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Published in final edited form as: Reprod Toxicol. 2022 Jun 6;111:178–183. doi: 10.1016/j.reprotox.2022.05.017

Interaction of Alcohol & Phosphatidic Acid in Maternal Rat Uterine Artery Function

Vishal D Naik 1, Jehoon Lee 2, Marcus O Orzabal 2, Jayanth Ramadoss 1,4,*
PMCID: PMC9670159  NIHMSID: NIHMS1849268  PMID: 35671880

Abstract

Alcohol has been demonstrated to impair maternal uterine arterial adaptations in Fetal Alcohol Spectrum Disorder (FASD) animal models. However, the exact mechanism remains inconclusive. We hypothesized that phosphatidic acid (PA), a direct target of alcohol metabolism, would alleviate alcohol-induced vascular dysfunction of the maternal uterine artery. Mean fetal weight, and crown-rump length of the alcohol administered rats were ~9% and 7.6% lower than the pair-fed control pups, respectively. Acetylcholine (Ach)-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P<0.05). Supplementation of 10−5 M PA reversed alcohol-induced vasodilatory deficit; no difference was detected after PA treatment between pair-fed control and alcohol groups (P=0.37). There was a significant interaction between PA concentrations and alcohol exposure (PA X Alcohol effect, P<0.0001). Pair-wise comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats, with % relaxation significantly improved at PA concentrations > 10−7 M (P<0.05). Alcohol significantly reduced vasodilatory P-Ser1177 endothelial nitric oxide synthase (eNOS) levels in the uterine artery (↓90.7%; P=0.0029). PA treatment significantly reversed P-Ser1177 eNOS level in alcohol uterine arteries (153.7%↑; P=0.005); following ex vivo PA, there was no difference in P-Ser1177 eNOS levels between Control and Alcohol. Neither alcohol treatment nor PA affected total eNOS levels. Our data provide the first evidence of the interaction of alcohol and PA in rat maternal uterine artery vascular function and demonstrates PA’s relationship with the eNOS system. Overall, the current study demonstrates that PA may be a promising therapeutic molecule of interest in alcohol-related gestational vascular dysfunction.

Keywords: Alcohol, Pregnancy, Phosphatidic Acid, FASD

Graphical Abstract

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1. Introduction

Fetal Alcohol Spectrum Disorder (FASD) is a hypernym encompassing a range of disorders that may occur in babies with developmental alcohol exposure [1]. Fetal Alcohol Syndrome (FAS), the most severe form of FASD, is associated with distinctive facial features, growth deficiency, and central nervous system damage [2, 3]. Despite dedicated and intense FASD research, the exact mechanism in the etiology of FASD remains inconclusive. While many studies have examined the teratogenic effects of alcohol on the brain, few studies have focused on the impact of alcohol on the maternal reproductive vascular system, specifically the uterine artery.

During pregnancy, the uterine artery experiences extensive remodeling and adaptations, including increased vasodilation, decreased vascular resistance, and increased blood flow to ensure adequate delivery of nutrients and oxygen to the developing fetus [4–8]. Studies exploring the effects of alcohol on uterine artery adaptations have reported that alcohol exposure leads to endothelium-dependent impairment of uterine artery vasodilation and altered spiral artery remodeling in rats, and decreased uterine blood flow in sheep [9–11]. Studies in human and animal models, both in vivo and in vitro, have reported systemic and reproductive vascular dysfunction via the endothelial nitric oxide synthase (eNOS) vasodilatory system, including the post-translational modification of phosphorylation sites P-Ser1177 eNOS [11–14]. However, the mechanism underlying alcohol-induced vascular dysfunction remains elusive, probably due to limited knowledge on direct molecular targets of alcohol.

One direct molecule, specifically affected by alcohol exposure, is phosphatidic acid (PA) [15, 16]. During alcohol exposure, PA is converted to Phosphatidylethanol (PEth); notably, PEth only forms in the presence of alcohol (ethanol) [17–20]. PA is a vital signaling molecule involved in the biosynthesis of various lipids and is an essential factor in determining cellular morphology and function [21–23]. We have previously reported an accumulation of PEth in the uterine arteries of maternal rats exposed to alcohol [24]. Since PEth replaces PA in proportion to the alcohol magnitude in the tissue, we conjectured PA would interact with alcohol and thus affect uterine artery function. While functional studies utilizing PA have been performed in studies outside the alcohol field, our study is the first to explore PA’s effects in pregnant rats’ uterine arteries following binge alcohol exposure. Since PA has been reported to affect membrane-bound protein function, and a study using bovine cells reported that eNOS selectively binds to anionic lipids [25], we inferred PA might affect UA eNOS activity indices as well. We herein, for the first time, have examined the concentration response of ex vivo PA on uterine artery vascular reactivity. We hypothesized that PA interacts with alcohol and that the addition of PA ex vivo will alleviate alcohol-induced vascular dysfunction of the maternal uterine artery via the eNOS system.

