Molecular Human Reproduction, Vol. 9, No. 12, pp. 765-774, 2003
© European Society of Human Reproduction and Embryology 2003; all rights reserved
Effects of hypoxia on endothelin-1 sensitivity in the corpus cavernosum
1Andrology Unit and 2Endocrinology Unit, Department of Clinical Physiopathology, 3Department of Anatomy, Histology and Forensic Medicine and 4Department of Pharmacology, University of Florence, V. le G. Pieraccini, 6, 50139 Florence, and 5Menarini Ricerche S.P.A., 00040 Pomezia (Roma), Italy
6 To whom correspondence should be addressed. e-mail: m.maggi{at}dfc.unifi.it
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The penis remains in a hypo-oxygenated, flaccid state for a large majority of the time. In this study, we investigated the effect of changing oxygen tension on the expression and functional activity of endothelin-1 (ET-1) receptors in the penis. Experiments were performed in rabbit and human corpora cavernosa (CC) as well as in human fetal penile tissue and cell cultures [human fetal penile endothelial cells (hfPECs) and human fetal smooth muscle cells (hfPSMCs)]. Endothelin A (ETA) receptors are expressed by both endothelial and muscular cells in all tissues investigated. Only penile endothelial cells express endothelin B (ETB) receptors, which are further turned on during experimental hypoxia. In addition, hypoxia also allows ETB expression in the muscular compartment without affecting ETA expression. This hypoxia-induced over-expression of ETB decreased the contractile activity of ET-1 and increased ETB-mediated relaxation. The latter was essentially related to increased ETB-mediated nitric oxide formation in hfPEC and even in hfPSMC. Hypoxia also induced a time-dependent down-regulation of RhoA and Rho kinase (ROK) expression which, in turn, participated in the decreased contractile activity of ET-1 in the hypoxic penile tissue. Accordingly, during hypoxia, an ROK inhibitor, Y27632, was less effective in relaxing ET-1-precontracted strips. In conclusion, prolonged (24 h) hypoxia stimulated several counter-regulatory mechanisms in penile tissue, including up-regulation of ETB and down-regulation of RhoA/ROK pathways, which may help to preserve CC hypo-oxygenation, allowing smooth muscle relaxation and, most probably, penile erection.
Key words: corpus cavernosum/endothelin/erection/hypoxia/Rho kinase
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Introduction |
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Endothelin-1 (ET-1) is expressed by endothelial and stromal cells of the human penis since the earliest stages of external genitalia development (Granchi et al., 2002) and is considered the most potent stimulator of trabecular smooth muscle cell (SMC) contractility (Andersson, 2001). ET-1 is at least two- to three-log units more potent than the
adrenergic agonists, such as phenylephrine (Phe) or noradrenaline, in stimulating rabbit (Holmquist et al., 1990; Gondre and Christ, 1998; Abeysinghe et al., 2002; Filippi et al., 2002a) and human (Holmquist et al., 1990; Christ et al., 1995; Kim et al., 1996; Filippi et al., 2002a) corpora cavernosa (CC) contractility and calcium fluxes in isolated SMC (Zhao and Christ, 1995; Abeysinghe et al., 2002; Filippi et al., 2002a). It is well established that in human (Granchi et al., 2002), rat (Ari et al., 1996) and bovine (Parkkisenniemi and Klinge, 1996) penile preparations the endothelin A (ETA) receptor subtype mediates the contractile effect of ET-1. On the other hand, the role of the endothelin B (ETB) in penile physiology has been less extensively studied. In animal models, ETB receptor activation induced a nitric oxide (NO)-dependent decrease in penile vascular tone (Ari et al., 1996; Parkkisenniemi and Klinge, 1996). Similar results have been reported in other vascular beds and are related to an ETB-mediated increase in endothelial nitric oxide synthase (NOS) activity (Haynes and Webb, 1998; Schiffrin and Touyz, 1998). However, ETB expression has never been demonstrated in penile endothelial cells (Bell et al., 1995; Parkkisenniemi et al., 2000).
