Down-regulation of the mineralocorticoid receptor by dexamethasone in an amphibian kidney cell line (A6).

The A6 cell line, derived from Xenopus kidney, is an in vitro model of cortico-steroid mediated transepitheial Na+ transport stimulation. We report the apparent down-regulation of mineralocorticoid receptor levels in A6 cells, in response to the presence of the synthetic glucorticoid dexamethasone in the culture medium. Mineralocorticoid receptor binding was suppressed to approximately 25% of control following 24-hour exposure to 10nM dexamethasone. Scatchard analysis of concentration-binding experiments show down-regulation of maximum binding capacity by Dex exposure with no alteration of MR affinity, i.e., alteration of MR number only. The effect is dose-responsive with half-maximal down regulation at 1nM. Maximal inhibition of binding occurred after 24-hours exposure to dexamethasone. The inhibitory effect of dexamethasone on MR binding was unique for the glucocorticoid, with no effect exhibited following similar treatment with an androgen, an estrogen, or a mineralocorticoid.

Renal and hepatic family 3A cytochromes P450 (CYP3A) in spontaneously hypertensive rats.

Troleandomycin (TAO), a selective family 3A cytochromes P450 (CYP3A) inhibitor, decreases enhanced in vivo corticosterone 6 beta-hydroxylation and blood pressure in spontaneously hypertensive rats (SHR). Corticosterone 6 beta-hydroxylation was measured in liver and kidney microsomes, to determine ontogeny and the effect of TAO on CYP3A activity at the organ level. SHR kidney CYP3A activity increased from 4 to 8 weeks, stabilized at 11 and 16 weeks, and was much higher than in control (Wistar-Kyoto, WKY) rats at all ages. Hepatic activity showed less consistency in strain difference. TAO produced a relatively large decrease in renal CYP3A activity compared with liver. Although renal CYP3A mRNA was not present in sufficient quantity for detection by northern blot analysis of total RNA, its presence was demonstrated in SHR by reverse transcriptase-polymerase chain reaction amplification. Correlations between renal CYP3A activity and systolic blood pressure in SHR and WKY rats with variations in age, strain and drug treatment are consistent with the role of the enzyme in the pathogenesis of blood pressure elevation in SHR.

Maternal environment defines blood pressure and its response to troleandomycin in spontaneously hypertensive rats.

Relationship between family-3A cytochrome P-450-dependent (troleandomycin inhibitable) and maternal environmental-dependent systolic blood pressure (SBP) was investigated in spontaneously hypertensive rats (SHR). Adult SHR nursed by foster or natural SHR mothers had indistinguishable SBP. Troleandomycin reduced 50% of Wistar-Kyoto (WKY)-SHR strain difference in SBP. SHR having WKY foster mothers had SBP similar to troleandomycin-reduced SHR levels, which was unaffected by troleandomycin. The two components of SBP elevation appear identical. Because observations of others demonstrated that WKY fostered to SHR show no SBP increase, the maternally dependent/troleandomycin-sensitive component of SBP elevation may reflect epistatic interaction between genes determining maternal differences and offspring sensitivity, respectively.

Progestin stimulation of lactate dehydrogenase in the human breast cancer cell line T-47D.

We have previously reported that physiological levels of progestins alone stimulate lactate dehydrogenase in a dose-responsive manner in the progesterone-receptor-rich human breast cancer cell line T-47D. Using isozyme electrophoresis, we have not found that lactate dehydrogenase isozyme 5 is the only isozyme detectable in these cells, as has been reported for other human breast cancer cells in long-term tissue culture. Upon treatment with progestins, isozyme 5 remains the only isozyme detectable. T-47D cells were plated in charcoal-stripped serum-containing medium and grown for 2 days before treatment with progestin. Lactate dehydrogenase stimulation then plateaued after around 2-3 days of treatment with progestin and was maintained until around day 5, following which a decline in enzyme activity occurred. The effect is specific for progestins, and inhibited by the anti-progestin RU-38486 (17 beta-hydroxy-11 beta-(4-dimethyl-aminophenyl-1)-17 alpha-(prop-1-ynil)-estra-4,9-dien-3-one). Experiments using actinomycin D and cycloheximide suggests that the effect is dependent on RNA and protein synthesis, respectively. Lactate dehydrogenase stimulation occurs regardless of the presence of the estrogenic pH indicator Phenol red, and of whether it was analyzed per mg DNA or per mg protein.

A progestin effect on lactate dehydrogenase in the human breast cancer cell line T-47D.

The human breast cancer cell line T-47D has high levels of progesterone receptor even in the absence of exogenously added estrogen. Because of this it is a good line in which to study aspects of progestin action. It has been shown by others that lactate dehydrogenase in MCF-7 cells is responsive to estrogen but not to progesterone. Other proteins in other systems have been found to be responsive to both estrogen and progesterone, often requiring priming by estrogen, presumably to produce sufficiently high quantities of progesterone receptor. Reasoning that lactate dehydrogenase in T-47D cells might be stimulated by progestins alone at physiological levels since these cells already have high levels of progesterone receptor, we now report that this is indeed the case.