Dysregulation of Amino Acid Transporters in a Rat Model of TLR7-Mediated Maternal Immune Activation.

Maternal immune activation (MIA) during pregnancy is linked to neurodevelopmental disorders in humans. Similarly, the TLR7 agonist imiquimod alters neurodevelopment in rodents. While the mechanisms underlying MIA-mediated neurodevelopmental changes are unknown, they could involve dysregulation of amino acid transporters essential for neurodevelopment. Therefore, we sought to determine the nature of such transporter changes in both imiquimod-treated rats and human placentas during infection. Pregnant rats received imiquimod on gestational day (GD)14. Transporter expression was measured in placentas and fetal brains via qPCR (GD14.5) and immunoblotting (GD16). To monitor function, fetal brain amino acid levels were measured by HPLC on GD16. Gene expression in the cortex of female fetal brains was further examined by RNAseq on GD19. In human placentas, suspected active infection was associated with decreased ASCT1 and SNAT2 protein expression. Similarly, in imiquimod-treated rats, ASCT1 and SNAT2 protein was also decreased in male placentas, while EAAT2 was decreased in female placentas. CAT3 was increased in female fetal brains. Consistent with this, imiquimod altered amino acid levels in fetal brains, while RNAseq demonstrated changes in expression of several genes implicated in autism. Thus, imiquimod alters amino acid transporter levels in pregnant rats, and similar changes occur in human placentas during active infection. This suggests that changes in expression of amino acid transporters may contribute to effects mediated by MIA toward altered neurodevelopment.

COVID-19 Vaccines and the Virus: Impact on Drug Metabolism and Pharmacokinetics.

This article reports on an American Society of Pharmacology and Therapeutics, Division of Drug Metabolism and Disposition symposium held at Experimental Biology on April 2, 2022, in Philadelphia. As of July 2022, over 500 million people have been infected with SARS-CoV-2 (the virus causing COVID-19) and over 12 billion vaccine doses have been administered. Clinically significant interactions between viral infections and hepatic drug metabolism were first recognized over 40 years ago during a cluster of pediatric theophylline toxicity cases attributed to reduced hepatic drug metabolism amid an influenza B outbreak. Today, a substantive body of research supports that the activated innate immune response generally decreases hepatic cytochrome P450 activity. The interactions extend to drug transporters and other organs and have the potential to impact drug absorption, distribution, metabolism, and excretion (ADME). Based on this knowledge, altered ADME is predicted with SARS-CoV-2 infection or vaccination. The report begins with a clinical case exploring the possibility of SARS-CoV-2 vaccination increasing clozapine levels. This is followed by discussions of how SARS-CoV-2 infection or vaccines alter the metabolism and disposition of complex drugs, such as nanoparticles and biologics and small molecule therapies. The review concludes with a discussion of the effects of viral infections on placental amino acid transport and their potential to impact fetal development. The session improved our understanding of the impact of emerging viral infections and vaccine technologies on drug metabolism and disposition, which will help mitigate drug toxicity and improve drug and vaccine safety and effectiveness. SIGNIFICANCE STATEMENT: Altered pharmacokinetics of small molecule and complex molecule drugs and fetal brain distribution of amino acids following SARS-CoV-2 infection or immunization are possible. The proposed mechanisms involve decreased liver cytochrome P450 metabolism of small molecules, enhanced innate immune system metabolism of complex molecules, and altered placental and fetal blood-brain barrier amino acid transport, respectively. Future research is needed to understand the effects of these interactions on adverse drug responses, drug and vaccine safety, and effectiveness and fetal neurodevelopment.

The Effect of Tobacco Use on the Expression of Placental Transporters in Alaska Native Women.

