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Effect of rat plasma lipoproteins on aldosterone production by rat zona glomerulosa cells in vitro
Short communication
710
*
t<+-stimulated
40’
* 0
400
Likw%n %&l*:t&l Concenttation (rtg per ml)
100
200
MO
400
t
‘00
Lipoprotein Cholesterol Concentration (ug per ml)
Fig. 1. EtTect of rat lipoproteins on aldosterone production by isolated rat zona glomerulosa cells incubated in KRBGA with O.Z:d BSA. Pooled data from two separate experiments with duplicate incubations presented as percent change from mean control for each experiment. Each point represents the mean *SE. n = 4. Upper panel, K’-stimulated steroidogenesis ([K*] = 8.6 mM): lower panel, basal steroidogenesis ([K’] ;r: 3.8 mM). Basal aldosterone production of control incubations for each experiment (ng per IO6 cells per h. mean & range of duplicate incubations): 23.2 f 3.6; 50.7 + 1.9. K’-stimulated aldosterone production of control incubations: 153 f 16; 242 + 13. *+ P e 0.05; **, P < 0.01, compared with control incubations.
Fig. 2. Eikct of rat lipoproteins on aldosterone production by isolated rat zona glomerulosa cells incubated in KRBGA with 4”,‘, BSA. Pooled data from three separate experiments with dupli~te incubations presented as percent change from mean control for each experiment. Each point represents the mean k SE, n given in parenthesis for each point. Upper panel, K+-stimulated steroidogenesis ([K’] = 8.6mM); lower panel. basal steroidogenesis ([K ‘1 = 3.8 mM). Basal aldosterone production of control intuitions for each experiment (ng per t06 cells per h, mean f range of duplicate incubations): 36.9 f 0.3; 13.8 f 0.1; 4.7 f 0.3. K+-stimulated aldosterone production of control incubations: 183 f 5; 45 + 6; 54 & 2. *, P c 0.05; **. P c 0.01, compared with control incubations.
medium (Fig, 2). LDL had no effect on basal aldoster-
during a 2 h incubation. This effect was seen for ail of five separate experiments. Gwynne and Hess[13] reported that both HDL (rat and human) and LDL (human) increased steroidogenesis by ACTH-stimuiated rat adrenocorticai ceils during a 20 h incubation; the effect of HDL was not evident during the first 8 h and the time course of the effect of LDL was not reported, Prior depletion of the adrenocortical cellular cholesterol ester stores was necessary before an effect of HDL on ACTH-stimulated steroidogenesis was demonstrable within the first hour of incubation [ 131. However, there is a need for caution in the extrapolation of data obtained using choiesterol depleted adrenai cells to the possible role of lipoproteins in steroidogenesis by adrenal cells with normal cholesterol ester stores. Cholesterol depleted adrenal ceils may utilize sources of cholesterol which make little contribution to steroidogenesis by adrenal cells with normal stores of cholesterol ester. In the present report, the stimuiatory effect of LDL on steroidogenesis was confined to K +-stimulated zona glomeruiosa cells. This is to be expected, given the much great& demand for cholesterol for steroidogenesis during K + -stimulation. The steroidogenesis
one production, but at concentrations of 50-6O~g cholesterol/ml, LDL consistentiy enhanced K+-stimulated aidosterone production (Figs 1 and 2). LDL concentrations of Ill pg cholesteroi/mi and greater had no effect on K*-stimuiated aidosterone production (Fig. 2). DISCUSSION
Several studies suggest that whereas both HDL and LDL play a role in the supply of cholesterol for steroidogenesis by the rat zona fasciculata cell in uivo[W-121, WDL is more important than LDL in this regard. However. in these studies[l&l2], the adrenal cells had been depleted of endogenous stores of cholesterol ester by prior treatment of the rats with 4-aminopyrazoio [3.4-apyrimidine, a drug which inhibits hepatic li~protein secretion. With respect to the effect of LDL on K+-stimulated aldosterone production by rat zona glomeruiosa cells. the present report is the first to describe the stimuiation by lipoproteins, of steroidogenesis by isolated adrenal cells with ‘normal’ stores of cholesterol ester
