Tissue-specific progesterone receptor-chromatin binding and the regulation of progesterone-dependent gene expression.

Progesterone receptor (PGR) co-ordinately regulates ovulation, fertilisation and embryo implantation through tissue-specific actions, but the mechanisms for divergent PGR action are poorly understood. Here we characterised PGR activity in mouse granulosa cells using combined ChIP-seq for PGR and H3K27ac and gene expression microarray. Comparison of granulosa, uterus and oviduct PGR-dependent genes showed almost complete tissue specificity in PGR target gene profiles. In granulosa cells 82% of identified PGR-regulated genes bound PGR within 3 kb of the gene and PGR binding sites were highly enriched in proximal promoter regions in close proximity to H3K27ac-modified active chromatin. Motif analysis showed highly enriched PGR binding to the PGR response element (GnACAnnnTGTnC), but PGR also interacted significantly with other transcription factor binding motifs. In uterus PGR showed far more tendency to bind intergenic chromatin regions and low evidence of interaction with other transcription factors. This is the first genome-wide description of PGR action in granulosa cells and systematic comparison of diverse PGR action in different reproductive tissues. It clarifies finely-tuned contextual PGR-chromatin interactions with implications for more targeted reproductive medicine.

Haemoglobin expression in in vivo murine preimplantation embryos suggests a role in oxygen-regulated gene expression.

Haemoglobin expression is not restricted to erythroid cells. We investigated the gene expression of the haemoglobin subunits haemoglobin, alpha adult chain 1 (Hba-a1) and haemoglobin, beta (Hbb), 2,3-bisphosphoglycerate mutase (Bpgm) and the oxygen-regulated genes BCL2/adenovirus E1B interacting protein 3 (Bnip3), solute carrier family 2 (facilitated glucose transporter), member 1 (Slc2a1) and N-myc downstream regulated gene 1 (Ndrg1) in the murine preimplantation embryo, comparing invivo to invitro gene expression. Relatively high levels of Hba-a1 and Hbb were expressed invivo from the 2-cell to blastocyst stage; in contrast, little or no expression occurred invitro. We hypothesised that the presence of haemoglobin invivo creates a low oxygen environment to induce oxygen-regulated gene expression, supported by high expression of Slc2a1 and Ndrg1 in invivo relative to invitro embryos. In addition, analysis of an invitro-derived human embryo gene expression public dataset revealed low expression of haemoglobin subunit alpha (HBA) and HBB, and high expression of BPGM. To explore whether there was a developmental stage-specific effect of haemoglobin, we added exogenous haemoglobin either up to the 4-cell stage or throughout development to the blastocyst stage, but observed no difference in blastocyst rate or the inner cell mass to trophectoderm cell ratio. We conclude that haemoglobin in the invivo preimplantation embryo raises an interesting premise of potential mechanisms for oxygen regulation, which may influence oxygen-regulated gene expression.

Females do not have increased risk of early or late mortality after isolated aortic valve replacement: results from a multi-institutional Australian study.

AIM: There is controversy regarding whether isolated aortic valve replacement (AVR) in women is associated with an increased risk of early and late mortality. The current study evaluates the impact of gender as an independent risk factor for early and late mortality after isolated AVR. METHODS: Data obtained between June 2001 and December 2009 by the Australasian Society of Cardiac and Thoracic Surgeons Cardiac Surgery Database Program was retrospectively analysed. Demographic, operative data and postoperative complications were compared between male and female patients using χ(2) and t-tests. Long-term survival analysis was performed using Kaplan Meier survival curves and the log rank test. Independent risk factors for short term and long term mortality were identified using binary logistic and Cox regression, respectively. RESULTS: Isolated aortic valve replacement was undertaken for 2790 patients in 18 Australian institutions; 41.9% were female. Female patients were generally older (mean age 72 vs. 66 years (P<0.001) and presented more often with hypertension (P<0.001) and obesity (P<0.001). They were less likely to present with cerebrovascular disease (P=0.018), renal failure (P=0.017) and non-elective presentation (P=0.017). Women were observed to have a lower 30-day mortality (1.7% vs. 2.1%) but there was no difference on univariate (P=0.490) or multivariate analysis (P=0.983). There was no difference in the incidence of early complications but women were more likely to require red blood cell transfusion (P<0.001). Long-term survival was comparable between men and women (P=0.662). CONCLUSION: Female patients undergoing isolated AVR do not have an increased risk of early and late mortality. Further investigation is required to delineate the impact of gender on early and late outcomes following AVR.

