Dopamine in the concentration 0.4 μg/mL abolishes protein synthesis rhythm in HaCaT keratinocytes and hepatocytes unlike noradrenaline or melatonin, which synchronize direct intercellular interactions and organize protein synthesis rhythm. Experiments with D2 dopamine receptors blocking agent metoclopramide (tserukal) in the concentration 2 μg/mL show that a disorganizing effect of dopamine is driven by the activation of D2 receptors, which block adenylyl cyclase and the efflux of calcium ions from internal depos according to the literature. It is shown that tserukal does not activate serotonin receptors in our experimental settings. Cellular interactions’ recovery during or after dopamine action is carried out by melatonin in the concentration 0.001 μg/mL. A recommendation to inject melatonin before dopamine administration for different medical indications is discussed.

The effect of inhibition of proteasome activity on direct cell-cell interactions in primary hepatocyte cultures was studied. The circahoralian rhythm of protein synthesis was a marker of cell-cell communication. The addition of the proteasome inhibitor MG132 at doses of 10 or 20 μM to the medium with hepatocyte cultures for 19 h resulted in a significant reduction in the total pool of 3H-leucine in cells. The incorporation of leucine into proteins changed slightly or negligibly, whereas the content of free labeled leucine in hepatocytes decreased. The rhythm of protein synthesis was distorted compared to the control. The rhythm was restored by external organizers, such as gangliosides and melatonin, as well as by enhancing the activity of protein kinases--the key factor in the organization of the rhythm of protein synthesis. A short-term (3-h) exposure to MG132 did not change the pool of leucine, but the rhythm of protein synthesis was also disturbed. Thus, protein catabolism affects cell-cell interactions organizing the rhythm of protein synthesis. Another factor of the downregulation of the rhythm of protein synthesis, the secretion of proteins from the hepatocytes in vivo, which was shown in vivo in many studies, was also revealed in our study when measuring the content of proteins stained with Coomassie Brilliant Blue G250 in the medium with hepatocyte cultures.

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.

Dopamine was injected intravenously (9 μg/kg) or intraperitoneally (15 μg/kg) to Wistar rats (3-4 months, 300-400 g). Hepatocytes were isolated 40 min after dopamine injection. Dense cultures were maintained on collagen-coated glasses. By the 5th hour, the circaholarian rhythm of protein synthesis in hepatocytes cultures was absent in the dopamine group, but was present in cultures from animals receiving physiological saline (NaCl). The rhythm-disorganizing effect of dopamine was reversible. The rhythm was observed in cultures of hepatocytes isolated 1 day after dopamine treatment. The effect of dopamine was abolished by melatonin. The protein synthesis rhythm was revealed in 5-h cultures of hepatocytes from rats receiving melatonin (32 ng/kg) 40 min after intraperitoneal injection of dopamine. The results of our in vitro experiments with addition of dopamine into the medium of cultured hepatocytes [1] suggest that dopamine in vivo produces a direct effect on liver cells. The observed changes are discussed taking into account the biochemical mechanisms for a direct cell–cell interaction and previously unknown properties of catecholamines.

We studied dense 24-hour cultures of rat hepatocytes in serum-free medium on collagen-coated slides. As before, a circahoralian rhythm of protein synthesis was observed in control cultures in a fresh medium. No rhythm was found after addition of 1-10 μM dopamine to the medium containing such cultures. The rhythm was observed after addition of 0.3 μM ganglioside to pretreated-dopamine cultures. Dopamine is likely to influence the conditioning of intercellular medium with gangliosides. Deficit of this endogenous synchronizing factor in the intercellular medium blocks self-organization of the protein synthesis rhythm. Thus, in contrast to previously studied norepinephrine and serotonin, as well as gangliosides, which organized the population rhythm of protein synthesis, dopamine disorganized the rhythm, impairing direct intercellular interactions.

A comparative study was performed of dense 5-hour cultures of rat hepatocytes and equal-density cultures of mesenchymal stromal cells (MSC) isolated from human adipose tissue of rat bone marrow. The cells were grown on collagen-coated class slides in serum-free medium. Unlike in hepatocytes, no rhythm of protein synthesis was initially revealed in MSC, but such a rhythm manifested itself when the culture medium was supplemented with melatonin (2 nM, 5 min). The results of experiments with cytoplasmic calcium chelator BAPTA-AM and protein kinase inhibitor H7 indicate that the mechanism of protein synthesis synchronization in MSC consists in calcium-dependent phosphorylation of cell proteins.

