Modulation of a renal estrogen receptor by protein kinase C.

In this paper we demonstrate the existence of specific, high affinity 17-beta estradiol binding in MDBK cells (a normal non-transformed renal cell line). Scatchard analysis revealed binding characteristics typical of the estrogen receptor (ER). Only unlabelled 17-beta estradiol, diethyl stilbestrol and estrone effectively competed with [3H] 17-beta estradiol for binding, other steroids did not compete. Short term TPA treatment of MDBK cells with TPA increased PKC activity and immunoreactivity and caused a transient increase in 17-beta estradiol binding, while longer term treatment with TPA decreased PKC activity and immunoreactivity. The inactive phorbol ester 4 alpha PDD was without effect on PKC activity and the ER. TPA did not affect the affinity of the ER for the nucleus nor did it increase degradation of the receptor. We hypothesize that the renal ER may be a substrate for PKC and its properties can be modified by PKC similarly to the vitamin D receptor.

Age-associated changes in the estrogen receptor-active proteases of female rabbit liver.

Female rabbit liver cytosol contains a receptor-modifying activity that converts the 250,000 estrogen receptor of liver and uterine cytosol to a 37,000 form. There is an age-dependent increase in this receptor-active protease and in the general protease activity of rabbit liver cytosol, measured with [14C]casein. Sephacryl S-200 chromatography of liver cytosol shows that in the young animal (5 weeks old) the major receptor-modifying activity elutes near the void volume, while in the older animal (13 weeks old) activities having lower molecular weights are present. The general protease activity elution profile is similar to the receptor-active protease profile for the 5-week-old rabbit but not the 13-week-old rabbit. The liver cytosol of the older animal has a high molecular weight protease active toward [14C]casein but not toward the estrogen receptor. The changes in the estrogen receptor forms and the receptor-modifying activity profiles of liver cytosol that occur during development in the rabbit suggest that receptor-modifying activity may initially be associated with the estrogen receptor to form a high molecular weight complex.

Factors influencing the uptake of cholesterol by isolated brush border membranes from rabbit small intestine.

The uptake of [3H] cholesterol by isolated brush border membranes from rabbit small intestine was studied under different conditions. In initial experiments the uptake from taurocholate-micellized suspensions was found to be greatly enhanced by Ca2+. Maximum stimulation was obtained with 10 mM Ca2+ and this condition was adopted for some of the subsequent experiments. In the presence of this cation, the uptake was too rapid to allow kinetic studies and only equilibrium values could be measured. Addition of lecithins to the micelles had an inhibitory effect on the equilibrium uptake in the presence of Ca2+ and on the rate of uptake in the absence of this cation which increased with acyl chain length. Admixture of the sterol with dipalmitoylphosphatidylethanolamine stimulated the uptake whereas admixture with dipalmitoylphosphatidylglycerol as well as with 1-monoolein alone or together with oleic acid had little or no effect on uptake in the presence of Ca2+. Again with this cation present, preincubation of membranes with the dipalmitoyl analogue of lecithin increased the equilibrium uptake of the sterol and abolished the inhibitory effect of the choline lipid. This inhibitory effect could be seen only with untreated membranes when lecithin and cholesterol were micellized together. In the absence of Ca2+, preincubation with dipalmitoylphosphatidylcholine had no marked effect on the rate of cholesterol uptake and the suppressive effect of lecithin on this rate could be seen even with lipid-treated membranes provided the lecithin and sterol were micellized together.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the uptake of fatty acids by brush border membranes of the rabbit intestine.