2. Methods

2.1. Treatment groups and alcohol dosing paradigm

All experimental procedures were in accordance with National Institutes of Health guidelines (NIH Publication No. 85–23, revised 1996), with approval by the Animal Care and Use Committee at Texas A&M University. Timed pregnant Sprague–Dawley rats purchased from Charles River (Wilmington, MA) arrived on GD 4 and were housed in a temperature-controlled room (23°C) with a 12:12-hour light-dark cycle. All Dams were allowed to acclimatize for one day before handling, weighing, and assignment into experimental groups. A nutritional pair-fed control group and a binge alcohol group were utilized for performing dual-chamber arteriography and Western blot. Binge alcohol group dams were acclimatized via a once-daily orogastric gavage of 4.5 g/kg ethanol (22.5% wt/v; peak blood alcohol concentration, 216 mg/dl) from GD 5–10, and advanced to a 6 g/kg alcohol dose from GD 11–19 (28.5% wt/v; peak blood alcohol concentration, 289 mg/dl) [26, 27]. The exposure regimen utilized in this study was modeled after reported alcohol consumption patterns in pregnant women and FASD animal models [28–30]. Daily feed intake of the alcohol rats was measured, and an equal amount of feed was given to pair-fed dams as a nutrition control. In addition, to control for the calories derived from alcohol, pair-fed control rats received a once-daily isocaloric maltose-dextrin. Rats were euthanized by decapitation while under isoflurane anesthesia.

2.2. Maternal and fetal weight measurements

Maternal weights, fetal weights, and crown-rump lengths (pair-fed control, n = 6; alcohol, n = 6) were measured on GD 20, one day after the last treatment on GD 19. Following euthanasia, the concepti were separated, and their weights and crown-rump lengths were measured.

2.3. Reagent preparation

HEPES-Bicarbonate Solution (HBS), pH 7.4 (NaCl 130 mM; KCl 4 mM; MgSO4.7H2O 2.5 mM; NaHCO3 4.05 mM; CaCl2, 2.4 mM; HEPES 10 mM; KH2PO4 1.18 mM; Glucose 6 mM; EDTA 0.024 mM) was prepared fresh on the day of experiment. PA (#840857C, Avanti Polar Lipids) was air-dried under a gentle stream of nitrogen gas and resuspended in 1% BSA to make a 10−2 M stock. Acetylcholine (Ach) and Thromboxane (Tbx) stocks were prepared in HBS.

2.4. Arteriography

Following euthanasia, the whole uterus was dissected and immediately put in ice-cold HBS for arteriography experiments (pair-fed control n=6; alcohol, n=6). Uterine artery functional assessments were performed following uterine artery isolation as described previously [11, 31–33]. Briefly, the uterine horn was transferred to a 200 mm petri dish with solidified sylguard to facilitate tissue isolation and cleaning in ice-cold HBS. A 3–5 mm segment of the primary uterine artery was dissected between arterial bifurcations from the approximate center of the uterine horn. Surrounding adipose and connective tissues were carefully removed from the uterine artery segment. Through the dual-chamber setup, arterial segments were mounted from both groups, ensuring identical treatment per experiment. The dual-chamber, with mounted vessels, was put in a closed enclosure with 37°C ambient temperature, and the cannulation setup was completed to allow continuous circulation of pre-warmed HBS bath. Intramural pressure was increased to 60 mm Hg until the vessels exhibited a myogenic tone (~ 15–20 min). Following equilibration in the buffer for another 15 min, the circulation buffer was changed to fresh HBS and warmed to 37°C. Intramural pressure was then increased to reflect in vivo pressures at 90 mm Hg, at which pressure the PA concentration response was performed. Vessels were pre-constricted with 10−7 M Tbx as determined previously [11]. Following this, 10−5 M Ach was added to maximally relax each vessel. This concentration of Ach was selected based on our previous data, which reported maximal difference in uterine artery vasodilation between the pair-fed control and alcohol administered rats at that dose [11]. Vessels that failed to get a myogenic tone or did not respond to Tbx or Ach treatment were discarded. The above treatment was followed by administration of three-fold increasing concentrations of PA from 10−10 M to 10−5 M. Vascular response was recorded using ionwizard software (Ionoptix LLC, MA, USA) for 5 min or until arterial diameter stabilized.