Prenatal tobacco use among Alaska Native (AN) women has decreased substantially over the past two decades. Previous research suggests that providing AN women with feedback regarding fetal exposure to tobacco may further promote cessation. Transporters in the placenta regulate fetal exposure to nutrients and xenobiotics, including compounds associated with tobacco use. We examined whether prenatal tobacco use impacts transporter expression in the placenta, and whether this is influenced by fetal sex, degree of tobacco exposure, or transporter genotype. At delivery, we obtained placental samples from AN research participants who smoked cigarettes, used commercial chew or iqmik (oral tobacco), or did not use tobacco during pregnancy. Transporter expression was evaluated using qRT-PCR and Western blotting and tested for correlations between transcript levels and urinary biomarkers of tobacco use. The impact of BCRP/ABCG2 and OATP2B1/SLCO2B1 genotypes on protein expression was also examined. Oral tobacco use was associated with decreased P-gp and increased MRP1, MRP3, LAT1, and PMAT mRNA expression. Transcript levels of multiple transporters significantly correlated with tobacco biomarkers in maternal and fetal urine. In women carrying male fetuses, both smoking and oral tobacco were associated with decreased P-gp. Oral tobacco was also associated with decreased LAT1 in women carrying female fetuses. BCRP and OATP2B1 genotypes did not appear to impact protein expression. In conclusion, prenatal tobacco use is associated with altered expression of multiple placental transporters which differs by fetal sex. As transcript levels of multiple transporters were significantly correlated with tobacco use biomarkers, eliminating prenatal tobacco use should alleviate these changes.

Impact of inflammation and infection on the expression of amino acid transporters in the placenta: A minireview.

Amino acid transporters expressed in the placenta help to regulate the transfer of amino acids from maternal to fetal circulation. Nutritional or hormonal factors are known to potentially impact the expression of amino acid transporters in the placenta. A relatively new field of inquiry has also demonstrated that inflammation, whether associated with infection or not, also alters the expression of amino acid transporters in the placenta. Indeed, studies over the past 15 years have demonstrated that malaria, viral and bacterial models of infection, preeclampsia, and direct administration of proinflammatory cytokines can alter placental amino acid transporter expression. While such studies have largely focused on System A and System L transporters, other transporters are also affected. p38 MAPK, STAT3, mTORC1, and AMPK signaling have all been implicated in these changes, but the underlying mechanism(s) remain to be fully elucidated. Furthermore, the implications of such changes warrant further investigation. This review will summarize studies that have investigated the impact of inflammation on placental amino acid transporter expression, identify questions that remain unanswered, and propose future areas of research to advance the field. As amino acid transporters are now being considered for drug targeting and drug delivery, furthering our understanding of the regulation of these transporters during disease states will be of increasing clinical value. Significance Statement While this is a relatively new field of research, multiple studies have demonstrated that inflammation alters placental amino acid transporter expression. This review will serve to summarize, for the first time, studies in this field and identify gaps in current knowledge as research in this area moves beyond identifying changes in transporter expression to investigating the implications of such changes and the mechanisms underlying them.

Viral model of maternal immune activation alters placental AMPK and mTORC1 signaling in rats.

INTRODUCTION: Maternal immune activation (MIA) is associated with neurodevelopmental disorders in offspring. We previously demonstrated that poly(I:C)-mediated MIA alters placental and fetal brain amino acid transporter expression in rats, which could potentially play a role in altered neurodevelopment; however, the mechanism(s) underlying these changes in amino acid transporter expression remain unknown. The objective of the current study was to investigate the mechanism(s) underlying poly(I:C)-mediated changes in the expression of the amino acid transporters in the placenta. METHODS: Pregnant rats received poly(I:C) on gestational day 14 and placentas were collected 6 h later. Mass spectrometry-based proteomics of placentas was performed followed by pathway enrichment analysis. Activation of mTORC1 and its upstream regulator, AMPK, was investigated using immunoblotting. Finally, the role of mTORC1 and AMPK in regulating the expression and localization of the amino acid transporters EAAT2 and ASCT1 was investigated in the human choriocarcinoma cell line JAR. RESULTS: The impact of poly(I:C) on the placental proteome was highly sexually dimorphic. While proteomics-based pathway enrichment analysis indicated enrichment of mTOR signaling in male placentas only, further investigation revealed inhibition of mTORC1 in both male and female placentas in addition to activation of AMPK. In vitro, activation of AMPK and inhibition of mTORC1 decreased membrane localization of EAAT2 and ASCT1. DISCUSSION: Poly(I:C)-mediated MIA activates AMPK and inhibits mTORC1 in rat placenta, both of which decrease expression and membrane localization of EAAT2 and ASCT1 in vitro. Thus, AMPK/mTORC1 signaling could be a novel treatment target for alleviating MIA-mediated changes in placental amino acid transport.

