Nutritional and gonadal effects on the intra-acinar profiles of low-Km and high-Km aldehyde dehydrogenase activity in rat liver

Total and low-Km aldehyde dehydrogenase (ALDH) activity was measured in 50-150 ng microdissected liver tissue samples of the entire sinusoidal length. High-Km ALDH activity was calculated by subtracting the low-Km ALDH values from the total ALDH activity. Enzyme activity was measured by a microchemical assay, using the oil-well technique with luminometric determination of NADH. The intra-acinar profiles of high-Km and low-Km ALDH activity could be demonstrated graphically for both male and female rats after 84 h of starvation, and after starvation and refeeding for 6 nights. In addition, the ALDH distribution patterns of juvenile, castrated, and castrated and testosterone-treated rats were determined. It could be demonstrated that starvation, and starvation followed by refeeding, lead to changes in enzyme activity which parallel the loss and regain of liver- and body-weight. The nutritional factors do not essentially alter the normal intra-acinar profiles. In juvenile rats, ALDH is lower by 30% in comparison with the controls, but sex-differences in the distribution profiles are not yet present. Castration has no effect on the amount of enzyme activity but the sex specific distribution profiles are less marked. The main effect of testosterone treatment is an elevation of low-Km ALDH in the perivenous zone. The characteristics of the intra-acinar profiles of high-Km and low-Km ALDH activity are discussed with respect to hepatic acetaldehyde oxidation and alcoholic liver damage.     

Active potassium transport in reticulocytes of high-K and low-K sheep

The kinetics of active K⁺ transport were studied in immature red blood cells cells from high-K⁺ and low-K⁺ sheep, particularly with respect to the effects of varying intracellular K⁺ concentration, [K]_i. Comparison was made with active transport, or pump, activity in mature high-K⁺ and low-K⁺ red cells. Reticulocytes from both types of sheep had much higher maximal active K⁺ influxes than did mature cells. In both types of reticulocytes, and in mature high-K⁺ cells as well, the pump was relatively insensitive to increasing [K]_i. In contrast, intracellular K⁺ markedly inhibited the pump in mature low-K⁺ cells. Active K⁺ transport in low-K⁺ reticulocytes, however, as in mature low-K⁺ cells, is stimulated by specific isoimmune anti-L serum. Therefore the K⁺ pumps of high-K⁺ and low-K⁺ reticulocytes have similar kinetic properties. Maturation of the red cells, involving inactivation of most of the pump activity in both cell types, results in mature high-K⁺ and low-K⁺ cells with K⁺ pumps of very different kinetic characteristics.     

Subcellular distribution and characterization of human liver aldehyde dehydrogenase fractions.

The subcellular distribution of human liver aldehyde dehydrogenases (E.C. 1.2.1.3) have been studied and the different types have been separated by ion exchange chromatography. The cytoplasmic fraction contained at least two chromatographically separable aldehyde dehydrogenases, which accounted for about 30% of the total activity. One of the cytoplasmic aldehyde dehydrogenases had a high K_m for aldehydes (in the millimolar range). A considerable part of the activity found in this fraction was due to an enzyme with a low K_m for aldehydes (in the micromolar range). It had properties similar to those of the mitochondrial main enzyme fraction, from where it may have originated as a contamination during subcellular fractionation. Specific betaine aldehyde and formaldehyde dehydrogenases were separated from these unspecific activities in the cytoplasmic fraction. In mitochondria, where more than 50% of the total aldehyde dehydrogenase activity was found, there was also evidence for slight high-K_m activity. The microsomal fraction contained only a high-K_m aldehyde dehydrogenase, which accounted for about 10% of the total activity.     

Sodium and potassium interactions with Na+-ATPase of inside-out membrane vesicles from high-K+ and low-K+ sheep red cells

Na⁺-ATPase of high-K⁺ and low-K⁺ sheep red cells was examined with respect to the sidedness of Na⁺ and K⁺ effects, using inside-out membrane vesicles and very low ATP concentrations (?2 μM). With varying amounts of Na⁺ in the medium, i.e., at the cytoplasmic surface, Na_cyt⁺, the activation curves show that high-K⁺ Na⁺-ATPase has a higher affinity for Na_cyt⁺ compared to low-K⁺. The apparent affinity for Na_cyt⁺ is also increased by increasing the ATP concentrations in high-K⁺ but not low-K⁺. With Na_cyt⁺ present, Na⁺-ATPase is stimulated by intravesicular Na⁺, i.e., Na⁺ at the originally external surface, Na_ext⁺, to a greater extent in low-K⁺ than high-K⁺. Intravesicular K⁺ (K_ext⁺) activates Na⁺-ATPase in high-K⁺ but not in low-K⁺ vesicles and extravesicular K⁺ (K_cyt⁺) inhibits low-K⁺ but not high-K⁺ Na⁺-ATPase. Thus, the genetic difference between high-K⁺ and low-K⁺ is expressed as differences in apparent affinities for both Na⁺ and K⁺ and these differences are evident at both cytoplasmic and external membrane surfaces.     

