cGMP-dependent Protein Kinase Type II Regulates Basal Level of Aldosterone Production by Zona Glomerulosa Cells without Increasing Expression of the Steroidogenic Acute Regulatory Protein Gene

There is considerable evidence supporting the role of calcium signaling in adrenal regulation of both aldosterone synthase (CYP11B2) and aldosterone production. However, there have been no studies that investigated the role played by the Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) in adrenal cells. In this study we investigated the role of CaMKK in adrenal cell aldosterone production. To determine the role of CaMKK, we used a selective CaMKK inhibitor (STO-609) in the HAC15 human adrenal cell line. Cells were treated with angiotensin II (Ang II) or K+ and evaluated for the expression of steroidogenic acute regulatory protein and CYP11B2 (mRNA/protein) as well as aldosterone production. We also transduced HAC15 cells with lentiviral short hairpin RNAs of CaMKK1 and CaMKK2 to determine which CaMKK plays a more important role in adrenal cell regulation of the calcium signaling cascade. The CaMKK inhibitor, STO-609, decreased aldosterone production in cells treated with Ang II or K+ in a dose-dependent manner. STO-609 (20μM) also inhibited steroidogenic acute regulatory protein and CYP11B2 mRNA/protein induction. CaMKK2 knockdown cells showed significant reduction of CYP11B2 mRNA induction and aldosterone production in cells treated with Ang II, although there was no obvious effect in CaMKK1 knockdown cells. In immunohistochemical analysis, CaMKK2 protein was highly expressed in human adrenal zona glomerulosa with lower expression in the zona fasciculata. In conclusion, the present study suggests that CaMKK2 plays a pivotal role in the calcium signaling cascade regulating adrenal aldosterone production.

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Roles of type I and type II isoenzymes of cyclic AMP-dependent protein kinase in steroidogenesis in rat adrenal cells

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0120195 ◽

1994 ◽

Vol 12 (2) ◽

pp. 195-202 ◽

Cited By ~ 8

Author(s):

B J Whitehouse ◽

D R E Abayasekara

Keyword(s):

Cyclic Amp ◽

Cell Function ◽

Zona Glomerulosa ◽

Zona Fasciculata ◽

Type I ◽

Type Ii ◽

Aldosterone Production ◽

Dependent Protein ◽

Glomerulosa Cells ◽

Zona Glomerulosa Cells

ABSTRACT The role played by cyclic AMP (cAMP)-dependent protein kinases (PKAs) in rat adrenal steroidogenesis has been investigated using cAMP analogues which show partial selectivity for the type I and type II PKA isoenzymes. These were aminohexylamino-cAMP (AHA-cAMP; selective for site 1 on type I PKA), N6-benzoyl-cAMP (BZ-cAMP; selective for site 2 on PKA types I and II) and 8-thiomethyl-cAMP (TM-cAMP; selective for site 1 on type II PKA). Positive cooperativity exists between the two nucleotide-binding sites, thus the presence of type I PKA was inferred when synergistic increases in corticosteroid production were obtained with AHA-cAMP plus BZ-cAMP and that of type II PKA when synergistic increases were obtained with TM-cAMP plus BZ-cAMP. The effects of AHA-cAMP, TM-cAMP and BZ-cAMP (10–100 μmol/l) on aldosterone production by glomerulosa cell preparations and corticosterone production by fasciculata/reticularis cell preparations were compared. Dose-related stimulation of steroid production was obtained with each cAMP analogue in both types of cell preparation. Experiments were performed using the cAMP analogues in combination at doses which gave minimal stimulation individually. Cells were incubated with AHA-cAMP (66 and 100 μmol/l) or TM-cAMP (15, 30 and 45 μmol/l) in the presence and absence of 15μmol BZ-cAMP/l. Synergistic responses were obtained with both analogue pairs in both cell types. The synergism ratio in fasciculata/reticularis cell preparations for the type I PKA selective pair of analogues (100 μmol AHA-cAMP/l plus 15μmol BZ-cAMP/l) was significantly higher (P<0·01) than that for the type II selective pair (45μmol TM-cAMP/l plus 15μmol BZ-cAMP/l; 7·9±1·2 (mean±s.e.m.) and 2·6±0·3 respectively). In zona glomerulosa preparations the ratio was higher (P<0·05) for the type II selective pair (1·6±0·1 for AHA-cAMP plus BZ-cAMP and 2·8±0·4 for TM-cAMP plus BZ-cAMP). The effects of 100μmol AHA-cAMP/l and 45μmol TM-cAMP/l on the response to ACTH (1 pmol/l–10 nmol/l) were examined. Synergistic responses were obtained in fasciculata/reticularis cells with both analogues in combination with low concentrations of ACTH (10 and 100 pmol/l). In zona glomerulosa cells only the addition of TM-cAMP (45 μmol/l) in combination with 10 pmol ACTH/1 gave rise to synergistic increases in aldosterone production, which suggests that there may be some compartmentalization of the cAMP-dependent pathway in these cells. The results indicate that both isoenzymes of PKA are present in rat adrenocortical cells and can play a part in the control of steroidogenesis. Type I PKA activity appears dominant in the control of zona fasciculata/reticularis cell function whereas modulation of type II PKA activity plays a more significant role in the responses of zona glomerulosa cells.

