Distribution and hormonal regulation of membrane progesterone receptors β and γ in ciliated epithelial cells of mouse and human fallopian tubes

Background  

The controlled beating of cilia of the fallopian tube plays an important role in facilitating the meeting of gametes and subsequently transporting the fertilized egg to its implantation site. Rapid effects of progesterone on ciliary beat frequency have been reported in the fallopian tubes of cows, but the identity of the receptors mediating this non-genomic action of progesterone is not known. We recently identified a member of the non-genomic membrane progesterone receptor family, mPR gamma, as a candidate for mediating these actions of progesterone. Here, we investigated the possible presence of a related receptor, mPR beta, in the fallopian tubes of mice and women as well as the possible hormonal regulation of mPR beta and gamma.     

Differential effects of UTP, ATP, and adenosine on ciliary activity of human nasal epithelial cells

The purinergic regulation of ciliary activity was studied using small, continuously superfused explants of human nasal epithelium. The P2Y(2) purinoceptor (P2Y(2)-R) was identified as the major purinoceptor regulating ciliary beat frequency (CBF); UTP (EC(50) = 4.7 microM), ATP, and adenosine-5'-O-(3-thiotriphosphate) elicited similar maximal responses, approximately twofold over baseline. ATP, however, elicited a post-peak sustained plateau in CBF (1.83 +/- 0.1-fold), whereas the post-peak CBF response to UTP declined over 15 min to a low-level plateau (1.36 +/- 0.16-fold). UDP also stimulated ciliary beating, probably via P2Y(6)-R, with a maximal effect approximately one-half that elicited by P2Y(2)-R stimulation. Not indicated were P2Y(1)-R-, P2Y(4)-R-, or P2Y(11)-R-mediated effects. A(2B)-receptor agonists elicited sustained responses in CBF approximately equal to those from UTP/ATP [5'-(N-ethylcarboxamido)adenosine, EC(50) = 0.09 microM; adenosine, EC(50) = 0.7 microM]. Surprisingly, ADP elicited a sustained stimulation in CBF. The ADP effect and the post-peak sustained portion of the ATP response in CBF were inhibited by the A(2)-R antagonist 8-(p-sulfophenyl)theophylline. Hence, ATP affects ciliary activity through P2Y(2)-R and, after an apparent ectohydrolysis to adenosine, through A(2B)AR.     

Levonorgestrel antagonism on estrogen-induced pituitary tumors is mediated by progesterone receptors

Using both IN VITRO and IN VIVO approaches, we studied the antagonism exerted by the synthetic progestin levonorgestrel on estrogen-induced prolactinomas, considering that levonorgestrel shows partial androgenic properties and that androgens inhibit estrogen-induced prolactin synthesis and secretion. In the tumors, binding of estrogens to their receptors was competed neither by progesterone receptor ligands nor by androgen receptor ligands, ruling out direct inhibitory effects of these drugs on tumor development. Progestin binding was competed by the progesterone receptor agonists progesterone and levonorgestrel, by the antagonist mifepristone, and also by the androgen dihydrotestosterone, whereas the androgen receptor antagonist hydroxyflutamide was a weak competitor. In addition, both progesterone receptor and androgen receptor ligands competed for binding to androgen receptors. In primary cultures of pituitary tumors, levonorgestrel decreased prolactin secretion, an effect that was blocked by mifepristone but not by hydroxyflutamide. IN VIVO results indicated that levonorgestrel inhibition of both estrogen-induced pituitary weight increment and hyperprolactinemia was reduced by mifepristone, whereas flutamide was unable to block levonorgestrel effects. Our results suggest that even when an interaction of levonorgestrel with androgen receptors in the tumors is possible, the antagonistic effects of levonorgestrel on tumor development and functionality are mediated by progesterone receptors.     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

TRPV4 channel is involved in the coupling of fluid viscosity changes to epithelial ciliary activity

