Prenatal Endotoxemia and Placental Drug Transport in The Mouse: Placental Size-Specific Effects

Lipopolysaccharide (LPS) in high doses inhibits placental multidrug resistance P-glycoprotein (P-gp - Abcb1a/b) and breast cancer resistance protein (BCRP - Abcg2). This potentially impairs fetal protection against harmful factors in the maternal circulation. However, it is unknown whether LPS exposure, at doses that mimic sub-lethal clinical infection, alters placental multidrug resistance. We hypothesized that sub-lethal (fetal) LPS exposure reduces placental P-gp activity. Acute LPS (n = 19;150 µg/kg; ip) or vehicle (n = 19) were given to C57BL/6 mice at E15.5 and E17.5. Placentas and fetal-units were collected 4 and 24 h following injection. Chronic LPS (n = 6; 5 µg/kg/day; ip) or vehicle (n = 5) were administered from E11.5–15.5 and tissues were collected 4 h after final treatment. P-gp activity was assessed by [³H]digoxin accumulation. Placental Abcb1a/b, Abcg2, interleukin-6 (Il-6), Tnf-α, Il-10 and toll-like receptor-4 (Tlr-4) mRNA were measured by qPCR. Maternal plasma IL-6 was determined. At E15.5, maternal IL-6 was elevated 4 h after single (p<0.001) and chronic (p<0.05) LPS, but levels had returned to baseline by 24 h. Placental Il-6 mRNA was also increased after acute and chronic LPS treatments (p<0.05), whereas Abcb1a/b and Abcg2 mRNA were unaffected. However, fetal [³H]digoxin accumulation was increased (p<0.05) 4 h after acute LPS, and maternal [³H]digoxin myocardial accumulation was increased (p<0.05) in mice exposed to chronic LPS treatments. There was a negative correlation between fetal [³H]digoxin accumulation and placental size (p<0.0001). Acute and chronic sub-lethal LPS exposure resulted in a robust inflammatory response in the maternal systemic circulation and placenta. Acute infection decreased placental P-gp activity in a time- and gestational age-dependent manner. Chronic LPS decreased P-gp activity in the maternal myocardium and there was a trend for fetuses with smaller placentas to accumulate more P-gp substrate than their larger counterparts. Collectively, we demonstrate that acute sub-lethal LPS exposure during pregnancy impairs fetal protection against potentially harmful xenobiotics in the maternal circulation.     

Estriol and estradiol interactions with the estrogen receptor in vivo and in vitro

The cytosolic estrogen receptor (calf uterus) bound to estradiol (E₂) at 0°C changes from a state with fast into a state with slow E₂ dissociation rates when placed at 28°C. This temperature accelerated transition in receptor affinity for its ligand takes place within 10 min at 28°C. Similarly, receptor bound to estriol (E₃) at 0°C changes, when heated, from a state with fast into a state with slow E₃ dissociation. The main difference between RE₂ and RE₃ was that E₃ dissociates from unheated 8S RE₃ and heat-transformed 5S RE₃ at a much faster rate than E₂ from RE₂;In the mature ovariectomized rat a slow dissociating 5S receptor estrogen complex is found in nuclei 1 h after injection of [³H]E₂ or [³H]E₃. In vitro dissociation of these 2 estrogens from this nuclear bound receptor formed in vivo takes place at rates similar to those from heat-transformed cytosolic RE₂ or RE₃ complexes.Addition of pyridoxal 5'-phosphate (PLP) to the slow-dissociating heat-transformed 5S estrogen receptor complexes causes rapid dissociation of E₂ or E₃; this effect is dose-dependent and is not due to disruption of 5S dimers, since after PLP addition RE₂; and RE₃ sediment unchanged as 5S dimers.The presence of a large excess of non-radioactive 4S RE₃ does not interfere with the temperature induced rapid transition of 4S R[³H]E₂ complexes from the state with fast into a state with slow E₂ dissociation kinetics.A model is presented to explain the temperature induced biphasic estrogen dissociation from the receptor. It is proposed that the low affinity 4S RE₂ monomer undergoes a temperature and estrogen dependent conformation change, such that the ligand is “locked” into the receptor's binding site. This conformational change results in the formation of a high affinity 4S monomer from which estrogen dissociates at a slower rate. This reaction is independent from subsequent 4S to 5S dimerization (transformation). The different rates of ligand dissociation from the low and high affinity 4S receptors reflect the different interactions (hydrophobic and hydrogen bonding) of E₂ and E₃ with the estrogen binding domain.     

