Effects of ovariectomy and chronic estradiol administration on pituitary-thyroid axis in adult rats

The effects of ovariectomy (Ovx) and of ovariectomy followed by chronic estradiol dipropionate administration (Ovx EDP) on the structure and function of the pituitary-thyroid axis were examined in the rat. Pituitary TSH cells and thyroid tissue were histologically, immunohistochemically and stereologically investigated. Serum TSH and T(4) levels were determined by RIA. Ovx did not affect pituitary weight, but subsequent treatment with EDP led to its more than two-fold increase (p<0.05). After ovariectomy, the cellular volume of pituitary TSH-immunoreactive cells increased by 28%, p<0.05 compared to sham-operated animals (SO). Treatment of Ovx rats with EDP partially reversed this change. However, the relative volume density of thyrotrophs decreased in comparison to the Ovx and SO groups (by 18% and 23%, p<0.05, respectively). No statistically significant differences in serum TSH levels were observed between the experimental groups. In thyroid tissue both peripheral and central follicles responded to Ovx and EDP treatments. Compared to SO rats, the relative volume densities of the follicles and colloid were increased (by 14% and 30%, p<0.05, respectively) in Ovx rats. Chronic EDP treatment of Ovx rats reversed these changes to the pre-ovariectomy state. Hyperplasia of thyroid follicular cells and a significant reduction (by 21%, p<0.05) of the serum level of T(4) were detected. In conclusion, estradiol deficiency and chronic treatment affected pituitary TSH cells and thyroid tissue. The sum effect of Ovx on the pituitary-thyroid axis was slightly stimulatory. Subsequent EDP treatment decreased thyroid functioning but at the same time preserved serum TSH at the control level.     

Ethanol metabolism by the rat heart and alcohol dehydrogenase activity.

Rat hearts perfused with oxygenated buffer containing [1-14C]ethanol metabolized small amounts of the ethanol to carbon dioxide. Very sensitive techniques are required to separate the resulting 14CO2 from the ethanol. This metabolism is not inhibited by levels of pyrazole which markedly inhibit NAD dependent liver alcohol dehydrogenase (EC 1.1.1.1). In vitro studies suggest that NADP functions as a cofactor for the rat heart alcohol dehydrogenase activity of crude heart homogenates. The kinetics parameters, the specific activity, and the pH dependence of the enzyme activity measured in these experiments suggest that it may have a minor role in ethanol metabolism by the rat.     

Liver alcohol dehydrogenase and aldehyde dehydrogenase in the Japanese: isozyme variation and its possible role in alcohol intoxication.

Forty autopsy livers from Japanese individuals were studied concerning alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) isozymes using electrophoretic and enzyme assay methods. A remarkably high frequency (85%) was found for the atypical ADH phenotype. The gene frequencies of ADH22 and ADH32 were .625 and .05, respectively. The usual ALDH phenotype showed two major isozyme bands, a faster migrating (low Km for acetaldehyde) and a slower migrating isozyme (high Km for acetaldehyde). Fifty-two percent of the specimens had an unusual phenotype of ALDH, which showed only the slower migrating isozyme. The usual phenotype was inhibited about 20%--30% by disulfiram and the unusual type up to 90%. Such a high incidence in the Japanese of the unusual phenotype, which lacks in the low Km isozyme, suggests that the initial intoxicating symptoms after alcohol drinking in these subjects might be due to delayed oxidation of acetaldehyde rather than its higher-than-normal production by typical or atypical ADH.     

Drosophila alcohol dehydrogenase activity in vitro and in vivo: Effects of acetone feeding

When adult Drosophila are placed on medium containing 0.5% acetone, their level of alcohol dehydrogenase activity drops rapidly. At the same time, the proportion of activity in the various electrophoretic forms of the enzyme shifts; most of the activity becomes localized in what is ordinarily a minor form of the enzyme. Moreover, the loss of enzyme activity occurs in vivo as well, as shown by sensitivity to ethanol poisoning, insensitivity to pentenol treatment, and inability to utilize ethanol as an energy source. These observations are discussed in light of a model advanced for the origin of the multiple forms of alcohol dehydrogenase in Drosophila.This research was supported by NIH Grants GM-18254 and ES-1527 and by DOE Contract No. EY-76-5-02-2965. Publication No. 1016 from the Department of Biology, Johns Hopkins University.     

Effects of ovariectomy and steroid replacement on GABAA receptor binding in female rat brain.

The specific binding of tritiated muscimol to gamma-aminobutyric acid (GABA) receptor sites was studied in distinct brain areas of female rats during different endocrine states. In diestrous rats with intact ovaries the highest receptor densities were found in the cortex (10.24 pmol/mg protein) and the lowest concentrations in the mediobasal hypothalamus (3.29 pmol/mg protein). Four weeks after removal of the ovaries, the number of binding sites was enhanced up to 2.4-fold in all brain areas investigated: the preoptic brain area, mediobasal hypothalamus, corticomedial amygdala, and cerebral cortex. The affinity of the binding sites remained unchanged. Substitution of estradiol and progesterone reduced the number of binding sites to values seen before ovariectomy. The induction of an afternoon surge of LH by estradiol that could be blocked by enhancing the GABAergic tone was accompanied by a distinct reduction in Bmax in the preoptic area in the morning. These results give evidence that ovarian hormones modulate GABAergic neurotransmission by regulation of GABAA receptor synthesis or degradation.     