The effect of sex hormones on the acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in rat liver

The intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity (PEPCK) was investigated in microdissected samples of livers from normal, castrated, castrated and estradiol- or testosterone-treated, and uncastrated and testosterone- or estradiol-treated male and female rats. The total PEPCK activity showed a marked sex dependency, with 1.8 times higher activity in males. The intra-acinar distribution profiles were also sex-dependent. The periportal-to-perivenous gradient was steeper in males. Castration resulted in an approximation of PEPCK activity and its acinar distribution pattern between the sexes due to a reduction in males and an increase in females. Estrogen treatment of castrated males had no further effect on PEPCK activity and its acinar gradient, whereas in ovariectomized animals the activity was reduced to levels near normal. Testosterone treatment of castrated male or female animals led to a marked increase in enzyme activity with a concomitant steepening of the acinar gradient. Administration of estradiol to normal male rats also led to a reduction in activity, together with a change in the acinar activity gradient. Testosterone treatment of normal females resulted in an induction of PEPCK activity which was most prominent in the periportal zone. The most drastic changes were observed in the perivenous zones. In all experiments a periportal-to-perivenous activity gradient persisted thus marking the periportal zone as the area with highest gluconeogenic capacity.     

The effects of starving and refeeding on the intra-acinar distribution pattern of alcohol-dehydrogenase activity in rat liver

Microquantitative determinations of ADH activity were carried out on the livers of male and female rats. The animals were either starved for 84 h, or starved and then refed with a carbohydrate-rich diet for 6 nights. When the enzyme activity is expressed in mumoles/min/g dry weight, fasting does not appear to alter liver ADH activity, while in starved and subsequently refed rats it is diminished by 20%. Microquantitative measurements of ADH activity in 50-150 ng lyophilized tissue samples, microdissected the whole way along the sinusoidal length, made the computer-aided plotting of intra-acinar distribution patterns possible. The results showed that, under the feeding conditions selected, only minor changes in the ADH activity profiles occur in the liver acinus. These are within the range of the standard deviations of the normal mean values. From these results it can be deduced that fasting and refeeding do not lead to specific inhibition or induction of liver ADH activity. - The decrease of ADH activity of total liver (mumol/min) per total body weight in starved rats is obviously the result of a loss of protein which affects the liver cells of all acinar zones almost equally.     

Acetaldehyde levels during ethanol oxidation: a diet-induced change and its relation to liver aldehyde dehydrogenases and redox states.

3 different diets that had previously been observed to cause large differences in blood acetaldehyde levels of rats administered ethanol were compared with respect to their influence on liver enzymes metabolizing alcohol, on ethanol elimination and on the ethanol-induced changes in the hepatic content of metabolites that reflect the cytosolic or the mitochondrial redox state of the nicotine-amide dinucleotide couple. The results demonstrate that an unknown dietary factor affects the activity of liver aldehyde dehydrogenase, especially that of the low-K_m enzyme. It is suggested that these enzyme activity changes are reflected in the observed alterations in acetaldehyde levels, which in turn may be associated with the magnitude of the shift in the mitochondrial redox state during ethanol oxidation.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Summary Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient.Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Effect of starvation and refeeding on autophagy and heterophagy in rat liver

Increase in the density of liver lysosomes after leupeptin administration was marked in starved rats but only slight in starved-refed rats. The levels of several intracellular enzymes in the liver lysosome fraction purified from leupeptin-treated rats were about 10 to 30 times more in starved rats than in refed rats. However, there was no difference between the intralysosomal levels of endocytosed FITC-labeled asialofetuin in starved and refed rats, indicating that refeeding after starvation markedly suppressed autophagy but not heterophagy in vivo. Immunohistochemical studies with cathepsin B and asialofetuin Fab'-peroxidase conjugates showed that refeeding after starvation markedly altered the cellular distribution of cathepsin B in the liver, resulting in a linear arrangement of the enzyme only on the periphery of hepatocytes. In contrast, endocytosed asialofetuin was found only in the periphery of hepatocytes of both starved and starved-refed rats. These results indicate that autophagy and heterophagy are regulated by different mechanisms in vivo.     