Angiotensin AT(4) receptors in the normal human prostate and benign prostatic hyperplasia.

The cellular localisation and expression of angiotensin AT(4) receptors was examined in the normal human prostate and benign prostatic hyperplasia (BPH) by quantitative in vitro autoradiography using [(125)I]-Ang IV. In the normal human prostate, AT(4) receptors were localised to the glandular epithelium. Interestingly, specific AT(4) receptor binding was significantly reduced in BPH compared to the normal prostate, as quantitated macroscopically (normal: 5038+/-476 dpm/mm(2), n=6 vs BPH: 2701+/-176 dpm/mm(2), n=6, P<0.001) and microscopically (normal: 7.28+/-0.36 grains/mm(2), n=6 vs BPH: 2.50+/-0.47 grains/mm(2), n=6, P<0.001). The findings of the present study demonstrate the presence of AT(4) receptors in the human prostate, being localised to the glandular epithelium, which suggest that the Ang IV/AT(4) system may play a role in the regulation of ionic transport and glandular secretion in the human prostate. The observation that AT(4) receptors appear reduced in BPH suggests that the AT(4) receptor may undergo agonist-induced receptor internalisation, possibly due to increased local tissue levels of Ang IV in BPH.

Angiotensin receptors: distribution, signalling and function.

Angiotensin II (Ang II) is a multi-functional hormone that plays a major role in regulating blood pressure and cardiovascular homoeostasis. The actions of Ang II are mediated by at least two receptor subtypes, designated AT(1) and AT(2). In addition, other angiotensin receptors have been identified which may recognize other angiotensin peptide fragments; however, until now only the AT(1) and AT(2) receptor have been cloned in animals or humans. Most of the well-described actions of Ang II, such as vasoconstriction, facilitation of sympathetic transmission, stimulation of aldosterone release and promotion of cellular growth are all mediated by the AT(1) receptor. Much less is known about the function of the AT(2) receptor, but recent studies suggest that it may play a role in mediating anti-proliferation, cellular differentiation, apoptosis and vasodilatation. In this review, we discuss recent advances in our understanding of Ang II receptors, in particular, their distribution, signalling and function.

Identification, distribution, and expression of angiotensin II receptors in the normal human prostate and benign prostatic hyperplasia.

The tissue distribution, cellular localization, and level of expression of angiotensin II (Ang II) receptors were examined in the normal human prostate and benign prostatic hyperplasia (BPH) by in vitro autoradiography, immunohistochemistry, and radioligand binding studies. In the normal human prostate, Ang II receptors were of the AT(1) subtype and localized predominantly to periurethral stromal smooth muscle. The AT(1) receptor antagonist losartan totally displaced specific [(125)I]-[Sar(1),Ile(8)]Ang II binding, in a concentration-dependent manner, whereas the AT(2) receptor antagonist PD123319 was without effect. There was no significant difference in receptor affinity, but AT(1) receptor density was markedly reduced in BPH compared with that in normal prostate. In rat prostate, Ang II (0.01-1 microM) produced a concentration-dependent increase in [(3)H]-noradrenaline release from sympathetic nerves. The findings of the present study suggest that angiotensin AT(1) receptors predominate in the human prostate. The high concentration of AT(1) receptors in the periurethral region suggests a role for Ang II in modulating cell growth, smooth muscle tone, and possibly micturition. Furthermore, down-regulation of AT(1) receptors in BPH may be due to receptor hyperstimulation by increased local levels of Ang II in BPH. Finally, Ang II may play a functional role in modulating sympathetic transmission in the prostate. These data support the novel concept that activation of the renin-angiotensin system may be involved in the pathophysiology of BPH.

Comparative in vivo effects of irbesartan and losartan on angiotensin II receptor binding in the rat kidney following oral administration.