In cultures of human keratinocytes HaCaT contained in a serum-free medium on glass, a circahoralian rhythm of protein synthesis was found similar to the one in hepatocytes in vitro. The intensity of the synthesis was determined by the inclusion of 3 H-leucine corrected for the pool of free marked leucine. Rhythm was studied in washed 1- or 2-day cultures after the change of the medium. The medium conditioned with keratinocytes HaCaT synchronized the rarefied hepatocyte cultures nonsynchronous in the control. Therefore, the keratinocytes liberate synchronizing factors into the medium. A BAPTA-AM chelator of calcium ions eliminates the protein synthesis rhythm both in dense hepatocyte cultures synchronous in the control and in the HaCaT keratinocyte cultures. The effect of the H7 inhibitor of protein kinases was analogous. Thus, both in keratinocytes and hepatocytes, self-synchronization of fluctuations of the intensity of protein synthesis takes place. The mechanism of self-synchronization is the calcium-depending phosphorylation of cell proteins.

В культурах кератиноцитов человека HaCaT, содержащихся в бессывороточной среде на стеклах, обнаружен околочасовой ритм синтеза белка, сходный с ритмом в гепатоцитах in vitro. Интенсивность синтеза определяли по включению 3-лейцина с поправкой на пул свободного меченого лейцина. Ритм изучали в отмытых 1- или 2-суточных культурах после смены среды. Среда, кондиционированная кератиноцитами HaCaT, синхронизировала разреженные несинхронные в контроле культуры гепатоцитов. Следовательно, кератиноциты выделяют в среду синхронизирующие факторы. Хелатор ионов кальция ВАРТА-АМ ликвидирует ритм синтеза белка как в плотных синхронных в контроле культурах гепатоцитов, так и в культурах кератиноцитов HaCaT. Аналогичным был эффект ингибитора протеинкиназ Н7. Таким образом, в кератиноцитах, как и в гепатоцитах, происходит самосинхронизация колебаний интенсивности синтеза белка. Механизм самосинхронизации – кальций-зависимое фосфорилирование клеточных белков.

Melatonin (5 nM) added to medium with primary hepatocyte cultures shifted the phase of circahoralian rhythm of protein synthesis and hence, can be a factor synchronizing fluctuations in protein synthesis and rhythm organizer in the hepatocyte population. Blockade of melatonin receptors with luzindole (20 nM) arrested rhythm organization of protein synthesis by melatonin. Prospects of studying biochemical mechanisms of protein synthesis rhythm organization with other drugs (calcium agonists, similarly to melatonin) are discussed.

Мелатонин, введенный внутрибрюшинно крысе, синхронизирует околочасовой ритм синтеза белка в первичных культурах гепатоцитов, выделенных из этой крысы через 1ч 40 мин после инъекции мелатонина и изученных через 1 или 2 сут. Эффективные синхронизирующие концентрации мелатонина – 0.01–0.02 мкг/кг веса крысы – на три порядка ниже доз мелатонина, используемых в клинической практике при лечении людей.

Мелатонин в наномолярных концентрациях синхронизирует ритм синтеза белка в первичных культурах гепатоцитов путем кальцийзависимой активации протеинкиназ. Синхронизирующий эффект мелатонина блокируется хелатором цитоплазматического кальция ВАРТА-АМ (20 мкМ), а также ингибитором протеинкиназ 1-(5-изохинолинсульфонил)-5-метилпиперазин дигидрохлоридом (40 мкМ). Таким образом, основное событие синхронизации гепатоцитов мелатонином – фосфорилирование белков, что ранее было нами показано также для ганглиозидов и биогенных аминов. Антагонист рецепторов мелатонина лузиндол (20 нМ) блокирует синхронизирующую функцию мелатонина.

Melatonin in nanomolar concentrations synchronizes protein synthesis in primary cultures of hepatocytes through calcium-dependent activation of protein kinases. The synchronizing effect of melatonin is blocked by the cytoplasmic calcium chelating agent BAPTA-AM (20 μM) as well as by the inhibitor of protein kinases 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine dihydrochloride (40 μM). Thus, protein phosphorylation is the key event in hepatocyte synchronization by melatonin, as we have demonstrated previously for gangliosides and biogenic amines. The antagonist of melatonin receptors luzindole (20 nM) blocks the synchronizing function of melatonin.

The effect of 1 to 1000 nM melatonin was studied on daily cultures of rat hepatocytes on slides in serum-free medium. The minimum melatonin concentration (1 nM) proved to synchronize protein synthesis in asynchronous sparse cultures of hepatocytes from rats of different age, and a circahoralian rhythm of protein synthesis was revealed in them. In dense weekly synchronous hepatocytes from old rats (2.5 years old with the weight of about 600 g), melatonin improved cell synchronization to the level of young animals. Melatonin treatment increased the mean rate of protein synthesis in rats of different age.