Lead Alters Growth and Reduces Angiotensin II Receptor Density of Rat Aortic Smooth Muscle Cells

Abstract Environmental lead (Pb2+) contributes a small but significant risk to human hypertension. It is postulated that the hypertensinogenic action of Pb2+ may be due, in part, to its direct action on vascular smooth muscle cells. To investigate this hypothesis, freshly isolated rat aortic smooth muscle (RASM) cells were propagated in defined media containing one of two Centers for Disease Control-based concentrations of Pb2+ (as lead citrate): 100 and 500 μg Pb2+/I (i.e., equivalent to 5.5 and 27.5 μg Pb2+/dl blood; designated 100-RASM and 500-RASM). Control (CON-RASM) cells received sodium citrate. 500-RASM cells exhibited suppressed propagation and fell out of propagation synchrony with CON-RASM cells: when CON-RASM cell approached confluence (~90%), 500-RASM cell density was 6.4% that of CON-RASM cell density. By contrast, 100-RASM cells exhibited marked hyperplasia albeit this was not apparent until passage 3 (p3). Overall, when p3-p6 CON-RASM cells approached confiuence, 100-RASM cell density was 107.6% greater than CON-RASM cell density. The protein content of CON-RASM and 100-RASM was not different, whereas that of 500-RASM cells was 29% greater than that of CON-RASM and 100-RASM cells. Phase-contrast microscopy revealed that 100 μg Pb2+/I converted normal spindle-shaped/ribbon-shaped RASM cells into less spread, cobblestone-shaped, neointimal-like cells. Immunocytochemical analysis revealed that 100-RASM cells lacked or had markedly fewer actin cables, characteristic of rapidly dividing cells. In addition, Pb2+-treated RASM cells exhibited altered membrane fatty acyl composition with a trend towards an increase (by as much as 50%) in membrane arachidonic acid. Interestingly, hyperplastic 100-RASM cells exhibited a 70.6% reduction in angiotensin II (Ang II) receptor concentration whereas the concentrations of α1- and β-adrenergic and atrial natriuretic peptide (ANP) receptors were not affected. In addition, in experiments designed to control for Pb2+-associated differences in RASM cell propagation, there was a concentration-dependent decrease in Ang II receptor concentration: for 100 and 500 μg Pb2+/I, Ang II receptor concentration was decreased 39.6% and 65.5%, respectively. Thus, although Pb2+, depending on its concentration, had contrasting effects on RASM cell propagation, it had a consistent, concentration-dependent inhibitory effect on Ang II receptor concentration. Recovery (r) from Pb2+ required at least two additional passages. At p71r, the enhanced propagation (+54%) and reduced Ang II receptor concentration (-49%) of 100r-RASM cells persisted. However, 500r-RASM cells exhibited a rebounded and enhanced (+37%) propagation with a concomitant reduction (-58%) in Ang II receptor concentration. These residual effects of Pb2+ were almost completely normalized with a second recovery passage (i.e., p82r). These effects of the Pb2+ appeared to be direct since conditioned medium from hyperplastic 100r- and 500r-RASM cells did not affect CON-RASM cell propagation or Ang II receptor parameters. Thus, depending on its concentration and duration in culture, Pb2+ can differentially alter vascular smooth muscle cell growth and can selectively reduce cellular Ang II receptor concentration.