SLC Neurotransmitter Transporters as Therapeutic Targets for Alcohol Use Disorder: A Narrative Review.

Alcohol use disorder (AUD) is 1 of the most prevalent of all substance use disorders and contributes significantly to global disease burden. Despite its prevalence, <10% of individuals with AUD receive treatment. A significant barrier to receiving treatment is a lack of effective pharmacotherapies. While 3 medications have been approved by the FDA for AUD (disulfiram, acamprosate, naltrexone), their efficacy remains low. Furthermore, a number of undesirable side effects associated with these drugs further reduce patient compliance. Thus, research into new effective pharmacotherapies for AUD is warranted. Due to their involvement in regulating synaptic neurotransmitter levels, solute carrier (SLC) transporters could be targeted for developing effective treatment strategies for AUD. Indeed, a number of studies have shown beneficial reductions in alcohol consumption through the use of drugs that target transporters of dopamine, serotonin, glutamate, glycine, and GABA. The purpose of this narrative review is to summarize preclinical and clinical studies from the last 2 decades targeting SLC neurotransmitter transporters for the treatment of AUD. Limitations, as well as future directions for expanding this field, are also discussed.

Impact of Viral Inflammation on the Expression of Renal Drug Transporters in Pregnant Rats.

Inflammation impacts the expression and function of drug transporters at term-gestation; however, the impact of inflammation on the expression of drug transporters at mid-gestation is largely unknown. Since renal drug transporters play a key role in the clearance of many drugs prescribed during pregnancy, our objective was to study the impact of the viral mimetic poly I:C on the expression of renal transporters in pregnant rats at mid-gestation. Poly I:C (10 mg/kg) or saline was administered intraperitoneally to pregnant Sprague-Dawley rats on gestational day 14. Expression of renal transporters was measured at 6, 24, and 48 h by qRT-PCR and Western blot. The mRNA levels of Mdr1a, Mrp4, Oct2, Octn1, Octn2, Mate1, Oat1-3, Urat1, Oatp4c1, Ent1, and Pept2 were significantly lower in the poly I:C group at 6 h. At 24 h, only the mRNA levels of Oct2, Oatp4c1, and Ent1 were decreased compared to saline. Poly I:C significantly decreased protein expression of Urat1 at 24 h, and P-gp, Oct2, Mate1, Oat1, Oat3 at 48 h,. Poly I:C imposed significant reductions in the expression of several key renal transporters at mid-gestation in pregnant rats. Thus, viral infection may impact renal excretion of drug transporter substrates, potentially leading to drug-disease interactions.

Poly(I:C) alters placental and fetal brain amino acid transport in a rat model of maternal immune activation.

PROBLEM: Maternal immune activation (MIA) during pregnancy is associated with increased chances of neurodevelopmental disorders including schizophrenia and autism spectrum disorder (ASD). However, the exact mechanism through which MIA contributes to altered neurodevelopment is unknown. Due to the important role that amino acids play in neurodevelopment, altered amino acid transport could play a role in neurodevelopmental disorders. Indeed, altered plasma concentrations of multiple amino acids have been reported in individuals with ASD or schizophrenia. Therefore, our objective was to determine whether virally mediated MIA induces changes in amino acid transporters in the placenta and fetal brain. METHOD OF STUDY: Pregnant rats were administered poly(I:C) on gestational day 14, and placental and fetal tissues were collected 6, 24, and 48 hours later. Amino acid transporter expression was measured in the placenta and fetal brain using qPCR, Western blotting, and Simple Western. Free amino acid concentrations in the fetal brain were quantified using HPLC. RESULTS: Poly(I:C) increased mRNA expression of several amino acid transporters in the placenta and fetal brain over these timepoints. Conversely, poly(I:C) imposed significant decreases in the protein expression of ASCT1 and EAAT2 in placenta and expression of SNAT5, EAAT1, and GLYT1 in fetal brain. Functional consequences of altered transporter expression were demonstrated through widespread changes in the concentrations of free amino acids in the fetal brains. CONCLUSION: Together, these results represent novel findings with the poly(I:C) MIA model and contribute to the understanding of how MIA during pregnancy potentially leads to neurodevelopmental disorders.