Effects of estradiol and progesterone on rat uterine ribonuclease inhibitor activity

The effects of estradiol (E2) and progesterone on rat uterine RNase inhibitor activity were investigated. RNase inhibitor activity was found to be most prominent in nuclear and cytosolic fractions of the uterus. Uterine RNase inhibitor activity in the 45,000 x g cytosolic fraction was stimulated by E2 over a 6-24 hr treatment period. Progesterone did not produce a significant change in the uterine cytosolic RNase inhibitor activity during the same period.     

Effects of alcohol consumption on mitochondrial aldehyde dehydrogenase isoenzymes in rat liver

The effects of long-term and short-term exposure of rats to ethanol on aldehyde dehydrogenase (ALDH) activity in the liver mitochondria were investigated. The specific activities of mitochondrial high Km ALDH and low Km ALDH after the prolonged administration of ethanol were both increased to levels about 2.5 times that of the control group. In contrast, high Km and low Km ALDH showed maximum activity 12 h after administration of a single large dose of ethanol, increasing 21 and 4.4 times, respectively, over the level in the control group. When ethanol was administered for a long time, the two ALDH isoenzyme levels showed approximately the same increase, while the high Km ALDH level was more significantly increased than the low Km ALDH level after a single large dose. These results suggest that the high Km ALDH level of the outer membrane was increased as a result of a transient increase in the level of acetaldehyde around the liver mitochondria after a single large dose of ethanol, and that high Km ALDH plays an important role in acetaldehyde metabolism. However, when ethanol was administered for a long time, the mitochondria were exposed to low concentrations of acetaldehyde over a long time, leading to an increase in levels of low and high Km ALDH in the matrix.     

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell. This revised version was published online in August 2006 with corrections to the Cover Date.     

Circadian rhythms of alcohol dehydrogenase and MEOS in the rat

The circadian peak in alcohol dehydrogenase (ADH) activity fell near the time of maximal blood ethanol clearance rates both in groups of rats injected with a single ethanol dose (acute group) and in rats continuously exposed to ethanol for 22 weeks (chronic group). However, at all timepoints investigated ADH activity levels were lower and fluctuated less in the chronic group than in either the acute or control (ethanol naive) groups. In contrast, activity levels of the microsomal ethanol oxidizing system (MEOS) revealed a prominent rhythm that was 180 degrees out of phase with the ADH rhythm in the chronic group, while MEOS activity showed very low levels in the acute and control groups and did not vary over the circadian span.     

Developmental changes in rat liver alcohol dehydrogenase

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 ± 0.4 μmole/min/g liver and 92 ± 20 μg/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 ± 0.002 μmole/min/mg protein and 2.0 ± 0.4 μg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.     

Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.     

Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

Hepatic aldehyde dehydrogenase activity in Peromyscus genetically deficient in alcohol dehydrogenase

1. Hepatic aldehyde dehydrogenase (ALDH) activity was measured in two strains of deer-mouse, Peromyscus maniculatus. 2. There is no difference in the subcellular distribution of ALDH activity in the two strains. Animals of AdhN/AdhN genotype, lacking liver alcohol dehydrogenase (ADH), had 90% of total ALDH activity in the mitochondrial fraction compared to 94% for the AdhF/AdhF animals with normal ADH activity. Almost all of the remaining ALDH activity was in the hepatic cytosol with less than 1% in the microsomal fraction. 3. By contrast, in mice (Mus musculus) 43% of total hepatic ALDH activity was found in the cytosolic fraction and 55% in the mitochondrial. 4. It was concluded that the subcellular distribution of hepatic ALDH activity in Peromyscus does not vary with the presence or absence of ADH and that this ALDH distribution is not similar to that reported for other rodents.     

The circadian rhythm of intra-acinar profiles of alcohol dehydrogenase activity in rat liver: a microquantitative study

Summary Using microquantitative measurements of alcohol dehydrogenase activity in microdissected samples of liver tissue along the sinusoidal length, the intra-acinar distribution profiles were studied in seven groups of female rats at different times during 24h with a light phase from 630h to 1830h. The mean values of alcohol dehydrogenase activity showed a circadian rhythm with a minimum at 13.30h and a maximum at 17.30h (p<0.0001). However, the intra-acinar gradients remained almost unchanged, indicating that increase and decrease in enzyme activity takes place simultaneously in all parts of the liver acinus. This observation, together with data from the literature, suggests that the circadian rhythm of alcohol dehydrogenase activity reflects variations in different liver cell consituents, rather than enzyme protein synthesis or proteolysis.