Behavior of RNA and protein synthesis during the acclimatization of the carp. Studies with isolated hepatocytes

1. Carp hepatocytes were isolated by dissociating the liver tissue with collagenase. The procedure yields viable cells with highly preserved ultrastructural and metabolic features. The isolated cells were able to self-aggregated and form tissue. 2. RNA and protein synthesis activity was significantly higher in the carp hepatocytes from summer acclimatized fish compared to the activity present in the cold adapted animals. 3. RNA synthesis assayed in carp hepatocytes suspensions obtained from summer and winter acclimatized fish exhibited a behaviour consistent with an inverse compensation to the cold acclimatization state, being apparently repressed, whereas protein synthesis did not show a compensatory activity.     

Temperature induced metabolic costs in carp, Cyprinus carpio L., during warm and cold acclimatization

The oxygen consumption of 6 carps was measured in a flow-through respirometer when water temperature was reduced from 23 to 7°C and increased from 23 to 33°C as well as from 11 to 32°C. The metabolic reaction of Cyprinus carpio L. was assessed at 3 levels: standard, routine and spontaneous. The Standard Metabolic Rate increased and the Q₁₀ decreased with rising temperatures. A quadratic relation was established between metabolic rates and temperature level. The enery exenditure above Standard Metabolic Rate induced by temperature changes was 6.65 ± 1.97 kJ kg^-0.8 d^-1 (x ± S.E., N = 6).
Costs for temperature acclimatization amounted to 29 %, 24 % and 9 % of the energy required for standard, routine and spontaneous action metabolism, respectively.

Zusammenfassung

Temperaturinduzierte Stoffwechselerhöhungen bei Karpfen , Cyprinus carpio L., während Warm- und Kaltakklimatisierung
Wir haben untersucht, wie der Stoffwechsel von Cyprinus carpio im Standard-, Routine- und Spontanniveau während einer Kalt- und Warmakklimatisierung im Bereich von 7–33 °C reagiert. Dieses Verhalten wurde mit Hilfe eines Durchfluß-Respirometers ermittelt. Die "Standard Metabolic Rate" nahm zu und der Q₁₀ verringerte sich mit steigender Temeratur.
Eine quadratische Beziehung besteht zwischen den Stoffwechselraten und dem Temperaturniveau. Die durch Temperaturänderun induzierte Stoffwechselerhöhung über die Standardrate wurde für den Meßbereich mit 6.65 ± 1.97 kJ kg^-0.8 d^-1 (x ± S.E., N = 6) ermittelt.
Der Anteil temperaturinduzierter Aufwendungen entsprach 29%, 24% und 9% des Standard-, Routine- und Spontanverbrauches an Energie.     

Effect of thyroxin on the functional state of the respiratory chain in liver mitochondria of the carp Cyprinus carpio during acclimatization to changes in water temperature

It has been demonstrated that oxidative phosphorylation in liver mitochondria of the carps which were kept within a week at 5 and 25 degrees C remains unaffected by the addition of 0.5 micron thyroxin to the incubation medium. Addition of thyroxin (0.5 micron) to mitochondrial suspension prepared from the liver of carps acclimated within a week at 20 and especially 25 degrees C, resulted in uncoupling of respiration and phosphorylation. Daily injections of thyroxin within a week (2 mu kg per 1 g of body weight) to carps at 20 and 25 degrees C resulted in the increase of the rate of non-phosphorylating oxidation and the decrease of oxidative phosphorylation in liver mitochondria. The increase of temperature of water from 5 to 30 degrees C decreases triiodthyronine content of the blood serum in the carp.     

Structural changes in the erythrocyte membrane of the trout Salmo irideus at seasonal acclimatization

Differences of thermostability were studied in red blood cells of the trout Salmo irideus differing in sex and age, as well as structural-dynamic characteristics of erythrocyte membrane proteins at seasonal acclimatization in the interval of reservoir water temperature of 0–19°C. An increase of resistance of erythrocytes to temperature lysis with elevation of the environmental temperature was revealed to be accompanied by a rise of the proteins segmental mobility and a decrease of intermolecular interactions in spectrin-actin cytoskeleton from the data of the ESR spin labeling method. Regulation of erythrocyte stability during acclimation was concluded to occur both changes of the fatty acids chain package at the variations of lipid composition and by changes of the cytoskeleton structural lability. Thereby this provides an increase of the bilayer firmness, on the one hand, while, on the other hand, a rise of elasticity and expansibility of the membrane on the whole, which increases resistance of cells to colloidal-osmotic hemolysis. Changes of concentration of oxygen dissolved in water, which are caused by temperature fluctuations, do not deem to be of crucial importance for structural stability of erythrocytes, as it can be compensated by another mechanism, specifically by changes of affinity of hemoglobin to oxygen.     