We recently observed that, in preparations of human fetal smooth muscle cells (hfPSMCs), prolonged hypoxia (more than 24 h) dramatically increased ETB receptor expression and responsiveness, as well as ET-1 gene and protein expression (Granchi et al., 2002). Understanding the role of hypoxia in the penis is relevant because it is an exceptional vascular bed, residing in a venous-like oxygen tension (25–40 mmHg) for at least 21 out of 24 h daily. Only during sleep- or sexual-related erections does the increased arterial blood flow allow CC oxygen tension to achieve arterial values of 
90–100 mmHg (Kim et al., 1993; Sattar et al., 1995; Nehra et al., 1996; Brown et al., 1997). There is evidence that such periodic oxygenation is important for the maintenance of normal penile function and that prolonged hypoxia can alter responsiveness to vasoactive agents (Kim et al., 1996, 1998; Moon et al., 1999; Angulo et al., 2003). This hypoxia-induced altered responsiveness may be due to a switch in receptor expression for vasoactive agents (Granchi et al., 2002) or to a change in the contractile machinery (Kim et al., 1996), favouring compensatory vasodilation. We report for the first time in this study the immunolocalization of ETA and ETB receptors in rabbit and human (fetal and adult) penile preparations as well as the effect of prolonged 24 h hypoxia on penile responsiveness to ET-1.
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Chemicals
Phenylephrine HCl (Phe), acetylcholine (ACh), Nw-nitro-L-arginine methyl ester hydrochloride (L-NAME), Dowex 50WX8-400 resin, L-arginine HEPES (sodium salt), HEPES (free acid), minimum essential medium (MEM), phosphate buffered saline (PBS), bovine serum albumin (BSA), EDTA, glutamine, antibiotics, collagenase type IV, reagents for immunohistochemistry and SDS–PAGE were purchased from Sigma (St Louis, MO). EGM-2-MV and EGM-2-MV SingleQuots" were purchased from Clonetics (BioWhittaker, Walkersville, MD) and plasticware for cell cultures was purchased from Falcon (Oxnard, CA); ET-1, the ETB-selective antagonist N-cis-2,6-dimethyl piperidinocarbonyl-
-MeLeu-D-Trp(MeOCO)-D-Nle-OHNa (BQ 788) and the ETB-selective agonist Suc-[Glu9, Ala11,15]-endothelin-1 (8, 21) (IRL1620) were purchased from Novabiochem (Switzerland). [3H]arginine was purchased from Amersham Life Science Ltd (Buckinghamshire, UK). Oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) and trans-4-[(1R)-1-aminoethyl]-N-4-pyridinylcyclohexanecarboxamide dihydrochloride (Y27632) were purchased from Tocris Cookson Ltd (Bristol, UK); polyclonal antibodies, anti-human ET-A (ETA) receptors and anti-human ET-B (ETB) receptors, purified rabbit IgG affinity, were purchased from Assay Design, Inc. (Ann Arbor, MI). The BM enhanced-chemiluminescence system was purchased from Roche Diagnostics (Milan, Italy). The protein measurement kit was from Bio-Rad Laboratories, Inc. (Hercules, CA). Antibody against RhoA (26C4) sc-418 was purchased from Santa Cruz Biotecnology, Inc. (Santa Cruz, CA). Antibody against Rho kinase (ROCK-1) was purchased from BD Transduction Lab (Heidelberg, Germany). The substances were dissolved daily in double-distilled water, and further dilutions to the final concentrations were made in Krebs solution.
Corpora cavernosal tissue preparations
CC were obtained from New Zealand white rabbits weighing 3 kg. The animals were killed by a lethal dose of pentobarbital. Tissue specimens were fresh frozen for RNA preparation. The penis was removed and the CC were carefully dissected free from the tunica albuginea. For in-vitro contractility studies, CC preparations were immediately placed and maintained in cold Krebs solution until the beginning of the experiments.
In-vitro contractility
The CCs were cut into three to four strips (0.2x0.2x0.7 cm). Strips were vertically mounted under 1.8 g resting tension in organ chambers containing 10 ml of Krebs solution at 37°C, gassed with 95% O2 and 5% CO2 at pH 7.4. The solution had the following composition (mmol/l): NaCl, 118; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; NaHCO3, 25; CaCl2, 2.5; glucose, 10. The preparations were allowed to equilibrate for at least 90 min; during this period the bath medium was replaced every 15 min. Changes in isometric tension were recorded on a chart polygraph.
Responses to Phe, to ET-1 and Y27632, such as the relaxant effect induced by different IRL1620 concentrations, were expressed as a percentage of the maximum KCl (80 mmol/l)-induced response, taken as 100%. The high potassium salt solution was made by equimolar substitution of sodium by potassium. Drug cumulative concentrations were added, at 7 min intervals, to the bath, to obtain a concentration-dependent curve. A 15–30 min pre-treatment with selected antagonists and/or inhibitors was performed before repeating the concentration–response curve for ET-1.
The effect of ET-1 was also tested in rabbit CC after endothelium deprivation in the absence and in the presence of a 24 h hypoxic pre-treatment. Hypoxic treatment was performed by incubating rabbit CCs in Tyrode solution in a humified incubator with 5% CO2 and 3% O2 for 24 h. Normoxic treatment was performed by incubating rabbit CCs in a Tyrode solution continuously bubbled with a 95% O2, 5% CO2 gas mixture during the 24 h incubation.
