Mitochondrial functions under hypoxic conditions: The steady states of cytochrome c reduction and of energy metabolism

1. The effect of low oxygen concentration on the oxidation-reduction states of cytochrome c and of pyridine nucleotide, on Ca²⁺ uptake, on the energy-linked reduction of pyridine nucleotide by succinate, and on the rate of oxygen consumption have been examined under various metabolic conditions, using pigeon heart mitochondria.

2. The oxygen concentration required to provide half-maximal reduction of cytochrome c (p50_c) ranges from 0.27 to 0.03 μM (0.2-0.02 Torr) depending upon the metabolic activity. There is a linear increase of the p50_c value with increasing respiratory rate.

3. The fraction of the normoxic respiration that is observed at p50_c is 70–90% under State 4 conditions, but is 30% under State 3 conditions.

4. The oxygen requirement for half-maximal reduction of pyridine nucleotide (p50_PN) varies less than p50_c, being 0.08 μM in State 3 and 0.06 μM in the uncoupled state.

5. The ability of the mitochondria to exhibit an energy-linked reduction of pyridine nucleotide by succinate disappears at an oxygen concentration of 0.09 μM (0.06 Torr). Below this oxygen concentration, endogenous Ca²⁺ begins to be released from the mitochondria. Thus, the critical oxygen concentration for bioenergetic function of mitochondria corresponds approximately to 50% reduction of pyridine nucleotide (p50_PN).     

The effects of in vivo and in vitro non-enzymatic glycosylation and glycoxidation on physico-chemical properties of haemoglobin in control and diabetic patients

The erythrocyte deformability, which is related to erythrocyte internal viscosity, was suggested to depend upon the physico-chemical properties of haemoglobin. In the present study we employed ESR spectroscopy in order to explore further the extent to which the in vivo or in vitro glycation and/or glycoxidation might affect haemoglobin structure and conformation. We revealed that under both in vivo and in vitro conditions the attachment of glucose induced a mobilization of thiol groups in the selected domains of haemoglobin molecules (the increased h₊₁/h₀ parameter of maleimide spin label, MSL; 0.377 ± 0.021 in diabetics vs 0.338 ± 0.017 in controls, n = 12, P < 0.0001). The relative rotational correlation time (τ_c) of two spin labels, TEMPONE and TEMPAMINE, respectively, in erythrocyte insides (5.22 ± 0.42 in diabetics, n = 21 vs 4.79 ± 0.38, n = 16 in controls, P < 0.005) and in the solutions of in vitro glycated haemoglobin, were increased. Neither oxidation nor crosslinking of thiol groups was evidenced in glycated and/or oxidized haemoglobin. In addition, erythrocyte deformability was found to be reduced in type 2 diabetic patients (6.71 ± 1.08, n = 28 vs 7.31 ± 0.96, n = 21, P < 0.015). In conclusion, these observations suggest that: the attachment of glucose to haemoglobin might have decreased the mobility of the Lys-adjacent Cys residues, thus leading to the increased h₊₁/h₀ parameter of MSL. Such structural changes in haemoglobin owing to non-enzymatic glycosylation may contribute to the increased viscosity of haemoglobin solutions (r = 0.497, P < 0.0035) and the enhanced internal viscosity of diabetic erythrocytes (r = 0.503, P < 0.003). We argue that such changes in haemoglobin, and consequently in red blood cells, might contribute to the handicapped oxygen release under tissue hypoxia in the diabetic state.     

Cortisol metabolism in vitro—III. Inhibition of microsomal 6β—hydroxylase and cytosolic 4-ene-reductase

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3,5β-tetrahydrocortisol; 3,5β-THF), while microsomal incubations generate upto 7 metabolites, including 6β-hydroxycortisol (6β-OHF), and 6β-hydroxycortisone (6β-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6β-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [³H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The K_m value for 6β-OHF and 6β-OHE formation was 15.2 ± 2.1 μM (mean ± SD; n = 4) and the V_max value 6.43 ± 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6β-hydroxylase was ketoconazole (K_i = 0.9 ± 0.4 μM; N = 4), followed by gestodene (K_i = 5.6 ± 0.6 μM) and cyclosporine (K_i = 6.8 ± 1.4 μM). Both betamethasone and dexamethasone produced some inhibition (K_i = 31.3 and 54.5 μ, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The K_m value for cortisol 4-ene-reductase was 26.5 ± 11.2 μM (n = 4) and the V_max value 107.7 ± 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (K_i = 17.8 ± 3.3 μM) and gestodene (K_i = 23.8 ± 3.8 μM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.     