Initial studies revealed that the uptake of palmitic acid and oleic acid into brush border membranes was similar when these were isolated from either whole small intestine, jejunum, or ileum. The uptake of these fatty acids was somewhat lower with membranes obtained from duodenum. Subsequent studies, all with membranes obtained from whole intestine, indicated an increase in binding with chain length of fatty acid of up to 16 carbons. Unsaturation decreased this uptake somewhat. Taurocholate and 1-palmitoyl lysolecithin had a moderate stimulatory effect on the binding of oleic acid and palmitic acid at concentrations of 10 and 0.5 mM, respectively, and inhibited at higher concentrations. Addition of 1.4 mM egg lecithin to the fatty acid - bile salt micelles, such that the lecithin - bile salt ratio was 0.2, decreased the uptake of fatty acids generally, but did not significantly affect the pattern of binding by membrane fractions isolated from different segments nor did it change the pattern of labelling when fatty acid chain length and unsaturation were varied. At lower concentrations, egg lecithin had little effect on the uptake of oleic acid, whereas dipalmitoyl phosphatidylcholine stimulated binding of both palmitic acid and oleic acid over the entire range of concentrations tested. Preincubation of the membranes with this saturated phospholipid stimulated the uptake of oleic acid, and addition of this choline lipid to the oleic acid - bile salt containing micelles did not substantially enhance fatty acid uptake in lipid-treated membranes. The binding of fatty acid was very rapid either in the presence or the absence of Ca2+, such that even in zero-time controls essentially equilibrium bindings were obtained.(ABSTRACT TRUNCATED AT 250 WORDS)

A 17 beta-hydroxysteroid dehydrogenase of female rabbit liver cytosol. Purification and characterization of multiple forms of the enzyme.

Multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver were identified. NAD-dependent and NADP-dependent enzyme activities were separated by affinity chromatography on agarose-immobilized Procion Red HE3B, and three forms of the NADP-dependent enzyme activity were purified by chromatofocusing. These three enzyme forms are charge isomers and have no quaternary structure. The enzymes catalysed the C-17 oxidoreduction of oestrogens and androgens; with all enzyme forms the activity towards androgens was higher than that toward oestrogens. The enzymes also exhibited 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. Comparison of the relative activities of the enzymes towards a number of oestrogen and androgen substrates revealed differences among the enzyme forms for both the oxidative and the reductive reactions. In particular, one enzyme form had a significantly lower Km for the 3 alpha-hydroxysteroid substrate and a higher 3 alpha-/17 beta-hydroxysteroid dehydrogenase activity ratio than the other two enzyme forms.

Age-dependent changes in the multiple forms of the soluble 17 beta-hydroxysteroid dehydrogenase of female rabbit liver.

The soluble NADP-dependent 17 beta-hydroxysteroid dehydrogenase activity of female rabbit liver increases with the age of the animal, the specific activity of the enzyme in the 56-day-old rabbit being 3 times that of the 28-day-old animal. The increase in activity is accompanied by a change in the molecular heterogeneity of the enzyme. Three forms (enzymes I, II and III) were identified in the liver cytosol of the 56-day-old female rabbit, whereas only one major form (enzyme IIIY) was present in the 28-day-old animal. Peptide maps of the four purified enzymes showed that there were minor differences in structure. The enzyme present in the liver of the 28-day-old rabbit was distinct from the three enzymes of the 56-day-old animal. All of the enzymes exhibited bifunctional activity, having 17 beta-hydroxysteroid dehydrogenase activity towards androgen and oestrogen substrates and 3 alpha-hydroxysteroid dehydrogenase activity towards androgens of the 5 beta-androstane series. The differences in substrate specificity of the enzymes paralleled their differences in structure. The data suggest that one enzyme (enzyme III) may have a special role in steroid metabolism during development in the female rabbit.

Studies on the mechanism of cholesterol uptake and on the effects of bile salts on this uptake by brush-border membranes isolated from rabbit small intestine.