The presence of functional endothelium was assessed by testing the vasodilator effect of ACh; the preparations in which ACh (1 µmol/l) reduced the tone by <40% were not used for the study. To remove endothelium, CC strips were rubbed between the thumb and index finger for 20 s (Saenz de Tejada et al., 1988). The lack of a relaxation response to ACh in preconstricted preparations indicated that the procedure was successful.
Immunohistochemistry
Immunohistochemical studies were carried out on deparaffinized and rehydrated sections as previously described (Crescioli et al., 2003). The specimens were subsequently exposed to a 0.3% hydrogen peroxide–methanol solution to quench endogenous peroxidase activity. Slides were rinsed in tap water, then immersed in EDTA (pH 8) and microwaved for 20 min at 350 W to enhance antigen exposure. The primary antibodies against ETA and ETB receptors were appropriately diluted (2 µg/ml) in PBS, added to the slides and incubated overnight at 4°C. Although these antibodies were raised against the human ET receptors they recognized equally well rabbit ET receptors as derived from Western blot studies (data not shown). Sections were rinsed in PBS, incubated with biotinylated secondary antibodies, and finally incubated with streptavidin–biotin peroxidase complex (LSAB kit; DAKO Corp., Carpinteria, CA). The development reaction of the product was performed using diaminobenzidine tetrahydrochloride as chromogen. Slides were washed in running tap water followed by dehydration and coverslip mounting. Controls were performed by processing slides lacking the primary antibodies or stained with the corresponding non-immune serum. The slides were evaluated and photographed using a Nikon Microphot FX microscope (Nikon, Tokyo, Japan).
Penile cell cultures
Human fetal penile cells were prepared from seven samples of fetal male external genitalia (11–12 weeks of gestation) obtained after spontaneous or therapeutic abortion. Legal abortions were performed in authorized hospitals, and certificates of consent were obtained. The Local Ethical Committee of the University Hospital gave approval for the use of human material (Azienda Ospedaliera Careggi, protocol no. 6783-04). Human fetal penile smooth muscle cells (hfPSMCs) were prepared as previously described (Granchi et al., 2002). For human fetal penile endothelial cells (hfPECs), penile tissues were cut and treated with 0.1 mg/ml bacterial collagenase for 1 h at 37°C. Fragments were then mechanically dispersed, collected, washed in PBS and seeded onto 60 mm diameter plastic dishes in microvascular endothelial cell growth medium-2 (EGM-2-MV; Clonetics)and supplemented with ‘ready-to-use’ aliquots of EGM-2-MV SingleQuots (containing FBS, hydrocortisone, hFGF-b, VEGF, IGF-I, ascorbic acid, hEGF and GA-1000) in a fully humidified atmosphere of 95% air and 5% CO2. As cells began to emerge (within 24–48 h), plates were observed under an inverted microscope to identify endothelial cell groups. Endothelial cells were then isolated using cloning cylinders, sterile silicon glue and 0.25% trypsin solution B (PBI, Milan, Italy) and plated onto gelatin-coated T25 flasks (Ikawa, Japan). Two hours later, non-adherent cells were removed, and adherent endothelial cells remained. After 4–5 days, hfPEC reached confluence and were split 1:3 in gelatin-coated T25 flasks and grown in the aforementioned EGM-2-MV medium. Cells from the primary cultures as well as from the following passages were utilized for characterization using specific markers. Immunocytochemical assays demonstrated that a few groups of cells (5% of total) from primary cultures were positive for von Willebrand factor (vWF), whereas cells obtained from gelatin-coated T25 flasks (second and third passages) were positive (>90%) for anti-vWF, anti-CD34 and anti-vimentin antibodies (Sigma), and negative for antibody anti-cytokeratin and anti- smooth muscle actin (Sigma).
HfPECs were used for experiments starting from the third passage and within the sixth passage. Experimental hypoxia was realized as previously described (Granchi et al., 2002).
RT–PCR
Total penile RNA was retrotranscribed and then amplified using the Super script One Step RT–PCR kit (Life Technologies, Milan, Italy). Briefly, total RNA (500 ng) was retrotranscribed for 30 min at 50°C, denatured for 2 min at 95°C and amplified for the appropriate number of cycles (35 cycles for eNOS; 23 cycles for GAPDH; 23 cycles for Rho kinase (ROK); 21 cycles for RhoA; 28 cycles for ETA and ETB; 23 cycles for -actin). The integrity of total RNA was verified by performing RT–PCR for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) housekeeping gene for human and -actin for rabbit. The sequences of the used primers are shown in Table I.
