Prolactin alters blood pressure by modulating the activity of endothelial nitric oxide synthase Albert S. Changa, Ruriko Granta, Hirofumi Tomitaa, Hyung-Suk Kima, Oliver Smithiesa,1, and Masao Kakokia,1 aDepartment of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599 Contributed by Oliver Smithies, September 12, 2016 (sent for review March 31, 2016; reviewed by Jason D. Heaney and Akihiro Tojo) Increased levels of a cleaved form of prolactin (molecular weight to those in the plasma of WT female mice in the last third of a 16 kDa) have been associated with preeclampsia. To study the normal pregnancy, accompanied by a decrease in BP and an in- effects of prolactin on blood pressure (BP), we generated male mice crease in nitric oxide (NO) production, likewise similar to those with a single-copy transgene (Tg; inserted into the hypoxanthine- occurring in females during pregnancy. When the Tg mice are fed a guanine phosphoribosyltransferase locus) that enables inducible diet containing IC3, plasma levels become threefold basal, BP in- hepatic production of prolactin and its cleavage product. The Tg is creases, and cardiac insufficiency develops. In the absence of en- driven by the indole-3-carbinol (I3C)-inducible rat cytochrome P450 dothelial NO synthase (eNOS), these changes did not occur. 1A1 promoter. When the Tg mice were fed normal chow (NC), plasma prolactin concentrations were comparable to those in female Results WT mice in the last third of pregnancy, and BP was lower than in WT Generation of Tg Mice Producing Prolactin in the Liver. Gene tar- mice (∼95 mm Hg vs. ∼105 mm Hg). When the Tg mice were fed geting, as described previously (9), was used to insert a single-copy chow containing IC3, plasma prolactin concentrations increased three- Tg into the Hprt locus of the X chromosome. The Tg is comprised fold, BP increased to ∼130 mm Hg, and cardiac function became of a copy of mouse cDNA for prolactin (Prl cDNA; Fig. 1A) driven markedly impaired. IC3 chow did not affect the WT mice. Urinary ex- by the rat cytochrome P450 1A1 promoter (PCyp1a1) (10), with the cretion of nitrite/nitrate and the amount of Ser1177-phosphorylated 3′-UTR of the bovine growth hormone gene (bGH) in place of its endothelial nitric oxide (NO) synthase (eNOS) were significantly natural 3′-UTR.MaleTgmicefedNChavegreaterthanWTex- greater in the Tg mice fed NC than in WT mice, as they are during pression of prolactin mRNA in the liver and a higher concentration pregnancy. However, when I3C was fed, these indicators of NO pro- of prolactin in the plasma (16.1 ± 1.1 ng/mL vs. 5.1 ± 0.6 ng/mL), duction became significantly less in the Tg mice than in WT mice. The indicating that the inducible Tg is leaky (Fig. 1 B and D). This basal effects of increased plasma prolactin were abolished by a genetic plasma prolactin concentration is close to that we observe in female absence of eNOS. Thus, a threefold increase in plasma prolactin is WT mice in the last third of pregnancy (15.3 ± 0.6 ng/mL). When sufficient to increase BP significantly and to markedly impair cardiac the Tg mice were fed I3C, hepatic prolactin mRNA levels were function, with effects mediated by NO produced by eNOS. We sug- markedly increased, and the plasma prolactin concentration be- gest that pregnant women with abnormally high prolactin levels may came approximately three times basal (Fig. 1 B and D). The ex- need special attention. pression of prolactin in the pituitary gland was not significantly altered by the Tg or by the I3C diet (Fig. 1C). hypertension | lactation | protein kinase B/Akt Opposite Changes in BP in the Tg Mice with Modestly and Substantially rolactin, a 23-kDa polypeptide hormone, is a potent multi- Increased Plasma Prolactin Levels. We found that the Tg mice fed NC Pfunctional cytokine with a broad range of biological effects, had a significantly lower BP than WT mice fed NC [WT (n = 9), including water and salt balance, lactogenesis, cell proliferation 108 ± 1 mm Hg vs. Tg (n = 11), 97 ± 3mmHg;P < 0.05; Fig. 2A and differentiation, testicular Leydig cell function, T-cell im- and Fig. S1A], but the heart rate was not significantly different munity, pancreatic β-cell function, hematopoiesis, and adipo- genesis (1). Prolactin is physiologically secreted mainly from the Significance anterior lobe of pituitary gland. The secretion is negatively regu- lated by dopamine and positively regulated by prolactin-releasing Prolactin is a hormone secreted by the pituitary gland that peptide (2) synthesized in the hypothalamus. The levels of pro- controls changes in the breast to enable milk production