Ionic effects on strain differences in hepatic cytosolic glucocorticoid receptor levels in mice.

Ionic conditions were varied during homogenization of adult livers and during incubation of hepatic cytosol with glucocorticoid, and the effects on the detection of differences between inbred strains of mice for receptor levels were studied. When homogenized directly in 0.01 M Tris or homogenized in distilled water and then buffered to either 0.05 M Tris or 0.01 M potassium phosphate, A/J glucocorticoid receptor levels were greater than those of BIO.A. However, when homogenized directly in 0.05 M Tris or buffered to 0.05 M potassium phosphate after homogenization in distilled water, A/J and BIO.A had similar receptor levels. When buffered to 0.01 M Tris after homogenization in distilled water, A/J receptor levels were greater than those of BIO.A but not significantly so. In Tris buffers, glucocorticoid receptor levels were higher when livers were homogenized directly in buffer than when liver homogenates were buffered after homogenization. This concentration effect is apparently due to the more rapid decay of glucocorticoid receptor in the lower molarity buffer. A reducing agent, thioglycerol, did not appear to affect either the rate of decay of the receptors or the measured level of receptor for BIO.A and A/J. C57BL/6J receptor levels were measured in Tris buffers and had a similar relationship to A/J as did BIO.A. A popular model for studies of cleft palate is glucocorticoid-induced cleft palate in mice (Fraser and Fainstat, '51). Inbred strains of mice differ significantly for susceptibility to glucocorticoid-induced cleft palate, and it has been suggested that genetic variation in amount of glucocorticoid receptor may be a part of the explanation for this variation. However, there has been considerable controversy about the direction and degree of difference among inbred mouse strains for amount of glucocorticoid receptor. Salomon and Pratt presented evidence that cultured, embryonic facial mesenchyme cells from a strain with high susceptibility to glucocorticoid-induced cleft palate, A/J, have about twice as many specific steroid receptors as do such cells from a low susceptibility strain, C57BU6 (Salomon and Pratt, '76). They also found a correlation of sensitivity to steroidinduced cleft palate with amount of steroidspecific receptor in cultured facial mesenchyme cells among several inbred strains of mice (Salomon and Pratt, '79). Goldman et al. ('77) provided data that this variation in the amount of steroid receptor was associated with H-2 genotype, but this conclusion was contested. We demonstrated that a difference between A/J and C57BIJ6J of similar direction and degree to that found in cultured facial mesenchyme cells could be detected for hepatic cytosolic receptors but that variation among alleles a t the H-2 locus does not affect these receptor levels (Butley et al., '78). Francke and Gehring ('80) mapped the glucocorticoid receptor locus to chromosome 18, not chromosome 17, which carries the H-2 locus. Hackney ('80) detected a specific glucocorticoid receptor in mouse fetal heads but found higher levels in the C57BU6J than in the A/J strain. Differences in the glucocorticoid used for the measurements were not responsible for these differences: triamcinolone acetonide (TA) was used with mouse livers and mouse fetal heads with quite opposite results. Since a large variety of factors including Ca + + (Arhyi and Niiray, '80), molybdate (Nielsen et al., '77b), alkaline phosAbbreviations used. TA, triamcinolone acetonide; ATP. adenosine tiphosphate, Dx, dexamethasone; %a, tris (hydromethyl) aminomethane. Received November 5. 1981; accepted June 23. 1982. Address reprint requests to Robert P. Erickenn, M.D., Department of Human Genetics, Box 015, University of Michigan Medical Schml, 1137 E. Catherine Street, Ann Arbor, MI 48109. 0040-3709/83/2701-0043$02.50 01983 ALAN R. LISS, INC 44 K.J. HARPER AND R.P. ERICKSON phatase (Nielsen et al., '77a), ATP (John and Moudgil, '79), pyridoxine (DiSorbo et al., '801, 5'-deoxypyridoxal (O'Brien et al., '801, pyridoxal 5'-phosphate (Dolan et al., '80)) and unidentified dialyzable factors (Sato et al., '80) are known to affect measurements of glucocorticoid receptors, we sought to determine some factors with an effect on measured levels of hepatic glucocorticoid receptors. Of several variables studied, varying ionic conditions were found to have the greatest effects. We have continued to use adult livers as our source of glucocorticoid receptors since 1) they provide sufficient material for biochemical characterization and 2) the strain difference found parallels that originally reported (Salomon and Pratt, '76) in cultured embryonic facial mesenchyme cells. MATERIALS AND METHODS