2.5. Immunoblotting

In a separate cohort of dams (pair-fed control, n=5; alcohol, n=5), following euthanasia, the uterine arteries were isolated by separating the vein, and cleaning the adipose tissues, followed by incubation in either HBS or HBS + PA for 1 min, before flash freezing for Immunoblot analysis. Following sacrifice on GD 20, uterine arteries from each horn were isolated and incubated in either HBS or HBS+10−5 PA solution for 1 min before flash freezing in liquid nitrogen and stored at −80°C until the day of use. Tissues were homogenized using a refrigerated bead homogenizer (Benchmark Scientific, NJ, USA) and quantified using BCA assay. Twenty (20) μg of sample protein was loaded onto 4–20% mini-protean TGX gel (Bio-rad, CA, USA). Following transfer to PVDF membrane, P-Ser1177 eNOS, total eNOS, and GAPDH were probed. Densitometry analysis was performed using AzureSpot software (Azure Biosystems, CA, USA).

3. Statistics

Maternal weight, fetal weight, and fetal crown-rump length were analyzed using Student’s t-test. Normality was tested using the Shapiro-Wilk normality test where appropriate. Uterine vascular response to PA was analyzed using two-way repeated measure ANOVA, followed by multiple comparisons using Fisher’s LSD. Data for the PA concentration response following ANOVA were reported as mean ± SEM. Non-linear regression curve fit was performed using a three-parameter equation, Y=Baseline + (Max Response-Baseline)/ 1+10 (LogEC50-X) to get the effective concentration (EC50). The data were considered significant if the P-value was < 0.05. All data were analyzed using Graphpad Prism (GraphPad Software, CA, USA).

4. Results

4.1. Growth Assessment

Maternal body weights did not differ between pair-fed control and alcohol-administered rats (Figure 1 A). Average fetal weight and crown-rump length of alcohol-administered rats were ~9 % lower (P = 0.0139) (Figure 1B) and 7.6% lower (P = 0.0033) (Figure 1C) than the pair-fed control pups, respectively, displaying the classic intrauterine growth restriction (IUGR) phenotypes observed in FAS diagnosis.

Figure 1. Effects of prenatal alcohol on fetal growth parameters.

Figure 1.

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Following gestational alcohol administration, fetal weight and crown-rump length were measured on gestational day (GD) 20, one day after the last alcohol exposure. (A) Average maternal weight was not different between pair-fed control (PF-Cont) and alcohol (Alcohol) groups. (B) Mean fetal weight in the alcohol group was decreased compared with pair-fed control (P = 0.0139). (B) Fetal crown-rump length in the alcohol group was decreased compared to the pair-fed control (P = 0.0033). Values are mean ± SEM, * indicates statistical significance, P < 0.05.

4.2. Ex-vivo PA supplementation significantly ameliorated alcohol-induced uterine vascular dysfunction

Uterine arteries were maximally constricted and 10−5 M Ach was added to record endothelium dependent relaxation. Ach-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats (P < 0.05, Figure 2A). We then selected a physiologic dose of 10−5 M PA (refer methods) and assessed if PA would reverse alcohol-induced deficits in uterine artery vasodilation. Supplementation of 10−5 M PA reversed alcohol-induced vasodilatory deficit and no differences were detected after PA treatment between pair-fed control and alcohol groups (P = 0.37) (Figure 2B). PA produced an additional ↑27.5% relaxation in the alcohol group, whereas no additional benefits of supplementation were observed in the Controls (↑5.92%), the PA effect on alcohol group was significantly higher compared to the pair-fed controls (P=0.0012; Figure 2C). Following dilation with 10−5 M Ach, PA was added ex vivo, and concentration-dependent response of the uterine artery was recorded to determine the minimal dose of PA required to ameliorate alcohol-induced dysfunction. Representative pressure arteriograph tracing from the pair-fed control and alcohol-administered rats showing a dose-dependent vasodilatory response of the pregnant rat uterine artery with cumulative increasing doses of PA is shown in Figure 3A. There was a significant interaction effect between PA concentrations and treatment groups (PA X Alcohol effect, P < 0.0001). Pair-wise multiple comparisons showed a concentration-dependent vasodilatory effect on uterine arteries of the alcohol-administered rats with % relaxation significantly enhanced at PA concentrations 10−7 M and above compared to the pair-fed controls (P < 0.05). This is expected as concentration-dependent vasodilation is already achieved in the control subjects and PA reverses alcohol-induced functional deficits.

