Исследовано влияние торможения активности протеасом на прямые межклеточные взаимодействия в первичных культурах гепатоцитов. Маркер межклеточных коммуникаций – околочасовой ритм синтеза белка. Введение ингибитора протеасом MG132 в дозах 10 или 20 мкМ в среду с культурами гепатоцитов на 19 час приводит к значительному снижению суммарного пула 3-лейцина в клетках. Включение лейцина в белки изменяется мало или не изменяется, падает содержание свободного меченого лейцина в гепатоцитах. Искажается сравнительно с контролем ритм синтеза белка. Ритм восстанавливается внешними организаторами – ганглиозидами и мелатонином, а также при усилении активности протеинкиназ – ключевого фактора организации ритма синтеза белка. Кратковременное 3-ч действие MG132 не изменяет пул лейцина, но ритм синтеза белка также нарушается. Таким образом, катаболизм белков влияет на межклеточные взаимодействия, организующие ритм синтеза белка. Другой фактор негативного контроля ритма синтеза белка – секреция белков из гепатоцитов, показанная in vivo во многих исследованиях, выявлена и в нашей работе при измерениях белков, окрашенных кумасси бриллиантовым синим G250 в среде с культурами гепатоцитов.

На первичных суточных культурах гепатоцитов крысы на стеклах в бессывороточной среде исследовано действие мелатонина в концентрации от 1 до 1000 нМ. Показано, что в минимальной дозе (1 нМ) мелатонин синхронизирует колебания синтеза белка в несинхронных разреженных культурах гепатоцитов крыс разного возраста и в них выявляется околочасовой ритм синтеза белка. В плотных слабо синхронизированных гепатоцитах старых крыс (возраст около 2.5 лет, вес около 600 г) мелатонин увеличивает степень синхронизации клеток до уровня культур молодых животных. После обработки мелатонином у крыс разного возраста повышается средний уровень синтеза белка.

The aim of the review is to attract attention to the importance of a dynamic approach to symptoms of health and pathology. In this regard, long-known approximately 24-hour or circadian rhythms (CR) are very prospective. However, according to recent studies, shorter approximately hourly rhythms (AHR) are of not less importance. Unlike CR, HR are not obtruded on the organism by periodical external influence of day-night pattern, but present cell's own fundamental property, a method of their metabolism. The fractal nature of HR has been substantiated; age-dependent changes in HR have been demonstrated; the importance of AHR for diagnostics and prognosis in gastroenterology and cardiology has been determined. To perform more clinical observations is an important task.

Circahoralian cellular rhythms have been revealed after the amendment of quantitative cytochemical methods initiated at the Department of Histology (Moscow State University) and Laboratory of Cytology (Institute of Animal Morphology, Russian Academy of Sciences). The first findings have been confirmed in many laboratories. Circumhoralian kinetics proved to be specific for various cell types (from bacteria to mammalian cells) and cellular functions. Independent physiological investigations found circahoralian rhythms for various tissue functions. Distribution of the rhythms as well as their possible nature and significance for tissue biology are reviewed.

Published data indicate that 1-3 microM adrenomimetic phenylephrine increases the concentration of calcium ions in the cytoplasm of cultured hepatocytes. We studied low-density cultures exhibiting no protein synthesis rhythm in the fresh medium and demonstrated that a 2 min action of 2 microM phenylephrine induces protein synthesis rhythm, i.e., synchronizes the synthesis oscillations in hepatocytes. A similar effect was observed for a selective inhibitor of the reticulum calcium pump, di-tert-butyl-benzohydroquinone, that increases the cytoplasm concentration of calcium ions by a receptor-independent mechanism. A calcium antagonist imipramine obviated the synchronization effect of phenylephrine. We propose that short-term changes in the cytoplasm concentration of calcium ions covering the whole cell population are among intracellular mechanisms of protein synthesis synchronization in hepatocytes in vitro.

Newborn rats were grown in litters of 8 (control), 4, or 16 individuals up to the day 21 of postnatal development and then transferred to usual keeping and feeding conditions similar for all groups. Liver and cerebellum were removed for the study at days 18, 45, and 90. At day 28, the body weight of animals from experimental groups differed by approximately three-fold, weight of liver, almost five-fold, and weight of the cerebellum, by 1.4 times. The body weight of animals also showed significant differences at day 90, however, the weight of the liver and cerebellum differed from the control only in animals that received excessive feeding. We demonstrate that in the control and in experimental rats, the intensity of the protein synthesis during the period from day 18 to day 90 shows similar changes, and it is always higher in the liver as compared with the cerebellum. Differences between fasting animals and animals given excess food generally were apparent mainly at day 90. Thus, feeding disturbances during the first weeks after birth result in uncompensated changes of animal growth and of the studied animal organs. These data are discussed in connection with perspectives of using this model to study specific aspects of three-dimensional organization of the nucleolar complex of hepatocytes and cerebellar neurons.

Changes in the activity and number of nucleoli has been studied in hepatocytes of the same rat before and after partial hepatectomy. In the latter case, the rate of ribosomal RNA (rRNA) synthesis was increased by a factor of 1.7, on the average. The synthesis was evaluated by incorporation of adenosine triphosphate in situ after alpha-amanitine treatment of squashed cells. In 2c, 4c and 8c nuclei, the number of labeled nucleoli was less than in nucleolonemic nucleoli stained with silver nitrate. These values were drawn together during stimulation of rRNA synthesis but the difference was often preserved especially for highly ploid nuclei. The lack of activity in part of nucleolonemic nucleoli was assumed. After stimulation of RNA synthesis, the number of nucleolonemic and satellite nucleoli decreased, while the size of preserved nucleoli was enlarged. The number of nucleoli did not correspond to the nuclear ploidy; their total size was proportional to the gene dosage. The mean level of rRNA synthesis was similar in nuclei of the same ploidy with two, three and four nucleoli. A great variability was detected in the rate of rRNA synthesis as well as in the number of nucleoloneme and satellite nucleoli in hepatocytes of the control and experimental rats. Some difference was observed in the label degree for nucleoli of the same nucleus.

Variability of cells is analyzed using data obtained in studies on polyploidy in the heart muscle and on ontogenetic changes in the number of myocytes. In normal myocardium, variation in the number and ploidy of muscle cells is observed. The types of genomic variation are similar to those found in populations of organisms, i. e., to hereditary and modifying variation. In studies on modifications of the myocyte genome, heart growth reserves were revealed, which manifest themselves in pathological states associated with myocardial hypertrophy. These reserves depend on the level of myocardial polyploidy that is determined during early postnatal development.

The rate of rRNA synthesis increased in hepatocytes within 14 h after partial hepatectomy. The increase, 1.7-fold on the average, was similar in diploid, tetraploid and octaploid nuclei. Under these conditions, the content and area of argentophilic (Ag-) nucleolar proteins were increased, although they varied between individual animals and nuclei of the same cell population. The changes of transcription and Ag proteins were not proportional. Thus, the Ag stain may characterize only the trend in activity of the ribosomal genes but not their exact state in hepatocytes.

A part of suspension with isolated hepatocytes was cooled and then warmed to normal temperature. In cultures formed with such cells, antiphase oscillations of the protein synthesis rate were detected concerning control cultures from another part the same hepatocyte suspension. Oscillations of protein synthesis were observed in the mixed cultures formed with equal parts of antiphase subpopulations of hepatocytes. The experiments established cell interactions in vitro resulted in synchronization of protein synthesis oscillations in different cells the same population.