Thermotropic behavior of major phospholipids from marine invertebrates: changes with warm-acclimation and seasonal acclimatization

The crystal-liquid crystal-isotropic melt phase transitions of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from muscle tissue of five species (actinia Metridium senile fimbriatum, mussel Crenomytilus grayanus, sea-urchin Strongylocentrotus intermedius, starfish Distolasterias nipon and the ascidian Halocynthia aurantium) of marine invertebrates, collected in winter at 0 degrees C and then acclimated to 18.5 degrees C for 5 days, were studied by differential scanning calorimetry and polarising microscopy. To elevate temperature from 0 to 18.5 degrees C, we used the rate of 4.5 degrees C/h. Although phase transitions of both phospholipids from animals collected in summer occurred already at temperatures below -1.7 oC (minimal temperature of seawater in winter), compensatory mechanisms resulted in a decrease by 29-43 oC in the phase transition temperature of PE in winter. Thermotropic behavior of PCs changed in various trends. However, the total heat of their phase transitions always decreased in winter compared with summer. For all species, except the mussel, the time of warm-acclimation was insufficient to adjust the thermotropic behavior of either phospholipid. Nevertheless, the unsaturation index decreased to achieve summer values, due primarily to decreased proportions of eicosapentaenate and docosahexaenate. The accumulation of arachidonate, during warm-acclimation, might be connected to the signalling properties of n-6 eicosanoids. Absence of effective homeoviscous mechanisms suggests that most of the studied marine invertebrates have very limited capacity to survive an acute temperature elevation, e.g. at the appearance of thermal currents.     

Variations of rat brown adipose tissue composition during cold acclimatization.

The modifications in weight and composition (lipids, proteins, water) of rat interscapular brown adipose tissue (BAT) were studied along the six first weeks of cold exposure and acclimatization. The variations of noreponephrine content was also investigated. During the first day of cold exposure, the major part of tissue lipids was released. During the following two days there was a fall in lipid and norepinehprine contents and uptake of water. Then, until the end of the first week a rapid repletion occurred. At that moment the relative pass of the tissue and the amounts of its principal components reached values which are not changed during the following weeks. We can conclude that the adaptative changes in the levels of BAT essential components are carried out at the end of the first week of cold exposure, long time before the non shivering thermogenesis is entirely effective.     

Observations on the ultrastructure of the carp liver (Cyprinus carpio L.)

The paper presents information about the fine structure of the sinusoids, the space of Dissé, the development of the bile canaliculi and some compartments of the hepatocytes in the liver of the carp. The sinusoids are covered completely by flat endothel cells. The plasma of these cells contains numerous vesicles. The endothelial cells possess plasmatic processes, which extend into the space of Dissé. The fine structure of the space of Dissé corresponds to that of mammals, the existence of fibrebundles included. The bile canaliculi don't develop intracellularly as described by other authors. They run intercellularly as in mammals, but they form diverticles which reach into the plasma sideways. The rough ER was found in two types. Outwardly the liver is limited by a layer of connective tissue existing in two different layers.     

Seasonal changes in hepatocyte ultrastructure correlated with the cyclic synthesis of secretory proteins in the winter flounder (Pleuronectes americanus)

Liver tissue was sampled from flounder (Pleuronectes americanus) throughout the year with the intention of documenting changes in the ultrastructure coincident with the production and secretion of antifreeze proteins. In the winter, hepatocytes are dedicated to the production of these proteins and, in the female, also reproductive proteins. In both sexes, liver cells in the summer contain abundant lipid and glycogen stores. In the female, there is a conspicuous hepatocyte transformation from a fat-filled cell in the summer to one with well-developed rough endoplasmic reticulum in the winter. Large amounts of rough endoplasmic reticulum (11.2 mg/gm) were recovered after subcellular fractionation of female wintertime liver. The increased appearance of secretory organelles and the high number of nucleolar profiles observed in winter animals is consistent with the elevated demand for protein secretion and synthesis in both sexes. The fractional volumes occupied by lipid droplets and mitochondria were different when comparisons were made between sex and season. Females contained a greater volume of lipid than did males, and summer animals contained more lipid than those in winter.     

Na, K-ATPase activity of the hepatocyte plasma membranes during disturbance of the bile-secreting function of the liver by inhibitors of protein synthesis

The infusion of the protein synthesis inhibitors depressed the liver bile secretion and Na, K-ATPase activity of the hepatocyte plasma membranes in rats. The significance of Na, K-ATPase in the mechanism of bile secretion is discussed.     

