Properties of low density lipoprotein binding by cultured swine granulosa cells

Johannes D Veldhuis, J. T. Gwynne, P. Azimi

Research output: Contribution to journalArticle

7 Citations (Scopus)

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 (K(d)) values for swine and human LDL of 1.1 and 3.6 μg/ml at 4 C, and 2.7 and 4.8 μg/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 μg/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 μg 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 μg/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 production (r = +0.88, P < 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. Moreover, rates of LDL degradation correlate with rates of progesterone biosynthesis, suggesting the functionally important nature of this pathway of cholesterol delivery in pig granulosa cells. Thus, the present in vitro system provides a useful model in which to appraise the endocrine regulation of LDL metabolism in granulosa cells. Such regulation is likely to be of physiological importance in the later stages of follicle maturation, when granulosa cells undergo terminal cytodifferentiation into mature luteal cells that require large quantities of lipoprotein-borne sterol substrate.

Original languageEnglish (US)
Pages (from-to)1067-1075
Number of pages9
JournalEndocrinology
Volume117
Issue number3
StatePublished - 1985
Externally publishedYes

Fingerprint

Granulosa Cells
LDL Lipoproteins
Swine
Progesterone
Lipoproteins
Apolipoproteins B
Sterols
Trypsin
Arginine
Pinocytosis
Luteal Cells
Povidone

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Veldhuis, J. D., Gwynne, J. T., & Azimi, P. (1985). Properties of low density lipoprotein binding by cultured swine granulosa cells. Endocrinology, 117(3), 1067-1075.

Properties of low density lipoprotein binding by cultured swine granulosa cells. / Veldhuis, Johannes D; Gwynne, J. T.; Azimi, P.

In: Endocrinology, Vol. 117, No. 3, 1985, p. 1067-1075.

Research output: Contribution to journalArticle

Veldhuis, JD, Gwynne, JT & Azimi, P 1985, 'Properties of low density lipoprotein binding by cultured swine granulosa cells', Endocrinology, vol. 117, no. 3, pp. 1067-1075.
Veldhuis, Johannes D ; Gwynne, J. T. ; Azimi, P. / Properties of low density lipoprotein binding by cultured swine granulosa cells. In: Endocrinology. 1985 ; Vol. 117, No. 3. pp. 1067-1075.
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N2 - 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 (K(d)) values for swine and human LDL of 1.1 and 3.6 μg/ml at 4 C, and 2.7 and 4.8 μg/ml at 37 C. In contrast, high density lipoprotein competed sparingly with [125I]iodo-LDL with an apparent half-maximally inhibitory concentration of 650 μg/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 μg 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 μg/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 production (r = +0.88, P < 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. Moreover, rates of LDL degradation correlate with rates of progesterone biosynthesis, suggesting the functionally important nature of this pathway of cholesterol delivery in pig granulosa cells. Thus, the present in vitro system provides a useful model in which to appraise the endocrine regulation of LDL metabolism in granulosa cells. Such regulation is likely to be of physiological importance in the later stages of follicle maturation, when granulosa cells undergo terminal cytodifferentiation into mature luteal cells that require large quantities of lipoprotein-borne sterol substrate.

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