The effect of bile salts and other surfactants on the rate of incorporation of cholesterol into isolated brush-border membranes was tested. At constant cholesterol concentration, a stimulatory effect of taurocholate was noticed which increased as the bile salt concentration was raised to 20 mM. Taurodeoxycholate was as effective as taurocholate at concentrations of up to 5 mM and inhibited at higher concentrations. Glycocholate was only moderately stimulatory whereas cholate was nearly as effective as taurocholate at concentrations above 5 mM. Other surfactants such as sodium lauryl sulfate and Triton X-100 were very inhibitory at all concentrations tried whereas cetyltrimethyl ammonium chloride was stimulatory only at a very low range of concentrations. These micellizing agents all caused some disruption of the membranes and the greater effectiveness of taurocholate in stimulating sterol uptake was partly relatable to the weaker membrane solubilizing action of this bile salt. Preincubation of membranes with 20 mM taurocholate followed by washing and exposure to cholesterol-containing lipid suspensions lacking bile salt, did not enhance the incorporation of the sterol. In the absence of bile salt the incorporation of cholesterol was unaffected by stirring of the incubation mixtures. Increasing the cholesterol concentration in the mixed micelle while keeping the concentration of bile salt constant caused an increase in rate of sterol incorporation. This increased rate was seen whether the cholesterol suspension was turbid, i.e., contained non-micellized cholesterol, or whether it was optically-clear and contained only monomers and micelles. When the concentration of taurocholate and cholesterol were increased simultaneously such that the concentration ratio of these two components was kept constant, there resulted a corresponding increase in rate of cholesterol uptake. The initial rates of cholesterol incorporation from suspensions containing micellar and monomer forms of cholesterol were much larger than from solutions containing only monomers of the same concentration. The rates of incorporation of cholesterol and phosphatidylethanolamine from mixed micelles containing these lipids in equimolar concentrations were very different. The results as a whole suggest at least for those experimental conditions specified in this study, that uptake of cholesterol by isolated brush-border membranes involves both the monomer and micellar phases of the bulk lipid and that the interaction of the micelles with membrane does not likely involve a fusion process.

The effect of phosphoglycerides on the incorporation of cholesterol into isolated brush-border membranes from rabbit small intestine.

The incorporation of cholesterol from bile salt micelles into brush-border membranes was found to be similar whether these originated from jejunum, ileum or whole small intestine. This incorporation, however, was appreciably lower in membranes obtained from duodenum. Studies pursued with membranes from whole small intestine revealed that dipalmitoyl or dioleoylphosphatidylcholine, when micellized together with the sterol and taurocholate markedly inhibited the incorporation. The didecanoyl and dilauroyl analogues of this lipid class were without significant effect. Preincubation of the membranes for 30 min at 37 degrees C with or without dipalmitoylphosphatidylcholine had no effect on cholesterol incorporation. Again in this case, suppression of sterol uptake could be seen only when phosphatidylcholine was admixed with the sterol. In contrast, dipalmitoyl and dilauroylphosphatidylethanolamines were found to be stimulatory rather than inhibitory. Addition of palmitic acid to the sterol-bile salt micelles had no effect on the uptake of cholesterol; however, this fatty acid could completely reverse the inhibition of cholesterol uptake by dipalmitoylphosphatidylcholine. The present study supports the conclusion that cholesterol incorporation into isolated brush-border membranes is governed largely by factors which affect the partitioning of the sterol out of the bile salt micelle.

Uptake of 17 beta-estradiol and its conjugates by isolated rabbit liver nuclei.

The present study compares the uptake and metabolism of 17 beta-estradiol, 17 beta-estradiol 3-glucoside, and 17 beta-estradiol 3-glucuronide by a highly purified preparation of rabbit liver nuclei. The uptake of the three estrogens was rapid and equilibration was reached within 60 s. The order of uptake was 17 beta-estradiol (64 fmol X mg protein -1) greater than 17 beta-estradiol 3-glucoside (10 fmol X mg protein -1) greater than 17 beta-estradiol 3-glucuronide (6.5 fmol X mg protein -1). Thin-layer chromatography of the estrogens taken up by rabbit liver nuclei indicated the presence of a beta-glucosidase activity associated with the nuclear preparation. The apparent Km value of this enzyme for 17 beta-estradiol 3-glucoside (3.5 microM) was about 10-fold higher when compared with the cytosolic enzyme. The uptake of the three estrogens was linearly proportional to the substrate concentration from 1 to 100 nM. No competition for uptake was observed among the steroids and the presence of diethylstilbestrol did not reduce the uptake of the steroids. These findings suggest that 17 beta-estradiol, 17 beta-estradiol 3-glucoside, and 17 beta-estradiol 3-glucuronide are taken up by nuclei by a nonsaturable diffusion process. The effect of cytosol on the uptake of estrogens by purified nuclei was also investigated. It was observed that cytosol reduced the uptake o 17 beta-estradiol but had little effect on that of its conjugates.

A highly sensitive method for identification of amino termini of proteins: application to multiple forms of poly(C)-avid ribonuclease and 17 beta-hydroxysteroid dehydrogenase.

