Anu Pekki

Subcellular location of androgen receptor in rat prostate, seminal vesicle and human osteosarcoma MG-63 cells

Location of the androgen receptor (AR) before and after dihydrotestosterone (DHT) administration was studied in 6 castrated and 2 normal male rats, as well as in MG-63 human osteosarcoma cell culture. Two days after castration, rats were injected with DHT and sacrificed 0, 6 and 24 h later. Cryosections of ventral prostate and seminal vesicle were stained with a polyclonal anti-AR antibody. Cultured MG-63 cells were also stained similarly. The intensity of immunoreaction was measured semiquantitatively by computer-assisted image analysis. In both normal and castrated rats, a positive reaction was seen mainly in the nuclei of epithelial cells and stromal cells of the prostate and seminal vesicle, as well as in those of smooth muscle cells of the seminal vesicle. AR immunoreactivity was up-regulated by DHT, it decreased clearly in both organs after castration. Nuclear AR and its up-regulation by androgen were also seen in MG-63 cells. At the immunoelectron microscopy, silver enhanced gold particles were predominantly found in the heterochromatin of cell nuclei. Treatment with DHT caused a decondensation of the heterochromatin and AR was more dispersed. Thus, AR appears to be nuclear independently of the ligand.

Contractions induced by potassium‐free solution and potassium relaxation in vascular smooth muscle of hypertensive and normotensive rats

1 Vascular contractions induced by K+‐free solution and relaxation responses following the return of K+ to the organ bath were studied in mesenteric arterial rings from spontaneously hypertensive rats (SHR) and normotensive Wistar‐Kyoto rats (WKY) with particular focus on the role of vascular adrenergic nerve‐endings and endothelium. 2 In endothelium‐denuded rings the omission of K+ from the incubation medium resulted in gradual contractions, the rate of which was slower in SHR than WKY. Nifedipine (1 μm) inhibited the contractions more effectively in SHR than WKY. 3 Adrenergic denervation in vitro with 6‐hydroxydopamine reduced the contractions induced by the K+‐free medium in endothelium‐denuded rings. The remaining contractions after denervation were markedly greater in SHR than WKY. 4 The presence of intact vascular endothelium attenuated the K+‐free contractions in both strains, the attenuation being smaller in SHR than WKY. NG‐nitro‐l‐arginine methyl ester (l‐NAME, 0.1 mm) and methylene blue (10 μm), but not indomethacin (10 μm), abolished the attenuating effect of endothelium on the K+‐free contractions. l‐Arginine (1 mm) reversed the effect of l‐NAME in WKY but not in SHR. 5 The re‐addition of K+ after full K+‐free contractions dose‐dependently relaxed the rings. The rate of this K+‐induced relaxation was significantly slower in SHR than WKY at all K+ concentrations (0.1–5.9 mm) studied, whether the endothelium or functioning adrenergic nerve‐endings were present or not. Ouabain (1 mm) totally inhibited the K+ relaxation in SHR but only partially in WKY. 6 Vascular smooth muscle contractions induced by high concentrations of potassium were comparable between the strains. The EC50 for noradrenaline‐induced contractions was lower in SHR than WKY, but the maximal forces did not differ significantly. 7 In conclusion, the contractile response in K+‐free solution more clearly differentiates vascular rings from SHR and WKY than the responses induced by the classical contractile agents noradrenaline and high concentrations of potassium. The depressant effect of the presence of intact endothelium on the K+‐free contractions, which was smaller in SHR than WKY, is mediated via the endothelium‐derived relaxing factor. Neurotransmitter release from vascular adrenergic nerve‐endings participates less in the K+‐free contractile response in SHR than WKY. Moreover, the contractile response is more dependent on calcium entry through nifedipine‐sensitive calcium channels in SHR than WKY. The greater K+‐free contractions of denervated endothelium‐denuded rings and the reduced K+ relaxation rate in SHR when compared to WKY suggest increased cell membrane permeability and decreased activity of vascular Na+, K+‐ATPase, respectively, in this type of genetic hypertension.