Short communication stimuiatory effect of LDL suggests that during K+-stimulation the rate of supply of cholesterol for steroidogenesis becomes rate-limiting, despite normal
cellular cholesterol stores. In contrast, during basal steroidogenesis where the demand for cholesterol is much lower, the addition of LDL caused no significant increase in steroidogenesis. The inhibition of steroidogenesis by VLDL and HDL demonstrated for zona glomerulosa cells incubated in KRBGA with 0.27(, BSA was largely prevented by the use of KRBGA with 4% BSA as incubation medium. These data suggest that the inhibition of zona glomerulosa cellular steroidogenesis by VLDL and HDL may be similar to that previously described for BSA [14]. Moreover, the steroidogenesis inhibitory properties of lipoproteins may mask any contribution they make to steroidogenesis through increased cholesterol delivery to the zona glomerulo~ cell. If such a steroidogenesis inhibitory property was shared by LDL, this might be responsible for the biphasic effect of LDL on K+-stimulated steroidogenesis. Acknowledgements--I am most grateful to Dr J. W. Funder and Professor A. E. Doyle for their assistance with these studies. and to MS Cherie Delamere for typing the manuscript.
REFERENCES
Brown M. S.. Kovanen P. T. and Goldstein J. L.: Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Reeenr Prog. Harm. Rex 35 (1979) 215-257. Haning R.. Tait S. A. S. and Tait J. F.: In eirro effects of ACTH. angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. Endocrinology 87 (1970) 1147-l 167.
S.B. I? 6-l
711
3. Have1 R. J., Eder H. A. and Bragdon 1. H.: The distribution and chemical com~sition of ultracentrifu~lly separated lipoprotein in human serum. J. clin. lnousr. u ( 1955) I j4j_ 1353. 4. Zlatkis A. and Zak B.: Study of a new cholesterol reagent. Arm/. Biochrm. 29 (1969) 143- 148. 5. Folch J.. Lees M. and Sloane Stanley G. H.: A simple method for the isolation and purification of total lipides from animal tissues. J. hiol. Chem. 226 (1957) 497-509. 6. Abell L. L., Levy B. B.. Brodie B. B. and Kendall F. E.: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. hi&. Chem. 195 (1952) 357-366. 7. Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. .I. hiol. Chem. 193 (1951) 265-275. 8. Fredlund P., Saltman S. and Catt K. J.: Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II. ~~~crino~ogy 97 (1975) 1577- 1586. 9. Dunnett C. W.: New tables for multiple comparisons with a control Biometrics 20 (1964) 482-493. 10. Andersen J. M. and Dietschy J. M.: Regulation of sterol synthesis in 15 tissues or rat. II. Role of rat and human high and low density plasma lipoproteins and of rat chylomicron remnants. J. biol. C&m. 252 (1977) 3652-3659. II. Andersen J. M. and Dietschy J. M.: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J. biol. Chem. 253 (1978) 90249032. IL. Balasubramaniam S., Goldstein J. L., Faust J. R.. Brunschede Cl. Y. and Brown M. S.: Linooroteinmediated regulation of 3-hydroxy-3-methyiglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J. biol. Chem. 252 (1977) 1771-1779. 13. Gwynne 1. T. and Hess B.: The role of high density li~proteins in rat adrenal cholesterol metabolism and steroidogenesis. f. bid. Cftem. 255 (1980) 10875-10883. l 14. Campbell D. J.: El&et of composition of incubation medium on aldosterone and corticosterone production by isolated rat zona glomerulosa cells. J. Endocr. 94 (1982) in press.
