| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1995: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1994: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Research Abstract |
The alpha_1-adrenergic response, measured as the agonist-induced generation of Ins (1,4,5) P_3, was predominant over the beta-adrenergic one, measured as the production of cAMP,in adult rat hepatocytes. During primary culture of the hepatocytes, the beta-adrenergic response was developed rapidly, whereas alpha_1-response was decreased gradually. Such receptor-subtype switching was not observed unless the cells were cultured under conditions favorable for cell growth, i.e. at low cell density (10^4 cells/cm^2). This switching was prevented progressively when the cells were grown at the cell density increased up to 20-fold or at low cell density in the presence of increasing amounts of liver plasma membranes. In this project, I have tried to isolate the growth-inhibitory factor(s).
Unfortunately, the isolation has not yet been succeeded. After many trials to prepare plasma membranes by the reported methods, it was noticed that some preparations had not only the growth-inhibitory activity
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but also the growth-stimulatory activity as reported. The difficulties to isolate the inhibitory factor(s) originate from unstability of the factor(s) : the inhibitory activity decreased during the primary culture of the hepatocytes within 24 hr ; the activity was ceased by repeated freeze-thawing process. The preparation that had the inhibitory activity was heat-unstable : the activity was abolished by treating it at 100ﾟC for 3 min. The result shows that the inhibitory factor might be a protein.
I ahve also tried to find the inhibitory materials which prevent the switching and cell growth. Retinoic acid is one of the candidates which showed that kind of the inhibitory effect. Retinoic acid can decrease gradually development of the switching after 6 hr from the onset of primary culture. The mechanism should be studied further. Another candidate is shphingomyelinase. The addition of this enzyme to the primary culture of hepatocytes caused inhibition of development of the switching. However, the problem is the contamination of the enzyme with much unknown proteins detected by SDS-PAGE and Coomasee Blue staining. Therefore, I did not check the enzyme effect more detail. Ganglioside GM_3, ceramides, atrial natriuretic peptides, and opioid peptides showed no effect on the switching.
In conclusion, I have found that the plasma membrane has both the inhibitory and stimulatory factors for development of the switching (and probably for growth). Another new finding is the effect of retinoic acid on the switching process. Isolation and characterization of the factors and elucidation of the mechanism of the inhibitory effect of retinoic acid may shed lights on the mechanism for cell growth. Less
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