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Northern blot analysis
Total RNA was extracted from cultured cells with Rneasy Mini Kit (Qiagen, Valencia, CA), whereas the Rneasy Midi Kit from the same source was used to extract total RNA from human tissues. RNA concentrations were determined by spectrophotometric analysis at 260 nm. For northern blot analysis 20 µg of total RNA were fractionated in a 1.2% agarose gel containing 8% formaldehyde. RNA was then transferred onto a nylon membrane (Hybond-n; Amersham, Milan, Italy) and baked at 80°C for 2 h. Membranes were prehybridized for 1 h and hybridized overnight at 65°C with Church and Gilbert buffer solution as described previously (Maggi et al., 1995). The probes for the detection of ROK and RhoA mRNA have been prepared from RT–PCR products, as previously described (Filippi et al., 2002b). The probes were labelled with deoxycytidine 5' [
-32P]triphosphate by a random priming kit (Roche Diagnostics) and chromatographed (Nu-Clean D25 disposable spun columns; IBI, New Haven, CT) before use. The hybridized nylon membranes were submitted to autoradiography using Hyperfilm-MP (Amersham) and a Kodak X-Omatic Regular intensifying screen at –80°C for various exposure times.
SDS–PAGE and western blot analysis
To evaluate the presence of ETA, ETB, RhoA and ROK, in both hfPECs and hfPSMCs, cultured cells were washed and scraped in PBS. After centrifugation, pellets were extracted in lysis buffer (20 mmol/l Tris, pH 7.4, 150 mmol/l NaCl, 0.25% NP-40, 1 mmol/l Na3VO4, 1 mmol/l PMSF) on ice for 2 h. After protein measurement, aliquots containing 30 µg of proteins were diluted by reducing 2x SB (Laemmli’s sample buffer: 62.5 mmol/l Tris pH 6.8, 10% glycerol, 2% SDS, 2.5% pyronin and 100 mmol/l dithioteithrol) and loaded onto 10% (for ETA and ETB) and 12% (for RhoA and ROK) polyacrylamide–bisacrylamide gels. After SDS–PAGE, proteins were transferred to nitrocellulose membranes. Membranes were blocked for 2 h at room temperature in 5% BSA–Tween Tris-buffered saline (TTBS) (0.1% Tween-20, 20 mmol/l Tris, 150 mmol/l NaCl), washed in TTBS and incubated for 2 h with rabbit anti-ETA, ETB antibodies (1:100 in 5% BSA–TTBS), mouse anti-RhoA antibody (1:1000 in 5% BSA–TTBS) and mouse anti-ROK antibody (1:250 in 5% BSA–TTBS), followed by peroxidase-conjugated secondary IgG (1:4000 in 5% BSA–TTBS). Finally, reacted proteins were revealed by a BM enhanced-chemiluminescence system (Roche Diagnostics).
NOS activity
NOS activity was tested in hfPSMC and hfPEC monolayers according to the previously described method (Filippi et al., 2001). HfPSMCs and hfPECs cultured in either normoxic or hypoxic conditions were used. Briefly, the enzyme activity was evaluated by measuring the amount of L-[3H]citrulline produced after administration of L-[3H]arginine. Equilibration for 20 min at 37°C with HEPES buffer was followed by cell incubation for 30 min with 10 µmol/l L-arginine and 20 min with 1 µCi of L-[3H]arginine. Cells were exposed to the tested drug for 5 min at 37°C, and then cold HEPES buffer was added to stop the reaction. Following the addition of ethanol and of 10 mM HEPES-sodium at pH 5.5, the amount of L-[3H]citrulline produced was assayed by liquid scintillation counting after elution through a resin column (Dowex AG50WX-8 activated sodium-form). NOS activity was measured as recovered radioactivity and expressed as counts per minute (c.p.m.) per milligram of protein. Each experiment was performed in duplicate.
Statistical analysis
Results are expressed as means ± SEM for n experiments. Statistical analysis was performed using the t-test for paired or unpaired data, with ANOVA followed by Fisher’s test to evaluate the differences among groups, and a P < 0.05 was taken as significant. Half-maximal response effective concentrations (EC50) and half-maximal response inhibitory concentration (IC50) values were calculated using the ALLFIT computer program (De Lean et al., 1978).