Response of heterogenous rat liver lysosome populations to starvation and refeeding

Starvation-induced alterations in liver lysosomes and their recovery pattern following refeeding were investigated. Fasting of adult rats for five days caused an increase in ‘free’ activities of acid hydrolyses in liver homogenates and loss in sedimentation of one of the heterogenous populations of lysosomes that could be isolated by differential centrifugation. Isopycnic sucrose gradient centrifugation revealed a decrease in the median and modal equilibration densities of all the forms of lysosomes in response to the dietary deprivation. Further, starvation also evoked a distinct bimodal distribution in a population that was rich in acid phosphatases, β-galactosidase and N-acetyl-βglucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.     

The intra-acinar distribution patterns of alcohol-dehydrogenase activity in the liver of juvenile, castrated and testosterone-treated rats

Rat liver alcohol dehydrogenase shows characteristic sex-differences with respect to activity and heterotopy. For the recognition of gonadal influences on the intra-acinar distribution patterns luminometric determinations of ADH activity were carried out on 50-150 ng lyophilized liver tissue samples which had been microdissected along the sinusoidal length. Juvenile rats of both sexes showed equally high alcohol dehydrogenase activity, which surpassed the adult values by a factor of 2 in males and 1.3 in females. The distribution pattern was rather flat, with a weak maximum at the beginning of the last third of the sinusoid. Castration of adult male and female rats resulted in an increase of alcohol dehydrogenase activity to around the prepubertal values. The intra-acinar profiles showed a gradual increase in activity from low periportal values to a peak near the perivenous zone. Only the hepatocytes directly adjacent to the efferent venule showed an even lower activity. Administration of testosterone to castrated animals had no effect on the ADH activity in males and resulted in only a slight decrease of enzyme activity in females. The intra-acinar distribution patterns showed an intermediary peak at the end of the second third of the sinusoidal length in males and a gradual increase of activity, beginning periportally, in the direction of the perivenous zone in females. The present findings on total activity of ADH and its distribution patterns in the liver are considered to be the result of complex hormonal alterations rather than a specific effect of testosterone.     

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.     

Effect of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus

We evaluated the effects of starvation and refeeding on digestive enzyme activities in juvenile roach, Rutilus rutilus caspicus. Fish were divided into four feeding groups (mean mass 1.68 ± 0.12 g). The control group was fed to satiation twice a day throughout the experiment with formulated diet (SFK). The other three groups were deprived of feed for 1(S1), 2(S2), and 3(S3) weeks, respectively, and then fed to satiation during the refeeding period. The results showed that trypsin specific activity was not affected significantly either by starvation or refeeding, in all experimental groups. Chymotrypsin specific activity did not change significantly in S1 fish during the experimental period. In S2 and S3 fish no significant changes were observed during the starvation period. Upon refeeding, the activity increased in S2 fish, while it decreased in S3 fish. Amylase specific activity decreased significantly during the starvation period in all experimental groups. Upon refeeding, the activity increased. Alkaline phosphatase specific activity did not change significantly during the experiment period in S3 fish, while it showed significant changes during the starvation and refeeding period in the S1 and S2 fish. Starvation also had a significant effect on the structure of the intestine.     

Serine dehydratase and tyrosine aminotransferase activities increased by long-term starvation and recovery by refeeding in rainbow trout (Oncorhynchus mykiss)

Here, we study a cycle of long-term starvation followed by refeeding in relation to the kinetics of serine dehydratase (SerDH) and tyrosine aminotransferase (TyrAT) in rainbow trout (Oncorhynchus mykiss). We determine SerDH- and TyrAT- specific activity at different substrate concentrations in liver and white muscle of juvenile trout starved for 70 days and then refed for 6 hr, 32 hr, 4 days, and 9 days. SerDH showed a hyperbolic kinetic with a K(m) for L-serine of 77.07+/-8.78 mM in the liver of control trout. After 70 days of starvation, the SerDH activity at saturate substrate concentration rose 100% over control. No significant changes were found in the K(m) values of the enzyme. After refeeding, the SerDH activity declined to control values. TyrAT also showed a hyperbolic kinetic with a K(m) for L-tyrosine of 1.86+/-0.12 and 2.55+/-0.57 mM in liver and white muscle, respectively. In starved trout, TyrAT activity in liver and white muscle was about 64 and 267%, respectively, higher than control. After 9 days of refeeding, the control values recovered, although, at 6 hr of refeeding, hepatic TyrAT activity was higher than that for starvation. This work shows that SerDH and TyrAT are present in rainbow trout and that the two enzymes have regulatory functions in the catabolism of their respective amino acids in this species.     