Effects of fetectomy on plasma estrogens and progesterone in monkeys (Macaca mulatta)

Effects of fetectomy on peripheral plasma levels of estrogens and progesterone were studied at 10 weeks (3 monkeys) and 16 weeks gestation (4 monkeys). Fetectomy was followed by a decrease in maternal peripheral plasma estradiol 17-β (E₂) at a time when E₂ levels remained elevated in intact pregnant monkeys. Estrone (E₁) levels, initially low at fetectomy (10 weeks), were maintained at similar low levels in contrast to elevated concentrations observed in normal animals during the final 30 days of pregnancy. In the absence of the fetus, progesterone (P) levels were similar to those of the normal pregnancies. After removal or delivery of the placenta, P levels decreased rapidly. Maternal hypophysectomy in one animal (10 weeks) produced a transient decrease in E₂ followed by recovery to control levels by 16 weeks. Subsequent fetectomy (16 weeks) was followed by a precipitous decline in maternal E₂ levels. In conclusion, results indicate: a fetal origin, possibly from adrenal precursors, for the major contribution of E₂ during the last 3 months and E₁ during the last month of gestation; and placental production of most of the P found in peripheral plasma of pregnant rhesus monkeys.     

Uptake,transfer and metabolism of prostaglandin E2 in the isolated perfused human placental cotyledon

(³H) PGE₂ uptake and transfer in the isolated perfused human placental cotyledon was assessed by a ingle pass paired isotope dilution technique utilising (¹⁴C) sucrose as an extracellular marker. Metabolism of (³H) PGE₂ was measured by analysing maternal and fetal effluents from perfused human placental cotyledons after bolus injection of (³H) PGE₂ into ither the maternal or fetal sides. Maximal uptake of (³H) PGE₂ was greater on the maternal (81 +/- 8%) than the fetal sides (42 +/- 12%) and showed saturation with increasing concentrations of PGE₂ only on the fetal side with an apparent Km of 12 +/- 4.9 nmol/l and v_max of 1.5 +/- 0.2 pmol/min/g. Total recoveries of (³H) PGE₂ were 84.6 +/- 11.8 % and 32.6 +/- 6.3 % of the injected dose after injection on the fetal and maternal sides respectively.Transferof (³H) PGE₂ was the same in both directions being 6.4 +/- 1.2 % of the injected dose in the fetal-maternal direction and 5.8 +/- 2.7 % of the injected dose in the maternal-fetal direction. Metabolism was greater on the maternal side (35% of injected (³H) PGE₂) thanthe fetalside(18% of injected (³H) PGE₂) and was principally to the 13,14-dihydro-15-keto-PGE₂ metabolite. Metabolism of (³H) PGE₂ after passage across the placenta was the same in both directions and was of the order of approximately 60%.     

Placental Transfer of Lactate,Glucose and 2-deoxyglucose in Control and Diabetic Wistar Rats

Placental transfer of lactate, glucose and 2-deoxyglucose was examined employing the in situ perfused placenta. Control and streptozotocin induced diabetic Wistar rats were infused with [U¹⁴C]-glucose and [³H]-2-deoxyglucose (2DG). The fetal side of the placenta was perfuseci with a cell free medium and glucose uptake was calculated in the adjacent fetuses. Despite the 5-fold higher maternal plasma glucose concentration in the diabetic dams the calculated fetal glucose metabolic index was not significantly different between the 2 groups. Placental blood flow was reduced in the diabetic animals compared with controls but reduction of transfer of [U¹⁴C]-glucose and [³H]-2-deoxyglucose and endogenously derived [¹⁴C]-Lactate to the fetal compartment, could not be accounted for by reduced placental blood flow alone. There was no significant net production or uptake of lactate into the perfusion medium that had perfused the fetal side of the placenta in either group. The plasma lactate levels in the fetuses adjacent to the perfused placenta were found to be higher than in the maternal plasma and significantly higher in the fetuses of the diabetic group compared with control group. In this model the in situ perfused placenta does not secrete significant quantities of lactate into the fetal compartment in either the control or diabetic group.     