Dysregulation of solute carrier transporters in malaria-infected pregnant mice.

AIMS: Malaria in pregnancy (MiP) alters the expression of ATP-binding cassette efflux transporters in maternal and foetal tissues, as well as the placenta. Malaria induces oxidative stress, and pregnancy is associated with arginine deficiency. We hypothesized that reducing oxidative stress during MiP by supplementation with L-arginine, a NO precursor, would attenuate transcriptional changes in a second superfamily of transporters, solute carrier (SLC) transporters, and improve pregnancy outcomes. METHODS AND RESULTS: Pregnant BALB/c mice receiving L-arginine (1.2%) in water, or water alone, were infected with Plasmodium berghei ANKA on gestational day 13 and sacrificed on gestational day 19. Compared to controls, the mRNA of numerous SLC transporters was downregulated in maternal and foetal tissues, as well as in the placentas of infected mice. While supplementation with L-arginine did improve foetal viability, it did not improve the mRNA expression of oxidative stress markers, transporters nor other indices of foetal and maternal health. Moreover, amino acid uptake transporters were downregulated upon infection, which could potentially contribute to decreased foetal birthweight. CONCLUSIONS: Malaria in pregnancy significantly alters the expression of SLC transporters in maternal and foetal tissues as well as the placenta, regardless of L-arginine supplementation. Further studies to investigate methods of reducing oxidative stress in MiP are warranted.

Battling the HIV/AIDS Epidemic: Triumphs and Barriers.

Worldwide, over 77 million people have been infected by human immunodeficiency virus (HIV) but its cure remains elusive. Once considered a fatal disease, advances in antiretroviral therapy (ART) have dramatically increased the life expectancy of infected persons. Much progress has been made in the development and utilization of combination ART and preventative pre-exposure prophylaxis products, however, numerous obstacles prevent eradication. Clinical pharmacologists along with world health organizations continue to play a key role in identifying and implementing strategies to combat this disease.

Resonance Raman studies on the flavohemoglobin of the protist Giardia intestinalis: evidence of a type I/II-peroxidase-like heme environment and roles of the active site distal residues.

Flavohemoglobins are microbial enzymes that counter nitrosative stress, but the details of their underlying enzymatic activities and structure-function relationships are not completely understood. These enzymes have been identified in Gram-negative bacteria, certain fungi, and the parasitic protist Giardia intestinalis (gFlHb) which, despite lacking the ability to make heme, encodes several hemeproteins. To gain knowledge about the biophysical properties of the active site of gFlHb, we used resonance Raman spectroscopy to probe the wild-type protein and variants at globin domain positions E11, E7, and B10 on the distal, ligand-binding side of the heme. The heme of gFlHb has a peroxidase-like environment resembling that of the well-characterized E. coli flavohemoglobin HMP. We provide evidence that gFlHb has two Fe-His stretching modes, a feature that also occurs in type I/II-peroxidases in which a proximal histidine with strong imidazolate character and a nearby carboxylic acid residue can exist as a tautomeric pair depending on the position of a shared proton. Characterization of the distal variants Tyr30Phe, Gln54Leu, and Leu59Ala shows that TyrB10 and GlnE7 but not LeuE11 are implicated in stabilisation of bound exogenous ligands such as CO and O2. Our work revealed that several biophysical properties of the heme active site of gFlHb are highly conserved compared to HMP and suggest that they are conserved across the flavohemoglobin family.