Figure 2. Percent relaxation of rat uterine arteries following Acetylcholine and Phosphatidic Acid.

Figure 2.

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(A) Ach-induced uterine artery relaxation was significantly impaired in uterine arteries of alcohol-administered rats in the absence of PA (P < 0.05). (B) Supplementation of 10−5 M PA reversed alcohol-induced vasodilatory deficit and no differences were detected after PA treatment between pair-fed control (PF-Cont) and alcohol (Alcohol) groups. (C) Additional magnitude of percent relaxation of the maternal uterine artery in the control and alcohol groups produced by addition of 10−5 M ex vivo PA. Data are expressed as mean ± SEM. Significance (*) was established a priori at P < 0.05

Figure 3. Ex vivo Phosphatidic acid (PA) reverses alcohol-induced dysfunction of the uterine artery in pregnant rats.

Figure 3.

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(A) Representative pressure arteriography tracing from the pair-fed control (PF-Cont) and alcohol-administered rats (Alcohol) showing a dose-dependent vasodilatory response of the pregnant rat uterine artery with cumulative increasing doses of PA. (B) Effect of accumulative doses of PA on uterine artery vasodilation in pair-fed control rats (PF-Cont) (●) and alcohol-administered rats (Alcohol) ( ). Significance (*) was established a priori at P < 0.05.

4.3. PA altered stimulatory Ser1177 eNOS phosphorylation

Alcohol significantly reduced (↓ 90.7 %) excitatory Ser1177 eNOS phosphorylation in the uterine artery (P = 0.0029) (Figure 4A). The phosphorylation level of excitatory Ser1177 eNOS was significantly reversed in PA-treated alcohol uterine arteries (153.7 % ↑, P = 0.005). In contrast, PA-treatment did not enhance the eNOS activity index any further in the pair-fed control uterine arteries. Thus, there was no difference in P-Ser1177 eNOS levels following PA treatment between the pair-fed control and alcohol uterine arteries. Total eNOS was not different between the control and the alcohol groups (Figure 4B). Further, PA did not affect total eNOS levels in either pair-fed control or alcohol uterine arteries.

Figure 4. Effect of ex vivo PA on phosphorylation level of Ser1177NOS and total eNOS expression.

Figure 4.

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(A) Alcohol significantly reduced excitatory Ser1177 eNOS phosphorylation in the uterine artery (*, P = 0.0029). The phosphorylation level of excitatory Ser1177 eNOS was significantly reversed in PA-treated uterine arteries in the alcohol group (A+PA) (#, P < 0.05). PA-treatment did not enhance the eNOS activity index any further in the pair-fed (PF) control uterine arteries (PF+PA) (B)Total eNOS was not different between the control and the alcohol groups. Further, PA did not alter total eNOS levels in either pair-fed control or alcohol uterine arteries. Data are expressed as mean ± SEM and as fold of GAPDH (loading control).

5. Discussion

Alcohol-induced impairment of uterine artery vasodilation during pregnancy can have damaging effects on the developing fetus. Currently, no published reports document strategic approaches to reverse alcohol exposure-induced uterine vascular dysfunction. Herein, we utilized phosphatidic acid (PA), a direct molecular target of alcohol metabolism, to study PA’s effect on uterine vascular function. This is the first study showing the interaction of phosphatidic acid and alcohol in agonist-mediated dilation of the uterine artery in alcohol-exposed maternal rats. Our study demonstrates that ex vivo PA ameliorates alcohol-induced uterine artery dysfunction. Further, ex vivo PA reverses alcohol-induced deficits in uterine artery eNOS activity index; a system reported previously to directly affect alcohol-induced vasodilation in our model.

5.1. Ex vivo PA supplementation ameliorates alcohol-induced uterine artery dysfunction

The uterine artery undergoes extensive adaptations during pregnancy to accommodate increased blood flow for adequate nutrient and gas exchange, ensuring the development of a healthy baby. We previously reported that chronic binge alcohol exposure significantly reduced endothelial-dependent vasodilation of the maternal uterine artery compared to pair-fed control rats [11]. The current study supplemented PA ex vivo and demonstrated significant improvement in vasodilatory deficits resulting from alcohol exposure. However, the control subjects did not exhibit any additional enhancement in vasodilation following PA administration; this is not surprising as the expected concentration-dependent vasodilation is already achieved in the control subjects. In support of these findings, a recent study showed a similar observation with an increased microvascular diameter and enhanced blood flow in the ischemic hindlimb muscle in a microbubble (dipalmitoylphosphatidylcholine [DPPC], dipalmitoyl phosphatidic acid [DPPA], dipalmitoyl phosphatidylethanolamine–polyethylene glycol 5000) cavitation model in rats, however, whether the effect was due to PA component of the microbubble was neither ascertained, nor studied [34]. Phosphatidic acid has been reported to play an essential role in agonist-induced contractions in rat resistant arteries and vascular smooth muscle cells relaxation [35–37].