Placental Aromatase Is Deficient in Placental Ischemia and Preeclampsia

IntroductionPreeclampsia is a maternal hypertensive disorder with uncertain etiology and a leading cause of maternal and fetal mortality worldwide, causing nearly 40% of premature births delivered before 35 weeks of gestation. The first stage of preeclampsia is characterized by reduction of utero-placental blood flow which is reflected in high blood pressure and proteinuria during the second half of pregnancy. In human placenta androgens derived from the maternal and fetal adrenal glands are converted into estrogens by the enzymatic action of placental aromatase. This implies that alterations in placental steroidogenesis and, subsequently, in the functionality or bioavailability of placental aromatase may be mechanistically involved in the pathophysiology of PE.MethodsSerum samples were collected at 32–36 weeks of gestation and placenta biopsies were collected at time of delivery from PE patients (n = 16) and pregnant controls (n = 32). The effect of oxygen tension on placental cells was assessed by incubation JEG–3 cells under 1% and 8% O₂ for different time periods, Timed-mated, pregnant New Zealand white rabbits (n = 6) were used to establish an in vivo model of placental ischemia (achieved by ligature of uteroplacental vessels). Aromatase content and estrogens and androgens concentrations were measured.ResultsThe protein and mRNA content of placental aromatase significantly diminished in placentae obtained from preeclamptic patients compared to controls. Similarly, the circulating concentrations of 17-β-estradiol/testosterone and estrone/androstenedione were reduced in preeclamptic patients vs. controls. These data are consistent with a concomitant decrease in aromatase activity. Aromatase content was reduced in response to low oxygen tension in the choriocarcinoma JEG–3 cell line and in rabbit placentae in response to partial ligation of uterine spiral arteries, suggesting that reduced placental aromatase activity in preeclamptic patients may be associated with chronic placental ischemia and hypoxia later in gestation.ConclusionsPlacental aromatase expression and functionality are diminished in pregnancies complicated by preeclampsia in comparison with healthy pregnant controls.     

Synthesis and evaluation of (17α,20E)21-[125I]Iodo-11-substituted-19-norpregna-1,3,5(10),20-tetraene-3,17β-diols: the influence of 11-stereochemistry on tissue distribution of radioiodinated estrogens

The 21-tri-n-butylstannyl derivatives of (17α,20E)-11α and β-methoxy-19-norpregna-1,3,5(10),20-tetraene-3,17β-diol were synthesized and characterized. These compounds, as well as the 11-unsubstituted compound were converted via electrophilic ipso radioiododestannylation to the corresponding 21[¹²⁵I]iodo analogs at the no-carrier-added level in 73–90% isolated radiochemical yields. The radiochemical 4c [IVαME₂, (17,20E)-21[¹²⁵I]iodo-11α-methoxy-19-norpregna-1,3,5(10) ,20-tetraene-3,17β-diol] was evaluated in immature female rats and the results compared to those previously reported for 4a (IVE₂) and 4b (IVβ ME₂) to determine the influence of 11-substitution on the ability of the compounds to function as estrogen receptor-seeking agents in vivo. The results indicated that the uptake of 11α-methoxy derivative in the target organ was substantially lower, of shorter duration, with a much smaller specific receptor binding component than the other two radioligands. The distribution profile of the three 17α-iodovinyl estrogens paralleled that previously reported for the corresponding 17α-ethynyl estrogens and this study suggests that the in vivo pharmacological results reported for the 17α-ethynyl estrogens may be used to predict the in vivo behavior of the corresponding 17α-iodovinyl analogs.     

Transport of folates at maternal and fetal sides of the placenta: lack of inhibition by methotrexate

