Prostaglandin E1 leads to penile erection, mainly via prostaglandin E2 (EP) receptors. This study aimed to identify the expression profile of EP receptor genes in human corpus cavernosum. Using the quantitative real-time reverse transcription polymerase chain reaction, the mRNA levels of EP receptor subtypes were measured. In addition, expressions of EP receptor subtype proteins were determined by immunohistochemical method. Among the four subtypes, EP4 receptor mRNA expression was the highest, and EP2 receptor mRNA followed, whereas EP1 and EP3 receptor mRNAs were hardly observed. Expression level of EP4 receptor mRNA was significantly higher than that of EP2 receptor mRNA. Expression of both EP2 and EP4 receptor proteins were clearly detected in the cavernous smooth muscle. These results may suggest that EP4 receptor plays an important role among four EP receptor subtypes for relaxation of smooth muscle in the human corpus cavernosum.
Treatment strategy for erectile dysfunction (ED) has been greatly changed by the launch of phosphodiesterase type 5 (PDE5) inhibitors, and PDE5 inhibitors have became the first line therapy for ED; however, PDE5 inhibitors do not have an effect for about 30% of ED patients.1 Hence, intracavernous injection (ICI) therapy has become the main treatment option in the second line therapy. Over the last two decades, ICI therapy using vasoactive drugs, such as papaverine, phentolamine and prostaglandin E1 (PGE1), has been established as an effective and safe treatment for ED.2 PGE1 is widely used for ICI therapy and as a pharmacotest for screening of arteriogenic ED. Although ICI with PGE1 is associated with a 73–94% success rate,3 there are side effects including pain in the penis, penile fibrosis and priapism. The prevalence of penile fibrosis was 25% after 1 year therapy with PGE1,3 and a previous study demonstrated that PGE1 had 40% cytotoxity to cultured human cavernosal cells at the therapeutic dose.4
In the relaxation of the smooth muscle, PGE1 works mainly via the prostaglandin E2 receptor (EP receptors). After combining to EP receptor, PGE1 acts by increasing the synthesis of cyclic-adenosine monophosphate (cAMP) as a second messenger, causing a decrease of intracellular calcium ion, and consequently eliciting cavernosal smooth muscle relaxation, vasodilation and erection.3
EP receptor has four subtypes, EP1, EP2, EP3 and EP4 receptors,5 and the work of all EP receptors has not been clarified. Hence, the purpose of this study was to identify the expression pattern of EP receptor genes in the human corpus cavernosum by using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR). Real-time RT-PCR is a sensitive and rapid tool for quantifying mRNA expression with small tissue quantity. In addition, expressions of proteins of EP receptor subtypes were determined by immunohistochemical method.
We obtained human penile cavernous tissues from six patients who underwent penile operations in our hospital. Details about the patients are summarized in Table 1. None of the patients had history of ED according to patient-oriented questionnaire. The protocol of the study adhered to the tenets of the Helsinki Declaration for experiments involving human tissue.
Isolation of total RNA
Tissues obtained were immediately frozen, and stored at −80°C in RNase-free container. Frozen cavernosum tissue was homogenized, and total RNA was extracted by a single-step method, based on guanidine isothiocyanate extraction, using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. Any DNA contamination was removed with DNase I treatment. Reverse transcription was carried out using 1st Strand cDNA Synthesis Kit for RT-PCR (Roche, Basel, Switzerland) with random primer. The cDNA was stored at −40°C until analysis.
Quantitative real-time PCR
The quantitative assessment of mRNA levels was performed using a detection system SmartCycler (Cepheid, Sunnyvale, CA, USA) and analyzed with SmartCycler software (Ver. 1.2d). SmartCycler dedicated to the real-time monitoring of fluorescent dyes such as SYBR Green I (Molecular Probes, OR, USA). This dye is added to the PCR mixture and is fluorescent only when bound to double-stranded DNA (dsDNA), allowing measurement of the progressive accumulation of the specifically amplified product in the course of the PCR.6
Specific primers were designed to amplify unique regions in the human EP receptor subtypes suitable for real-time PCR (Table 2). The length of the amplicons were kept as close as possible to 200 bp, and the melting temperature of the primers was chosen between 60 and 62°C. Specificity was checked in the BLAST search (http://www.ncbi.nlm.nih.gov/blast/).
