👤 A Okanlawon

We have examined the effects of increasing doses of chloroquine (CQ), on transferrin secretion in primary cultures of immature rat Sertoli cells (SC) grown on a reconstituted basement membrane (Matrigel) in bicameral chambers. SC cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64cm2/well on Matrigel covered Millicell-HA filters. CQ at concentrations ranging from 0.04-1.0 microM was added to the basal compartment of the bicameral system from day 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter TER in untreated controls was 50 Ohms/cm2 on day 1, and increased progressively to 80 Ohms/cm2 by day 7 and plateaued until day 12. On the seventh day of culture, CQ was introduced into the basal chamber During the 4 days of the experiment, the secretion of transferrin decreased with time. Maximal transferrin secretion by SC was detected during the initial 2 day collection period. During the subsequent collection period, CQ (1 microM) decreased significantly transferrin secretion by SC, while 0.04 microM CQ did not affect transferrin secretion. The polarized secretion of transferrin in response to CQ was also studied. During both collection periods there was no significant difference between controls and 0.04 microM CQ cultures in the ratio of apical to basal transferrin secretion. In the 1 microM culture medium, CQ diminished significantly the ratio of apical to basal transferrin secretion. These observations demonstrate the heterogenous effects of lower doses of CQ on immature rat SC in cultures. Show less

We investigated the effect of CQ, an antimalarial drug with antiprotease activity, and NH4Cl, a related amines on the development of intercellular tight junctions in cultured immature rat Sertoli cells. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64 cm2/well on Matrigel-covered Millicell-HA filters. CQ (1 microM and 2 microM) or NH4Cl (6.25 mM and 12 mM) was added to the outer (basal) compartment of the bicameral system either on day 1 or day 7 of the culture. Formation of tight junctions was monitored by measurement of the transepithelial resistance (TER) at 24 hr intervals using an impedance meter. TER in untreated controls was 50 omega/cm2 on day 1, increased progressively to 80 omega/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with CQ showed dose-dependent progressive increase in TER until day 12, reaching 191 omega/cm2 in cells treated with 1 microM concentration. In cells treated with CQ starting from day 7 of culture onwards, TER patterns were similar to those noted following exposure to chloroquine from day 1. Also in cultures containing NH4Cl, in comparison to the control, the increase in TER was significantly higher. These observations demonstrate that CQ and HN4Cl promote tight junction formation between immature rat Sertoli cells invitro suggesting that antiproteases may be involved in the formation of blood-testis barrier. Show less

Adjoining immature Sertoli cells in the seminiferous epithelium form a tight junctional complex leading to the development of the blood-testis barrier. Protease and antiprotease activities have been implicated in the process of formation of tight junctions. Here, we report the effect of chloroquine, an antimalarial drug with antiprotease activity, on the development of intercellular tight junctions in cultured immature rat Sertoli cells. For positive control, the classical lysosomotropic agent ammonium chloride was used. Sertoli cells were seeded in serum-free defined medium at a density of 3 x 10(6) cells/0.64-cm2 well on Matrigel-covered Millicell-HA filters. Chloroquine at concentrations ranging from 25 to 100 microM was added to the outer chamber of the bicameral system on either day 1 or 7 of the culture. The formation of the tight junction was monitored by the measurement of the transepithelial resistance (TER) at 24-hour intervals using an impedance meter. TER in untreated controls was 50 ohms/cm2 on day 1; it increased progressively to 80 ohms/cm2 by day 7 and plateaued until day 12. The cells treated from day 1 with chloroquine also showed a dose-dependent progressive increase in TER until day 9, reaching 225 ohms/cm2 in cells treated with the 100 microM concentration. In comparison to controls, the increase in TER was significantly higher. In cells treated with chloroquine starting from day 7 of culture onwards, there was no observable difference in TER from the untreated control. These observations demonstrate that chloroquine and ammonium chloride increase the TER of immature Sertoli cells in the bicameral chamber. Show less