Pregnenolone

About: Pregnenolone is a research topic. Over the lifetime, 3539 publications have been published within this topic receiving 126444 citations. The topic is also known as: (3b)-3-hydroxy-Pregn-5-en-20-one & 3-Hydroxypregn-5-en-20-one.

Angiotensin II Activates Cholesterol Ester Hydrolase in Bovine Adrenal Glomerulosa Cells through Phosphorylation Mediated by p42/p44 Mitogen-Activated Protein Kinase

Abstract: In adrenal glomerulosa cells, the stimulation of aldosterone biosynthesis by angiotensin II (Ang II) occurs via activation of the Ca2+ messenger system, increased expression of the steroidogenic acute regulatory protein, and enhanced transfer of cholesterol to the inner mitochondrial membrane. We examined here whether Ang II affects the activity of cholesterol ester hydrolase (CEH), also named hormone-sensitive lipase, the enzyme recruiting cholesterol from intracellular pools, in bovine adrenal glomerulosa cells. In bovine adrenal tissue, CEH activity was detected with characteristics similar to those reported in other tissues (Michaelis constant = 46.3 +/- 6.7 microM, n = 3; maximal velocity = 1 nmol/mg.min). This activity was significantly enhanced in isolated bovine glomerulosa cells challenged for 2 h with 10 nM Ang II (to 149 +/- 11% of controls, n = 3). Similarly, 25 microM forskolin raised CEH activity to 151 +/- 5% of controls (n = 3). This increase in activity of CEH was not due to an increase in the amount of enzyme protein but was associated with an increased phosphorylation of the enzyme to 337 +/- 33% of controls (n = 9, P < 0.0001). Potassium ion (K+) and forskolin also stimulated [32P]orthophosphate incorporation, although to a lesser extent (to 157 +/- 18% and 186 +/- 25% of controls, respectively). On SDS-PAGE, the majority of this radioactivity was associated with a species of 172 kDa, corresponding to a CEH dimer. Both Ang II-induced CEH phosphorylation and pregnenolone production were significantly reduced (to 47 +/- 6% and 50 +/- 8% of controls with Ang II alone, respectively) in the presence of PD098059, an inhibitor of p42/p44 MAPK. Indeed, Ang II challenge led to a rapid 32P incorporation into p42/p44 MAPK. These results demonstrate that, in addition to its known effects on intramitochondrial cholesterol transfer, Ang II also promotes aldosterone biosynthesis by rapidly increasing cholesterol supply to the outer mitochondrial membrane.

Atrial natriuretic peptide inhibits calcium-induced steroidogenic acute regulatory protein gene transcription in adrenal glomerulosa cells.

Abstract: Atrial natriuretic peptide (ANP) is a potent inhibitor of mineralocorticoid synthesis induced in adrenal glomerulosa cells by physiological agonists activating the calcium messenger system, such as angiotensin II (Ang II) and potassium ion (K+). While the role of calcium in mediating Ang II- and K(+)-induced aldosterone production is clearly established, the mechanisms leading to blockade of this steroidogenic response by ANP remain obscure. We have used bovine adrenal zona glomerulosa cells in primary culture, in which an activation of the calcium messenger system was mimicked by a 2-h exposure to an intracellular high-calcium clamp. The effect of ANP was studied on the following parameters of the steroidogenic pathway: 1) pregnenolone and aldosterone production; 2) changes in cytosolic ([Ca2+]c) and mitochondrial ([Ca2+]m) Ca2+ concentrations, as assessed with targeted recombinant aequorin; 3) cholesterol content in outer mitochondrial membranes (OM), contact sites (CS), and inner membranes (IM); 4) steroidogenic acute regulatory (StAR) protein import into mitochondria by Western blot analysis; 5) StAR protein synthesis, as determined by [35S]methionine incorporation, immunoprecipitation, and SDS-PAGE; 6) StAR mRNA levels by Northern blot analysis with a StAR cDNA; 7) StAR gene transcription by nuclear run-on analysis. While clamping Ca2+ at 950 nM raised pregnenolone output 3.5-fold and aldosterone output 3-fold, ANP prevented these responses with an IC50 of 1 nM and a maximal effect of 90% inhibition at 10 nM. In contrast, ANP did not affect the [Ca2+]c or [Ca2+]m changes occurring under Ca2+ clamp or Ang II stimulation in glomerulosa cells. The accumulation of cholesterol content in CS (139.7 +/- 10.7% of control) observed under high-Ca2+ clamp was prevented by 10 nM ANP (92.4 +/- 4% of control). Similarly, while Ca2+ induced a marked accumulation of StAR protein in mitochondria of glomerulosa cells to 218 +/- 44% (n = 3) of controls, the presence of ANP led to a blockade of StAR protein mitochondrial import (113.3 +/- 15.0%). This effect was due to a complete suppression of the increased [35S]methionine incorporation into StAR protein that occurred under Ca2+ clamp (94.5 +/- 12.8% vs. 167.5 +/- 17.3%, n = 3). Furthermore, while the high-Ca2+ clamp significantly increased StAR mRNA levels to 188.5 +/- 8.4 of controls (n = 4), ANP completely prevented this response. Nuclear run-on analysis showed that increases in intracellular Ca2+ resulted in transcriptional induction of the StAR gene and that ANP inhibited this process. These results demonstrate that Ca2+ exerts a transcriptional control on StAR protein expression and that ANP appears to elicit its inhibitory effect on aldosterone biosynthesis by acting as a negative physiological regulator of StAR gene expression.