Human renal tissue was used as a positive standard since expressions of EP1 through EP4 have been confirmed.7 This normal renal tissue was obtained under informed consent to the patient with renal cell cancer at the time of nephrectomy. Total RNA was extracted from the renal tissue and cDNA was synthesized by RT. Next, standard PCR was carried out by using specific primer pairs, EP1, EP2, EP3, EP4 and β-actin (Table 2). Agarose gel electrophoresis was carried out, and the amplification of the target product was confirmed by size fractionation. Amplified DNA was extracted from the agarose gel using QIAEX II Gel Extraction Kit (Qiagen), and that concentration was measured. The DNA was serially diluted from 100 pmol to 0.1 fmol, and used for derivation of the standard curve. To correct for differences in cDNA load between the different samples, the target PCR may be normalized to a reference PCR, involving a selected endogenous housekeeping gene. We selected β-actin as the housekeeping gene.8 The serial dilutions of positive standard and samples were made to react on the same PCR condition. TaKaRa Ex Taq R-PCR Version (Takara biomedical, Shiga, Japan) was used as a PCR premix reagent, according to the manufacturer's instructions. This reagent contained Taq enzyme. In total, 10 pmol of each primer was added, and cDNA samples and buffer water as negative control were added in 2 μl volume. Then the final reaction mixture was made in a total volume of 25 μl. The PCR conditions were as follows. An initial step of 95°C for 30 s was used to activate Taq polymerase. Cycling conditions were: melting step at 95°C for 10 s and annealing-extension at 60°C for 20 s, with 40–50 cycles. All reactions were performed in duplicate by using each set of primers. With the progress of the PCR, amplification curve was drawn, and the number of PCR cycles which reached a threshold concentration was found automatically by SmartCycler software. Then a calibration curve was made from the amplification curve of the standards, and the initial concentration of sample was estimated9 (Figure 1). The above PCR was carried out by using each primer pair.
The ratio of EP receptor subtypes over β-actin was calculated to account for variability in the initial concentration of the total RNA and the conversion efficiency of the RT step.10 Data are expressed as mean±s.e. Using Student's t-test, P<0.05 was considered statistically significant.
Tissues obtained were immediately kept in Tissue-Tek OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA) and stored at −80°C until cryostat sectioning. Tissues were cut at 7 μm and mounted onto aminopropyl-triethoxysilane (APS)-coated glass slides (Matsunami glass, Osaka, Japan). After air drying, slides were fixed in acetone for 10 min at 4°C. Next procedure was performed using ENVISION kit (Dako Corp., Carpinteria, CA, USA), according to the manufacture's protocols. Endogenous peroxidase activity was blocked with 0.03% hydrogen peroxide, and nonspecific staining was blocked with blocking reagent X0909 (Dako). After washing with Tris-buffered saline (TBS), slides were incubated for 120 min at 37°C with specific antibodies for EP1 (Catalog No. 101740), EP2 (No. 101750), EP3 (No. 101760) and EP4 (No. 101775, c-terminal antibody) receptor (Cayman chemical Co., Ann Arbor, MN, USA). These antibodies were diluted 1:100. For the negative control, the primary antibody was replaced by preimmune rabbit serum. After washing with TBS, slides were incubated with a peroxidase labeled anti-rabbit antibody for 60 min at 37°C. After washing with TBS, the color reaction was performed by the addition of substrate solution 3-amino-9-ethylcarbazole (AEC). This reaction was stopped by washing, and the slides were counterstained with hematoxylin for 40 s and finally mounted in Glycergel (Dako) mounting medium. Finally, we examined AEC immunolabeling results with a light microscope.
Positive staining of antibody for each subtype (EP1–EP4) has been confirmed using normal human renal tissue as a positive standard since expressions of EP1 through EP4 have been confirmed.7
Quantitative real-time PCR
Standard PCR was carried out with the RNA extracted from normal human renal tissue, using each primer. Agarose gel electrophoresis was carried out to confirm that the PCR products were of the expected size. Using these PCR products from the kidney as positive standard, quantitative real-time PCR was performed with a sample twice each. The real-time detection of dsDNA allows construction of a melting curve at the end of the PCR run by ramping the temperature of the sample from 60 to 95°C while continuously collecting fluorescence data. The melting curves show a single peak and the melting temperatures for EP1, EP2, EP3, EP4 and β-actin were 93, 85, 88, 90 and 90°C. Agarose gel analysis showed a single band at the expected size (Figure 2). Both melting curves and agarose gel analysis of all EP receptors and β-actin did not reveal any accumulation of primer dimer.11 Negative control experiments without samples did not yield any product. Calibration curve was drawn from an amplification curve of positive standard, and the r2 was 0.998 for EP1, 0.997 for EP2, 0.999 for EP3, 0.996 for EP4 and 0.998 for β-actin. According to the data of the calibration curve, we estimated the initial concentration of samples. The ratio of EP receptor subtypes over β-actin was calculated to account for variability in the initial concentration of the total RNA and the conversion efficiency of the RT step. So, expression of each mRNA of EP1, EP2, EP3 and EP4 was quantified. Results of two times procedure were approximately similar, and the average was calculated in Table 3.