We examined the ability of the new non-peptide angiotensin II receptor antagonist irbesartan to inhibit AT(1) receptors in vivo in the rat kidney following oral administration, compared with the prototype drug losartan. Male Sprague-Dawley rats (250-300 g) were gavaged with either irbesartan or losartan at doses of 1, 3, 10, 30 or 100 mg/kg, or with corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood was collected and the kidneys were removed. The effects of irbesartan and losartan on angiotensin II receptor binding were determined by quantitative in vitro autoradiography using the specific radioligand (125)I-[Sar(1),Ile(8)]angiotensin II. High levels of angiotensin II receptor binding in the rat kidney were demonstrated in the glomeruli and inner stripe of the outer medulla, which was attributed to AT(1) receptors. At 1 h after dosing, irbesartan (1-100 mg/kg) and losartan (1-30 mg/kg) significantly inhibited AT(1) receptor binding in all anatomical areas of the kidney, in a dose-dependent manner, with a maximal effect at 100 mg/kg and 30 mg/kg respectively. For a 10 mg/kg dose, inhibition of AT(1) receptor binding was maximal around 1-2 h after oral administration of losartan, whereas maximal binding occurred between 2 and 8 h for irbesartan; both drugs produced persistent tissue blockade at 24h. In radioligand binding studies, irbesartan, losartan and EXP3174 (1x10(-10) to 1x10(-5) M) displaced (125)I-[Sar(1),Ile(8)]angiotensin II binding from renal AT(1) receptors in a concentration-dependent manner, with a rank order of potency of irbesartan>EXP3174>losartan. The concentration required to displace 50% of radioligand binding (IC(50)) by irbesartan, EXP3174 and losartan was 1.00+/-0.2 nM, 3.5+/-0.4 nM and 8.9+/-1.1 nM respectively. In conclusion, the findings of the present study suggest that irbesartan and losartan produce effective and sustained inhibition of AT(1) receptors in vivo in the kidney following oral administration. However, irbesartan appears less potent, with respect to dosage, than losartan in vivo, despite having a higher affinity for AT(1) receptors in vitro. The reason for this apparent discrepancy is unclear, but it may reflect the slower onset of action of irbesartan and its rate of tissue accessibility. Inhibition of angiotensin II receptors in target tissues such as the kidney may represent an important action of AT(1) receptor antagonists, which may contribute to the beneficial effects of these agents in the clinical setting.

In vivo inhibition of angiotensin receptors in the rat kidney by candesartan cilexetil: a comparison with losartan.

The present study examined the in vivo effects of candesartan cilexetil compared with losartan on angiotensin II (Ang II) receptor binding in the rat kidney after oral administration. Male Sprague-Dawley rats (250 to 300 g) were gavaged with candesartan cilexetil or losartan in doses of 0.1, 0.3, 1, 3, 10, or 30 mg/kg, or corresponding vehicle. Rats were killed at 0, 1, 2, 8, or 24 h after drug administration, trunk blood collected, and kidneys removed. The effects of candesartan cilexetil and losartan on Ang II receptor binding were determined by quantitative in vitro autoradiography using the radioligand [125I]-[Sar1,Ile8] Ang II. Ang II receptor binding in the kidney was mainly due to AT1 receptors with high levels of binding localized to the inner stripe of the outer medulla and glomeruli in cortical regions. Candesartan cilexetil (0.1 to 30 mg/kg) inhibited Ang II receptor binding to all anatomical sites of the kidney, in a dose-dependent manner. Losartan (0.1 to 30 mg/kg) also produced dose-dependent inhibition of Ang II receptor binding but was approximately 10- to 30-fold less potent than candesartan cilexetil. Inhibition of Ang II receptor binding was near maximal about 1 h after administration of candesartan cilexetil (10 mg/kg) or losartan (10 mg/kg), with both drugs producing persistent blockade at 24 h despite plasma renin activity and plasma drug concentrations returning to near normal levels. In vitro, candesartan, losartan, and EXP3174 (1 x 10(-10) to 1 x 10(-5) mol/L) displaced [125I]-[Sar1,Ile8] Ang II binding from AT1 receptors in the kidney in a concentration-dependent manner with a rank order of potency of candesartan > EXP3174 > losartan. The concentration required to displace 50% of radioligand binding (IC50) by candesartan, EXP3174, and losartan was 0.9+/-0.1 nmol/L, 3.4+/-0.4 nmol/L, and 8.9+/-1.1 nmol/L, respectively. In conclusion, the findings of the present study suggest that candesartan cilexetil is more potent than losartan in antagonizing AT1 receptors in the kidney in vivo. Nonetheless, both candesartan cilexetil and losartan produce rapid, complete, and sustained blockade of AT1 receptors in the rat kidney. Tissue blockade of Ang II receptors in target organs, such as the kidney, may contribute to the beneficial effects of Ang II receptor antagonists as antihypertensive agents.