5.2. PA reversed alcohol-induced post-translational modification of eNOS

We and others have previously reported gestational alcohol-exposure induced disruption of endothelial nitric oxide-mediated vasodilation of the maternal uterine artery [7, 11, 32]. We previously reported significant decreases in P-Ser1177 eNOS levels in the uterine arteries of alcohol-administered rats. As expected, in this study, alcohol significantly decreased phosphorylation of Ser1177 eNOS; however, ex vivo supplementation with PA resulted in a significant increase in excitatory Ser1177 eNOS phosphorylation levels in the uterine arteries of alcohol-administered rats, thus reversing alcohol effects on eNOS post-translational modification. Following PA treatment, the phosphorylation levels of Ser1177 eNOS of the alcohol-exposed uterine arteries were not different from those in the pair-fed control uterine arteries. While a direct link between PA and eNOS phosphorylation cannot be concluded from this study, PA has been reported to activate Protein Phosphatase-1 and Protein Phosphatase-2A in vitro [38], and studies utilizing PLD-2 (upstream of PA) knock-out mice have reported decreased eNOS activity [39], decreased vascular smooth muscle cell migration, plasma membrane remodeling, and membrane protein activation [40, 41] PA is an anionic lipid primarily found on the lipid bilayer and plays prominent role as a modulator of membrane shape and protein phosphorylation [42]. It has been reported using bovine eNOS/sf9 cell system that eNOS selectively binds to anionic lipids; further, amongst various acidic phospholipids, PA has the highest affinity to eNOS peptides [25, 43], demonstrating the importance of PA for eNOS binding to the lipid bilayer. Alcohol has been reported to significantly alter lipid profile and lipid ionization in rats [44]. Interestingly, we showed accumulation of PEth in the uterine arteries of alcohol-exposed maternal rats [24]. The accumulation of PEth on the uterine arteries of alcohol-treated rats may thus affect eNOS-lipid bilayer interaction and decrease eNOS mediated vasodilation. Since PEth is only found in alcohol-treated animals, this may explain why PA significantly improved eNOS mediated vasodilation of alcohol-exposed uterine arteries but not the control uterine arterial vasodilation (Figure 5). This is the first study showing the direct effect of ex vivo PA supplementation on eNOS phosphorylation in uterine arteries of pregnant rats and complete reversal of alcohol effects on excitatory eNOS post-translational modification in the uterine artery.

Figure 5. Plausible Model: PA and alcohol interaction in rat uterine artery.

Figure 5.

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In the presence of alcohol, hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD) is interrupted and switched to transphosphatidylation of PC. This results in Phosphatidylethanol (PEth) formation instead of phosphatidic acid (PA). PA is located in the lipid bilayer of the uterine arteries. PEth accumulates in the uterine arteries of alcohol-exposed pregnant rats [24]. eNOS, a membrane-bound protein, interacts with anionic lipids such as PA. Alcohol decreases phosphorylation of excitatory Ser1177eNOS, resulting in attenuated endothelium-dependent vasodilation, possibly due to PEth-induced homeostatic imbalance of the anionic PA-eNOS interaction required for post-translational modification of eNOS. PA reverses alcohol/PEth induced dysregulation of the eNOS system, resulting in reversal of uterine artery vasodilation of alcohol-exposed rats.

5.3. Perspectives:

In summary, this manuscript provides the first evidence of the interaction of alcohol and PA (a direct molecular target of alcohol) in maternal uterine artery vascular function. PLD2 and PA are likely targets of interest in multiple therapeutic intervention studies, including systemic inflammation, vascular injury, and hypertension. PA has been proven safe in both short and long-term studies in pregnant rat models [45]. This study provides promising data showing PA-mediated reversal of alcohol-induced vascular dysfunction. Considering the safety and direct link between PA and alcohol, future studies would examine the effects of prenatal PA supplementation in pregnant rats exposed to alcohol and examine effects on classic FAS phenotypes, including growth, neurodevelopmental, and behavioral outcomes. Overall, the current study explores a potentially promising therapeutic agent of interest in FASD treatment.