EP receptor mRNA expressions showed interindividual differences, and average of six samples were calculated. EP receptor mRNA expressions were as follows; EP1/β-actin=8.89±6.74 × 10−7, EP2/β-actin=1.81±0.85 × 10−2, EP3/β-actin=5.34± 5.26 × 10−4, EP4/β-actin=1.12±0.45. As written in Table 3, EP4 receptor mRNA expression in the corpus cavernosum of all six cases was the highest among four subtypes, followed by EP2 receptor, and mRNA expressions of EP1 and EP3 receptors were hardly detected. EP4 receptor mRNA level was significantly higher than EP2 receptor mRNA level (P<0.05).
Light microscopic immunolabeling results are shown in Figure 3. Using antibodies against EP1, EP2, EP3 and EP4, expression of both EP2 and EP4 receptor proteins were clearly detected in cavernous smooth muscle; however, neither EP1 nor EP3 receptor protein could be detected. The negative control was not stained using any one of antibody. Furthermore, the staining density of EP4 receptor protein was considered higher than that of EP2 (Figure 3). The distribution of EP2 and EP4 proteins was similar. Either endothelial cells or smooth muscle cells of blood vessels in the cavernous tissue was hardly stained. Tunica albuginea of corporum cavernosorum was not stained. Immunohistochemical finding was similar among six samples.
This expression pattern of protein of EP receptor subtypes was well reflected by expression pattern of mRNA of EP receptor subtypes.
PGE2 shows very diverse actions in various kinds of organs and tissues. In diverse actions, some cannot be easily correlated. For example, PGE2 causes contraction or relaxation in smooth muscle and it works acceleratively or restrainingly in transportation of water and sodium ion. In this way, diverse actions can be explained by the fact that PGE2 receptor consists of four subtypes: EP1 through EP4. The four EP receptor subtypes differ in structures, binding profiles, and signal-transduction pathways, which couple to EP receptors via G-protein.5 EP2 and EP4 receptors activate adenylate cyclase through Gs and stimulate increases in the intracellular concentration of cAMP. Activation of EP1 receptor is associated with increases in intracellular calcium concentration, and EP3 receptor generally couples to Gi and inhibits increases in intracellular cAMP levels. As for the function of subtypes of EP receptor to the smooth muscle, it is clarified that EP2 and EP4 work for relaxation and that EP1 and EP3 work for contraction. However, the role of EP4 in penile erection remains to be clarified.
In the present study, we analyzed the expression and regional distribution of mRNAs and proteins of four EP receptor subtypes in human corpus cavernosum tissues. This combined approach confirmed the presence and different expression patterns of both messages and proteins of four EP receptor subtypes. The result shows that EP1 and EP3 receptor mRNAs were low or below the detection limit as measured, and EP2 and EP4 receptor mRNAs were mainly observed. In spite of the possibility of individual deviations, EP4 mRNA showed about 60 times higher expression levels than EP2 receptor mRNA. As for the protein expression by immunohistochemistry, a similar expression pattern was shown. This result of protein expression experiment may support the result of the gene expression. Our present result is the first and important evidence of quantified mRNA manifestation of EP receptors in the human corpus cavernosum.