Mitochondrial function in seasonal acclimatization versus latitudinal adaptation to cold in the lugworm Arenicola marina (L.)

Previous studies in marine ectotherms from a latitudinal cline have led to the hypothesis that eurythermal adaptation to low mean annual temperatures is energetically costly. To obtain more information on the trade-offs and with that the constraints of thermal adaptation, mitochondrial functions were studied in subpolar lugworms (Arenicola marina L.) adapted to summer cold at the White Sea and were compared with those in boreal specimens from the North Sea, either acclimatized to summer temperatures or to winter cold. During summer, a comparison of mitochondria from subpolar and boreal worms revealed higher succinate oxidation rates and reduced Arrhenius activation energies (Ea) in state 3 respiration at low temperatures, as well as higher proton leakage rates in subpolar lugworms. These differences reflect a higher aerobic capacity in subpolar worms, which is required to maintain motor activity at low but variable environmental temperatures--however, at the expense of an elevated metabolic rate. The lower activity of citrate synthase (CS) found in subpolar worms may indicate a shift in metabolic control within mitochondria. In contrast, acclimatization of boreal lugworms to winter conditions elicited elevated mitochondrial CS activities in parallel with enhanced mitochondrial respiration rates. With falling acclimation temperatures, the significant Arrhenius break temperature in state 3 respiration (11 degrees C) became insignificant (5 degrees C) or even disappeared (0 degrees C) at lower levels of Arrhenius activation energies in the cold, similar to a phenomenon known from hibernating vertebrates. The efficiency of aerobic energy production in winter mitochondria rose as proton leakage in relation to state 3 decreased with cold acclimation, indicated by higher respiratory control ratio values and increased adenosine diphosphate/oxygen (ADP/O) ratios. These transitions indicate reduced metabolic flexibility, possibly paralleled by a loss in aerobic scope and metabolic depression during winter cold. Accordingly, these patterns contrast those found in summer-active, cold-adapted eurytherms at high latitudes.     

Change in the ultrastructure of hepatocyte contacts in mechanical deformations of liver tissue]

Rat liver specimens were deformed by compression between two parallel plates up to the state of plastic deformation to study with the electron microscope. The mechanical deformation was followed by a destruction of intercellular contacts and separation of hepatocytes. According to the pattern of destruction, all the contact elements can be divided into two groups. 1. Simple junction, desmosome, intermediate junction which exibit a wide range of structural changes prior to the moment of destruction. In this case, the process of cell separation proceeds in the adhesive way. 2. Tight and gap junctions which exibit a higher structural stability. In this case the process of cell separation appears to be of the cohesine nature.     

Spatiotemporal changes in Ha-ras p21 expression through the hepatocyte cell cycle during liver regeneration.

The protein product of the ras oncogene, Ha-ras (p21), is thought to be an important regulator of cell growth. The cytoplasmic relocalization of p21 in the cell during the cell cycle suggests a transient signaling role for this protein in association with its signal transduction function. Because of the importance of this role we examined spatial patterns in vivo of p21 expression at the protein and mRNA levels in hepatocytes during compensatory growth in rat liver following partial hepatectomy. A low level of p21 was immunolocalized on the cytoplasmic membrane of nonregenerating hepatocytes. The level of hepatic p21 increased significantly and without spatial restriction within the liver from 36 to 60 hr after partial hepatectomy (PH). p21 was localized in the cytoplasm of dividing hepatocytes and on the hepatic cytoplasmic membrane. The elevated p21 level decreased and was found mainly on hepatocyte plasma membranes by 96 hr after PH. Immunogold electron microscopy showed p21 localized over mitochondrial membranes and nuclei in nondividing regenerating hepatocytes. Approximately 50% of nonregenerating hepatocytes show nuclear localization of p21. This percentage changes with time following PH. The decrease in nuclear localization was accompanied with an increase in the low number of hepatocytes which demonstrated cytoplasmic localization in nondividing hepatocytes in regenerating liver. Flow cytometric analysis revealed a significant increase of p21 at 36 hr after PH which was 12 hr after the initial induction of ras mRNA. ras mRNA level increased 1.5-fold at 24 hr after PH and a maximum twofold induction was observed at 48 hr. Cell-cycle analysis of regenerating hepatocytes indicated a synchronized first peak of cell division 36-40 hr after PH. Dual parameter flow cytometry revealed that the level of p21 in hepatocytes in S phase and G2/M phase of the cell cycle was significantly higher than that in G0/G1 phase during regeneration. These findings suggest that p21 is important for the progression of regenerating hepatocytes to S phase and then to G2/M phase.