Autoregulation of the ciliary beat frequency (CBF) has been proposed as the mechanism used by epithelial ciliated cells to maintain the CBF and prevent the collapse of mucociliary transport under conditions of varying mucus viscosity. Despite the relevance of this regulatory response to the pathophysiology of airways and reproductive tract, the underlying cellular and molecular aspects remain unknown. Hamster oviductal ciliated cells express the transient receptor potential vanilloid 4 (TRPV4) channel, which is activated by increased viscous load involving a phospholipase A(2)-dependent pathway. TRPV4-transfected HeLa cells also increased their cationic currents in response to high viscous load. This mechanical activation is prevented in native ciliated cells loaded with a TRPV4 antibody. Application of the TRPV4 synthetic ligand 4alpha-phorbol 12,13-didecanoate increased cationic currents, intracellular Ca(2+), and the CBF in the absence of a viscous load. Therefore, TRPV4 emerges as a candidate to participate in the coupling of fluid viscosity changes to the generation of the Ca(2+) signal required for the autoregulation of CBF.     

Rapid inhibition of Ca2+ influx by neurosteroids in murine embryonic sensory neurones

The non-genomic role of neuroactive steroids on [Ca2+]i transients induced by GABA receptor activation was investigated in cultured dorsal root ganglia (DRG) neurones at embryonic stage E13. [Ca2+]i measurements were performed with Fura-2 fast fluorescence microfluorimetry. Application of the GABAA receptor agonist muscimol (Musci) evoked an increase in [Ca2+]i, confirming the excitatory effect of GABA at this embryonic stage. The muscimol-induced [Ca2+]i response was inhibited by progesterone (Proges) and its primary metabolite allopregnanolone (Allo) in a rapid, reversible and dose-dependent manner. These calcium transients were suppressed in the absence of external Ca2+ or in the presence of Ni2+ + Cd2+ suggesting an involvement of voltage-activated Ca2+ channels. In contrast, none of these steroids affected the resting [Ca2+]i nor exhibited any inhibitory effect on 50 mM KCl-induced [Ca2+]i increases. In view of the well-established potentiation of GABAA receptor by direct binding of neurosteroids, the inhibitory effects described in this study seem to involve distinct mechanisms. This new inhibitory effect of progesterone is observed at low and physiological concentrations, is rapid and independent of RU38486, an antagonist of the classic progesterone receptor, probably involving a membrane receptor. Using RT-PCR, we demonstrated the expression of progesterone receptor membrane component 1 (Pgrmc1), encoding 25-Dx, a membrane-associated progesterone binding protein in DRG neurones at different stages of development. In conclusion, we describe for the first time a rapid effect of progestins on embryonic DRG neurones involving an antagonistic effect of progesterone and allopregnanolone on GABAA receptors.     

Rapid effects of progesterone on ciliary beat frequency in the mouse fallopian tube

Background  

The physiological regulation of ciliary beat frequency (CBF) within the fallopian tube is important for controlling the transport of gametes and the fertilized ovum. Progesterone influences gamete transport in the fallopian tube of several mammalian species. In fallopian tubes isolated from cows, treatment with 20 micromolar progesterone caused a rapid reduction of the tubal CBF. The aims of this study were to establish methodology for studying fallopian tube CBF in the mouse, as it is an important model species, and to investigate if progesterone rapidly affects the CBF of mice at nM concentrations.     

Molecular properties and preclinical pharmacology of JNJ-1250132, a steroidal progesterone receptor modulator that inhibits binding of the receptor to DNA in vitro

Progesterone receptor modulators have diverse potential therapeutic uses, including the treatment of endometriosis, uterine fibroids and breast cancer. Here we describe the molecular properties and preclinical pharmacology of a new steroidal progestin antagonist, JNJ-1250132. The compound is a high affinity ligand for the progesterone receptor, possessing cross-reactivity with other steroid receptors comparable to that of steroidal antagonists such as mifepristone. It inhibits progestin-inducible alkaline phosphatase gene expression in T47D human breast cancer cells, and also inhibits their in vitro proliferation. It inhibits gestation in rats and progesterone-dependent endometrial transformation in rabbits with efficacies comparable to mifepristone. Like mifepristone, it is a glucocorticoid antagonist in vivo. In cell-free DNA binding assays, the compound inhibits binding of the human progesterone receptor to a progesterone response element, and thus is similar to onapristone in this regard. In contrast, as judged by proteolytic analysis, JNJ-1250132 induces a receptor conformation more similar to that induced by mifepristone, which promotes receptor binding to DNA. Therefore, JNJ-1250132 has unique effects on the progesterone receptor that may translate into a novel clinical profile.     