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Figure 1 shows the immunolocalization of ET-1 receptor subtypes in the rabbit and human penis. Results obtained in human fetal external genitalia are also shown. We found a diffuse expression of the ETA receptor subtype in all the penile tissue examined. The endothelial and SMCs of the blood vessels and CC were extensively labelled in both the adult (rabbit and human) and in the fetal penis. Conversely, ETB receptor positivity was almost exclusively confined to the endothelial layer. Only a faint positivity was observed in scattered stromal cells.
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Figure 2 shows the effect of increasing concentrations of ET-1 in rabbit CC strips exposed for 24 h to a normal oxygen tension or to an experimental condition of hypoxia (3% O2). In preparation with endothelium (Figure 2A), ET-1 induced the previously reported (Filippi et al., 2002a; Granchi et al., 2002) dose-dependent increase in tension with EC50 = 19 ± 2.3 nmol/l and Emax = 96.9 ± 2.5%. Removal of the endothelium (Figure 2B) did not affect the EC50 (23 ± 1.7 nmol/l) but significantly increased the Emax (145.2 ± 2.9%, P < 0.0001). The experimental hypoxia significantly blunted the Emax values for ET-1 in preparations with (48.8 ± 2.0%, P < 0.0001) and without (90.4 ± 4.0%, P < 0.0001) endothelium, when compared with their respective normoxic controls. The ETB antagonist, BQ788, completely rescued this hypoxia-induced ET-1 hypo-responsiveness in preparations with endothelium (Emax = 89.2 ± 2.3%, P < 0.05 versus normoxia). The ETB block significantly restored ET-1 responsiveness (Emax = 122.66 ± 2.6%, P < 0.0001 versus hypoxia) in hypoxic preparations without endothelium but did not completely restore it to the normoxic values (P < 0.0001 versus normoxia). Conversely, responsiveness to Phe was not affected by changing O2 tension, irrespective of the presence or absence of intact endothelium (Figure 2C).
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To further investigate the role of ETB receptors during hypoxia, we studied the effect of an ETB agonist, IRL1620, on KCl pre-contracted rabbit CC incubated for 24 h at different oxygen tension. IRL1620 dose-dependently relaxed pre-contracted strips. However, during hypoxia (3% O2), ETB receptor-induced relaxation was significantly enhanced (Figure 2D). These findings suggest that hypoxia-induced hypo-responsiveness to ET-1 in rabbit CC is at least partially related to an up-regulation of relaxing ETB receptors. To test this hypothesis, we studied ET-1 receptor subtype expression and distribution in rabbit CC preparations exposed for 24 h to a normoxic or hypoxic (3% O2) environment by RT–PCR and immunohistochemistry (Figure 3). As previously noted in hfPSMCs (Granchi et al., 2002), specific transcripts for the ETB receptors were increased after hypoxia also in rabbit CC, while the ETA ones were unchanged (Figure 3A). A semi-quantitative analysis of multiple experiments indicated that hypoxia induced a statistically significant increase in ETB (P < 0.001) but not in ETA receptors. The morphological changes induced by hypoxia on the distribution of ET-1 receptors in rabbit CC are shown in the lower part of Figure 3. Trabecular stromal cells and SMCs of the blood vessels which, during normoxia, were virtually negative for ETB receptors (Figures 3C and 1E), after 24 h of hypoxia became immuno-positive for ETB (Figure 3D). On the other hand, the pattern of ETA staining did not change between normoxia (Figure 3F) and hypoxia (Figure 3G). Similar results were obtained in at least two other independent experiments. Method control for either ETB or ETA receptors, obtained by omitting the primary antibodies, are reported in Figure 3E and H, for ETB and ETA respectively.
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Because it is generally assumed that ETB-induced relaxation is mediated by the activation of NO transmission, we tested the effect of inhibitors of NO formation (100 µmol/l L-NAME) or action (1 µmol/l ODQ) on hypoxia-induced ET-1 hypo-responsiveness in rabbit CCs. We found that, after 24 h of hypoxia, both L-NAME and ODQ completely restored ET-1 sensitivity to the normoxic control (Figure 4). This finding indicates that, during hypoxia, the decreased contractile effect of ET-1 is mediated by increased NO production and/or activity in penile cells, most probably mediated by the previously described ETB receptor up-regulation (Granchi et al., 2002 and present study). To verify this hypothesis, we studied ETB protein expression and NOS activity in both hfPECs and hfPSMCs obtained from human fetuses. In basal conditions, ETB signal was detectable in endothelial cells but not in SMCs. Conversely, after 24 h hypoxia, ETB-specific bands became apparent in both types of penile cells (Figure 5A). This effect, again, is specific for the ETB receptors, because ETA-specific bands did not change in either hfPECs or hfPSMCs (Figure 5B).
