Seasonal variations of DNA-synthesis in intestinal epithelial cells of hibernating animals--2. DNA-synthesis in intestinal epithelial cells of ground squirrel (Citellus undulatus) during autumn and late hibernation season

1. DNA-synthesis was found to be gradually reduced in the intestinal crypt cells of ground squirrel during autumn at the time of preparation for hibernation and gradually rose during late hibernation season in February-March at the time of preparation for arousal. 2. A conclusion is made on the seasonal variations in cell replacement of true hibernators.     

The intestinal epithelial cells of ground squirrel (Citellus undulatus) accumulate at G2 phase of the cell cycle throughout a bout of hibernation

1. The mitotic index was found to be greatly reduced in the intestinal crypt cells of ground squirrel during bout of hibernation. The percentage of mitosis rose abruptly at least 2 hr after arousal. 2. An increase in the number of G2 cells was found in the intestinal tract of ground squirrel during bouts of hibernation. 3. The conclusion can be drawn that the cells are progressing steadily through the cell cycle. The cells accumulate at G2 in hibernation. 4. It was assumed that the block in G2 prevents the cells from possible damage in mitosis under hypothermia accompanying hibernation and, therefore, it represents an adaptive reaction.     

Protein synthesis in cardiac cells and the ultrastructural dynamics of cardiomyocytes of hibernating animals during the hibernation cycle

A significant increase in protein synthesis correlating with ultrastructural dynamics of cardiomyocyte organelle convertions has been demonstrated in cardiomyocytes of ground squirrel during arousal from hibernation. In hibernating ground squirrels, the ultrastructure of protein-synthesizing organelles and of the cardiomyocyte nucleus points out to the readiness of cells to active synthesis of proteins. In the perinuclear area of cardiomyocytes abundant ribosomes, elements of endoplasmic reticulum and Golgi complex, mitochondria and high-energy substrates--glycogen and lipid inclusions--are seen. The cardiomyocyte nuclei are large, with highly convoluted borders and abundant pores, their nucleolar structure is granular, the chromatin is mainly diffuse. The potency of cardiomyocyte protein-synthesizing system of hibernating ground squirrels is realized every time at periodical arousals during hibernation. The role of cyclic changes of protein synthesis rate in adaptation of cells of hibernating mammals to functioning at various temperatures is discussed.     

Seasonal changes in phosphorylation of myosin regulatory light chains and C-protein in myocardium of hibernating ground squirrel Citellus undulatus

A comparative study concerning the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of hibernating ground squirrel Citellus undulatus during the periods of hibernation and activity was carried out. During hibernation, regulatory light chains of ground squirrel were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averages 40-45% of their total amount. The extent of phosphorylation of the cardiac C-protein during hibernation is about two times higher than that in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.     

Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts

We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p < 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p < 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.     

Induction of host nuclear DNA synthesis in coccidia-infected chicken intestinal cells

When Eimeria maxima (gamonts) infects villus epithelial cells of the chicken duodenum there is extensive cellular enlargement with no alteration in nuclear size. Feulgen DNA microspectrophotometric measurements indicated that the infected host-cell nucleus contains the same amount of DNA as an uninfected cell nucleus. Evidence is presented to indicate that second generation schizonts of E. necatrix develop in crypt epithelial cells that are displaced/migrate into the lamina propria. The developing parasite causes cellular and nuclear hypertrophy in these cells as does E. tenella in cecal cells of the chicken. In these two cases nuclear enlargement is accompanied by induced rounds of DNA synthesis in the host-cell. Analyses indicated that the DNA content of enlarged nuclei does not fall into classes that correspond to a geometric series 2:4:6:8:16: etc. times the DNA content of a 2C equivalent, and that nuclear size and DNA content in infected cells are not significantly correlated. Autoradiographic studies on E. necatrix infected chicks administered ³H-thymidine show that DNA synthesis takes place in the nuclei of cells containing all developing stages but not mature schizonts, and that this synthesis is not a continuous process. The data suggest that intestinal cells that are capable of undergoing cell division and therefore additional rounds of DNA synthesis, can be induced by coccidial infection in the absence of concomitant cell division.     

