Properties of low density lipoprotein binding by cultured swine granulosa cells.

Abstract

The properties of low density lipoprotein (LDL) binding to ovarian cells were investigated in cultured swine granulosa cells in serum-free monolayer cultures. Swine and human LDL bound with high affinity and specificity, with apparent dissociation constant (Kd) values for swine and human LDL of 1.1 and 3.6 micrograms/ml at 4 C, and 2.7 and 4.8 micrograms/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 micrograms/ml. Binding of LDL was dependent upon arginine residues within the apoprotein B moiety, since covalent modification of LDL with 1,2-cyclohexanedione markedly reduced its ability to compete for binding or degradation and to support progesterone biosynthesis. This specific, high affinity saturable binding of LDL to pig granulosa cells exhibited a maximal binding capacity of 0.7 micrograms LDL protein/mg DNA, which corresponds to approximately 5500 LDL receptors per cell. The relative time course of LDL binding, internalization, and degradation by swine granulosa cells was assessed by examining trypsin-releasable (surface-bound) and trypsin-resistant (internalized) [125I]iodo-LDL. At 37 C, granulosa cells exhibited a rapid increase in surface-bound lipoprotein, followed by delayed but subsequently progressive increases in internalized and degraded LDL. LDL degradation by swine granulosa cells was a saturable, temperature- and time-dependent process, with half-maximal degradation occurring at a concentration of 16 micrograms/ml LDL. This correlates closely with the half-maximal concentration of LDL that stimulates progesterone secretion. Degradation of [125I]iodo-LDL was not attributable to bulk fluid-phase pinocytosis, since the cellular ingestion of an impermeant probe, [125I]iodo-polyvinylpyrrolidone, occurred at 1/40 the rate of lipoprotein degradation. In addition, degradation of [125I]iodo-LDL was specifically inhibited by excess unlabeled LDL, decreased by prior exposure of granulosa cells to the soluble sterol, 25-hydroxycholesterol, and antagonized by the lysosomotrophic agent, chloroquine. Moreover, in separate experiments, rates of degradation of LDL were found to be significantly correlated with progesterone one production (r = +0.88, P less than 0.01). In summary, swine granulosa cells exhibit specific, high affinity, saturable, and low capacity receptors for homologous and heterologous LDL. These LDL recognition sites on ovarian cells depend upon cyclohexanedione-sensitive arginine residues with the apoprotein B moiety for effective binding, internalization, and functional (steroidogenic) responses.(ABSTRACT TRUNCATED AT 400 WORDS)