In this study, the experimental procedure of mRNA expression was quantitative real-time PCR method. Real-time RT-PCR can do more correctly quantitative analysis than classic RT-PCR. Quantification of mRNA by the real-time RT-PCR method is used widely, and it has been established.11
Concerning expression of EP receptor in human corpus cavernosum, there are some previous reports using pharmacological method. Angulo et al.12 reported that EP2 receptor and/or EP4 receptor were related to relaxation of smooth muscle in human corpus cavernosum. In this pharmacological experiment, butaprost was used as the EP2 selective agonist by this study. However, butaprost methyl ester does not show selectivity between EP2 and EP4.13 Therefore, it is not clear as to which one of EP receptor subtype (EP2 or EP4) works predominantly in relaxation of human corpus cavernosum by this report.12 In addition to butaprost, sulprostone, which has agonistic activity for both EP1 and EP3, was used in this report, and did not lead to contraction of human corpus cavernosum.12 Moreland et al.14 reported that human corpus cavernosum tissues and cultured smooth muscle cells express EP1, EP2 and EP3 receptors by RNase protection assay and pharmacological methods. They used several agonists for EP1 and/or EP3, and elicited only weak contraction of human corpus cavernosum tissues, and they have found the expression of EP2, EP3I and EP3II receptor mRNA. The data of functional experiment are thought to be almost similar to our data; however, the data of RNase protection assay were different from our data. They have found the expression of EP2, EP3I and EP3II receptor mRNA; however, the expression of EP1 and EP4 receptor mRNA was not determined. But before, Moreland et al.15 reported that human corpus cavernosum express both EP2 and EP4 receptor mRNA. In addition, there are eight isoforms of EP3 receptor.16 Most EP3 isoforms are coupled to Gi and inhibit cAMP. However, EP3II isoform can also couple to Gs and can increase cAMP.14 Thus, the work of EP3 isoforms is complicated, and it is not understood well about pharmacological reaction of EP3 isoforms. Our results of the higher expression of EP4 receptor mRNA did not contradict the previous reports and might provide stronger activity of EP4 on the human cavernosum tissue. In addition, our result of little expression of both EP1 and EP3 supports the result that sulprostone did not elicit contraction of human corpus cavernosum. Based on our data, it may become clear that EP4 receptor mRNA expression is predominant. Moreover, it may play an important role in the erectile function of human corpus cavernosum among four EP receptor subtypes.
Until recent years, selective EP4 agonist could not be developed, and the pharmacological research of EP4 receptor has been unsuccessful. However, newly developed EP4 agonist AE1-437 and AE1-734 as the prodrug17, 18, 19 might enable the study of the action of EP4. In the human corpus cavernosum, functional study using newly developing EP4 agonists and antagonists is required to resolve the function of EP4 receptor, resulting in future drug development for ED patients.
Many tissues reveal expression of EP4 mainly on thymus, lung, heart, ileum, kidney, uterus and immune system. On the other hand, expression of EP2 is not found in as many tissues as EP4, but it is found in the kidney, vessel, immune system and so on.5 EP4 receptor reveals agonist-dependent homologous desensitization; however, EP2 receptor does not.20 According to reactivity for metabolic product and a difference of desensitization, transient action of PGE2 is shown by EP4 receptor, and persistent action is estimated to be shown by EP2 receptor.21 It may be reasonable that EP4 receptor subtype expresses at higher level than EP2 in the human cavernosum tissue, from these functional standpoints. According to this hypothesis, agonists working for both EP2 and EP4 may become more efficient and promising drug with aspect of prolonged erection, in addition to EP4 agonist as a new therapy.
ICI with PGE1 is an invasive method for drug administration; hence, orally-active EP4 selective agonist (and dual-acting agent for EP2 and EP4) will be a good alternative or second line therapy for PDE5 inhibitors. Unfortunately, pharmacological examination could not be performed, because of limited amount of available human tissue. Functional experiment using human tissue is strongly required.
By using a combination of genetic and immunohistochemical approaches, we identified the EP4 receptor as the predominant PGE2 receptor subtype in the human corpus cavernosum.
These results suggest that EP4 receptor may work as an important subtype among EP receptors for relaxation of smooth muscle in the human corpus cavernosum, resulting in penile erection. New safe and specific agonists for EP4 receptor will be waited for ED patients who do not respond to PDE5 inhibitors.
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This work was supported in part by the Grant in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to Dr M Takeda (No. 14370508, 15639014 and 16659436), and to Dr I Araki (No. 16591588).
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Komuro, M., Kamiyama, M., Furuya, Y. et al. Gene and protein expression profiles of prostaglandin E2 receptor subtypes in the human corpus cavernosum. Int J Impot Res 18, 275–281 (2006). https://doi.org/10.1038/sj.ijir.3901408
- EP receptor subtype
- corpus cavernosum
- quantitative RT-PCR
- erectile dysfunction
Current Sexual Health Reports (2006)