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effects of insulin on aldosterone production in rat zona glomerulosa cells

Life Sciences ◽

10.1016/0024-3205(92)90062-t ◽

1992 ◽

Vol 50 (23) ◽

pp. 1781-1787 ◽

Cited By ~ 23

Author(s):

Dan Petrasek ◽

Gerard Jensen ◽

Michael Tuck ◽

Naftali Stern

Keyword(s):

Zona Glomerulosa ◽

Aldosterone Production ◽

Glomerulosa Cells ◽

Zona Glomerulosa Cells

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Inhibition of aldosterone release by hypoxia in vitro: interaction with carbon monoxide

Journal of Applied Physiology ◽

10.1152/jappl.1994.76.2.689 ◽

1994 ◽

Vol 76 (2) ◽

pp. 689-693 ◽

Cited By ~ 3

Author(s):

H. Raff ◽

B. Jankowski

Keyword(s):

Carbon Monoxide ◽

Angiotensin Ii ◽

Zona Glomerulosa ◽

Michaelis Constant ◽

Aldosterone Synthase ◽

Aldosterone Production ◽

Glomerulosa Cells ◽

In Vitro Interaction ◽

Zona Glomerulosa Cells

We have demonstrated that the aldosteronogenic pathway of the zona glomerulosa is unusually sensitive to modest changes in PO2 (Michaelis constant for O2 approximately 95 Torr). The current study evaluated the interaction of CO (the classic ligand for P-450 enzymes) and the decreases in O2 on aldosteronogenesis in vitro. Bovine adrenocortical zona glomerulosa cells were incubated for 2 h and stimulated with either adenosine 3′,5′-cyclic monophosphate (cAMP) or angiotensin II. Ten and 20% CO led to significant decreases in cAMP- and angiotensin II-stimulated aldosteronogenesis. The combination of 20% CO and moderate decreases in PO2 (from approximately 140 to approximately 100 Torr) led to an interactive decrease in aldosterone production. The conversion of corticosterone to aldosterone catalyzed by aldosterone synthase, which is the site of O2 sensitivity, was not significantly inhibited by CO. We conclude that the aldosterone pathway is not exceptionally sensitive to CO compared with other steroidogenic pathways. This observation suggests that the unique O2-sensitive properties of the aldosterone pathway located primarily within aldosterone synthase may not reside in its CO binding site (i.e., heme).

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THE EFFECT OF SEROTONIN AND POTASSIUM ON CORTICOSTERONE AND ALDOSTERONE PRODUCTION BY ISOLATED ZONA GLOMERULOSA CELLS OF THE RAT ADRENAL CORTEX

Australian Journal of Experimental Biology and Medical Science ◽

10.1038/icb.1972.81 ◽

1972 ◽

Vol 50 ◽

pp. 833-846 ◽

Cited By ~ 35

Author(s):

Sylvia AS Tait ◽

JF Tait ◽

JES Bradley

Keyword(s):

Adrenal Cortex ◽

Zona Glomerulosa ◽

Aldosterone Production ◽

Glomerulosa Cells ◽

Zona Glomerulosa Cells

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Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells

Life Sciences ◽

10.1016/0024-3205(92)90518-t ◽

1992 ◽

Vol 51 (14) ◽

pp. 1157-1163

Author(s):

Doris Kurscheid-Reich ◽

Lutz Hegemann ◽

Stefan Wohlfeil

Keyword(s):

Protein Kinase ◽

Kinase Inhibitors ◽

Zona Glomerulosa ◽

Protein Kinase Inhibitors ◽

Aldosterone Production ◽

Glomerulosa Cells ◽

Zona Glomerulosa Cells

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O2 dependence of pregnenolone and aldosterone synthesis in mitochondria from bovine zona glomerulosa cells

Journal of Applied Physiology ◽

10.1152/jappl.1995.78.5.1625 ◽

1995 ◽

Vol 78 (5) ◽

pp. 1625-1628 ◽

Cited By ~ 9

Author(s):

H. Raff ◽

B. Jankowski

Keyword(s):

Energy Production ◽

Zona Glomerulosa ◽

Mitochondrial Enzymes ◽

Isolated Mitochondria ◽

Aldosterone Production ◽

Glomerulosa Cells ◽

Air Control ◽

Electron Transport Proteins ◽

Zona Glomerulosa Cells

Hypoxia in vivo results in a decrease in aldosterone not accounted for by extra-adrenal controllers. We have demonstrated that aldosteronogenesis but not cortisol synthesis in the whole cell is O2 sensitive. In the intact glomerulosa cell, this sensitivity is located in the late pathway step catalyzed by conversion of corticosterone to aldosterone (P-450aldo), whereas the early pathway catalyzed by conversion of cholesterol to pregnenolone (P-450scc) is not inhibited until PO2 is very low. Because P-450aldo and P-450scc are mitochondrial enzymes that depend on the same NADPH-specific electron transport proteins, we hypothesized that O2 sensitivity would be independent of energy production and expressed in isolated mitochondria. We measured the conversion of exogenous 25(OH)-cholesterol to pregnenolone and of exogenous corticosterone to aldosterone in the presence of cyanoketone in mitochondria isolated from bovine zona glomerulosa cells and exposed to an experimental gas (1–100% O2) vs. a room air control. Pregnenolone production was not affected until PO2 was < 35 Torr and decreased to almost nil when PO2 was < 30 Torr. In contrast, aldosterone production increased under hyperoxia and decreased under moderate decreases in O2. The conversion of corticosterone to aldosterone was maintained at approximately 50% of control, even when PO2 was < 20 Torr. The sensitivity of the aldosterone pathway to changes in O2 within the physiological range appears to reside in the mitochondrial late pathway (i.e., P-450aldo) and is not significantly influenced by cytosolic regulators of steroidogenesis or by limitation of reducing equivalents.

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