Marion Schoenl

Interaction of thyroid hormone and sex steroids at the rabbit reticulocyte membrane in vitro: Control by 17β-estradiol and testosterone of thyroid hormone-responsive Ca2+-ATPase activity

Physiological concentrations (10(-10) M) of L-thyroxine and triiodo-L-thyronine were found in vitro to enhance Ca2+-ATPase activity in reticulocyte-enriched red cell membranes from female rabbits and to inhibit this enzyme in the male reticulocyte. Cross-incubation experiments with reticulocyte-enriched red cells and plasma from the opposite sex demonstrated that this sex-specific membrane response to thyroid hormone was transferable by plasma. Similar experiments with intact reticulocytes exposed to physiological concentrations (10(-11) M) of testosterone and 17 beta-estradiol indicated that the plasma factors were the sex steroids. That is, incubation in vitro with testosterone converted female-source reticulocytes to male-type responsiveness to thyroid hormone (inhibition of Ca2+-ATPase activity); incubation with estradiol converted male-source reticulocyte-enriched red cells to female-type responsiveness (stimulation by iodothyronines of membrane Ca2+-ATPase activity). Similar results were obtained when reticulocyte ghosts were incubated with testosterone and 17 beta-estradiol prior to determination of membrane enzyme activity. Etiocholanolone (5 beta-androstan-3 alpha-ol-17-one) and testosterone were equipotent, but 5 alpha-dihydrotestosterone had little activity in this system. Estrone and estradiol were equipotent, but estriol had no permissive effect on the stimulation by iodothyronine of reticulocyte membrane Ca2+-ATPase activity. Expression of thyroid hormone action in vitro on Ca2+-ATPase activity in the rabbit reticulocyte is determined at the membrane level by testosterone and estrogen. The structure-activity relationships of the sex steroids for this membrane action are different than those reported for nuclear actions of the steroids.

Dissociable and non-dissociable cytoplasmic protein-thyroid hormone interactions

Abstract Dog kidney cytosol contains a high molecular weight (50 000–70 000) and a low molecular weight (approx. 6000) thyronine-binding protein. Low molecular weight cytosol thyronine-binding protein has not been previously recognized in cytoplasm. Binding of thyroxine (tetraiodothyronine, T4) by the low molecular weight protein has a half-time of association of more than 24 h and accounts for 32% of bound cytoplasmic tetraiodothyronine after 48 h of incubation. Binding of labeled tetraiodothyronine and triiodothyronine by this moiety is non-dissociable in the presence of 1 · 10−5 M unlabeled tetra- or triiodothyronine. The low molecular weight protein exists in a dispersed and apparently aggregated form; the latter elutes in the void volume on Sephadex G-100 and its generation is minimized by 2 mM Ca2+. This binding protein elutes in a fraction which has a high A260nm : A280nm ratio, is pentose enriched (orcinol method) and which, because of these characteristics and low susceptibility to digestion by nuclease, is postulated to be a ribosylated cytoplasmic protein or polypeptide. Binding of tetra- and triiodothyronine by the high molecular weight protein has a half-time of association of 2 h and is saturable. Displacement of labeled triiodothyronine from this cytosol thyronine-binding protein is more readily effected with excess unlabeled tetra- than with triiodothyronine, indicating the absence of a triiodothyronine-specific cytosol thyronine-binding protein site. 3,3′,5′-Triiodothyronine (reverse triiodothyronine) is bound with low avidity. Uptake of high molecular weight protein by isolated kidney cell nuclei cannot be demonstrated. Binding of tetraiodothyronine by cytosol proteins is independent of pH in the pH range 6.8–8.9, but binding of triiodothyronine is minimized at pH 7.4 and enhanced at alkaline pH to the point of equivalency of tetra- and triiodothyronine binding at pH 8.9. At concentrations of tetraiodothyronine calculated to exist intracellularly, essentially all soluble fraction tetraiodothyronine is bound to cytosol thyronine-binding protein, restricting access of this iodothyronine to binding sites in nucleus and mitochondria. Cytosol removes labeled tetra- and triiodothyronine previously reacted in vitro with isolated cell nuclei; such removal is a linear function of cytosol protein concentration and is blocked by saturation of cytosol thyronine-binding protein with unlabeled iodothyronines. Only the high molecular weight protein accounts for unbinding by cytosol of nuclear hormone.