Ultrastructural changes in Paneth cells during hibernation in the ground squirrel Spermophilm lateralis

Summary The ultrastructure of Paneth cells from jejuno-ileal segments of the small intestine of the ground squirrel, S. lateralis, was examined under normal euthermic conditions and during the profoundly depressed metabolic conditions of natural hibernation. Paneth cells obtained from hibernating animals gave evidence of markedly reduced activity when compared to Paneth cells from euthermic animals. In hibernating animals, the nuclei were smaller, with less prominent nucleoli and with an increased proportion of heterochromatin. In hibernating animals, the rough endoplasmic reticulum was fragmentary and poorly organized, in contrast to the typical arrangement of concentric lamellae seen in euthermic animals. Although the total number of ribosomes was decreased in hibernating animals, there were proportionally more free ribosomes than in euthermic animals. Paneth cells from hibernating animals also contained a greater number of apical secretory granules which were smaller and more variable in electron density than granules from control animals. These ultrastructural features indicate that during hibernation the Paneth cell is relatively quiescent.Supported by U.S.P.H.S. grants RR 05411 and RR 05583 from the N.I.H.     

Phosphorylation of myosin light chains and C-protein in the myocardium of hibernating ground squirrel Citellus undulatus

A comparative study was made of the extent of phosphorylation of myosin regulatory light chains and C-protein from the left ventricle of the hibernant ground squirrel Citellus undulatus during the periods of hibernation and activity. During hibernation, the light chains were found to be completely dephosphorylated. In active animals, the share of phosphorylated light chains averaged 40–45%. The extent of cardiac C-protein phosphorylation in hibernation was about twice higher than in the active state. Seasonal differences in phosphorylation of the two proteins of ground squirrel myocardium are discussed in the context of adaptation to hibernation.     

Induction of summer hibernation in the 13-lined ground squirrel shown by comparative serum transfusions from arctic mammals.

A “trigger” substance was again indicated to be present in sera of hibernating animals. Sera from the hibernating 13-lined ground squirrel, hibernating woodchuck, hibernating Arctic ground squirrel, and hibernating Arctic marmot were all capable of inducing the 13-lined ground squirrel to hibernate in the summer, a season when that species would normally be active. The hibernation trigger is thus not species specific. It is effective whether drawn from these two Arctic species of hibernators or drawn from these two species of hibernators from the midwestern states. The normothermic Arctic marmot appears to have an “anti-trigger” substance in its serum in the summer, which impedes fall hibernation in the transfused 13-lined ground squirrel. This is similar to the anti-trigger observed in the summer serum of active 13-lined ground squirrels and active woodchucks. With respect to hypothermia, it was induced in Artic marmots and in Arctic foxes at Point Barrow, Alaska, in summer. Though in such cases body temperatures fell significantly (as in hibernation), no trigger was recovered from their hypothermic sera that could be shown to be capable of inducing summer hibernation in the ground squirrel. Neither was anti-trigger found in the serum of hypothermic experimentals. These latter experiments thus suggest that the release of trigger into the blood during hibernation is dependent on a mechanism more complex than simply lowering body temperature.     

DNA synthesis in human duodenal biopsies maintained in organ culture

Incorporation of 3H-thymidine during organ culture was studied in duodenal biopsies from 14 patients. Pulse-label at various intervals disclosed active incorporations during the first 2 h in culture. Labelling index declined to low levels at 3-4 h. Thereafter incorporation increased again and persisted throughout the rest of the culture period of 11 h. The DNA synthesis rate of crypt cells between 4 and 11 h in culture was calculated in 5 patients after pulse-label and continuous labelling of explants in parallel culture. The rate of entry into DNA synthesis was about 24 cells per 1,000 crypt cells per hour in flat, coeliac biopsies, versus 9-13 in controls, Gluten did not influence DNA synthesis rate, whereas wheat germ lectin inhibited DNA synthesis. Counting of the total number of mitoses and labelled fraction of mitoses disclosed active crypt cell renewal in flat, coeliac biopsies. In normal-appearing biopsies no mitoses were labelled, indicating delayed exit from S-phase or long duration of G2-phase in these explants.     