Interaction of the renin-angiotensin system, bradykinin and sympathetic nerves with cholinergic transmission in the rat isolated trachea.

1. The present study was undertaken to investigate the interaction of the renin-angiotensin system (RAS), bradykinin and the sympathetic nervous system with cholinergic transmission in the rat airways. Experiments were performed on epithelium-intact and epithelium-denuded preparations of rat isolated trachea which had been incubated with [3H]-choline to incorporate [3H]-acetylcholine into the cholinergic transmitter stores. Tracheal preparations were subjected to electrical field stimulation (trains of 1 ms pulses, 5 Hz, 15 V) and the stimulation-induced (S-I) efflux taken as an index of transmitter acetylcholine release. 2. In both epithelium-intact and epithelium-denuded tracheal preparations, the alpha 2-adrenoceptor agonist UK14304 (0.1 and 1 microM) inhibited the S-I efflux, in a concentration-dependent manner. The inhibition of S-I efflux produced by UK14304 (1 microM) was antagonized by the selective alpha 2-adrenoceptor antagonist idazoxan (0.3 microM). Idazoxan (0.3 microM) alone had no effect on the S-I efflux. 3. Angiotensin II (0.1 and 1 microM) was without effect on the S-I efflux in either epithelium-intact or epithelium-denuded tracheal preparations. When angiotensin-converting enzyme was inhibited by perindoprilat (10 microM), angiotensin II (1 microM) was also without effect on the S-I efflux. Similarly, in the presence of idazoxan (0.3 microM), to block prejunctional alpha 2-adrenoceptors, angiotensin II (0.1 and 1 microM) did not alter the S-I efflux. When added alone, perindoprilat (10 microM) did not alter the S-I efflux. 4. In epithelium-denuded preparations, bradykinin (0.01-1 microM) inhibited the S-I efflux. In epithelium-intact preparations, there was also a tendency for bradykinin (0.1 and 1 microM) to inhibit the S-I efflux but this was not statistically significant. However, when angiotensin-converting enzyme and neutral endopeptidase were inhibited by perindoprilat (10 microM) and phosphoramidon (1 microM), respectively, bradykinin (1 microM) significantly inhibited the S-I efflux in epithelium-intact preparations as well as in epithelium-denuded preparations. The inhibition of the S-I efflux produced by bradykinin, in the combined presence of perindoprilat (10 microM) and phosphoramidon (1 microM), was unaffected by the additional presence of the cyclo-oxygenase inhibitor indomethacin (10 microM) and/or the nitric oxide synthase inhibitor NG-nitro-L-arginine (100 microM), in either epithelium-intact or epithelium-denuded preparations. 5. In conclusion, the findings of the present study suggest that airway parasympathetic nerves are endowed with alpha 2-adrenoceptors which subserve inhibition of transmitter acetylcholine release. Under the present conditions, however, transmitter acetylcholine release is not subject to transneuronal modulation by noradrenaline released from adjacent sympathetic nerves in the airways. Moreover, angiotensin II and perindoprilat do not appear to modulate acetylcholine release from parasympathetic nerves of the airways. In contrast, bradykinin inhibits acetylcholine release from airway parasympathetic nerves but this action of bradykinin is limited by the activity of epithelial angiotensin-converting enzyme and/or neutral endopeptidase. The inhibitory action of bradykinin on cholinergic transmission in the airways does not appear to involve the liberation of prostaglandins or nitric oxide.

Hypothalamic cuts suggest a brain site for the antigonadotrophic action of melatonin in the Syrian hamster.

Thrice daily melatonin injections inhibit reproductive functions in pinealectomized male hamsters, as measured by sex organ weights and plasma testosterone levels. Total or anterior, but not posterior, hypothalamic deafferentation prevented the antigonadotrophic effect of melatonin. The findings suggest a site of action of melatonin within the central nervous system.

Pineal melatonin rhythm: reduction in aging Syrian hamsters.

The melatonin content measured by radioimmunoassay of the pineal gland over a 24-hour period (a light:dark cycle of 14 hours of light and 10 of darkness) was compared in young and old female and male Syrian hamsters. The young animals of both sexes exhibited roughly an eightfold rise in pineal melatonin during the dark phase of the cycle, whereas in the old hamsters the nocturnal rise in melatonin was almost completely absent. The results indicate a marked drop in pineal biosynthetic activity in the aging hamster.