A radiochemical method for the determination of the amino terminus on very small amounts (0.5-5 nmol) of protein is described. The high sensitivity of the method is achieved by using undiluted 1-fluoro-2,4-dinitro-[3,5-3H]benzene [( 3H]Dnp-F) as the labelling reagent under conditions in which a maximum amount of radioactive label is incorporated. Chemical homogeneity is achieved by reacting with excess unlabelled Dnp-F. High recovery is obtained by adding Dnp-albumin as carrier protein. A mixture of Dnp 14C-labelled amino acids is added prior to hydrolysis and identification of the amino terminus is made on the basis of the 3H/14C ratios of the separated Dnp-amino acids. The method was tested on insulin, pancreatic ribonuclease, and lysozyme which gave high 3H/14C ratios only in the expected amino-terminal amino acids. Application to multiple forms of poly(C)-avid ribonuclease gave only amino-terminal lysine. Two of four putative isozymes of 17 beta-hydroxysteroid dehydrogenase had serine as the amino terminus while the other two had aspartic acid or asparagine.

The effects of high pass filtered speech on speech discrimination.

This study was designed to determine the intelligibility of speech at two intensity levels using two different conditions (broad band and high pass filtered speech) and to determine the vowels and consonants most affected by the conditions imposed. The ten subjects were all stimulated with 50 words of the CID W-22 series at two levels of intensity (30 db SL and 15 dB SL) each in the filtered and broad-band (unfiltered) conditions. The results tended to indicate that the type of condition (filtered or unfiltered) did not affect the speech comprehension until the intensity of the filtered signal approached 15 dB SL. It was also determined that consonants and vowels identification were only slightly affected by filtering if the intensity of the signal is above 15 dB SL.

Comparative studies of the 17 beta-estradiol receptors in rabbit liver, kidney and uterus.

The cytosol 17 beta-estradiol receptors from rabbit kidney, liver and uterus, compared under identical experimental conditions, were similar in terms of their pH-activity profiles, dependence on incubation temperature, sensitivity to sulfhydryl reagents and steroid specificity. 17 beta-[3H]-Estradiol binding was saturable with all three tissues, having an apparent dissociation constant of 4 X 10(-10)M. The binding of 17 beta-[3H]-estradiol in kidney, liver and uterus was inhibited by estrogens, including estrogen conjugates, but not by testosterone, progesterone or cortisol. The 17 beta-estradiol receptors of liver, kidney and uterus exhibited significant differences with respect to their chromatographic behaviour on heparin-Sepharose. Furthermore, a comparison of their sucrose density gradient centrifugation patterns showed that the 17 beta-[3H]-estradiol-receptor complex of liver and kidney sedimented at 3-4 S in both low and high ionic strength media, while the uterine receptor sedimented at 7-8 S in low ionic strength media and at 4-5 S in high ionic strength media. When the liver and uterine cytosol fractions were combined the uterine receptor was altered and sedimented at 3-4 S in low ionic strength media.

Interaction of lipids with intestinal brush border membrane preparations.

Conditions for uptake of lipids by rabbit intestinal brush border membrane preparations were investigated. A variety of lipids were found to be incorporated, including choline and ethanolamine phosphatides as well as cholesterol, diglyceride, and fatty acid. The incorporation of those lipids tested was enhanced by Ca2+ and other divalent cations but not by monovalent cations. The optimal Ca2+ concentration was approximately 10 mM. The uptake varied with lipid and membrane protein concentration and proceeded at rates which were too rapid to measure under several assay conditions tried. Incorporations were decreased substantially outside the pH range of 6.5-8.0. The effect of one lipid, phosphatidylcholine, on the structural appearance of the membrane fraction was examined by electron microscopy. No free or surface-bound lipid structures could be detected and the membrane fractions appeared to be unchanged after uptake.

A 3(17) alpha-hydroxysteroid dehydrogenase of female rabbit kidney cytosol. Purification and characterization of multiple forms of the enzyme.