Folate (pteroylglutamate) and methotrexate rapid (seconds) uptake by the trophoblast was investigated from either the maternal or fetal circulations of the isolated dually-perfused guinea-pig placenta. Tissue uptake was measured by using a single-circulation paired-tracer (³H-test and ¹⁴C-extracellular marker) technique. [³H]Folate uptakes were 80 and 52% (mean) in perfusates without unlabelled folate, on maternal and fetal sides, respectively. There was negligible ³H-tracer backflux into the circulation up to 6 min probably due to metabolic sequestration. [³H]Methotrexate uptakes were about 85 and 22% on maternal and fetal sides, respectively; however these uptakes were followed by rapid and complete backflux of the label. Specific transplacental transfer of [³H]folate or [³H]methotrexate in either direction was not detectable within 5–6 min. At the brush-border side (maternal) uptake of [³H]folate was highly inhibited by 100 nM unlabelled folate or its reduced form, methyltetrahydrofolate (the main form in plasma); however, equimolar methotrexate (an antifolate chemotherapeutic agent) failed to produce any inhibition of folate uptake. Our findings demonstrate that on both sides of the placenta a high-affinity transport system exists for trophoblast uptake of folate compounds. For methotrexate, either a separate transport system may exist or methotrexate may have a very low affinity for the folate system. These results are distinct from the findings reported in mouse L1210 leukemia cells.     

[3H]A-69024: A non-benzazepine ligand for in vitro and in vivo studies of dopamine d1 receptors

[³H]A-69024 has been prepared as a radioligand for studying the dopamine D₁ receptor. [³H]A-69024 binds to rat striatal membranes with a K_D = 14.3 ± 3.2 nM (mean ± SEM; n = 3) and B_max = 63.5 ± 12.8 fmol/mg wet tissue (1.8 ± 0.3 pmol/mg protein). This ligand binds to only one site with a Hill coefficient close to unity. The in vivo biodistribution of [³H]A-69024 showed a high uptake in the striatum (5.9 %ID/g) at 5 min followed by clearance. As a measure of specificity, the striatum/cerebellar ratio reached a maximum of 6.7 at 30 min post-injection. Pre-treatment with the D₁ antagonist R(+)SCH 23390 (1 mg/kg) reduced this ratio to unity. The dopamine antagonist (+)butaclamol and unlabeled A-69024 inhibited striatal uptake by 70 and 51%, respectively. Spiperone (D₂/5-HT_2A) and ketanserin (5-HT_2A/5-HT_2C) at doses of 1 mg/kg had no inhibitory effect on [³H]A-69024 uptake in the striatum; however, increased uptake of [³H]A-69024 by > 30% in the whole brain was observed. The selectivity and affinity of [³H]A-69024 suggests that this non-benzazepine radioligand may be useful for in vitro and in vivo studies of the dopamine D₁ receptor.     

High-affinity binding sites for prostaglandin E on human lymphocytes.

Binding sites on human lymphocytes for prostaglandins were examined by incubating cells with [³H]prostaglandin (PG) A₁, E₁, E₂, F_1α, and F_2α. Specific reversible binding for [³H]PGE₁ and E₂ was found with a K_d of ~2 × 10^?9M and a B max of ~200 binding sites per cell, assuming uniform distribution. We detected no specific binding of [³H]PGA₁, F_1α, or F_2α to lymphocytes. Also, the addition of 10- to 1000-fold greater amounts of unlabeled PGA, F_1α, or F_2α did not inhibit the binding of [³H]PGE. The time course of [³H]PGE binding appeared to be bimodal with one component complete within 5 min at 37 °C and another component of binding increasing over a 40-min incubation. We feel that the rapid component of binding may represent cell surface receptors for PGE while the slower component may represent a specific uptake mechanism for PGE into the cell. Glass adherent cells had fewer binding sites than nonadherent cells. Preincubation of the cells overnight resulted in a loss of binding sites.     

A role for prostaglandins in the regulation of the placental blood flows

A model is proposed for the regulation of the placental blood flows to the near-term pregnancy. The model has three features. 1) The maternal uterine and fetal placental tissues can synthesize constrictor and dilator prostaglandins. 2) Prostaglandins can cross the placenta. 3) There must exist a prostaglandin which has a vasodilating action in one of the placental circulations and a vasoconstricting action in the other circulation.Evidence is provided to indicate that in the sheep, prostaglandin E₂ (PGE₂) can cross the placenta and has a vasodilating action in the uterine placental circulation and a vasoconstricting action in the umbilical placental circulation.The placenta and the lung are compared and PGE₂ is shown to have similar actions in each of these organs.     

