Angiotensin-II-induced cell hypertrophy: potential role of impaired proteolytic activity in cultured LLC-PK1 cells

BACKGROUND: Angiotensin II-induced hypertrophy of both mesangial and tubular cells has been shown to be caused by enhanced protein synthesis. There are no data about its role on protein breakdown. Therefore, protein turnover and proteolytic activities were investigated in LLC-PK1 cells. METHODS: Protein turnover was measured by determining the incorporation and release of [14C]phenylalanine; collagenolytic and gelatinolytic activities were assayed by using fluorogenic peptidyl substrates. RESULTS: Angiotensin II (10(-8)-10(-6) M) exerted a dose-dependent inhibition of collagenolytic and gelatinolytic activities, associated with reduction of protein degradation rate. In addition angiotensin II stimulated protein synthesis in the cells. These combined effects on protein turnover resulted in an increase in both cell size and cell protein content (31.7% after 48 h). However, the rise of cell protein content was only partly (48.0%) prevented by the protein synthesis inhibitor cycloheximide (10(-5)M), which supports the role of decreased protein degradation in the angiotensin-II-induced cell hypertrophy. The angiotensin-II-induced effects on proteolytic activities as well as on cell protein content could be abolished by coincubation with the angiotensin II type I-receptor antagonist DuP 753 (10(-6)M). The calcium-channel blocker verapamil (10(-6)M) ameliorated the impairment of collagenolytic activity. On the contrary the calcium ionophore A23187 (10(-6)M) mimicked the action of angiotensin II on this enzyme activity (control 34.5 +/- 1.9; angiotensin II 24.0 +/- 2.0; A23187 23.0 +/- 2.2 and angiotensin II+verapamil, 33.8 +/- 2.6 pmol/min/micrograms DNA). The role of cytosolic [Ca2+] in the actions of angiotensin II could be finally shown by a dose-dependent rise which was partly blunted by verapamil. CONCLUSION: The angiotensin-II-induced hypertrophy in LLC-PK1 cells is caused not only by enhanced protein synthesis but also by reduced protein degradation. The concomitant decline of collagenolytic and gelatinolytic activities may contribute to the accumulation of extracellular matrix, and presumably also to cell hypertrophy. These effects are obviously mediated via angiotensin II type I receptors and seem to be [Ca2+] dependent.