A mammalian hydroxysteroid dehydrogenase having both 3 alpha- and 17 alpha-enzyme activity is described for the first time. Multiple forms of this 3(17) alpha-hydroxysteroid dehydrogenase are present in the cytosol of female rabbit kidney. Four forms of this enzyme were resolved by a purification procedure which included DEAE-cellulose chromatography and isoelectric focusing and three of the isolated enzymes were homogeneous on the basis of polyacrylamide gel electrophoresis. The purified enzymes exhibited 17 alpha-enzyme activity toward both androgen and estrogen substrates and 3 alpha-enzyme activity toward androgens of the 5 alpha-androstane series. In general, the 3 alpha-enzyme activity was greater than the 17 alpha-enzyme activity and the latter activity was greater toward androgens than estrogens. There were differences in substrate specificity among the enzyme forms. In particular, with estrogen substrates one of the forms displayed a high specificity toward 17 alpha-estradiol 3-glucuronide.

Comparison of the multiple forms of the soluble 3(17) alpha-hydroxysteroid dehydrogenases of female rabbit kidney and liver.

Multiple forms of the soluble 17 alpha-hydroxysteroid dehydrogenases of female rabbit liver and kidney having similar purification characteristics and isoelectric points were compared with regard to their relative rates of oxidation and reduction of estrogen and androgen substrates, their kinetic parameters and their primary structures. All of the enzyme forms exhibited both 3 alpha- and 17 alpha-enzyme activity toward androgen substrates and the oxidation of the 17 alpha-hydroxysteroid epitestosterone was competitively inhibited by the 3 alpha-hydroxysteroid androsterone. The most basic enzyme forms from liver and kidney had similar relative activities toward estrogen and androgen substrates in the oxidative direction but differed in their activities in the reductive direction. Major differences in the peptide maps of these enzymes were observed. The less basic enzyme forms from the two tissues had similar activities toward estrogen substrates but differed considerably in their relative activities toward androgens. Only minor differences were observed in the peptide maps of these enzymes.

Estrone beta-glucosidase activity in human placenta.

The 105 000g supernatant from human placental homogenates, prepared in the presence of sodium taurocholate and Cutscum, contained beta-glucosidase activity towards estrone glucoside as well as towards 4-methylumbelliferyl glucoside (4-MU-glucoside) and glucocerebroside. After partial purification, the estrone glucosidase was found to be active only after the addition of negatively charged phospholipid, whereas the other beta-glucosidases did not exhibit this requirement. The estrone glucosidase was separated from the 4-MU-glucosidase by chromatography on Sephadex G-200 with 0.1% sodium taurocholate in the eluting buffer. The estrone glucosidase was mainly contained in material with a pI of 4.7, while the 4-MU-glucosidase was distributed in fractions with pI values of 4.7 and 6.2 to 6.4. The partially purified estrone glucosidase had a pH optimum of 5.8, as distinct from that of 6.4 found for the 4-MU-glucosidase, and differed markedly from the 4-MU-glucosidase in its response to treatment with heat, sulfhydryl reagents, and detergents. Its sensitivity to changes in pH differed from those reported for glucocerebrosidase.

Mechanism of glucose transfer to steroids by rabbit liver microsomes.

The formation of the 3-glucosides of estrone, 17alpha-estradiol, or 17beta-estradiol by washed rabbit liver microsomes in the absence of UDP glucose (UDPG) was increased by the addition of crude lipid extracts of rabbit or pig liver and of dolichol or retinol. The reaction was stimulated by Mn2+, Mg2+, and Ca2+. Direct transfer of glucose to the steroid from added UDPG also occurred by a mechanism which was inhibited by metal ions. The two transferases can be separated from each other into the 105000 g pellet and supernatant respectively by treatment with detergents.

A comparison of two response-reinforcement methods in pure-tone testing of the retarded.

The purpose of this study was to determine whether a visual reward method or play audiometry is a better motivator in eliciting the lowest possible pure-tone thresholds from a group of Down's syndrome adults. Thresholds were obtained at 250, 500, 2000, 4000, and 6000 Hz for both procedures using a modified ascending method. The method of testing was constructed such that each of the two procedures served as a comparison for validity of the other. The level of significant difference between the play audiometry and the visual reward method was determined using the Sign Test. In each case, there appeared to be a significant difference between the two tests, but the difference was attributed to the order of administration of the tests and practice rather than by the tests themselves. The data obtained suggests that more accurate auditory thresholds may be determined by administering two pure-tone tests and accepting the second as most valid.