Interaction of gender and dietary protein on renal growth and the renal growth hormone—insulin-like growth factor axis

The female kidney tends to be smaller, have a lower glomerular filtration rate, and be less susceptible to glomerulosclerosis than the male kidney. Insulin-like growth factor-I (IGF-I) is a peptide growth factor that appears to be important for normal and adaptive kidney growth. The purpose of this study was to compare the kidney growth response of the male and female rat kidneys to increased dietary protein intake and to see whether differences in IGF-I production or receptor expression might underlie any gender differences seen. Male (M) and female (F) Munich-Wistar rats (6 to 9 weeks of age) were randomized to isocaloric diets containing either 20% (NP) or 50% (HP) protein and studied after 3 and 14 days. In the male rat, wet kidney weight was significantly increased with HP at both day 3 (M-HP 1028+/-21 mg vs M-NP 891+/-19 mg, p < 0.01) and day 14 (M-HP 1499+/-41 mg vs M-NP 1246 +/-37 mg, p < 0.01). In contrast in the female rat, while there was evidence of initial increased growth at day 3 in the kidneys of F rats fed HP (F-HP 788+/-39 mg vs F-NP 650+/-23 mg, p < 0.01), this difference was not sustained at 14 days (F-HP 961+/-67 mg vs F-NP 931+/-71 mg, p = NS). At day 3, kidneys of both male and female rats fed HP exhibited an increase in total protein but not DNA content. The kidneys of male rats showed increased protein/DNA ratios in the medulla and inner cortex, whereas in the kidneys of female rats, the increase in protein/DNA ratio was confined to the cortex. After 14 days of HP ingestion, the kidneys of male rats showed increases in total kidney content of both DNA and protein, and protein/DNA ratios returned to control values in whole kidney, inner cortex, and medulla. In contrast, in the kidneys of female rats, not only was overall growth response reduced, but neither total kidney protein content nor DNA content was increased. Increased protein/DNA ratios were seen in inner cortex and in outer and inner medulla, similar to that seen at day 3 in the kidneys of male rats. Neither baseline plasma (M-NP 793+/-10 ng/ml, F-NP 704+/-32 ng/ml, p = NS) nor kidney IGF-I content (M-NP 520+/-55 ng/gm tissue, F-NP 506+/-54 ng/gm tissue, p = NS) differed between male and female rats fed NP diets. Both male and female rats showed a comparable increase in kidney IGF-I after 3 days of HP ingestion, and kidney IGF-I returned to control values by 14 days. There was no significant difference in the number or affinity of glomerular IGF-I receptors between male and female rats. In conclusion, we have shown that in the adult male rat, an increase in dietary protein ingestion results in a sustained increase in kidney size that is initially consistent with a hypertrophic response but subsequently shows elements of hyperplasia. In contrast, in the female rat, although there was evidence of the initial hypertrophic (and IGF-I) responses to increased dietary protein, the increase in kidney size was not sustained. However, these profound gender-based differences in the growth response to dietary protein did not appear to be due to differences in kidney expression of IGF-I or its receptors.

Early abnormal development of calmodulin gene expression and calmodulin-resistant Ca2+-ATPase activity in avian dystrophic muscle

We have reported previously that the pectoralis muscle from three month-old dystrophic chickens with signs of myopathy exhibits increased calmodulin content, elevated calmodulin-specific mRNA (Biochem. Biophys. Res. Commun. 137:507-512, 1986), and reduced sarcoplasmic reticulum (SR) Ca2+-ATPase activity in response to calmodulin exposure in vitro (Clin. Res. 34: 725A, 1986). To determine the early time sequence for development of these abnormalities, we have studied muscle from embryos and post-hatched chickens at various ages. Quantitated by dot blot analysis, there was an approximate two-fold increase in calmodulin-specific mRNA in dystrophic muscle as early as 13 days ex ovo which was maintained throughout development up to three months ex ovo. Similarly, Ca2+-ATPase activity measured in SR membranes from chickens as early as 13 days post-hatch was also found to be resistant to stimulation in vitro by exogenous calmodulin, whereas the enzyme from normal muscle was calmodulin-stimulable. These findings suggest that the genetic lesion expressed in the avian dystrophic animal model involves the loss of normal control of intracellular calcium metabolism early in the maturation of the affected musculature and prior to appearance of disease signs.

Identification and properties of myocardial myoglobin as a binder of iodothyronines

Gel filtration of dog myocardial cytosol previously incubated with [125I]T4 or [125I]T3 revealed hormone binding in three fractions, one of which, M-2, was presumptively identified as myoglobin by absorbance maximum, molecular weight and specific immunodiffusion. Gel chromatography of purified horse or dog myoglobins incubated with labeled T3 or T4 resulted in coelution of the myoglobin and iodothyronine peaks. Excess unlabeled thyroid hormone displaced no more than 25% of tracer bound to myoglobin. Acid-acetone fractionation of myoglobin into heme and globin, and subsequent precipitation of the heme, localized hormone binding to the heme moiety. Hematin (ferric state heme) in solution was also shown to bind thyroid hormone. Added to human sera which were then subjected to T3 or T4 radioimmunoassay, myoglobin reduced detectable, endogenous iodothyronine by 77 and 26%, respectively. The myoglobin effect was concentration dependent. Heart myoglobin, like hemoglobin in the erythrocyte, is a cytoplasmic heme protein responsible for a major fraction of binding of intracellular iodothyronine. The nature of the interaction between iodothyronines and the heme prosthetic group is unclear.