Glutamic Acid as Enhancer of Protein Synthesis Kinetics in Hepatocytes from Old Rats.

Dense cultures of hepatocytes from old rats (~2 years old, body weight 530-610 g) are different from similar cultures of hepatocytes from young rats by the low amplitude of protein synthesis rhythm. Addition of glutamic acid (0.2, 0.4, or 0.6 mg/ml) into the culture medium with hepatocytes of old rats resulted in increase in the oscillation amplitudes of the protein synthesis rhythm to the level of young rats. A similar action of glutamic acid on the protein synthesis kinetics was observed in vivo after feeding old rats with glutamic acid. Inhibition of metabotropic receptors of glutamic acid with α-methyl-4-carboxyphenylglycine (0.01 mg/ml) abolished the effect of glutamic acid. The amplitude of oscillation of the protein synthesis rhythm in a cell population characterizes synchronization of individual oscillations caused by direct cell-cell communications. Hence, glutamic acid, acting as a receptor-dependent transmitter, enhanced direct cell-cell communications of hepatocytes that were decreased with aging. As differentiated from other known membrane signaling factors (gangliosides, norepinephrine, serotonin, dopamine), glutamic acid can penetrate into the brain and thus influence the communications and protein synthesis kinetics that are disturbed with aging not only in hepatocytes, but also in neurons.

Glutamic Acid - Amino Acid, Neurotransmitter, and Drug - Is Responsible for Protein Synthesis Rhythm in Hepatocyte Populations in vitro and in vivo.

Primary cultures of rat hepatocytes were studied in serum-free media. Ultradian protein synthesis rhythm was used as a marker of cell synchronization in the population. Addition of glutamic acid (0.2 mg/ml) to the medium of nonsynchronous sparse cultures resulted in detection of a common protein synthesis rhythm, hence in synchronization of the cells. The antagonist of glutamic acid metabotropic receptors MCPG (0.01 mg/ml) added together with glutamic acid abolished the synchronization effect; in sparse cultures, no rhythm was detected. Feeding rats with glutamic acid (30 mg with food) resulted in protein synthesis rhythm in sparse cultures obtained from the rats. After feeding without glutamic acid, linear kinetics of protein synthesis was revealed. Thus, glutamic acid, a component of blood as a non-neural transmitter, can synchronize the activity of hepatocytes and can form common rhythm of protein synthesis in vitro and in vivo. This effect is realized via receptors. Mechanisms of cell-cell communication are discussed on analyzing effects of non-neural functions of neurotransmitters. Glutamic acid is used clinically in humans. Hence, a previously unknown function of this drug is revealed.

[Blockade of proteasome activity disturbs the rhythm of synthesis of the protein marker of direct cell-cell interactions].

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.

Administration of dopamine to rats disorganizes the rhythm of protein synthesis in hepatocytes.

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.

Effect of protein synthesis rhythm-organizing signal persists for a day after single administration of melatonin to rat.

Melatonin administered to rat intraperitoneally organizes ultradian rhythm of protein synthesis in hepatocytes that persists for 1 day after exposure to the synchronizing signal. Hepatocytes were isolated 1 day after melatonin administration and cultured on coverslips in a serum-free medium. In 24 h in culture, the kinetics of protein synthesis was analyzed. In our previous experiments, we detected a rhythm in cells isolated in 1.5 h, but not in 3 days after melatonin administration to the rat. We have found that synchronization of oscillations of the protein synthesis intensity in vivo persists over 1 day after rat exposure to melatonin. Phenylephrine, an efficient synchronizer of protein synthesis in vitro, does not organize the rhythm in vivo.

Dopamine disorganizes the rhythm of protein synthesis disrupting self-organization of hepatocytes in vitro.

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.