The role of cytokines in the regulation of Leydig cell P450c17 gene expression

Cytokines produced by immune-activated testicular interstitial macrophages (TIMs) may play a fundamental role in the local control mechanisms of testosterone biosynthesis in Leydig cells. We investigated whether in vivo immune-activation of TIMs can modulate Leydig cell steroidogenesis. To immune activate TIMs in vivo, mice were injected intraperitoneally (i.p.) with lipopolysaccharide (LPS, 6 mg/kg). TIMs and Leydig cells were purified for RNA analysis. LPS treatment resulted in a 47-fold increase in interleukin-1β (IL-1β) mRNA in TIMs. P450c17 mRNA levels in the Leydig cells from the same animals, decreased to less than 10% compared to control. The effect of LPS on IL-1β and P450c17 mRNA levels was reversible on both TIMs and Leydig cells, respectively. To determine if the effect of LPS on P450c17 was mediated by a possible decrease in pituitary LH secretion, mice were co-injected with LPS and hCG. Treatment with hCG did not change the effect observed with LPS alone, in TIMs or in Leydig cells. In vitro, LPS treatment of TIMs resulted in marked induction of IL-1β mRNA expression. In parallel, in vitro treatment of Leydig cells with recombinant IL-1 resulted in a dose-dependent inhibition of P450c17 mRNA expression and testosterone production. These data demonstrate that LPS treatment, in vivo and in vitro, induced IL-1 gene expression in TIMs, and that IL-1 inhibits P450c17 mRNA in vitro. Therefore, we suggest that immune-activation of TIMs might have caused the observed inhibition of P450c17 gene expression in Leydig cells in vivo.     

Uterine and serum cytokine arrays in the mouse during estrus

Cytokines govern uterine immunology and embryo receptivity and are increasingly recognized for their embryotrophic roles. While supplementing culture media with cytokines may improve embryo development/viability in vitro, little is known about their physiological profiles in vivo, and hence which are likely to be uterine immunoregulators and embryotrophins. Therefore, this study profiled 23 cytokines in uterine fluid and serum from individual naturally cycling estrous mice. Samples were analyzed by fluid-phase multiplex immunoassays for interleukin (IL)-1, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-17, eotaxin, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon (IFN)-γ, keratinocyte-derived chemokine (KC), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 MIP)-1β regulated upon activation, normal T-cell expressed and secreted (RANTES) and tumor necrosis factor (TNF)-. There was a marked divergence in cytokine concentrations between uterine fluid and serum. The former was dominated by G-CSF, eotaxin, KC and IL-1, and had significantly higher levels of IL-1β, IL-2, IL-3, IL-4, IL-6, IL-9, GM-CSF, MIP-1, MIP-1β and RANTES. Serum had significantly higher IL-12 (p40), IL-12 (p70), IL-17 and IFN-γ concentrations. No significant differences in IL-5, IL-10, IL-13, MCP-1 or TNF- profiles were noted. These data indicated a strict compartmentalization of uterine cytokines, with G-CSF as a major cytokine at estrous. Results are discussed with respect to immune cell function, post-coital paternal antigen processing, estrous cyclicity, and endometrial angiogenesis, cell turnover and differentiation.     

Viscosity of locust bean, guar and xanthan gum solutions in the Newtonian domain: a critical examination of the log (ηsp)o-log c[η]o master curves

The viscosity in the low shear rate Newtonian domain of three biopolymers, locust bean gum, guar gum and xanthan gum was studied as a function of temperature and of polymer concentration in various aqueous solvents. The intrinsic viscosities [η]_o of both galactomannans are not modified in the presence of 10 or 40% sucrose. In this case, a master curve relating the Newtonian specific viscosity (η_sp)_o, to the reduced concentration c[η]_o is obtained and allows (in good agreement with theoretical conjectures), two critical concentrations C* and C** to be defined, from which the value of the expansion coefficient may be estimated. For xanthan, as expected for a polyelectrolyte, [η]_o depends strongly on salt concentration and on added sucrose and the results did not obey the above-mentioned master curve. However, it is shown that (η_sp)_o depends only on xanthan concentration whenC > C**, and then it is assumed that chain dimensions have attained their unperturbed values whatever the solvent. Considering that both types of chains, random coils (galactomannans) and semi-rigid (xanthan) should give the same (η_sp)_o-C[η]_o master curve for C > C** when [η]_o is replaced by its unperturbed counterpart [η]_θ, a method for estimating [η]_θ for the xanthan sample is proposed. In conclusion, the numerous exceptions to the widely accepted (η_sp)_o vs C[η]_o “universal” behaviour are mainly ascribed to significant differences in expansion coefficient values which depend on both the polymer and the solvent.     