Protein-bound oligosaccharides of Semliki Forest virus

Semliki Forest virus was grown in BHK cells and labeled in vivo with radio-active monosaccharides. promnase digenst of the virus chromatographer on Bio-Gel P 6 revealed glycopeptides of A-type and B-type. (For the nomenclature see Johnson J. and Clamp J.R. (1971) Biochem. J. 123, 739–745) The former was labeled with [³H]fucose, [³H]galactose, [³H]mannose and [¹⁴C]glucosamine, the latter only with [³H]mannose and [¹⁴C]glucosamine. The three envelope glycoproteins E₁, E₂ and E₃ were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to pronase digestion. The glycoproteins E₁ and E₃ revealed glycopeptides of A-type. E₂ revealed glycopeptides of B-type. E₂ yielded additionally a glycopeptide (M_r3100) which was heavily labeled from [³H]galactose, but only marginally from [¹⁴C]glucosamine, [³H]fucose and [³H]mannose. Wether this glycopeptide belongs to the A-type or not remains uncertain. The apparent molecular weights of the A-type units measured by gel filtration were 3400 in E₁ and 4000 in E₃; the B-type unit of E₂ had an apparent molecular weight of 2000. Combined with the findings of our earlier chemical analysis these data suggast that E₁ and E₃ contain on the average one A-type unit; E₂ probably contains one 3100 dalton unit plus one or two B-type units.     

A study on the stability of an estrogen-protein conjugate in vivo and under in vitro conditions

[³ H] Estradiol-17β-succinyl bovine serum albumin conjugate ([³H]-E₂-BSA) was synthesized with a specific activity of 1.92 × 10⁷ cts/min/mg. The conjugate was administered iv to ovariectomized rats and the quantity of free [³H] steroid in the uterus was determined. Radioactive material was detected in all of the subcellular fractions of the uterus and identified as estradlol-17β. Similar subcellular distribution of the radioactivity was observed when [³H]E₂-BSA was added $\text{in vitro}$ to uterine homogenate. Free estradlo1-17β was released when the conjugate was Incubated with rat uterine homogenate or with serum. The results of the present study suggest that E₂-BSA is hydrolyzed $\text{in vivo}$ and under $\text{in vitro}$ conditions. It is recommended that the stability of a hormone-protein conjugate be established before use.     

Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography

Background

Complex but common maternal diseases such as diabetes and obesity contribute to adverse fetal outcomes. Understanding of the mechanisms involved is hampered by difficulty in isolating individual elements of complex maternal states in vivo. We approached this problem in the context of maternal diabetes and sought an approach to expose the developing fetus in vivo to isolated hyperglycemia in the pregnant rat.

Methodology and Principal Findings

We hypothesized that glucose infused into the arterial supply of one uterine horn would more highly expose fetuses in the ipsilateral versus contralateral uterine horn. To test this, the glucose tracer [¹⁸F]fluorodeoxyglucose (FDG) was infused via the left uterine artery. Regional glucose uptake into maternal tissues and fetuses was quantified using positron emission tomography (PET). Upon infusion, FDG accumulation began in the left-sided placentae, subsequently spreading to the fetuses. Over two hours after completion of the infusion, FDG accumulation was significantly greater in left compared to right uterine horn fetuses, favoring the left by 1.9±0.1 and 2.8±0.3 fold under fasted and hyperinsulinemic conditions (p<10⁻¹¹ n = 32-35 and p<10⁻¹² n = 27–45) respectively. By contrast, centrally administered [³H]-2-deoxyglucose accumulated equally between the fetuses of the two uterine horns. Induction of significant hyperglycemia (10³ mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained.

Conclusions and Significance

This approach exposes selected fetuses to localized hyperglycemia in vivo, minimizing exposure of the mother and thus secondary effects. Additionally, a set of less exposed internal control fetuses are maintained for comparison, allowing direct study of the in vivo fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health.     