Highlights.

  • Alcohol impairs maternal uterine artery vasodilation in the FASD rat model.

  • Ex vivo Phosphatidic acid (PA) treatment reversed alcohol-impaired UA dysfunction.

  • Ex vivo PA treatment reversed alcohol-induced reduction of eNOS activity indices.

  • PA is a promising therapeutic molecule in alcohol-related gestational UA dysfunction.

Acknowledgments:

Funding:

This study was supported by National Institutes of Health [HL151497 (JR), AA23520 (JR), AA23035 (JR)]

Footnotes

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Declaration of interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

References

  • [1].Sokol RJ, Fetal Alcohol Spectrum Disorder, JAMA 290(22) (2003) 2996. [DOI] [PubMed] [Google Scholar]
  • [2].Jones K, Smith D, Recognition Of The Fetal Alcohol Syndrome In Early Infancy, The Lancet 302(7836) (1973) 999–1001. [DOI] [PubMed] [Google Scholar]
  • [3].Bertrand J, Floyd LL, Weber MK, D.o.B.D. Fetal Alcohol Syndrome Prevention Team, N.C.o.B.D. Developmental Disabilities, C.f.D.C. Developmental Disabilities, Prevention, Guidelines for identifying and referring persons with fetal alcohol syndrome, MMWR Recomm Rep 54(RR-11) (2005) 1–14. [PubMed] [Google Scholar]
  • [4].Rosenfeld CR, Distribution of cardiac output in ovine pregnancy, Am J Physiol 232(3) (1977) H231–235. [DOI] [PubMed] [Google Scholar]
  • [5].Caton D, Kalra PS, Endogenous hormones and regulation of uterine blood flow during pregnancy, Am J Physiol 250(3 Pt 2) (1986) R365–369. [DOI] [PubMed] [Google Scholar]
  • [6].Magness RR, Shaw CE, Phernetton TM, Zheng J, Bird IM, Endothelial vasodilator production by uterine and systemic arteries. II. Pregnancy effects on NO synthase expression, Am J Physiol 272(4 Pt 2) (1997) H1730–1740. [DOI] [PubMed] [Google Scholar]
  • [7].Bird IM, Zhang L, Magness RR, Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function, American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 284(2) (2003) R245–R258. [DOI] [PubMed] [Google Scholar]
  • [8].Mandala M, Osol G, Physiological remodelling of the maternal uterine circulation during pregnancy, Basic Clin Pharmacol Toxicol 110(1) (2012) 12–18. [DOI] [PubMed] [Google Scholar]
  • [9].Falconer J, The effect of maternal ethanol infusion on placental blood flow and fetal glucose metabolism in sheep, Alcohol Alcohol. 25(4) (1990) 413–416. [PubMed] [Google Scholar]
  • [10].Cook JL, Zhang Y, Davidge ST, Vascular function in alcohol-treated pregnant and nonpregnant mice, American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 281(5) (2001) R1449–R1455. [DOI] [PubMed] [Google Scholar]
  • [11].Naik VD, Davis-Anderson K, Subramanian K, Lunde-Young R, Nemec MJ, Ramadoss J, Mechanisms Underlying Chronic Binge Alcohol Exposure-Induced Uterine Artery Dysfunction in Pregnant Rat, Alcoholism: Clinical and Experimental Research 42(4) (2018) 682–690. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Bird IM, Zhang LB, Magness RR, Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function, Am J Physiol-Reg I 284(2) (2003) R245–R258. [DOI] [PubMed] [Google Scholar]
  • [13].Kulandavelu S, Whiteley KJ, Qu DW, Mu JW, Bainbridge SA, Adamson SL, Endothelial Nitric Oxide Synthase Deficiency Reduces Uterine Blood Flow, Spiral Artery Elongation, and Placental Oxygenation in Pregnant Mice, Hypertension 60(1) (2012) 231–238. [DOI] [PubMed] [Google Scholar]
  • [14].Magness RR, Krishnamurthy P, Sheppard C, Bird IM, Angiogenic factors increase nitric oxide synthase (ecNOS) expression and prostacyclin (PGI(2)) production in ovine uterine and fetoplacental arteries., Biol Reprod 56 (1997) 556–556. [Google Scholar]
  • [15].Kobayashi M, Kanfer JN, Phosphatidylethanol Formation via Transphosphatidylation by Rat Brain Synaptosomal Phospholipase D, Journal of Neurochemistry 48(5) (1987) 1597–1603. [DOI] [PubMed] [Google Scholar]