Action of endogenous steroid inhibitors of brain aromatase relative to fadrozole

To study mechanisms of aromatase inhibition in brain cells, a highly effective non-steroidal aromatase inhibitor (Fadrozole; 4-[5,6,7,8-tetra-hydroimidazo-(1,5-a)-pyridin-5-yl] benzonitrile HCl; CGS 16949A) was compared with endogenous C-19 steroids, known to be formed in the preoptic area, which inhibit oestrogen formation. Using a sensitive in vitro tritiated water assay for aromatase activity in avian (dove) preoptic tissue, the order of potency, with testosterone as substrate was: Fadrozole (K_i < 1 × 10⁻⁹ M) > 4-androstenedione 5-androstanedione > 5-dihydrotestosterone (K_i = 6 × 10⁻⁸ M) > 5β-androstanedione > 5β-dihydrotestosterone (K_i = 3.5 × 10⁻⁷ M) > 5-androstane-3, 17β-diol (K_i = 5 × 10⁻⁶ M) > 5β-androstane-3β,17β-diol. Five other steroids, 5β-androstane-3,17β-diol, 5-androstane-3β,17β-diol, progesterone, oestradiol and oestrone, showed no inhibition at 10⁻⁴ M. The kinetics indicate that endogenous C-19 steroids show similar competitive inhibition of the aromatase as Fadrozole. Mouse (BALB/c) preoptic aromatase was also inhibited by Fadrozole. We conclude that endogenous C-19 metabolites of testosterone are effective inhibitors of the brain aromatase, and suggest that they bind competitively at the same active site as Fadrozole.     

Beta2-adrenoceptor agonists induce the mammalian clock gene, hPer1, mRNA in cultured human bronchial epithelium cells in vitro

The mammalian Per1 gene is one of the most important components of circadian clock function of the suprachiasmatic nucleus and peripheral tissues. We examined whether the β₂-adrenoceptor agonists, procaterol and fenoterol, induce human Per1 mRNA expression in human bronchial epithelium. The in vitro stimulation of β₂-adrenoceptor agonists in BEAS-2B cells led to a remarkable increase in the level of hPer1 mRNA. Moreover, fenoterol or procaterol induced the phosphorylation of CREB in BEAS-2B cells as verified by immunoblot analysis. β₂-adrenoceptor agonists induced human Per1 mRNA expression by the signaling pathways of cAMP-CREB in BEAS-2B cells.     

Aldosterone synthase activity in the Y-1 adrenal cell line

The Y-1 adrenal cell line was shown to produce 20-dihydroaldosterone from deoxycorticosterone. This compound was identified by GC-MS by comparison with the previously synthesized reference compound. Two other 18-hydroxylated metabolites were identified as 11β,18-dihydroxy-20-dihydroprogesterone from endogenous cholesterol and 18-hydroxy-20-dihydro-11-dehydrocorti-costerone from DOC. The conditions necessary for the synthesis of these compounds are culturing in 20% serum-supplemented medium and repeated incubations with the substrate. The production of 11β-hydroxylated steroids and that of 18-oxygenated steroids is stimulated differently by ACTH and angiotensin II suggesting the expression of two different enzymes, cytochrome P-450_11β and cytochrome P-450_aldo The Y-1 cell line can secrete either 11β-hydroxylated steroids characteristic of the glucocorticoid pathway or 18-oxygenated steroids characteristic of the mineralocorticoid pathway, which in vivo are generally produced in two different zones of the adrenal cortex. This cell line should be an interesting model for the study of the molecular mechanisms regulating the expression of these two enzymes involved in the final steps of the steroidogenic pathways.     

