Lead enters Rcho-1 trophoblastic cells by calcium transport mechanisms and complexes with cytosolic calcium-binding proteins

Within the placenta, a specialized Ca 2+ transport pathway develops in trophoblasts to promote growth of the fetus and hypothetically to enhance fetal uptake of Pb 2+. This hypothesis could not be tested until a method to monitor Pb 2+ influx by indo-1 fluorescence quench became available. We have applied this new method to cultured undifferentiated and differentiated Rcho-1 trophoblastic cells. Pb 2+ concentrations of 1 and 10 μM are equivalent to blood levels of 20 and 200 μg/dl in pregnant women. Over this range, Pb 2+ uptake increased with time and concentration in medium containing 1 mM Ca 2+ but was greater in Ca 2+-omitted solutions. Activation of capacitative Ca 2+ entry (CCE) with thapsigargin, an endoplasmic reticulum (ER) Ca 2+ pump inhibitor, increased Pb 2+ uptake, while inhibition of CCE by La 3+ decreased influx. Parathyroid hormone-related peptide (PTHrP) stimulates the synthesis of Ca 2+-binding proteins (CaBPs), as well as Ca 2+ transporters, during trophoblastic differentiation. Pretreatment for 72 h with PTHrP increased Pb 2+ uptake by undifferentiated Rcho-1 cells but had little effect on the quench in differentiated cells, probably due to their greater content of CaBPs which competed for Pb 2+-binding with indo-1. This competition was most evident in differentiated cells when 1 μM Pb 2+ caused an initial quench, followed by a rise in fluorescence. This rise was not inhibited by thapsigargin, thereby ruling out sequestration into the ER and leaving complexation of Pb 2+ by CaBPs as the most plausible interpretation. We conclude that trophoblasts have the ability to clear Pb 2+ from the maternal circulation and deliver it to the fetus.