Effect of gestational age on mRNA and protein expression of polyspecific organic cation transporters during pregnancy

Abstract

Polyspecific organic cation (OC) transporters play important roles in the disposition of clinically used drugs, including drugs used during pregnancy. Pregnancy is known to alter the expression of drug-metabolizing enzymes and transporters, but its specific effect on OC transporters has not been well defined. Using quantitative polymerase chain reaction and liquid chromatography coupled with tandem mass spectrometry targeted proteomics, we determined the effect of pregnancy and gestational age on mRNA and protein expression of major OC transporters in the kidney, liver, and placenta in mice with timed pregnancies. Human organic cation transporter 3 (hOCT3) expression was further investigated in human placentas from the first and second trimesters and at term. Our results showed that pregnancy had a marginal effect on renal mouse organic cation transporter 1/2 (mOct1/2) expression but significantly reduced mouse multidrug and toxin extrusion transporter 1 (mMate1) expression by 20%-40%. Hepatic expression of mOct1 and mMate1 was minimally affected by pregnancy. Human and mouse placentas predominantly expressed OCT3 with little expression of OCT1/2, MATE1/2, and plasma membrane monoamine transporter (PMAT). The hOCT3 protein in first and second trimester and term placentas was quantified to be 0.23 ± 0.033, 0.38 ± 0.072, and 0.36 ± 0.099 fmol/μg membrane protein, respectively. In contrast with the moderate increase in hOCT3 protein during human pregnancy, mOct3 expression in the mouse placenta was highly dependent on gestational age. Compared with gestational day (gd) 10, placental mOct3 mRNA increased by 37-fold and 46-fold at gd 15 and 19, leading to a 56-fold and 128-fold increase in mOct3 protein, respectively. Our study provides new insights into the effect of pregnancy on the expression of polyspecific OC transporters and supports an important role of OCT3 in OC transport at the placental barrier.

Fig. 1.

mRNA and protein expression of mouse OC transporters in kidney (A and C) and liver (B and D) at different gestational ages. Total RNA isolated from the kidneys and livers of nonpregnant (virgin) and pregnant mice at gd 10, 15, and 19 was reverse transcribed, and the mRNA levels of six OC transporters were determined by qRT-PCR. The relative protein expression of individual transporters across different gestational age was measured in the membrane protein prepared from kidney and liver using LC-MS/MS. The mRNA expression is normalized to mGusb in kidney and to mGapdh in liver. Data are expressed as mean ± S.D. from 3–6 mice at each gestational age. *Statistically significantly different from expression at nonpregnant state (P < 0.05).

Fig. 2.

mRNA expression of various OC transporters in (A) human term placenta and (B) mouse placenta at gd 19. Total RNA was isolated from human term placentas (n = 6) and mouse placentas at gd 19 (n = 3). cDNA was synthesized by reverse transcriptase, and mRNA levels of hOCT1-3/mOct1-3, hMATE1/2/mMate1/2, and hPMAT/mPmat were determined by qRT-PCR. The results were normalized to hGUSB or mGapdh and represent mean ± S.D.

Fig. 3.

mOct3 mRNA and protein expression in mouse placenta at different gestational ages. (A) Total RNA isolated from the placenta of nonpregnant (virgin) and pregnant mice at gd 10, 15, and 19 was reverse transcribed, and the mRNA levels of Oct3 were determined by semiquantitative reverse-transcriptase PCR and qRT-PCR. (B) Relative protein expression of mOct3 in isolated membrane protein of mouse placentas was measured using LC-MS/MS. The mRNA levels determined by qRT-PCR were normalized to mGapdh. Data are mean ± S.D. (n = 3 per group). *Statistically significantly different from the expression at gd 10 (P < 0.05).

Fig. 4.

Quantification of hOCT3 mRNA (A) and protein (B) in human placentas at different gestational stages and correlation analysis of mRNA and protein expression (C). Total RNA was isolated from first trimester (T1, n = 11), second trimester (T2, n = 16), and term (n = 6) placentas, and the mRNA levels were determined by qRT-PCR and normalized to hGUSB. Membrane protein was isolated from T1, T2, and term placentas (n = 6 for each gestational age placenta). The hOCT3 protein levels were quantified using a LC-MS/MS method. Each data point represents an individual placenta. The mRNA and protein expressions of hOCT3 were correlated using linear regression.