Quinapril treatment and arterial smooth muscle responses in spontaneously hypertensive rats

1 The effects of long‐term angiotensin‐converting enzyme inhibition with quinapril on arterial function were studied in spontaneously hypertensive rats, Wistar‐Kyoto rats serving as normotensive controls. 2 Adult hypertensive animals were treated with quinapril (10 mg kg−1 day−1) for 15 weeks, which reduced their blood pressure and the concentrations of atrial natriuretic peptide in plasma and ventricular tissue to a level comparable with that in normotensive rats. 3 Responses of mesenteric arterial rings in vitro were examined at the end of the study. Compared with normotensive and untreated hypertensive rats, responses to noradrenaline were attenuated in hypertensive animals on quinapril, both force of contraction and sensitivity being reduced. Quinapril also attenuated maximal contractions but not sensitivity to potassium chloride. Nifedipine less effectively inhibited vascular contractions in normotensive and quinapril‐treated than in untreated hypertensive rats. 4 Arterial relaxation responses by endothelium‐dependent (acetylcholine) and endothelium‐independent (sodium nitrite, isoprenaline) mechanisms were similar in normotensive and quinapril‐treated rats and more pronounced than in untreated hypertensive rats. 5 Cell membrane permeability to ions was evaluated by means of potassium‐free solution‐induced contractions of endothelium‐denuded denervated arterial rings. These responses were comparable in normotensive and quinapril‐treated rats and less marked than in untreated hypertensive rats. 6 Intracellular free calcium concentrations in platelets and lymphocytes, measured by the fluorescent indicator quin‐2, were similar in normotensive and quinapril‐treated rats and lower than in untreated hypertensive rats. 7 In conclusion, quinapril treatment improved relaxation responses and attenuated contractions in arterial smooth muscle of hypertensive rats. These changes may be explained by diminished cytosolic free calcium concentration, reduced cell membrane permeability, and alterations in dihydropyridine‐sensitive calcium channels following long‐term angiotensin‐converting enzyme inhibition.

Subcellular location of unoccupied and occupied glucocorticoid receptor by a new immunohistochemical technique

Recent immunohistochemical studies suggest that the unoccupied glucocorticoid receptor (GR) is cytoplasmic and that the ligand causes its translocation into the target cell nucleus. The subcellular location of GR is especially interesting in that other members of the steroid receptor superfamily appear to be nuclear. The intracellular distribution of GR was studied immunohistochemically using a new freeze-drying and vapor fixation method which eliminates the protein diffusion and redistribution possibly caused by liquid fixation techniques. We used two monoclonal antibodies against rat liver GR. Dried samples of the adrenalectomized rat brain and uterus were fixed in p-benzoquinone vapor for 3 h at 60 degrees C and embedded in paraffin. Sections were stained with a biotinylated mouse monoclonal GR antibody using the avidin-biotin-peroxidase complex. Both unoccupied and occupied GR were found in the nucleus of the target cells, fibroblasts in the uterus and nerve cells in the cortex of the brain. The staining was saturated with the cytosol of cos cells transfected with GR. No cytoplasmic staining was seen even 2 days after adrenalectomy. In conclusion we propose that GR is also located in the nucleus independently of occupation.

Mechanisms of action of sex steroid hormones: Basic concepts and clinical correlations

The review deals with the clinically important aspects of the basic mechanisms of sex steroid hormones. Steroids can act through two basic mechanisms: genomic and non-genomic. The classical genomic action is mediated by specific intracellular receptors, whereas the primary target for the non-genomic one is the cell membrane. Many clinical symptoms seem to be mediated through the non-genomic route. Furthermore, membrane effects of steroid and other factors can interfere with the intranuclear receptor system inducing or repressing steroid-and receptor-specific genomic effects. These signalling pathways may lead to unexpected hormonal or anti-hormonal effects in patients treated with certain drugs. Steroid receptors (SRs) are members of a large family of nuclear transcription factors that regulate gene expression by binding to their cognate steroid ligands, to the specific enhancer sequences of DNA (steroid response elements) and to the basic transcription machinery. SRs are phosphoproteins, which are further phosphorylated after ligand binding. The role of phosphorylation in receptor transaction is complex and may not be uniform to all SRs. However, phosphorylation/dephosphorylation is believed to be a key event regulating the transcriptional activity of steroid receptors. SR activities can be affected by the amount of SR in the cell nuclei, which is modified by the rate of transcription and translation of the SR gene as well as by proteolysis of the SR protein. There is an auto- and heteroregulation of receptor levels. Some of the SRs appear to bind specific protease inhibitors and exhibit protease activity. The physiological significance of this weak proteolytic activity is not clear. Some SRs are expressed as two or more isoforms, which may have different effects on transcription. Receptor isoforms are different translation or transcription products of a single gene. Isoform A of the progesterone receptor is a truncated form of PR isoform B originating from the same gene, but it is able to suppress not only the gene enhancing activity of PR-B but also that of other steroid receptors. From the clinical point of view, it is important to note that the final hormonal effect in a target tissue is dependent on the cross talk between different nuclear steroid receptors and on expression of receptor isoforms.