The IL-2A receptor pathway and its role in lymphocyte differentiation and function

Murine myeloid cells are developed from hemopoietic stem/progenitor cells. Different types of progenitor cells have variable differentiation potentials. Among the ten main types of cells differentiated from lymphoid progenitor cells, regulatory T cells (Tregs), an important cell subpopulation regulating immune and inflammatory responses, arise from the hematopoietic stem cells in the bone marrow. Tregs then differentiate into T lymphocytes and migrate to the thymus and finally generate Treg subsets, which are subsequently activated and regulated by inflammatory cytokines in the peripheral blood. Tregs also have different phenotypes and immunomodulatory functions. The cytokine interleukin-2/interleukin-2 receptor (IL-2/IL-2R) pathway is an important regulatory signaling pathway of Tregs. Besides, different types of CD4⁺ and CD8⁺ cells have different immune effects in the absence of IL-2. IL-2R consists of three subunits, α chain (CD25), β chain (CD122), and γ chain (CD132). Different subunit combinations have different effects on the activation of immune cells. Multiple studies have shown that IL2RA deficiency has various effects on the immune function in mice. This article reviews the subunit composition and signaling pathway of IL-2R, the classification of Tregs in a murine myeloid cell line and the regulatory effect of IL-2/IL-2R on them, the regulatory impact and signaling mechanism of IL-2/IL-2R on CD4⁺/CD8⁺ lymphocyte differentiation, the primary manifestations and molecular mechanism of immune dysfunction in IL-2- and IL-2R-deficient mice, soluble IL-2Rα as a biomarker for diagnosis, prognosis and therapeutic efficacy of treatment in immune system disorders, and the development and clinical application of IL-2 mutants.     

靶向人白介素-6蛋白的治疗性单克隆抗体研究进展*

白介素-6(interleukin-6,IL-6)作为一种多效的细胞因子,参与机体内众多生理与病理过程。研究表明,IL-6首先与自身受体(IL-6R、gp130)形成异源六聚体复合物,进而激活下游信号转导通路,最终发挥生物学功能。 IL-6信号通路异常活化及功能失调与多种疾病密切相关,如自身免疫疾病、慢性炎症、恶性肿瘤等。另外IL-6的异常表达在新型冠状病毒肺炎(COVID-19)细胞因子风暴综合征(CSS)中也扮演重要角色。一般而言,阻断IL-6信号通路上的各关键节点,均可用于IL-6相关疾病的治疗。有别于阻断IL-6R或gp130等公共受体分子,阻断IL-6蛋白的治疗性单克隆抗体特异性更高,在临床研究中,部分品种显示出其独有的治疗特点及有益的疗效。现阶段只有1个靶向IL-6蛋白的单克隆抗体药物获美国FDA批准上市,以及超过8个治疗性单克隆抗体在临床研究阶段。重点对国内外靶向人IL-6蛋白的治疗性单克隆抗体及其临床应用进行综述。     

In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI

The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1β induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h prior to fertilization. Two experiments were performed, each using three maturation media as the experimental treatments. Medium 1 was pure follicular fluid from subordinate follicles. Medium 2 was medium 1 plus 50 ng/ml recombinant human IL-1β. Medium 3 was pure follicular fluid collected from mares administered crude equine gonadotropin (CEG). Medium 4 was medium 2 plus 50 ng/ml of recombinant human IL-1 receptor antagonist. Media 1, 2 and 3 were compared in experiment 1. In experiment 2, media 1, 2 and 4 were compared. After maturation, metaphase II oocytes were submitted to microinjection and assessed for signs of fertilization. In experiment 1, 101 oocytes were evaluated. The rate of polar body extrusion was 66, 51 and 68% and the proportions of normally fertilized oocytes after ICSI were 40, 18 and 38% for media 1, 2 and 3, respectively. In experiment 2, 122 oocytes were evaluated. The rate of polar body extrusion was 55, 48 and 42% and the proportions showing normal fertilization after ICSI were 14, 25 and 29% for media 1, 2 and 4, respectively. There was no positive effect of IL-1β on maturation in both experiments, but the fertilization rate and percentage of embryos reaching four-cell were low in the presence of IL-1β, indicating that this cytokine may interfere with fertilization and early embryo development.     