  • [16].Luginbühl M, Weinmann W, Butzke I, Pfeifer P, Monitoring of direct alcohol markers in alcohol use disorder patients during withdrawal treatment and successive rehabilitation, Drug Testing and Analysis 0(0) (2019). [DOI] [PubMed] [Google Scholar]
  • [17].Alling C, Gustavsson L, Månsson JE, Benthin G, Änggård E, Phosphatidylethanol formation in rat organs after ethanol treatment, Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 793(1) (1984) 119–122. [DOI] [PubMed] [Google Scholar]
  • [18].Bakhireva LN, Savage DD, Focus On: Biomarkers of Fetal Alcohol Exposure and Fetal Alcohol Effects, Alcohol Research & Health 34(1) (2011) 56–63. [PMC free article] [PubMed] [Google Scholar]
  • [19].Baldwin AE, Jones J, Jones M, Plate C, Lewis D, Retrospective assessment of prenatal alcohol exposure by detection of phosphatidylethanol in stored dried blood spot cards: An objective method for determining prevalence rates of alcohol consumption during pregnancy, The International Journal of Alcohol and Drug Research 4(2) (2015) 131–137. [Google Scholar]
  • [20].Jastrzębska I, Zwolak A, Szczyrek M, Wawryniuk A, Skrzydło-Radomańska B, Daniluk J, Biomarkers of alcohol misuse recent advances and future prospects, Przegląd Gastroenterologiczny 11(2) (2016) 78–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [21].Wang XM, Devalah SP, Zhang WH, Welti R, Signaling functions of phosphatidic acid, Progress in Lipid Research 45(3) (2006) 250–278. [DOI] [PubMed] [Google Scholar]
  • [22].Athenstaedt K, Daum G, Phosphatidic acid, a key intermediate in lipid metabolism, European Journal of Biochemistry 266(1) (1999) 1–16. [DOI] [PubMed] [Google Scholar]
  • [23].English D, Cui Y, Siddiqui RA, Messenger functions of phosphatidic acid, Chemistry and Physics of Lipids 80(1–2) (1996) 117–132. [DOI] [PubMed] [Google Scholar]
  • [24].Naik V, Lunde-Young R, Ramire J, Lee J, Ramadoss J, Distribution of Phosphatidylethanol in Maternal and Fetal Compartments After Chronic Gestational Binge Alcohol Exposure, Alcoholism: Clinical and Experimental Research 44(1) (2020) 264–271. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [25].Venema RC, Sayegh HS, Arnal J-F, Harrison DG, Role of the Enzyme Calmodulin-binding Domain in Membrane Association and Phospholipid Inhibition of Endothelial Nitric Oxide Synthase *, Journal of Biological Chemistry 270(24) (1995) 14705–14711. [DOI] [PubMed] [Google Scholar]
  • [26].Davis-Anderson KL, Wesseling H, Siebert LM, Lunde-Young ER, Naik VD, Steen H, Ramadoss J, Fetal regional brain protein signature in FASD rat model, Reprod Toxicol 76 (2018) 84–92. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [27].Subramanian K, Naik VD, Sathishkumar K, Yallampalli C, Saade GR, Hankins GD, Ramadoss J, Chronic binge alcohol exposure during pregnancy impairs rat maternal uterine vascular function, Alcohol Clin Exp Res 38(7) (2014) 1832–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [28].Church MW, Gerkin KP, Hearing disorders in children with fetal alcohol syndrome: findings from case reports, Pediatrics 82(2) (1988) 147–154. [PubMed] [Google Scholar]
  • [29].Cudd TA, Chen W-JA, West JR, Fetal and Maternal Thyroid Hormone Responses to Ethanol Exposure During the Third Trimester Equivalent of Gestation in Sheep, Alcoholism: Clinical and Experimental Research 26(1) (2002) 53–58. [PubMed] [Google Scholar]
  • [30].Thomas JD, Sather TM, Whinery LA, Voluntary exercise influences behavioral development in rats exposed to alcohol during the neonatal brain growth spurt, Behav Neurosci 122(6) (2008) 1264–1273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [31].Naik VD, Lunde-Young ER, Davis-Anderson KL, Orzabal M, Ivanov I, Ramadoss J, Chronic binge alcohol consumption during pregnancy alters rat maternal uterine artery pressure response, Alcohol 56 (2016) 59–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [32].Mateev S, Sillau AH, Mouser R, McCullough RE, White MM, Young DA, Moore LG, Chronic hypoxia opposes pregnancy-induced increase in uterine artery vasodilator response to flow, American Journal of Physiology-Heart and Circulatory Physiology 284(3) (2003) H820–H829. [DOI] [PubMed] [Google Scholar]