Hormone-dependent changes in A and B forms of progesterone receptor

The influence of different estrogen and/or progesterone treatments on concentrations of A and B forms of progesterone receptor (PR-A and PR-B) in the different cell types of chick oviduct was studied. A semiquantitative immunohistochemical assay for cellular PR concentrations was developed using a computer-assisted image analysis system. The staining intensity of nuclear PR in the basal layer of epithelial cells, glandular, smooth muscle and mesothelial cells was analysed separately using two monoclonal antibodies, PR6 and PR22. The measured concentrations of PR varied between different cell types and from cell to cell. A significant decrease in PR concentration, as noted by a decrease in staining intensity, was observed in all cell types studied 2 or 6 h after a single injection of progesterone with or without simultaneous estrogen administration. The decrease was also verified with immunoblotting and an immunoenzymometric assay (IEMA) for chicken PR. After down-regulation the concentration of PR recovered to the control level within 48 h after progesterone or estrogen administration. Estrogen administration alone was observed to cause changes in the concentration of PR-A only, having little or no effect on PR-B concentration depending on the cell type studied. These findings indicate that estrogen and progesterone cause cell-specific changes not only to the total concentration of PR but also to the cellular ratio of PR-A and PR-B.

Effect of celiprolol therapy on arterial dilatation in experimental hypertension

1 It has recently been suggested that therapy with β‐adrenoceptor blockers reduces peripheral arterial resistance via enhanced vascular dilatation. Therefore, we studied the effects of celiprolol, which is a specific β1‐antagonist that has a weak β2‐agonist action, on arterial tone in spontaneously hypertensive rats (SHR) and Wistar‐Kyoto (WKY) rats. 2 Two doses of celiprolol (5 and 50 mg kg−1 day−1) were administered to the SHR, while the WKY rats received only the higher dose of the drug. During the 12‐week treatment period the higher dose attenuated the increase in blood pressure by approximately 20 mmHg in SHR, whereas the lower dose was without significant antihypertensive effect. Celiprolol therapy did not affect blood pressure in the normotensive WKY rats. 3 Responses of mesenteric arterial rings in vitro were examined at the end of the study. Interestingly, endothelium‐mediated relaxations of noradrenaline (NA)‐precontracted rings to acetylcholine (ACh) in the absence and presence of the cyclo‐oxygenase inhibitor, diclofenac, were equally enhanced in both celiprolol‐treated SHR groups. The nitric oxide synthase inhibitor NG‐nitro‐L‐arginine methyl ester (L‐NAME) practically abolished the relaxations to ACh in all SHR irrespective of whether they had received celiprolol, whereas in WKY rats L‐NAME only attenuated the responses to ACh. However, no differences were found between the SHR groups in relaxations to ACh when hyperpolarization of smooth muscle was prevented by precontractions induced by 50 mM KCl. Vasorelaxation of NA‐precontracted rings to the exogenous nitric oxide donor, nitroprusside, was also moderately augmented in both celiprolol‐treated SHR groups, while the relaxation to β‐adrenoceptor agonist, isoprenaline, remained equally impaired in all SHR whether or not they had received celiprolol. No differences were observed between the two WKY groups in the responses to ACh, nitroprusside or isoprenaline. 4 Contractile sensitivity of mesenteric arterial rings to the receptor‐mediated agonists, NA and 5‐hydroxytryptamine, was comparable in all study groups. 5 In conclusion, SHR treatment with either the low or the higher dose of celiprolol was accompanied by enhancement of both endothelium‐dependent and endothelium‐independent nitric oxide‐mediated arterial relaxation, possibly via a hyperpolarization mechanism. Interestingly, this effect appeared to be independent of the reduction in blood pressure.

