Inositol-Exchange Activity in Human Primordial Placenta

Abstract

Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn²⁺ and Mg²⁺ concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn²⁺ concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg²⁺, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn²⁺ concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.

Keywords: human placenta; inositol; phosphatidylinositol; phosphatidylinositol exchange enzyme; phosphatidylinositol synthase; placentation; trophoblast.

Figure 1

Regulation of phosphatidylinositol synthesis.

Figure 2

Intracellular distribution of inositol incorporation. (A) Specific radioactivity of crude membrane (Mb), microsomes (MS) and cytosolic (Cyt) [3H] inositol incorporation in phosphatidyl inositol. Samples were incubated for 60 min at 37 °C, Ph = 7.4, 2.5 μCi/mL [3H] inositol. (B) Distribution of total radioactivity. Total activity was calculated from protein concentration and tissue volume. (n = 6 ± SD).

Figure 3

[3H]inositol incorporation of human primordial placenta with Mn^2+.400 mg trophoblast tissue was incubated for 60 min at 37 °C, pH = 7.4, 2.5 μCi/mL [3H]inositol with Mn²⁺. (n = 6 ± SD).

Figure 4

Effect of GTP, GIDP and GDP in phosphatidylinositol synthesis. Trophoblast microsomes were incubated for 60 min at 37 °C with 10 mM Mg²⁺ in pH = 7.4, 2.5 μCi/mL [3H]inositol; 0.1 mM GTP, GIDP, GDP were added at 0 and 30 min. (n = 3 ± SD).

Figure 5

(A) Incorporation of [3H]inositol in phosphatidylinositol in microsomes of trophoblast with Mg²⁺. Microsomes were incubated with 2.5 μCi/mL [3H]inositol for 60 min at 37 °C in pH = 7.4, with Mg²⁺. (B) Incorporation of [3H]inositol in phosphatidylinositol in microsomes of trophoblast with Mn²⁺. Microsomes were incubated with 2.5 μCi/mL [3H]inositol in pH = 7.4 for 60 min at 37 °C with Mn²⁺. (C) Incorporation of [3H]inositol in phosphatidylinositol in microsomes of trophoblast with Mg²⁺ and 10 mM Mn²⁺. Microsomes were incubated with 2.5 μCi/mL [3H]inositol in pH = 7.4 for 60 min at 37 °C with Mg²⁺ and 10 mM Mn²⁺. (n = 6 ± SD).