Interleukin-1

Interleukin-I (IL-1) is the prototypic pro-inflammatory cytokine. There are two forms of IL-1, IL-1 and IL-Iβ and in most studies, their biological activities are indistinguishable. IL-1 affects nearly every cell type, often in concert with another pro-inflammatory cytokine, tumor necrosis factor (TNF). Although IL-1 can upregulate host defenses and function as an immunoadjuvant, IL-1 is a highly inflammatory cytokine. The margin between clinical benefit and unacceptable toxicity in humans is exceedingly narrow. In contrast, agents that reduce the production and/or activity of IL-1 are likely to have an impact on clinical medicine. The synthesis, processing, secretion and activity of IL-1, particularly IL-Iβ, are tightly regulated events. A unique aspect of cytokine biology is the naturally occurring IL-1 receptor antagonist (IL-1Ra). IL-IRa is structurally similar to IL-Iβ but lacking agonist activity is used in clinical trials to reduce disease severity. In addition, regulation of IL-1 activity extends to low numbers of surface receptors, circulating soluble receptors and a cell surface “decoy” receptor to down-regulate responses to IL-Iβ. This review updates the current knowledge on IL-1.     

体外模拟鳞杯伞子实体碱溶性多糖的消化与酵解特性

以热水浸提后的鳞杯伞Clitocybe squamulosa子实体残渣为原料,进行二次利用,浸提碱溶性多糖.通过模拟体外消化与厌氧发酵实验,探究鳞杯伞子实体碱溶性多糖的消化特性以及对肠道内短链脂肪酸含量的影响.结果 表明:体外模拟唾液和胃肠液消化后,多糖的官能团结构特征没有发生显著性改变,但碱溶性多糖的块状结构解体,碎...     

Relative stability of chlorophyll complexes in vivo

The time course of aerobic photobleaching of various chlorophyll-protein complexes in vivo at high light intensities was studied with isolated Aspidistra elatior chloroplasts.

1. 1. C_a680 bleaching starts with the onset of irradiation and, initially, proceeds linearly with time. Washing the chloroplasts causes a nearly constant increase of the bleaching rate throughout the experiment.

2. 2. C_a670 does not appreciably, if at all, bleach initially; subsequently, bleaching proceeds linearly with time and at a slightly higher rate than that for C_a680. Washing makes C_a670 bleach concomitantly with the onset of illumination, and at a nearly constant rate.

3. 3. Bleaching at 665 nm is likely to start only after a relatively long period of illumination. Washing shows no effects during this period. Once bleaching has started, washing causes its rate to increase.

4. 4. No indication of the occurrence of “short-wave” chlorophyll a forms other than C_a670 and C_a665 was obtained.

5. 5. C_b bleaching starts concomitantly with illumination at a low rate. The rate increases more or less exponentially with time. Washing enhances bleaching in two steps.

6. 6. The importance of the results is discussed.

Hetero-polynuclear complexes containing bridging diphenylphosphino-alkenes and -alkynes. The crystal structure of an Rh2{μ−ν:ν−P(Ph2) (CH2)3C≡CR}Co2 complex

The phosphinoalkenes Ph₂P(CH₂)_nCH=CH₂ (n= 1, 2, 3) and phosphinoalkynes Ph₂P(CH₂)_n C≡CR (R = H, N = 2, 3; R = CH₃, N = 1) have been prepared and reacted with the dirhodium complex (η−C₅H₅)₂Rh₂(μ−CO) (μ−η²−CF₃C₂CF₃). Six new complexes of the type (ν−C₅H₅)₂(Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃)L, where L is a P-coordinated phosphinoalkene, or phosphinoalkyne have been isolated and fully characterized; the carbonyl and phosphine ligands are predominantly trans on the Rh---Rh bond, but there is spectroscopic evidence that a small amount of the cis-isomer is formed also. Treatment of the dirhodium-phosphinoalkene complexes with (η−CH₃C₅H₄)Mn(CO)₂thf resulted in coordination of the manganese to the alkene function. The Rh₂---Mn complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂P(CH₂)₃CH=CH₂} (η−CH₃C₅H₄)Mn(CO)₂] was fully characterized. Simi treatment of the dirhodium-phosphinoalkyne complexes with Co₂(CO)₈ resulted in the coordination of Co₂(CO)₆ to the alkyne function. The Rh₂---Co₂ complex [(η−C₅H₅)₂Rh₂(CO) (μ−η¹:η¹−CF₃C₂CF₃) {Ph₂PCH₂C≡CCH₃}Co₂(CO)₂], C₃₇H₂₅Co₂F₆O₇PRh₂, was fully characteriz spectroscopically, and the molecular structure of this complex was determined by a single crystal X-ray diffraction study. It is triclinic, space group (C_i¹, No. 2) with a = 18.454(6), B = 11.418(3), C = 10.124(3) Å, = 112.16(2), β = 102.34(3), γ = 91.62(3)°, Z = 2. Conventional R on |F| was 0.052 fo observed (I > 3σ(I)) reflections. The Rh₂ and Co₂ parts of the molecule are distinct, the carbonyl and phosphine are mutually trans on the Rh---Rh bond, and the orientations of the alkynes are parallel for Rh₂ and perpendicular for Co₂. Attempts to induce Rh₂Co₂ cluster formation were unsuccessful.     

