Internalization and Degradation of Human Chorionic Gonadotropin in Ovine Luteal Cells: Effects of Inhibitors of Transglutaminase*

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Abstract
Suspensions of ovine luteal cells were used to study the effects of inhibitors of transglutaminase on the internalization of [125I]hCG. Ovine corpora lutea were collected from ewes during the midluteal phase of the estrous cycle, dissociated into cell suspensions with collagenase, incubated with [125I]hCG for 5–7 min, and washed five times to remove radioactivity which was not bound to receptor. The cells were then incubated for 6– 12 h in the presence of bacitracin (1 mM), ethylamine (1–5 DIM), methylamine (5–15 mM), or dansylcadaverine (100–500 μM) to determine the effects of these inhibitors of transglutaminase on the rates of internalization and degradation of the receptorbound [125I]hCG and/or progesterone secretion. None of the transglutaminase inhibitors tested inhibited the internalization of [125I]hCG significantly, while all of them inhibited the degradation of internalized hormone. In some cases, inhibition of the degradation of internalized hormone resulted in a buildup of radioactivity intracellularly; in other cases, it did not. Ethylamine, methylamine, and dansylcadaverine also inhibited the secretion of progesterone and were clearly toxic to the luteal cells. Thus, it was not possible to determine if the inhibition of [125I]hCG degradation was due to a specific effect of inhibition of transglutaminase activity or to the cytotoxic effects of these compounds. The effects of chloroquine (10 mM), an inhibitor of lysosomal activity, and Concanavalin A (50 μg/ml) on the internalization or degradation of [125I]hCG were also examined. Chloroquine inhibited the degradation of internalized [125]hCG, but did not affect the rate of internalization. Concanavalin A stimulated the rate of internalization of [125I]hCG and also inhibited the degradation of hormone. Neither compound affected the rate of progesterone production or cell viability. It was concluded from these studies that inhibitors of transglutaminase do not inhibit the internalization of the [125I]hCG bound to luteal cells. The mechanisms involved in the internalization of receptorbound proteins may be different for luteal cells and fibroblasts. The mechanisms involved in the internalization of hCG may also be different from those for as-macroglobulin and epidermal growth factor.

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