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As shown in Figure 5C, treatment of hfPECs with a selective ETB agonist (IRL1620, 100 nmol/l) increased NOS activity in either normoxia (29% increase, P < 0.05) or hypoxia, although the latter effect was more pronounced (112% increase, P < 0.001). L-NAME administration completely blocked IRL1620 effect in either normoxia (P < 0.02) or hypoxia (P < 0.001) and even decreased NOS activity below the basal levels (normoxia, P < 0.05; hypoxia, P < 0.001). This indicates that IRL1620 stimulates NOS activity through ETB in hfPECs, with a more sustained effect during hypoxia. In normoxic hfPSMCs (Figure 5D), which are virtually negative for ETB receptors (Granchi et al., 2002 and present study), NOS activity was not regulated by IRL1620. However, during hypoxia, IRL1620 increased NO formation in these cells (34%, P < 0.001), an effect that was completely blocked by the simultaneous incubation with L-NAME (P < 0.001; Figure 5D). The latter result indicates that NOS is expressed not only in endothelial cells, but also in SMCs. To verify this point we performed RT–PCR on total mRNA from hfPECs and hfPSMCs, using specific primers for endothelial nitric oxide synthase (eNOS). We found that the eNOS gene is expressed not only in penile endothelial cells, but also in penile SMCs (Figure 5D, insert).
Results obtained in rabbit CC and in human fetal penile cells clearly indicate that decreased ET-1 contractility during prolonged hypoxia is due to an ETB-mediated increase in NO production from endothelial cells. Although hypoxia also induced an increased expression and functional activity of ETB receptors in the smooth muscular compartment (Granchi et al., 2002 and present study), the ETB block did not completely rescue ET-1 sensitivity during hypoxia in endothelium-deprived CC preparations (Figure 2B). This indicates that prolonged hypoxia in penile SMCs activated other counter-regulatory mechanisms, besides an up-regulation of ETB receptors. Therefore, we tested, during experimental hypoxia, the effect of an ROK inhibitor, Y27632, in rabbit CC strips pre-contracted with ET-1, in both preparations with and without a functional endothelium. As previously reported (Figure 2), hypoxic preparations responded less to ET-1 than the normoxic counterparts, both in the presence (Figure 6A) and in the absence (Figure 6B) of a functional endothelium. Y27632 dose-dependently relaxed all the preparations, although to a different extent. This is more evident (see inserts to Figure 6A and B) after normalization of the maximal response to the highest dose of ET-1 employed (100 nmol/l). In the presence of a functional endothelium, Y27632 completely relaxed ET-1-stimulated CC strips with similar IC50 values in both hypoxic and normoxic preparations (shared IC50 = 51 ± 1.1 nmol/l; Figure 6A). In rabbit CC strips deprived of endothelium and in normoxic conditions, Y27632 still induced a complete relaxation, but with an IC50 significantly higher than intact strips (IC50 = 353.7 ± 54 nmol/l, P < 0.001 versus normoxic preparation with endothelium). The hypoxic state significantly blunted the relaxant effect of Y27632 on endothelium-deprived, ET-1 pre-contracted CC strips (normoxia Emax = 102.9 ± 2.2; hypoxia Emax = 76.3 ± 3.1; P < 0.0001), without significant (P = 0.56) changes in the IC50 values (shared IC50 = 445 ± 142 nmol/l). Because we performed cumulative dose–response curves with the specific ROK inhibitor, Y27632, a decreased responsiveness of the penile muscular cells to Y27632 during hypoxia should indicate decreased enzyme availability. Preliminary experiments indicated that during hypoxia the protein expression of RhoA and of its regulated kinase, ROK, was down-regulated in smooth muscle but not in endothelial cells (Figure 7A and B). Therefore, we investigated the gene expression of RhoA and its regulated kinase, ROK, in hfPSMCs exposed to experimental hypoxia (1.5% O2) by RT–PCR. Amplification with RhoA- and ROK-specific primers resulted in 244 and 512 bp products respectively, which were apparently reduced after prolonged hypoxia (Figure 8A). Northern blots were further performed with cDNA probes prepared from RhoA and ROK cDNA fragments amplified by PCR. A single major band of the expected molecular weight for RhoA (2.2 kb) and ROK (6.6 kb) was obtained. Expression of both genes apparently increased as a function of time, irrespective of the different oxygen tension (Figure 8B), most probably an effect of serum deprivation. We found that hypoxia did not change RhoA or ROK gene expression at the earliest times investigated (up to 9 h; Figure 8B). Conversely, later on, hypoxia significantly reduced Rho-A (24 h: 26%, P < 0.05, n = 6; 48 h: 44%, P < 0.001, n = 4; Figure 8C) and ROK (24 h: 34%, P < 0.001, n = 6; 48 h: 35%, P < 0.05, n = 4; Figure 8D) mRNA abundance.