710
*
t<+-stimulated
40’
* 0
400
Likw%n %&l*:t&l Concenttation (rtg per ml)
100
200
MO
400
t
‘00
Lipoprotein Cholesterol Concentration (ug per ml)
Fig. 1. EtTect of rat lipoproteins on aldosterone production by isolated rat zona glomerulosa cells incubated in KRBGA with O.Z:d BSA. Pooled data from two separate experiments with duplicate incubations presented as percent change from mean control for each experiment. Each point represents the mean *SE. n = 4. Upper panel, K’-stimulated steroidogenesis ([K*] = 8.6 mM): lower panel, basal steroidogenesis ([K’] ;r: 3.8 mM). Basal aldosterone production of control incubations for each experiment (ng per IO6 cells per h. mean & range of duplicate incubations): 23.2 f 3.6; 50.7 + 1.9. K’-stimulated aldosterone production of control incubations: 153 f 16; 242 + 13. *+ P e 0.05; **, P < 0.01, compared with control incubations.
Fig. 2. Eikct of rat lipoproteins on aldosterone production by isolated rat zona glomerulosa cells incubated in KRBGA with 4”,‘, BSA. Pooled data from three separate experiments with dupli~te incubations presented as percent change from mean control for each experiment. Each point represents the mean k SE, n given in parenthesis for each point. Upper panel, K+-stimulated steroidogenesis ([K’] = 8.6mM); lower panel. basal steroidogenesis ([K ‘1 = 3.8 mM). Basal aldosterone production of control intuitions for each experiment (ng per t06 cells per h, mean f range of duplicate incubations): 36.9 f 0.3; 13.8 f 0.1; 4.7 f 0.3. K+-stimulated aldosterone production of control incubations: 183 f 5; 45 + 6; 54 & 2. *, P c 0.05; **. P c 0.01, compared with control incubations.
medium (Fig, 2). LDL had no effect on basal aldoster-
during a 2 h incubation. This effect was seen for ail of five separate experiments. Gwynne and Hess[13] reported that both HDL (rat and human) and LDL (human) increased steroidogenesis by ACTH-stimuiated rat adrenocorticai ceils during a 20 h incubation; the effect of HDL was not evident during the first 8 h and the time course of the effect of LDL was not reported, Prior depletion of the adrenocortical cellular cholesterol ester stores was necessary before an effect of HDL on ACTH-stimulated steroidogenesis was demonstrable within the first hour of incubation [ 131. However, there is a need for caution in the extrapolation of data obtained using choiesterol depleted adrenai cells to the possible role of lipoproteins in steroidogenesis by adrenal cells with normal cholesterol ester stores. Cholesterol depleted adrenal ceils may utilize sources of cholesterol which make little contribution to steroidogenesis by adrenal cells with normal stores of cholesterol ester. In the present report, the stimuiatory effect of LDL on steroidogenesis was confined to K +-stimulated zona glomeruiosa cells. This is to be expected, given the much great& demand for cholesterol for steroidogenesis during K + -stimulation. The steroidogenesis
one production, but at concentrations of 50-6O~g cholesterol/ml, LDL consistentiy enhanced K+-stimulated aidosterone production (Figs 1 and 2). LDL concentrations of Ill pg cholesteroi/mi and greater had no effect on K*-stimuiated aidosterone production (Fig. 2). DISCUSSION
Several studies suggest that whereas both HDL and LDL play a role in the supply of cholesterol for steroidogenesis by the rat zona fasciculata cell in uivo[W-121, WDL is more important than LDL in this regard. However. in these studies[l&l2], the adrenal cells had been depleted of endogenous stores of cholesterol ester by prior treatment of the rats with 4-aminopyrazoio [3.4-apyrimidine, a drug which inhibits hepatic li~protein secretion. With respect to the effect of LDL on K+-stimulated aldosterone production by rat zona glomeruiosa cells. the present report is the first to describe the stimuiation by lipoproteins, of steroidogenesis by isolated adrenal cells with ‘normal’ stores of cholesterol ester
Short communication stimuiatory effect of LDL suggests that during K+-stimulation the rate of supply of cholesterol for steroidogenesis becomes rate-limiting, despite normal
cellular cholesterol stores. In contrast, during basal steroidogenesis where the demand for cholesterol is much lower, the addition of LDL caused no significant increase in steroidogenesis. The inhibition of steroidogenesis by VLDL and HDL demonstrated for zona glomerulosa cells incubated in KRBGA with 0.27(, BSA was largely prevented by the use of KRBGA with 4% BSA as incubation medium. These data suggest that the inhibition of zona glomerulosa cellular steroidogenesis by VLDL and HDL may be similar to that previously described for BSA [14]. Moreover, the steroidogenesis inhibitory properties of lipoproteins may mask any contribution they make to steroidogenesis through increased cholesterol delivery to the zona glomerulo~ cell. If such a steroidogenesis inhibitory property was shared by LDL, this might be responsible for the biphasic effect of LDL on K+-stimulated steroidogenesis. Acknowledgements--I am most grateful to Dr J. W. Funder and Professor A. E. Doyle for their assistance with these studies. and to MS Cherie Delamere for typing the manuscript.