after lactin in the serum, urine, and amniotic fluids are significantly the baby is born. In some mothers with pregnancy-related high higher in patients with preeclampsia than in subjects with normal – blood pressure (BP), the concentration of prolactin in the blood pregnancy (3 5), suggesting that prolactin is involved in the is higher than normal, but whether this causes the high BP or is pathogenesis of pregnancy-associated hypertension. However, a consequence of it is uncertain. To answer this question, we animal experiments have shown inconsistent effects of prolactin have generated experimental mice that produce prolactin in on blood pressure (BP). Thus, acute i.v. infusion of prolactin in- the liver when we feed them a substance, indole-3-carbinol creased BP in rabbits (6), but, when ovine prolactin was chronically (IC3), that is found in broccoli. When fed normal chow, the mice given i.p. via an osmotic minipump in rats, BP was decreased (7). It are well, but, when fed IC3, they develop high BP and heart has also been reported that chronic prolactin infusion caused an problems. This suggests that pregnant women with abnor- increase in urinary sodium, potassium, and water excretion, but no mally high prolactin levels may need special attention. significant changes in arterial pressure, in rats (8). To study the chronic effects of different plasma concentrations Author contributions: M.K. designed research; A.S.C., R.G., H.T., H.-S.K., and M.K. per- of prolactin on BP and general well-being, we have generated formed research; H.-S.K. and M.K. analyzed data; and O.S. and M.K. wrote the paper. male mice with a single-copy transgene [Tg; inserted into the Reviewers: J.D.H., Baylor College of Medicine; and A.T., University of Tokyo. hypoxanthine-guanine phosphoribosyltransferase (Hprt) locus] The authors declare no conflict of interest. that enables hepatic production of prolactin and its cleavage Freely available online through the PNAS open access option. product when the mice are fed indole-3-carbinol (I3C), a xeno- 1To whom correspondence may be addressed. Email: [email protected] or biotic agent naturally present at high levels in broccoli and [email protected]. similar vegetables. The Tg is leaky, and, when the mice are fed This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. normal chow (NC), it leads to basal prolactin levels comparable 1073/pnas.1615051113/-/DCSupplemental. 12538–12543 | PNAS | November 1, 2016 | vol. 113 | no. 44 www.pnas.org/cgi/doi/10.1073/pnas.1615051113 Downloaded by guest on September 28, 2021 A B P = NS P = 0.01 a. ΔHprt 3 4 5 6 7 8 9 6000 ** ( ) 3000 * Liver prolactin 0 mRNA (%WTNC) 8888 NC I3C NC I3C WT Tg C NS NS b. bGH Prl cDNA 3’UTR 200 PCyp1a1 PHprt 1 2 3 100 0 8 8 mRNA (%WTNC) mRNA Pituitary prolactin 8888 Homologous recombination NC I3C NC I3C WT Tg c. P = NS P = 0.01 Prl cDNA Hprt D 60 ** PCyp1a1 PHprt 1 2 3 4 5 6 7 8 9 30 * (ng/ml) 0 8888 Plasma prolactin NC I3C NC I3C 6 WT Tg Fig. 1. Generation of drug-inducible hypermorphic mice for prolactin. (A) The gene-targeting strategy (*P < 0.05 and **P < 0.01 vs. WT; NS, not significantly different). (a) The target locus, into which the exogenous Tg is introduced by homologous recombination, is the mutated Hprt gene (ΔHprt) in the E14TG2a cells. (b) The targeting vector contains the rat Cyp1a1 promoter, mouse prolactin (Prl) cDNA, and the 3′-UTR of bGH.(c) The resulting locus after homologous recombination. The prolactin expression now is controlled by the Cyp1a1 promoter, which can be induced by I3C. Coding sequences of the prolactin gene are shown as black columns. (B) The mRNA levels of prolactin in the liver of WT and the Tg mice with and without I3C at the age of 12 wk. Coding sequences of the prolactin gene are shown as black columns. (C) The mRNA levels of prolactin in the pituitary gland are not changes by the Tg or by the administration of I3C. (D) Plasma levels of prolactin of WT and the Tg mice with and without I3C. between Tg and WT [WT (n = 9), 635 ± 39 beats per minute (bpm) NC. The I3C diet increased total prolactin in the Tg mice but not vs. Tg (n = 11), 561 ± 17 bpm; Fig. 2B and Fig. S1A]. in the WT mice (Fig. 2G). A faint band corresponding to the When the Tg mice were fed chow containing 3.0% I3C (wt/wt), 16-kDa form of prolactin is visible in the plasma sample from the their BP was markedly higher than that in WT mice fed the I3C diet Tg mouse fed IC3, but whether this is because the cleaved form is [WT (n = 9), 106 ± 2 mm Hg vs. Tg (n = 11), 132 ± 3mmHg;P < now a greater proportion of the total or is just a reflection of the 0.01; Fig.