Possible mechanism of the reorganization of the cardiomyocyte plasma membrane in the suslik in the hibernation-arousal-wakefulness cycle

Studies have been made on the ultrastructure of cardiomyocytes during hibernation and arousal of the ground squirrel C. undulatus. It was found that the number of elements of the rough endoplasmic reticulum, Golgi complex, vesicles and ribosomes increases in the perinuclear areas of cardiomyocytes during arousal of animals. These areas are saturated with lipid inclusions and mitochondria. Numerous vesicles and fringed bubbles were found near the plasma membrane which has many caveolae. These findings may indicate the intense metabolism of the membrane material between plasmalemma and cytoplasmic vesicles. Possible mode of rapid reorganization of the sarcolemma and changes in its functional properties during hibernation-arousal stages are suggested. It is concluded that apart from structural and functional properties which are acquired by cells during preparation of animals to hibernation and which exhibit only small changes during the whole period of hibernation, cyclic changes in plasmalemma structure and function occur during arousal of the ground squirrels.     

The effect of hibernation on lipids of the liver mitochondrial fraction in the Yakut ground squirrel <Emphasis Type="Italic">Spermophilus undulatus</Emphasis>

We demonstrated that the level of phospholipids in the liver mitochondrial fraction is increased by 60% during the winter hibernation season in the Yakut ground squirrel S. undulatus; the phospholipid composition in sleeping animals is characterized by an increase in phosphatidylethanolamine compared with summer animals. A sharp increase in the level of cholesterol, as well as fatty acid, monoglycerides, and diglycerides was found in the mitochondrial fraction of hibernating ground squirrels in relation to summer ground squirrels. Functional changes during hibernation concern the level of phosphatidylserine (the growth in sleeping animals compared with active animals). Seasonal modification of the lipid composition of the liver mitochondria (particularly, an increase in the level of cholesterol) can play a role in the resistance of mitochondria to the seasonal increase in the level of fatty acids in the liver. Lipids of the liver mitochondrial fraction are involved in the ground squirrel adaptation to the hibernation season.     

Ultrastructure of cells from the liver-endocrine pancreas system in hibernating animals

In hibernated ground squirrel (Citellus erythrogenys) during various periods of hibernation morphological changes in organs participating in insurance of energoplastic homeostasis (liver, endocrine part of the pancreas) have been followed. At the beginning of hibernation certain signs of functional strain in the liver are observed-shortening and swelling of the endoplasmic reticulum (ER) canaliculi, decreases of glycogen resources and activation of the lysosomal apparatus, as well as certain features demonstrating its decreased activity--shortening cisterns in the Golgi complex, poorly manifested granular ER, tightening of mitochondrial matrix. At a deep hibernation, the signs of strain disappear, ultrastructure of hepatocytes corresponds to a new lower level of functioning in comparison to that in the active state and at the beginning of hibernation. During these periods the structure of endocrine cells in the pancreas demonstrates certain changes in the character of functioning of the cells. Ultrastructural transformations in the liver and endocrine part of the pancreas in the ground squirrel are mutually correlated.     

Membrane lipids and morphology of brain cortex synaptosomes isolated from hibernating Yakutian ground squirrel

Synaptosomes were isolated from Yakutian ground squirrel brain cortex of summer and winter hibernating animals in active and torpor states. Synaptosomal membrane cholesterol and phospholipids were determined. The seasonal changes of synaptosomal lipid composition were found. Synaptosomes isolated from hibernating Yakutian ground squirrel brain cortex maintained the cholesterol sphingomyelin, phosphatidylethanolamine, lysophosphatidylcholine, cardiolipin, phosphatidylinositol and phosphatidylserine contents 2.5, 1.8, 2.6, 1.8, 1.6, and 1.3 times less, respectively, and the content of phosphatidylcholine twice as much as the one in summer season. The synaptosomal membrane lipid composition of summer animals was shown to be markedly different from that as hibernating ground squirrels and non-hibernating rodents. It is believed that phenotypic changes of synaptosomal membrane lipid composition in summer Yakutian ground squirrel are the important preparation step for hibernation. The phosphatidylethanolamine content was increased in torpor state compared with winter-active state and the molar ratio of cholesterol/phospholipids in synaptosomal membrane of winter torpor ground squirrels was lower than that in active winter and summer animals. These events were supposed to lead to increase of the synaptosomal membrane fluidity during torpor. Synaptosomes isolated from torpor animals have larger sizes and contain a greater number of synaptic vesicles on the synaptosomal profile area. The synaptosomal membrane lipid composition and synaptosome morphology were involved in phenotypic adaptation of Yakutian ground squirrel to hibernation.     