Enhanced 5-HT<Subscript>2A</Subscript> Receptors in Brain Stem and ALDH Activity in Brain Stem and Liver: 5-HT<Subscript>2A</Subscript> Regulation on ALDH in Primary Hepatocytes Cultures In Vitro

Brain serotonin (5-HT) modulates the neural effects of ethanol. In the present study, we investigated the changes in 5-HT level, 5-HT_2A receptor binding and aldehyde dehydrogenase (ALDH) activity in brain stem and liver of ethanol treated rats and 5-HT_2A regulation on ALDH in hepatocyte cultures in vitro. The 5-HT content in the brain stem and liver significantly decreased with an increased 5-HIAA/5-HT ratio in the ethanol treated rats compared to control. Scatchard analysis of [³H] (±)2,3-dimethoxyphenyl-1-[2-(-4-piperidine)-methanol] [³H] MDL 100907 against ketanserin in brain stem of ethanol treated rats showed a significant increase in B _max without any change in K _d compared to control. The competition curve for [³H] MDL 100907 against ketanserin fitted one-site model in both control and ethanol treated rats with unity as Hill slope value. A significant increase in V _max of ALDH activity in liver and a significant decrease in K _m in liver and brain stem of ethanol treated rats compared to control was observed. In 24 h culture studies, an increase in enzyme activity was observed in cells in medium with 10% ethanol. The elevated ALDH activity in ethanol treated cells was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT. Ketanserin, an antagonist of 5-HT_2A, reversed the effect of 5HT on 10% ethanol induced ALDH activity in hepatocytes. Our results showed that there was a decreased 5-HT content with an enhanced 5-HT_2A receptor and aldehyde dehydrogenase activity in the brain stem of alcohol treated rats and in vitro hepatocyte cultures. The enhanced ALDH activity in ethanol supplemented hepatocytes was reversed to control level in presence of 10⁻⁵ and 10⁻⁷ M 5-HT.     

Decreased dopamine D2 receptor function in cerebral cortex and brain stem: their role in hepatic ALDH regulation in ethanol treated rats

Ethanol exerts numerous pharmacological effects through its interaction with various neurotransmitters. The dopaminergic pathway is associated with cognitive, endocrine, and motor functions, and reinforcement of addictive substances or behaviours. Aldehyde dehydrogenase (ALDH) is a vital enzyme involved with alcohol metabolism and detoxification. In the present study, we investigated the role of cerebral cortex and brain stem dopamine D₂ receptors in the functional regulation on ALDH enzyme activity, in ethanol administrated rats. Two groups of rats were selected viz. control and alcoholic. Cerebral cortex, brain stem and the liver dopamine content was decreased significantly (P < 0.05, 0.05, 0.001, respectively) and homovanillic acid/dopamine (HVA/DA) ratio has significantly increased (P < 0.05, 0.001 and 0.001), respectively in ethanol treated rats when compared to control. Scatchard analysis of [³H]YM-09151-2 binding to synaptic membrane preparations of cerebral cortex and brain stem showed a significant decrease (P < 0.001, 0.05, respectively) in B _max in ethanol treated rats compared to control and the K _d also decreased significantly (P < 0.05). The ALDH analysis showed a significant increase (P < 0.05) in V _max in cerebral cortex, plasma and liver of experimental rats when compared with control without having significant change in brain stem but with decreased K _m (P < 0.001). Our results suggest that decreased function of dopamine mediated through DA D₂ receptor in the cerebral cortex and brain stem enhanced the brain, plasma and liver ALDH activity in ethanol treated rats. This ALDH regulation has significance to correct alcoholics from addiction due to allergic reaction observed in aldehyde accumulation.     

Response of heterogenous rat liver lysosome populations to starvation and refeeding.