Progesterone receptor is constitutively expressed in chicken intestinal mesothelium and smooth muscle

We have previously shown that progesterone receptor (PR) is expressed in the mesothelium of the chick oviduct and ovary and in the smooth muscle cells of the oviduct and the bursa of Fabricius. Here, we investigated the presence of PR in different parts of the peritoneum and abdominal organs using an immunohistochemical staining based on monoclonal antibodies against chicken PR. In 4-week-old sexually immature chicks, PR expression was located in the mesothelial cells of different parts of the peritoneum, in a thin layer of muscle cells of the ileum and throughout the muscle tissue of the colon and cloaca. In chicks of the same age treated with estrogen, PR was demonstrated similarly in the peritoneum and in the smooth muscle cells of the ileum, colon and cloaca. Using 25-week-old mature chickens, PR was also detected in identical tissues. Immunoblotting of the cloacal cytosol revealed the B form, but no A form of PR, both of which were found in the oviduct samples. Muscle cells of the duodenum and jejunum were not found to contain PR. Estrogen treatment was not needed to stimulate the production of PR in any of the tissues examined. We therefore conclude that the B form of PR is constitutively expressed in the mesothelial cells in different parts of the peritoneum and also in the smooth muscle cells of the ileum, colon and cloaca.

Progesterone receptor and hsp90 are not complexed in intact nuclei

In hypotonic cell extract (cytosol), unliganded progesterone receptor (PR) is known to form an oligomeric complex with heat shock protein 90 (hsp90), and this complex does not bind to DNA. Since ligand binding has been shown to render the complex less stable in vitro, it has been proposed that ligand binding regulates DNA binding and receptor activity in vivo by altering the stability of the oligomeric complex. However, there is no direct evidence as to whether this oligomeric complex is present in vivo. The present study addressed this problem. First, we used an immunoelectron-microscopic technique and monoclonal antibodies to ascertain the location of PR and hsp90 in chick oviduct cells. Hsp90 was found in the cytoplasm and PR in the nucleus. To study the relative affinities of the PR and hsp90 antibodies, we then constructed a chimeric protein (PR-hsp90), which was expressed in the HeLa cells. Both hsp90 and PR antigens of the chimera were detected in the nuclei with the same intensity, which indicates that the antibodies have equal sensitivities in detecting their antigens. This suggests that if significant amounts of nuclear hsp90 were present in intact cells, it should have been detected by our method. Our results indicate that the PR does not exist in vivo as an oligomeric, nonDNA-binding form in the cell nuclei and that the oligomeric form found in tissue extracts is possibly formed during tissue processing.

Only a small portion of the cytoplasmic progesterone receptor is associated with Hsp90 in vivo

In cell extracts all of the nonliganded steroid receptor molecules are found as an oligomeric complex with Hsp90 and other proteins. In previous studies we have shown that Wild‐type Hsp90 and progesterone receptor (PR) are located in different cell compartments (Tuohimaa et al. [1993] Proc. Natl. Acad. Sci. USA 90:5848–5852). In the present work we studied whether PR and Hsp90 can efficiently associate provided they are present in the same cell compartment. The association of Hsp90 with PR in vivo was studied by nuclear cotranslocation and immunohistochemistry with an antibody (αD) which can distinguish between the oligomeric and dissociated form. Upon expression of a cytoplasmic mutant of PR with Wild‐type (cytoplasmic) Hsp90 and Wild‐type (nuclear) PR with NLS‐Hsp90 (a Hsp90 with a nuclear localization signal), we noted that the epitope of αD in PR was exposed in both cases. Also, in vivo crosslinking and treatment of cells with substances which stabilize the oligomeric complex in vitro were inefficient in demonstrating or inducing a similar oligomeric receptor form detectable in vitro in cell homogenates. However, when the cytoplasmic PR mutant (ΔPR) was coexpressed with a nuclear form of Hsp90 (NLS‐Hsp90), a portion of PR was cotranslocated into the nucleus. This would indicate that steroid receptors are indeed associated with Hsp90 in intact cells, but the Hsp90‐associated receptor pool represents only a small portion of the receptors. This suggests that the majority of oligomeric complexes seen in cell extracts are formed during cell fractionation. J. Cell. Biochem. 74:458–467, 1999.