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Korean Circ J. 1994 Jun;24(3):486-493. Korean. In Vitro.
Kim SS , Han DS , Lee HC , Park YT , Suh PG .

BACKGROUND: Cardiac hypertrophy is an adaptive mechanisms in response to an increased cardiac work load. Alterations in gene expression play an important role in this adaptive process. Recent investigations have indicated that the alpha-1 adrenergic stimulation in vitro induces hypertrophic change of neonatal cardiomyocytes. The signalling mechanisms of this alpha-1 agonist induced cardiomyocyte hypertrophy are largely unknown. however, recent evidence favors an effector pathway that involves phospholipase C(PLC) mediated hydrolysis of phosphatidylinositol 4,50 bisphosphate. It should be recognized that the demonstration of enhanced phosphoinositol turnover in the presence of alpha-1 adrenergic agonist in vitro does not necessarily imply that a similar response is operative in vivo. Furthermore, the role of subtypes of phospholipase C in this system should be determined. In this context, we produced in vivo cardiac hypertrophy by repeated injection of alpha-1 adrenergic agonist, phenylephrine, and tried to evaluate any change of phospholipase C subtypes by immunohistochemistry and immunoblotting technique and also measured the phosphatidylinositol hydrolyzing activity of the enzyme. METHOD: To produce cardiac hypertrophy, we injected phenylephrine 12mg/kg i.p. to the 28 female S-D rats weighing 150-250g daily for 5 days. This measures produced 22% increase of heart weight/body weight ratio. After 5 days. rats were sacrificed and hearts were rapid excised and freezed for next procedure. The immunohistochemical stainings of myocardium were carried out using monoclonal antibodies against PLC-beta1,-gamma1,-delta1 with Avidine-Biotin Complex method. Immunoblotting was done with monoclonal anti-PLC-gamma1 antibody after immnoprecipitation. The activity of PLC-gamma1 was determined in the assay mixture containing [3H] phosphatidylinositol of 20,000 cpm. The reaction was performed by incubating with resuspended immunoprecipttol of 20,000 cpm. The reaction was performed by incubating with resuspended immunoprecipitate for 10 min and supernatant was collected for -scintillation counting. RESULTS: Immunohistochemical staining demonstrated increased staining of PLC-gamma1 in the phenylephrine induced hypertrophied heart as compared with normal control heart. PLC-beta1 and-o1 did not showed any change. Elghteen out of 20 hypertrophied cardiac tissue(90%) demonstrated increased expression of the PLC-gamma1 compared with control heart tissue in immunoblotting. [3H] PI hydrolyzing activity of PLC-gamma1 in the immunoprecipitates of the hypertrophied hearts(4650+/-614 cpm) were increased consistently in 6 samples as compared with control normal hearts (2387+/-651 cpm). CONCLUSION: In the present experiments we demonstrated that Phospholipase C-gamma1 was overexpressed compared with control normal heart of rat by immunohistochemistry and immunoblotting technique and showed that the activity of this isoenzyme was elevated. Our findings of increased PLC-gamma1 expression in the alpha1-adrenergic agonist induced cardiac hypertrophy tissue suggest that the phosphatidylinositol signalling pathway is important in the genesis of cardiac hypertrophy and the isoenzyme of PLC-gamma1 may play a central role in this mechanism.

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