Inhibitory non-genomic effects of progesterone on Na+ absorption in epithelial cells from Xenopus kidney (A6)

The effect of the steroid hormone progesterone on transepithelial sodium transport was measured in confluent monolayers of the A6-cell line from Xenopus kidney. Apical permeabilization with Amphotericin B enabled us to measure the Na+/K+-pump current, and current-fluctuation analysis was used to analyze changes in apical channel density and gating characteristics. Basolateral progesterone (22.2 microM) had a rapid inhibitory effect on the Na+/K+-pump current, and a corresponding decrease in Na+ channel density. The effect occurred within some minutes and took about 50 min to reach a new steady state, in which 45% of the macroscopic current (ISC) was inhibited. Progesterone also inhibits the hypo-osmotic stimulation of Na+ channels which occurs in untreated monolayers. Compared with the known effects of adrenal steroids, our results show a rapid inhibitory action of a steroid hormone on Na+ absorption. The time profile of the progesterone effect suggests, at least in the first minutes, a non-genomic action of progesterone.     

Non-genomic effects of progestins--inhibition of cell growth and increased intracellular levels of cyclic nucleotides

The anti-proliferative effect of progestins was studied in human transformed cell lines from the uterine cervix (C-4I, C33A and Me-180). Progestins caused a concentration-dependent inhibition of proliferation. The maximum tested concentration (2.6-3.2 microM) inhibited C-4I cell growth by the following order of potency: progesterone (56%) > medroxyprogesterone (38%) > megestrol acetate (25%). The sensitivity, expressed as I(25) (the concentration that caused 25% inhibition of growth), showed the same order: progesterone (7.7 nM) > medroxyprogesterone (78 nM) > megestrol acetate (570 nM). The intracellular levels of cGMP and cAMP were elevated and the cellular export of these cyclic nucleotides was inhibited by a similar order of potency. The C-4I cell line was devoid of progesterone-, estrogen-, androgen- and glucocorticoid-receptors. In addition, the antiprogestins mifepristone, onapristone and ZK-112993 did not block the anti-proliferative effect of progesterone. On the other hand, antiprogestins (2.3 nM) appeared to have some progesterone-like ("mimetic") activity with inhibition of C-4I cell growth; mifepristone (11%), onapristone (12%) and ZK-112993 (16%). The observed effects of progestins and antiprogestins on C-4I cells were also presented in C33A cells (16% androgen receptor positive) and Me-180 cells (22% progesterone receptor positive, 9% androgen receptor positive and 17% glucocorticoid receptor positive). This study suggests that a non-genomic mechanism contributes to the anti-proliferative effect of progestins.     

Ciliary activity in the oviduct of cycling, pregnant, and muscarinic receptor knockout mice

The transport of the oocyte and the embryo in the oviduct is managed by ciliary beating and muscular contractions. Because nonneuronally produced acetylcholine influences ciliary beating in the trachea via the muscarinic receptors M2 and M3, we supposed that components of the cholinergic system may also modulate ciliary activity in the oviduct. To address this issue, we analyzed the expression profile of muscarinic receptors (CHRMs) in the murine oviduct by RT-PCR and assessed ciliary beat frequency (CBF) and cilia-driven particle transport speed (PTS) on the mucosal surface of opened oviductal segments in correlation with histomorphological investigations. RT-PCR of laser-assisted microdissected epithelium revealed expression of Chrm subtypes Chrm1 and Chrm3. In opened isthmic segments, particle transport was barely seen, correlating with a significantly lower number of ciliated cells compared to the ampulla. In the ampulla, basal PTS and CBF were high (71 μm/sec and 21 Hz, respectively) both in cycling and pregnant wild-type mice and in mice with targeted deletion of the Chrm genes Chrm1, Chrm3, Chrm4, and Chrm5. In contrast to the trachea, where basal ciliary activity was low and largely enhanced by muscarinic stimulation, muscarinic agonists and antagonists did not affect the high ampullar PTS. Our results imply that this high oviductal autonomous ciliary activity is independent from the intrinsic cholinergic system and serves to maintain optimal clearance of the tube throughout all stages of the estrous cycle and early pregnancy.     

