell lines. ACTH induced an increase in mouse ACTH-R mRNA in Y-1 cells that was time and dose dependent, increasing 6-fold over basal levels following exposure to 10(-8) M ACTH for 19-24 h. The amount of human ACTH-R mRNA in H295 cells increased 2-4-fold following a 24 h exposure to 10(-8) M ACTH, 1 mM dbcAMP, or 10(-5) M Forskolin. Treatment of H295 cells with angiotensin II (A-II) was found to dramatically increase the level of ACTH-R mRNA. These data indicate that regulation of ACTH-R mRNA levels is at least one mechanism by which ACTH and A-II elevate the number of ACTH binding sites in the adrenocortical cells.

Adrenal Cortex↗
PubMed1994

The NCI-H295 cell line: a pluripotent model for human adrenocortical studies.

The human adrenal cortex is a complex endocrine organ that secretes mineralocorticoids, glucocorticoids and adrenal androgens. These steroids arise from morphologically and biochemically distinct zones of the adrenal gland. Studying secretion of these distinct steroid hormones has, in the past, required the isolation of cells from each of the adrenocortical zones. Indeed, the lack of a human adrenocortical cell line retaining the ability to produce any of the major adrenal steroid products has slowed studies on normal and abnormal adrenal function. This obstacle has now been largely overcome with the availability of H295 cells, which represents the first adrenocortical cell line to maintain the ability, under specified conditions, to produce all the adrenocortical steroids (i.e., mineralocorticoids, glucocorticoids, and adrenal androgens). Thus, H295 cells appear to act as pluripotent adrenocortical cells capable of being directed to produce each of the zone-specific steroids. The H295 cell line should prove to be of value in studying the molecular and biochemical mechanisms controlling adrenal steroidogenesis.

3-Hydroxysteroid Dehydrogenases↗
PubMed1994

Both low sodium and high potassium intake increase the level of adrenal angiotensin-II receptor type 1, but not that of adrenocorticotropin receptor.

Angiotensin-II (AII), a component of the renin-angiotensin system, is the major factor that regulates the formation of aldosterone in the adrenal cortex zona glomerulosa (ZG). The activity of this system is increased by an increase in potassium intake or a decrease in sodium intake. Using immunoblotting analysis, we determined whether these ions affect the expression of type 1 AII receptors (AT1) and compared the results thus obtained with the AT1 receptor mRNA levels. We also studied the interrelation among AII, AT1 receptors, cytochrome P450 aldosterone synthase (P450c18), and plasma aldosterone levels in rats fed a normal diet or a low sodium or high potassium diet with or without captopril, an inhibitor of angiotensin-converting enzyme, for 7 days. The effects of ions on the level of ACTH receptor mRNA were also analyzed. We found that a low sodium intake increased plasma aldosterone levels from 5.5 to 236 ng/dl and led to 2.3- and 3.7-fold increases in the levels of adrenal ZG AT1 receptor protein and AT1 receptor mRNA, whereas a 11.8-fold increase was found in the level of P450c18 mRNA. Captopril almost completely reversed these effects. We have shown that a high potassium intake increased plasma aldosterone levels to 25.9 ng/dl and also led to 1.84- and 1.95-fold increases in the level of ZG AT1 receptor protein and AT1 receptor mRNA, whereas the ZG P450c18 mRNA level was increased 3.5-fold. The plasma aldosterone level of animals fed a high diet of potassium and captopril was still higher than that in control animals at 16.6 ng/dl, and the levels of ZG AT1 receptor and P450c18 mRNAs were only slightly less than those of the high potassium groups, indicating that captopril did not efficiently block aldosterone formation under these conditions. ACTH receptor mRNA levels remain unaffected by either low sodium or high potassium intake. Collectively, these results indicate that the increased aldosterone secretion induced by low sodium or high potassium intake involves concomitant increases in AT1 receptor and P450c18 mRNAs, which are effectively translated into their respective proteins, and that the expression of both proteins is mediated in part by AII.

Aldosterone↗
PubMed1994

Regulation of type 1 angiotensin II receptor messenger ribonucleic acid expression in human adrenocortical carcinoma H295 cells.

We have studied the hormonal regulation of type 1 angiotensin-II receptor (AT1-R) mRNA expression and [125I]angiotensin-II ([125I]AII) binding in human adrenocortical carcinoma H295 cells, which exhibit predominantly AT1-subtype receptors. Activation of the cAMP signaling pathway with forskolin or (Bu)2cAMP caused a rapid decrease in AT1-R mRNA levels (decreased 65% within 3 h). This preceded a time-dependent (maximal, 70% within 12 h) and dose-dependent (IC50, 2 microM forskolin) loss of [125I]AII binding together with decreased phosphoinositidase-C activation (72% decrease) on subsequent AII challenge. Thus, the decreases in AT1-R mRNA levels and functional receptor expression parallel each other in response to activation of protein kinase-A. AII treatment also caused a rapid loss in AT1-R mRNA (maximal, 80% decrease within 3 h), but 48-h treatment caused both [125I]AII binding and the subsequent phosphoinositidase-C response to decrease by only 6% (P < 0.05) and 22% (P < 0.05), respectively. The effect of AII on AT1-R mRNA levels was fully reproduced by the combination of calcium ionophore (A23187) and phorbol ester (12-O-tetradecanoylphorbol 13-acetate), suggesting that AII action was through protein kinase-C and possibly other Ca(2+)-sensitive protein kinases. The effect of AII, but not forskolin, was reversed by treatment in the presence of cycloheximide. In conclusion, control of AT1-R expression is differentially regulated by adenylate cyclase and phosphoinositidase-C signaling pathways, which act at multiple levels in human adrenocortical cells.

Adrenal Cortex Neoplasms↗