  • [33].Marshall SA, Senadheera SN, Jelinic M, O’Sullivan K, Parry LJ, Tare M, Relaxin Deficiency Leads to Uterine Artery Dysfunction During Pregnancy in Mice, Front Physiol 9 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [34].Qiu S, Li D, Wang Y, Xiu J, Lyu C, Kutty S, Zha D, Wu J, Ultrasound-Mediated Microbubble Cavitation Transiently Reverses Acute Hindlimb Tissue Ischemia through Augmentation of Microcirculation Perfusion via the eNOS/NO Pathway, Ultrasound in Medicine & Biology 47(4) (2021) 1014–1023. [DOI] [PubMed] [Google Scholar]
  • [35].Ohanian J, Ollerenshaw J, Collins P, Heagerty A, Agonist-induced production of 1,2-diacylglycerol and phosphatidic acid in intact resistance arteries. Evidence that accumulation of diacylglycerol is not a prerequisite for contraction, Journal of Biological Chemistry 265(15) (1990) 8921–8928. [PubMed] [Google Scholar]
  • [36].Bhugra P, Xu YJ, Rathi S, Dhalla NS, Modification of intracellular free calcium in cultured A10 vascular smooth muscle cells by exogenous phosphatidic acid, Biochem Pharmacol 65(12) (2003) 2091–8. [DOI] [PubMed] [Google Scholar]
  • [37].Lassegue B, Alexander RW, Clark M, Akers M, Griendling KK, Phosphatidylcholine Is a Major Source of Phosphatidic-Acid and Diacylglycerol in Angiotensin-Ii-Stimulated Vascular Smooth-Muscle Cells, Biochem J 292 (1993) 509–517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [38].Chalfant CE, Kishikawa K, Mumby MC, Kamibayashi C, Bielawska A, Hannun YA, Long Chain Ceramides Activate Protein Phosphatase-1 and Protein Phosphatase-2A: Activation Is Stereospecific And Regulated By Phosphatidic Acid, Journal of Biological Chemistry 274(29) (1999) 20313–20317. [DOI] [PubMed] [Google Scholar]
  • [39].Nelson RK, Ya-Ping J, Gadbery J, Abedeen D, Sampson N, Lin RZ, Frohman MA, Phospholipase D2 loss results in increased blood pressure via inhibition of the endothelial nitric oxide synthase pathway, Scientific Reports 7(1) (2017) 9112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [40].Tsukahara T, Tsukahara R, Haniu H, Matsuda Y, Murakami-Murofushi K, Cyclic phosphatidic acid inhibits the secretion of vascular endothelial growth factor from diabetic human coronary artery endothelial cells through peroxisome proliferator-activated receptor gamma, Molecular and Cellular Endocrinology 412 (2015) 320–329. [DOI] [PubMed] [Google Scholar]
  • [41].Wang Z, Cai M, Tay LWR, Zhang F, Wu P, Huynh A, Cao X, Paolo GD, Peng J, Milewicz DM, Du G, Phosphatidic acid generated by PLD2 promotes the plasma membrane recruitment of IQGAP1 and neointima formation, The FASEB Journal 33(6) (2019) 6713–6725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [42].Kooijman EE, Burger KN, Biophysics and function of phosphatidic acid: a molecular perspective, Biochim Biophys Acta 1791(9) (2009) 881–8. [DOI] [PubMed] [Google Scholar]
  • [43].Matsubara M, Titani K, Taniguchi H, Interaction of calmodulin-binding domain peptides of nitric oxide synthase with membrane phospholipids: Regulation by protein phosphorylation and Ca2+-calmodulin, Biochemistry-Us 35(46) (1996) 14651–14658. [DOI] [PubMed] [Google Scholar]
  • [44].Li H, Xu W, Jiang L, Gu H, Li M, Zhang J, Guo W, Deng P, Long H, Bu Q, Tian J, Zhao Y, Cen X, Lipidomic signature of serum from the rats exposed to alcohol for one year, Toxicology Letters 294 (2018) 166–176. [DOI] [PubMed] [Google Scholar]
  • [45].Who, Toxicological Evaluation Of Some Food Colours, Enzymes, Flavour Enhancers, Thickening Agents, And Certain Food Additives, Ammonium Salts Of Phosphatidic Acid, International Programme On Chemical Safety - World Health Organization, 1974.

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