Gonadotropin and prostaglandins binding sites in nuclei of bovine corpora lutea

Highly purified nuclei isolated from bovine corpora lutea showed marked enrichment of NAD pyrophosphorylase, a marker for this organelle. Rough endoplasmic reticulum and lysosomal markers were undetectable, whereas plasma membrane and Golgi markers were detectable but not enriched in nuclei. These highly purified nuclei exhibited specific binding with ¹²⁵I-labeled human choriogonadotropin, [³H]prostaglandin E₁ and [³H]prostaglandin F_2α. However, these bindings were only 15.4% (human choriogonadotropin), 7.9% (prostaglandin E₁) and 8.9% (prostaglandin F_2α) of the plasma membrane binding observed under the same conditions. Washing of nuclei and plasma membranes twice with buffer containing 0.1% Triton X-100 resulted in gonadotropin and prostaglandin F_2α binding site and 5′-nucleotidase (EC 3.1.3.5) losses from nuclei that were different from those observed for plasma membranes. More importantly, the washed nuclei exhibited 44% (human choriogonadotropin), 21–26% (prostaglandins) of original specific binding despite virtual disappearance of 5′-nucleotidase activity. The nuclear membranes isolated from nuclei, specifically bound ¹²⁵I-labeled human choriogonadotropin and [³H]prostaglandin F_2α to the same extent or significantly more ([³H]prostaglandin E₁, P < 0.05) than nuclei themselves, despite the marked losses of chromatin. In summary, our data suggest that gonadotropin and prostaglandins bind to nuclei and that this binding was intrinsic and was primarily associated with the nuclear membrane.     

Quantification of in vivo binding of [3H]RX 821002 in rat brain: Evaluation as a radioligand for central α2-adrenoceptors

On the basis of its established in vitro characteristics, [³H]RX 821002 was evaluated in rats as an in vivo radioligand for central α₂-adrenoceptors. Estimates for in vivo binding potential, obtained by compartmental analyses of time-radioactivity data, ranged between 1.9 for hypothalamus and 0.2 for cerebellum, with a regional distribution in brain which was similar to that observed in vitro. Selectivity and specificity of the signal were checked by predosing with either the α₂-antagonists, idazoxan or yohimbine, the α₂-agonist, clonidine, or the α₁-antagonist, prazosin. Pretreatment of the rats with the selective neurotoxin, DSP-4, had no significant effect on [³H]RX 821002 binding, suggesting that the majority of labelled sites were situated post-junctionally. The studies indicate that [³H]RX 821002 can be used experimentally as an in vivo marker for central α₂-adrenoceptors. The size and rate of expression of the specific signal encourage the development and assessment of [¹¹C]RX 821002 for clinical PET studies.     

Effects of oestriol: Preliminary results on receptor kinetics in target tissues of postmenopausal women

This in vivo investigation was done to study the effects of intravaginal oestriol (E₃) administration on endometrial, myometrial and vaginal tissue of normal postmenopausal women.All women received intravaginal E₃-suppositoria (containing 0.5 mg E₃) once a day for 3 weeks prior to hysterectomy. The medication was continued until the day of operation. At the time of operation both uterine and vaginal tisue was obtained.The receptor content in the cytosol was measured by a multiple point-dextran-coated-charcoal assay using [³h]e₂ and [³H]ORG-2058 as ligands. The receptor content in the nucleus was measured by incubating purified whole nuclei in 10 nM [³h]e₂ for 18 h at 0°C. We have shown that under these conditions there is a total exchange of all occupied receptors.Preliminary data on 4 patients are available. Vaginal cytology clearly showed an increase of the maturation value. Oestrogen receptor concentrations in the cytosol of all three tissues studied were lower than those obtained in untreated women, suggesting nuclear transformation of the receptor as a consequence of treatment. The nuclear E₂ receptor levels cannot be compared with normal women yet.Progesterone receptors in endometrial and myometrial cytosol seemed to be higher than those in untreated women, indicating effects of the treatment. In the human, vaginal progesterone receptor cannot be used as a marker for oestrogenic stimulation because only exceptionally could their presence be detected in either treated or untreated women.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

Effect of CO2 on labelling of nerve and liver cells by nucleic acid precursors

In vivo experiments in mice demonstrated that 5% CO₂ content in the air inhaled did not change the labelling in autoradiograms from animals injected with [³H]uridine, [³H]orotic acid, [³H]hypoxanthine, [³H]lysine or [³H]cytidine. At 20% CO₂ content there was a significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine, but not following [³H]lysine; there was no labelling of nerve cells with [³H]orotic acid or [³H]hypoxanthine, but a control group was not included. The labelling of choroid plexus and hepatocytes was independent of the CO₂ concentration. A comparison of in vivo and in vitro experiments at 20% CO₂ content showed a similar significant decrease in labelling of brain cells with [³H]uridine and [³H]cytidine. It is concluded that a metabolic change is the most appropriate explanation of the CO₂ effect.