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This is the first report about the immunolocalization and activity of ET-1 receptors in the human and rabbit penis and their modulation during hypoxia, a rather common condition for penile cells.
Although the presence of both ETA and ETB receptors in the human penis has been previously demonstrated by binding (Saenz de Tejada, 1991) and RT–PCR (Granchi et al., 2002) studies on human CC homogenates, their exact localization and function has never been investigated. Confirming previous data in the rat (Bell et al., 1995) and rabbit (Chang et al., 2003), we found that the human adult and fetal penis also expresses ETA receptors. The ETA receptor is localized in the endothelial and SMCs, where it probably mediates the well characterized calcium mobilization (Filippi et al., 2002a) and consequent in-vivo (Dai et al., 2000; Kim et al., 2002) and in-vitro (Granchi et al., 2002; Kim et al., 2002) contractile effect of ET-1. We now provide original evidence that ETB receptors are also present and functionally active in the human penis and mainly localized in the endothelial compartment. Although it is generally accepted that endothelial cells of several vascular beds express the ETB receptor and that this receptor is coupled with NO formation (Bagnato and Spinella, 2002; Davenport, 2002), its presence in the penis was suspected (Ari et al., 1996) but never demonstrated (Bell et al., 1995; Parkkisenniemi et al., 2000). We found that not only adult but also fetal penile endothelial cells express ETB and respond to ETB stimulation with increased NOS activity. It is possible that the endothelial ETB receptor plays a physiological relaxant role, because in rabbit penile strips, IRL1620 relaxed pre-contracted preparations and removal of the endothelium increase the contractile response to ET-1. On the other hand, there was only a faint labelling for ETB in the muscular compartment of both rabbit and human (adult and fetal) penis. Accordingly, isolated hfPSMCs were virtually negative for ETB (Granchi et al., 2002 and present study). In addition, hfPSMCs were not responsive to IRL1620 in terms of NO production, although, as previously demonstrated (Block et al., 1989), eNOS gene expression was detectable by RT–PCR. This scenario changes consistently after prolonged (24 h) hypoxia. In hypoxic hfPSMCs, ETB protein is expressed and IRL1620 significantly increases NOS activity. This finding agrees with the previously described hypoxia-induced up-regulation of ETB receptor in hfPSMCs (Granchi et al., 2002) and with the present report of increased ETB gene expression and protein staining in hypoxic rabbit CC. In particular, SMCs of the rabbit penis, almost negative for ETB in normoxic conditions, become positive after 24 h hypoxia. However, the most dramatic changes were observed in the endothelial compartment. There was a 4-fold increase in IRL1620-induced NOS activity in hypoxic hfPECs, when compared with normoxic controls. According to this hypoxia-induced up-regulation of ETB sensitivity, the contractile effect of ET-1 is drastically reduced in hypoxic CC strips and completely restored by blocking either NO formation (L-NAME) and activity (ODQ) or ETB receptors (BQ788). Our data, although based on qualitative methods such as immunohistochemistry, and on results in cultured cells, suggest that, during normoxia, ETB is prevalently expressed by the endothelium, while, during hypoxia, it is also expressed by the smooth muscle. Hence, ETB receptors in penile cells are regulated by oxygen tension and coupled to NO formation, which, in turn, mediates relaxation and vasodilation.