Effects of albuterol enantiomers on ciliary beat frequency in ovine tracheal epithelial cells.

beta(2)-Adrenergic agonists stimulate ciliary beat frequency (CBF), an integral part of mucociliary clearance. To evaluate the differential effects of albuterol enantiomers and their racemic mixture on ciliary function, CBF and intracellular calcium were measured at room temperature from single ovine airway epithelial cells with use of digital videomicroscopy. Baseline CBF was 7.2 +/- 0.2 (SE) Hz (n = 80 measurements). R-albuterol (10 microM to 1 mM) stimulated CBF in a dose-dependent manner to maximally 24.4 +/- 5.4% above baseline. Racemic albuterol stimulated CBF to maximally 12.8 +/- 3.6% above baseline, a significantly lower increase compared with R-albuterol alone, despite identical R-enantiomer amounts in both groups. Simultaneous recordings of intracellular calcium concentration and CBF from single cells indicated that the CBF increase in response to R-albuterol was mediated through beta-receptors and stimulation of protein kinase A, in a calcium-dependent and -independent fashion. S-albuterol had a negligible effect on CBF and did not change intracellular calcium. Together, these results suggest that R-albuterol is more efficacious than racemic albuterol in stimulating CBF. Thus S-albuterol may interfere with the ability of R-albuterol to increase CBF.     

Mechanosensitivity of mouse tracheal ciliary beat frequency: roles for Ca2+, purinergic signaling, tonicity, and viscosity

Mechanosensitivity is hypothesized to participate in the regulation of ciliary beat frequency (CBF) in airway epithelia. To investigate this hypothesis, CBF in excised mouse trachea was monitored (microscopy image analysis) while varying mucosal shear (perfusate velocity and/or viscosity; planar flow). CBF increased within minutes of step increase to steady shear stress as small as 10(-3) Pa and decreased within minutes of shear reduction (相似文献   

Membrane-bound progesterone receptor expression in human aortic endothelial cells.

Observational studies demonstrate that estradiol and progesterone affect vasoreactivity. In animal studies, progesterone treatment causes immediate relaxation of precontracted arteries with inhibition of calcium influx in vascular endothelial and smooth muscle cells, suggesting a non-genomic mechanism of action. In this study we investigated the presence of novel membrane-bound progesterone receptors in human aortic endothelial cells and correlated the expression with cell-cycle stage. Western blotting analysis with an antibody directed to the hormone-binding domain of the classic progesterone receptors shows predominant bands at 100 and 60 kD, whereas analysis with an antibody to the DNA-binding region shows only the 100-kD band. In contrast, classic nuclear progesterone receptors B and A are identified at 116 and 94 kD in similarly processed T47D cells. Both novel bands localize to the membrane fraction after differential centrifugation. Plasma membrane-bound progesterone receptor was further shown with immunofluorescent antibody and ligand-binding studies in a small percentage of human aortic endothelial cells. Fluorescent activated cell sorting demonstrated that approximately 8% of the human aortic endothelial cells expressed a plasma membrane progesterone receptor and that a greater percentage of the expressing cells were in the G2/M-phase of the cell cycle. Treatment with progesterone conjugated to BSA did not show any significant cell-cycle changes. Plasma membrane-bound progesterone receptor in vascular endothelial cells may regulate the non-genomic actions of progesterone, and expression of the receptor appears to vary with cell cycle stage.     