Changes in tight junctions of rat intestinal crypt cells associated with changes in their mitotic activity.

The freeze-fracture appearance of tight junctions between rat duodenal crypt cells was studied in normal, mitotically suppressed, and mitotically enhanced animals. In normal animals crypt cell tight junctions present a pleomorphic appearance. The population includes junctions resembling postmitotic junctions of the intestinal villus, junctions composed largely or completely of particle chains, and regions at the cell apex in which junctions are absent for 3-4 micron distances laterally. Mitotic suppression by inhibition of DNA synthesis with cytosine arabinoside results in the disappearance of pleomorphism and crypt tight junctions progressively come to resemble those of the intestinal villus. With recovery from the drug and further synchronization with Colcemid, the crypt cells undergo a mitotic burst, and all varieties of unusual junctional configurations are observed with increased frequency.     

Thyroid hormone levels in the peripheral blood of the ground squirrel Citellus parryi during winter hibernation

In the Arctic ground squirrel Citellus parryi, thyroxine and triiodothyronine concentrations in the peripheral blood have been determined by RIA in various seasons and during a short hibernation period. It was shown that during hibernation (December-February), hormonal concentration is higher than during homoiothermal period (August-October). The highest concentration of the thyroid hormones was found in hibernating animals in January. The data obtained indicate that thyroid hormones are involved in control of hibernation of the species investigated.     

The Van't Hoff rule in studying various phases of hibernation of the squirrel Citellus undulatus Pallas. Physico-chemical route to cold adaptation

Taking into account the van't Hoff's law the rates of relative erythrocyte hemolysis in ground squirrel Citellus undulatus Pallas during different phases of hibernation were first studied by the method of acid erythrograms with some modifications. The temperature of the erythrogram registration (8 degrees C) models the body temperature in hibernation, and temperature of 35 degrees C, corresponds to the body temperature of the awakened ground squirrel. The positions of the erythrogram maxima for the ground squirrel during short-term arousal and during hibernation coincide for each temperature studied: 8 or 35 degrees C. Therefore, the increase in HCl concentration at 8 degrees C reflects an increase in the stability of erythrocyte membranes or a decrease in the rate of relative hemolysis for the ground squirrel during hibernation. Thus, the adaptive response of erythrocytes in hibernating ground squirrel is revealed. The correlation of the rates of physicochemical processes in vitro with the rates of physiological ones in vivo is shown using the ratio of the rates mentioned above for the short-term arousal and for hibernation. First the physicochemical way of cold adaptation due to the van't Hoff's law is proved.     

The hormonal content of the hypophyseal-adrenocortical system in the blood of the suslik Citellus undulatus at different seasons of the year

Radioimmune assay has been made of the content of ACTH and cortisol in the peripheral blood of the ground squirrel Citellus undulatus parryi at various seasons and during a short hibernation period. Within annual cycle, negative relationship was observed between ACTH and cortisol levels. Cortisol content decreases from Autumn to Winter; the content of ACTH in the blood plasma, measured during periodic arousals, increases progressively during hibernation. Concentration of both hormones significantly decreases in sleeping animals, increasing during arousals. At the beginning of hibernation period, cortisol content gradually decreases. During self-warming of arousing animals, cortisol content remains low. The decreased production of cortisol in hibernating animals is presumably associated with the increase in the threshold of sensitivity of the adrenal receptors to stimulating effect of ACTH. The role of glucocorticoids and ACTH in the metabolism of hibernating animals is discussed.     

Spin label evidence for the role of lysoglycerophosphatides in cellular membranes of hibernating mammals.

The phospholipid composition of ground squirrel heart muscle changes during hibernation: more lysoglycerophosphatides are found in the hibernating state than in the active state. Phase transitions inferred from spin label motion occur in the usual manner typical of mammalian mitochondria for the mitochondria and mitochondrial lipids from active squirrels. However, a conspicuous absence of a spin label-detectable phase transition is observed in equivalent preparations from hibernating animals. The addition of lysolecithin to preparations from active squirrels removes the break and induces a straight line in the Arrhenius plot. The lack of a spin label-detectable phase transition in hibernating animals, therefore, is attributed to an increased content of lysoglycerophosphatides present in the phospholipids during hibernation.