The contractile effect of ET-1 is also significantly blunted in endothelium-deprived CC strips during 24 h hypoxia. However, in this experimental condition, BQ788 only partially restores ET-1 sensitivity. This implies that hypoxia induces, in addition to the ETB up-regulation, other molecular changes impairing ET-1 responsiveness in CC SMCs. ET-1-induced contractility is not only related to an increase in intracellular calcium concentration and myosin light chain kinase (MLCK) activation but also to the abundance and function of a calcium-sensitizing pathway involving the geranylgeranylated protein RhoA and its downstream target ROK. ROK is a serine/threonine kinase that phosphorylates the regulatory subunit of smooth muscle myosin phosphatase, inhibiting its activity, and thereby allowing MLCK activity independently of changes in cytosolic calcium levels (Wettschureck and Offermanns, 2002). RhoA-dependent calcium sensitization constitutes a major component of the sustained contractile tone of the CC, allowing the penis to reside in its flaccid (resting) state (Chitaley et al., 2001; Mills et al., 2001). Interestingly, both RhoA and ROK are apparently more expressed and active in penile tissue than in other SMCs (Chitaley et al., 2001; Wang et al., 2002). We found that, after 24 h hypoxia, rabbit CC strips deprived of the endothelium and pre-contracted with ET-1 are less responsive to the ROK inhibitor Y27632 than the normoxic strips. Especially, during hypoxia, the Emax, but not the IC50, for Y27632 is reduced, suggesting a lower availability of kinases. Because the sensitivity to Y27632 relaxation of oxytocin-induced contraction increased at term pregnancy in rat myometrium parallel with an up-regulation of RhoA and ROK expression (Tahara et al., 2002; Cario-Toumaniantz et al., 2003), we hypothesize that the decreased sensitivity to Y27632 induced by hypoxia may be caused by a down-regulation of ROK or its main regulator, RhoA, expression. Therefore, we measured ROK and RhoA gene expression at different times and found that after prolonged hypoxia (24 h), but not before, ROK and RhoA expression is reduced. At this latest time their relative protein expression is also reduced. Although mechanisms of activation of RhoA and ROK have been extensively studied in several tissues including the penis (reviewed in Chitaley et al., 2003), little is known about modulation of the expression in these kinases. RhoA and ROK gene and protein expression is increased in the penis in some experimental pathological conditions such as diabetes (Chang et al., 2003) and hypogonadism (Wingard et al., 2003). Recently, it has been demonstrated that NO/cGMP kinase positively regulates RhoA expression in SMCs through stimulation of rhoA transcription and protein stability (Sauzeau et al., 2003). In addition, rats chronically treated with an NOS inhibitor showed a 70% decrease in rhoA gene expression in the aorta (Sauzeau et al., 2003). The same authors suggest that a tonic release of NO is absolutely necessary to maintain RhoA expression in vascular SMCs (Sauzeau et al., 2003). Accordingly, we found that removing the endothelium in rabbit CC, i.e. the main source of NO, significantly decreases Y27632-induced relaxation. Y27632 hypo-responsiveness is further increased by chronic hypoxia in endothelium-deprived preparations, probably because NOS activity is reduced in the penis (Kim et al., 1993), as well as in other tissues (Kantrow et al., 1997; Whorton et al., 1997; Murata et al., 2001) during hypoxia, leading to decreased NO and cGMP availability. Therefore, it is possible that prolonged hypoxia, possibly through inhibition of NO formation, is associated with a counter-regulatory reduction of the expression of RhoA/ROK-dependent calcium sensitization, which allows decreased responsiveness to contractile agents, such as ET-1, relaxation and a compensatory increase in blood flow. It is interesting to note that these two hypoxia-related counter-regulatory mechanisms are induced simultaneously and only after protracted exposure to low oxygen tension. The up-regulation of NO-coupled endothelial (and smooth muscle) ETB receptors, induced by low oxygen tension, also helps to induce vasodilation and re-oxygenation. We hypothesize that when more NO is formed, because of ETB over-expression and re-oxygenation, the relative block to ROK pathway expression will be removed and the normal contractile, flaccid, state restored. The penis in the flaccid state is exposed to a relative hypoxia (20–40 mmHg) which is randomly interrupted by sudden increases in oxygen tension (80–100 mmHg) linked to sexual activity and to spontaneous nocturnal erections (Kim et al., 1993). Sex-related and unrelated (nocturnal) erections are proposed to have a protective role on the trabecular tissue, by increasing tissue oxygenation and NO synthesis (Kim et al., 1993, 1998) and decreasing pro-fibrotic factors, such as TGF-ß1 (Daley et al., 1996a,b; Nehra et al., 1996). Hence, there is the concept that ‘erections may be good for preserving erections’ (Moreland, 1998). Interestingly, penile erections have been measured by ultrasound as early as at the 11–12th weeks of gestation (Pedreira et al., 2001) and discrete episodes of nocturnal penile erections are clearly evident also at term gestation (Shirozu et al., 1995). Our results, showing that anti-hypoxic NO-related (ETB) and unrelated (RhoA and ROK) pathways are switched on to decrease the contractile effect of ET-1 after more than physiological (24 h) hypoxia, might help in the understanding of how the penis protects itself from protracted hypoxia.
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The authors wish to thank Mrs Mary Forrest for manuscript revision. This research was supported by grants from NicOx (Sophia Antipolis Cedex, France). This paper was supported by grants from LILLY ITALIA (Sesto Fiorentino, Florence, Italy) and by a grant from COFIN2002-MIUR (Progetti di Ricerca Scientifica di Rivalente Interesse Nazionale).
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Submitted on April, 2003; resubmitted on July 22, 2003. accepted July 28, 2003
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