The non-genomic effects on Na+/H+-exchange 1 by progesterone and 20alpha-hydroxyprogesterone in human T cells

Progesterone is an endogenous immunomodulator and can suppress T-cell activation during pregnancy. We have previously shown that the non-genomic effects of progesterone, especially acidification, are exerted via plasma membrane sites and suppress cellular genomic responses to mitogens. This study aimed to show that acidification is due to a non-genomic inhibition of Na(+)/H(+)-exchange 1 (NHE1) by progesterone and correlate this with immunosuppressive phytohemagglutinin (PHA)-induced T-cell proliferation. The presence of amiloride-sensitive NHE 1 was identified in T cells. The activity of NHE1 was inhibited by progesterone but not by 20alpha-hydroxyprogesterone (20alpha-OHP). Furthermore, 20alpha-OHP was able to compete with progesterone and release the inhibitory effect on the NHE1. The inhibition of NHE1 activity by progesterone-BSA demonstrated non-genomic action via plasma membrane sites. Finally, co-stimulation with PHA and progesterone or amiloride, (5-(N, N-dimethyl)-amiloride, DMA), inhibited PHA-induced T-cell proliferation, but this inhibition did not occur with 20alpha-OHP and PHA co-stimulation. However, when DMA was applied 72 h after PHA stimulation, it was able to suppress PHA-induced T-cell proliferation. This is the first study to show that progesterone causes a rapid non-genomic inhibition of plasma membrane NHE1 activity in T cells within minutes which is released by 20alpha-OHP. The inhibition of NHE1 leads to immunosuppressive T-cell proliferation and suggests that progesterone might exert a major rapid non-genomic suppressive effect on NHE1 activity at the maternal-fetal interface in vivo and that 20alpha-OHP may possibly be able to quickly release the suppression when T cells circulated away from the interface.     

Making human nasal cilia beat in the cold: a real time assay for cell signalling

Human nasal epithelium must adapt to cold climates, and yet, in vitro, human ciliary beat frequency (CBF) is zero at 4 degrees C. Similarly, hibernating mammals do not die of pneumonia despite a core body temperature as low as 6 degrees C, implying that cilia continue to beat. Here, we show that protein kinase C (PKC) and Ca(2+)/calmodulin-dependent kinase II (CaMKII) regulate the profile of human nasal CBF in response to rising temperature from 4 degrees C. Onset of ciliary beat was at 10 degrees C in Medium 199, 7 degrees C in the presence of the PKC activator phorbol 12-myristate 13-acetate (PMA), the calcium ionophore ionomycin, or the CAMKII blocker myristoylated autocamtide-2 related inhibitory peptide (MACI), and 6 degrees C for the myristoylated peptide PKC inhibitor EGF-R Fragment 651-658 (MyrPKCI). During cell warming to 32 degrees C, the thermal profile was sigmoid in all solutions except those containing MACI+PMA. Surprisingly, cilia continued to beat despite 4 degrees C and were significantly more responsive to rising temperature with either MACI+PMA, or MACI+MyrPKCI. Our data suggest that CaMKII and PKC regulate the thermal slope and profile of CBF in vitro, and that when these protein kinases are manipulated, cilia can continue to beat despite hypothermia. These findings may relate to adaptive responses to cold climates.     

糖皮质激素非基因组效应的研究

在过去的几十年间,人们认为糖皮质激素（glucocorticoid,GC）仅仅是通过改变基因的表达来发挥其生理作用,这个过程需要几个小时来完成。然而,近年来越来越多的证据表明GC对激素分泌、神经元兴奋性、机体行为及细胞形态、糖类代谢等具备快速效应,这些过程往往在数秒钟或者分种内完成,这种作用机制被称为GC的非基因组作用机制。GC的非基因组作用主要可能通过两种不同的机制起作用：（1）通过细胞膜上或者细胞质内结构未知的糖皮质激素受体（glucocorticoid Recptor,GR）来发挥非基因组作用,即为特异性非基因组效应,（2）GC主要通过改变细胞膜理化作用来发挥效应。也称为非特异性非基因组效应（non-specific nongenomic effects,NSNE）。本文通过阐述近年来GC的非基因组的作用的最新研究进展并且讨论了这些非基因组作用临床治疗过程中的联系。对糖皮质激素基因组和非基因组作用机制的深入了解有助于指导我们在临床合理用药并减少其副作用。     