Estrogen-like effect of a Cimicifuga racemosa extract sub-fraction as assessed by in vivo, ex vivo and in vitro assays

Black cohosh (Cimicifuga racemosa) is used in the treatment of painful menstruation and menopausal symptoms. Data about the nature of the active compounds and mechanism(s) of action are still controversial, chiefly with respect to its estrogenic activity.

This work aimed to assess the possible estrogenic activity of a commercial dry hydro-alcoholic extract of C. racemosa and its hydrophilic and lipophilic sub-fractions on in vivo, ex vivo, and in vitro assays.

In a yeast estrogen screen, only the lipophilic sub-fraction was able to activate the human estrogen receptor , with a lower potency but comparable efficacy to that of 17 β-estradiol.

Neither the total extract nor the lipophilic sub-fraction showed an in vivo uterotrophic effect in 21-day-old rats. Uterine tissues obtained ex vivo from C. racemosa treated animals were generally much less sensitive to oxytocin, prostaglandin F_2, and bradykinin than tissues obtained from estradiol valerate treated rats.

The lipophilic sub-fraction, instead, induced a dose-dependent inhibitory activity on the in vitro response to oxytocin, prostaglandin F_2, and bradykinin of uterine horns from naïve 28-day-old rats, with a potency rate close to 1:30 of that of 17 β-estradiol.

Reported results confirm the effectiveness of C. racemosa in menstrual distress and further emphasize the possibility that lipophilic constituents bind to an as yet not identified estrogen receptor, likely inversely involved in inflammation.     

Competitive product inhibition of aromatase by natural estrogens

In order to better understand the function of aromatase, we carried out kinetic analyses to asses the ability of natural estrogens, estrone (E₁), estradiol (E₂), 16-OHE₁, and estriol (E₃), to inhibit aromatization. Human placental microsomes (50 μg protein) were incubated for 5 min at 37°C with [1β-³H]testosterone (1.24 × 10³ dpm ³H/ng, 35–150 nM) or [1β-³H,4-¹⁴C]androstenedione (3.05 × 10³ dpm ³H/ng, ³H/¹⁴C = 19.3, 7–65 nM) as substrate in the presence of NADPH, with and without natural estrogens as putative inhibitors. Aromatase activity was assessed by tritium released to water from the 1β-position of the substrates. Natural estrogens showed competitive product inhibition against androgen aromatization. The K_i of E₁, E₂, 16-OHE₁, and E₃ for testosterone aromatization was 1.5, 2.2, 95, and 162 μM, respectively, where the K_m of aromatase was 61.8 ± 2.0 nM (n = 5) for testosterone. The K_i of E₁, E₂, 16-OHE₁, and E₃ for androstenedione aromatization was 10.6, 5.5, 252, and 1182 μM, respectively, where the K_m of aromatase was 35.4 ± 4.1 nM (n = 4) for androstenedione. These results show that estrogens inhibit the process of andrigen aromatization and indicate that natural estrogens regulate their own synthesis by the product inhibition mechanism in vivo. Since natural estrogens bind to the active site of human placental aromatase P-450 complex as competitive inhibitors, natural estrogens might be further metabolized by aromatase. This suggests that human placental estrogen 2-hydroxylase activity is catalyzed by the active site of aromatase cytochrome P-450 and also agrees with the fact that the level of catecholestrogens in maternal plasma increases during pregnancy. The relative affinities and concentration of androgens and estrogens would control estrogen and catecholestrogen biosynthesis by aromatase.