Starvation-induced alterations in liver lysosomes and their recovery pattern following refeeding were investigated. Fasting of adult rats for five days caused an increase in 'free' activities of acid hydrolyses in liver homogenates and loss in sedimentation of one of the heterogenous populations of lysosomes that could be isolated by differential centrifugation. Isopycnic sucrose gradient centrifugation revealed a decrease in the median and modal equilibration densities of all the forms of lysosomes in response to the dietary deprivation. Further, starvation also evoked a distinct bimodal distribution in a population that was rich in acid phosphatases, beta-galactosidase and N-acetyl-beta-glucosaminidase. Realimentation of starved animals for 10 days was found to restore the enzyme levels and the sedimentation characteristics to normal profiles.     

Effects of starvation and refeeding a high carbohydrate diet on the intra-acinar distribution pattern of phosphoenolpyruvate carboxykinase activity in the liver of male and female rats

Phosphoenolpyruvate carboxykinase activity in rat liver was shown to be heterotopically distributed within the acinus under varying feeding conditions. Highest values of PEPCK activity were found in the periportal zone of the acinus from where it decreased continuously towards the perivenous zone. 84 h of starvation resulted in an increase of activity, which was most prominent in the perivenous zone, but nevertheless resulted in a steeper gradient. Refeeding of starved rats with a high carbohydrate diet for 6 nights led to a decrease in PEPCK activity which was most prominent in the periportal zone, but almost negligible in the perivenous zone, resulting in a further change in the activity gradient. Sex-dependent differences for total PEPCK activity were found i) in controls, where the activity was lower in females, ii) after starvation, where the induction was much higher in females, and iii) after refeeding of starved rats, where the activity in females remained higher compared to that of the controls. Differences in the intra-acinar localization of the activity in dependence of the sex were registrated in the control group and in starved rats. Livers from female rats contained a higher periportal/perivenous ratio compared to males. In starved and starved and refed animals the periportal/perivenous ratios were almost the same in both sexes.     

Rate of synthesis and degradation of rat liver phosphoenolpyruvate carboxykinase in the different thyroid states

The effect of hypo-, eu-, and hyperthyroidism on the relative rate of rat liver phosphoenolpyruvate carboxykinase (PEP-ck) synthesis and degradation was studied in vivo by a radioimmunological technique. In hypo- and euthyroid rats, starvation induced an identical increase in PEP-ck synthesis and activity. Hyperthyroidism led to a significant acceleration in enzyme synthesis and correspondingly enhanced enzyme levels. PEP-ck degradation rate, as measured by the double pulse-labeling technique, was not affected by different thyroid states in starved rats ($\text{t}\text{1}\text{2}\text{~ 8}\text{h}$). Hypo-, eu-, and hyperthyroid starved rats responded to glucose refeeding with a rapid and similar PEP-ck deinduction, followed by a concomitant decrease in enzyme activity.     

Long-term effects of plant hormones on K+ levels and transport in young wheat plants of different K+ status

Long-term effects of 1-naphtaleneacetic acid (NAA), benzyladenine (BA), gibberellic acid (GA₃), abscisic acid (ABA) and ethylene on K⁺ levels, K⁺ uptake and translocation to the shoot were studied in young wheat plants (Triticum aesticum L. cv. Martonvásári-8) grown at different K⁺ supplies. Na⁺ levels and K⁺/Na⁺ selectivity were also investigated. Both in shoots and roots, NAA, BA and ABA decreased K⁺ and Na⁺ levels more effectively in high-K⁺ plants than in low-K⁺ plants. GA, and ethylene did not influence K⁺ and Na⁺ levels. K⁺/Na⁺ selectivity in roots of low-K⁺ plants was increased in favour of K⁺ by BA, NAA and to a lesser extent by ABA. In high-K⁺ plants only BA increased the K⁺/Na⁺ ratio, whereas the effects of the other hormones were the opposite (NAA) or less pronounced (ABA). K⁺(⁸⁶Rb) uptake was inhibited by NAA and BA in low-K⁺ plants but not in high-K⁺ plants. K⁺(⁸⁶Rb) uptake was inhibited throughout by 10 μM ABA. K⁺(⁸⁶Rb) translocation to the shoot was influenced by the hormones similarly to the uptake patterns, with the exception of ABA, which inhibited translocation in low-K⁺ plants but not in high-K⁺ plants. The results show that hormonal effects may quantitatively and qualitatively be modified by K⁺ levels in the plant and that internal K⁺ concentration may play a role in the mechanisms regulating the effects of NAA, BA and ABA but probably not in those of GA₃ or ethylene.