Ovum transport in the rat oviductal ampulla in the absence of muscle contractility

Ovum transport in mammalian oviducts involves two main effectors: ciliary motility and muscle contractility. To study the relative contribution of cilia to ovum transport in the rat, we blocked smooth muscle activity with isoproterenol, a beta-adrenergic agonist, and measured transport rates of surrogate ova in situ. Transport rates before isoproterenol administration were 0.04 mm/s in the cephalic ampulla and 0.03 mm/s in the caudal ampulla; rates were unchanged after administration of isoproterenol. To determine if isoproterenol affected ciliary activity, we measured ciliary beat frequency with laser-scattering spectroscopy over the effective isoproterenol dosage. Isoproterenol did not cause a significant change in ciliary beat frequency. Our results show that in the rat oviductal ampulla, ciliary motion is capable of transporting ova in the absence of muscle contractility.     

Vasoactive intestinal peptide stimulates ciliary motility in rabbit tracheal epithelium: modulation by neutral endopeptidase.

We studied the effect of vasoactive intestinal peptide (VIP) on ciliary activity in rabbit cultured tracheal epithelium by a photoelectric method in vitro. Administration of VIP (10(-7) M) elicited an increase in ciliary beat frequency (CBF) from the baseline values of 970 +/- 52 to 1139 +/- 75 beats/min (mean +/- S.E., P less than 0.01). This ciliostimulatory effect was dose-dependent, with the maximal increase and EC50 value being 17.4 +/- 1.0% (P less than 0.05) and 6.10(-11) M, respectively. The VIP-induced increase in CBF was abolished by pretreatment of cells with [4-Cl-D-Phe6, Leu17]-VIP, a VIP receptor antagonist. The neutral endopeptidase inhibitor phosphoramidon (10(-5) M) potentiated the effect of VIP, so that the CBF dose-response curve for VIP was shifted to lower concentrations by 0.5 log U. The administration of VIP increased cyclic AMP levels in epithelial cells, an effect that was also potentiated by phosphoramidon. These results suggest that VIP may interact with its specific receptors and stimulate airway ciliary activity probably through the activation of adenylate cyclase, and that neutral endopeptidase may play a role in modulating this effect of VIP.     

Uterine epithelial morphology and progesterone receptors in a mifepristone-treated viviparous lizard Pseudemoia entrecasteauxii (Squamata: Scincidae) during gestation

Structural and functional changes to the uterus associated with maintenance of pregnancy are controlled primarily by steroid hormones such as progesterone. We tested the hypothesis that progesterone regulates uterine structural changes during pregnancy in the viviparous skink, Pseudemoia entrecasteauxii, by treating pregnant females with the progesterone receptor antagonist mifepristone at different stages of pregnancy. Expression and distribution of progesterone receptor was determined using Western blot and immunohistochemistry. During early pregnancy, mifepristone treatment resulted in altered uterine epithelial cell surface morphology and high embryo mortality, but did not affect females at mid and late stages of pregnancy. Females treated with mifepristone in early pregnancy exhibited abnormal uterine epithelial cell morphology such as lateral blebbing and presence of wide gaps between cells indicating loss of intercellular attachment. Chorioallantoic membranes of the embryo were not affected by mifepristone treatment. Two isoforms (55 kDa and 100 kDa) of progesterone receptor were identified using immunoblots and both isoforms were localized to the nucleus of uterine epithelial cells. The 55 kDa isoform was expressed throughout pregnancy, whereas the 100 kDa isoform was expressed during mid and especially late pregnancy. In P. entrecasteauxii, mifepristone may prevent successful embryo attachment in early pregnancy through its effects on uterine epithelial cells but may have little effect on pregnancy once the maternal-embryo structural relationship is established.