REFERENCES
Brown M. S.. Kovanen P. T. and Goldstein J. L.: Receptor-mediated uptake of lipoprotein-cholesterol and its utilization for steroid synthesis in the adrenal cortex. Reeenr Prog. Harm. Rex 35 (1979) 215-257. Haning R.. Tait S. A. S. and Tait J. F.: In eirro effects of ACTH. angiotensins, serotonin and potassium on steroid output and conversion of corticosterone to aldosterone by isolated adrenal cells. Endocrinology 87 (1970) 1147-l 167.
S.B. I? 6-l
711
3. Have1 R. J., Eder H. A. and Bragdon 1. H.: The distribution and chemical com~sition of ultracentrifu~lly separated lipoprotein in human serum. J. clin. lnousr. u ( 1955) I j4j_ 1353. 4. Zlatkis A. and Zak B.: Study of a new cholesterol reagent. Arm/. Biochrm. 29 (1969) 143- 148. 5. Folch J.. Lees M. and Sloane Stanley G. H.: A simple method for the isolation and purification of total lipides from animal tissues. J. hiol. Chem. 226 (1957) 497-509. 6. Abell L. L., Levy B. B.. Brodie B. B. and Kendall F. E.: A simplified method for the estimation of total cholesterol in serum and demonstration of its specificity. J. hi&. Chem. 195 (1952) 357-366. 7. Lowry 0. H., Rosebrough N. J.. Farr A. L. and Randall R. J.: Protein measurement with the Folin phenol reagent. .I. hiol. Chem. 193 (1951) 265-275. 8. Fredlund P., Saltman S. and Catt K. J.: Aldosterone production by isolated adrenal glomerulosa cells: stimulation by physiological concentrations of angiotensin II. ~~~crino~ogy 97 (1975) 1577- 1586. 9. Dunnett C. W.: New tables for multiple comparisons with a control Biometrics 20 (1964) 482-493. 10. Andersen J. M. and Dietschy J. M.: Regulation of sterol synthesis in 15 tissues or rat. II. Role of rat and human high and low density plasma lipoproteins and of rat chylomicron remnants. J. biol. C&m. 252 (1977) 3652-3659. II. Andersen J. M. and Dietschy J. M.: Relative importance of high and low density lipoproteins in the regulation of cholesterol synthesis in the adrenal gland, ovary, and testis of the rat. J. biol. Chem. 253 (1978) 90249032. IL. Balasubramaniam S., Goldstein J. L., Faust J. R.. Brunschede Cl. Y. and Brown M. S.: Linooroteinmediated regulation of 3-hydroxy-3-methyiglutaryl coenzyme A reductase activity and cholesteryl ester metabolism in the adrenal gland of the rat. J. biol. Chem. 252 (1977) 1771-1779. 13. Gwynne 1. T. and Hess B.: The role of high density li~proteins in rat adrenal cholesterol metabolism and steroidogenesis. f. bid. Cftem. 255 (1980) 10875-10883. l 14. Campbell D. J.: El&et of composition of incubation medium on aldosterone and corticosterone production by isolated rat zona glomerulosa cells. J. Endocr. 94 (1982) in press.