sign in sign up
<<
Home , Prostaglandin E, Prostaglandin E1, Sulprostone, Thromboxane receptor

International Journal of Impotence Research (2003) 15, 362–368 & 2003 Nature Publishing Group All rights reserved 0955-9930/03 $25.00 www.nature.com/ijir Functional E (EP) receptors in human penile corpus cavernosum

RB Moreland1,4, N Kim1, A Nehra2, I Goldstein1 and A Traish1,3*

1Department of Urology, Boston University School of Medicine, Boston, Massachusetts, USA; 2Department of Urology, Mayo Clinic and Foundation, Rochester, Minnesota, USA; and 3Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA

In this study, we have characterized functional EP receptors in human corpus cavernosum (HCC) tissue and in HCC cells (SMC). Using RNase protection assays, we determined expression of EP2, EP3I and EP3II mRNA. In organ bath preparations of HCC tissue strips, PGE1 caused dose-dependent relaxation at concentrations below 300 nM. At concentrations greater than 300 nM, PGE1 caused contraction. Addition of the EP1/EP2/EP3 AH6809 inhibited this contraction and facilitated further relaxation through concentrations above 1 lMof PGE1. The EP1/EP3 receptor selective 17-phenyltrinor-PGE2 caused dose-dependent contraction that was partially attenuated by SC51322, an EP1 selective antagonist. In cultures of HCC SMC, PGE1 stimulated cAMP accumulation in a dose-dependent manner. Interestingly, AH6809 significantly attenuated PGE1-induced cAMP accumulation. , a selective EP3 receptor agonist, induced weak contractions in HCC tissue strips but augmented forskolin-induced cAMP synthesis in HCC SMC. The data in this study suggest that HCC and cultured smooth muscle cells express EP1, EP2 and EP3 receptors. These receptors mediate their responses via different biochemical pathways and are expected to have different responses in regulating smooth muscle tone. Thus, we suggest that the ultimate response in erectile tissue to various is the integration of responses elicited by individual EP receptor subtypes to a given . International Journal of Impotence Research (2003) 15, 362–368. doi:10.1038/sj.ijir.3901042

Keywords: PGE1; EP receptor; vascular smooth muscle; corpus cavernosum;

4,5 Introduction PGF2a and A2. In addition, corpus cavernosum smooth muscle cells (SMC) in culture have been shown to synthesize PGD2, PGE2 and are synthesized from 20 carbon unsa- PGF2a, all of which are regulated by oxygen turated fatty acids (the most common being arachi- tension.6 donic acid) and are local acting autacoids that Of these eicosanoids, only PGE1 has been reported mediate a variety of effects including smooth muscle to relax penile cavernosal smooth muscle.7–10 1–3 relaxation and contraction. These substances, Further, , the precursor of PGE2, which include (PGE), mediate their can relax human corpus cavernosum (HCC) smooth effects on smooth muscle by interaction with muscle in the presence of specific G- coupled receptors. The corpus antagonists, a process blocked by the prostaglandin 10 cavernosum synthesizes (PGI2), PGE2, G/H synthase inhibitor indomethacin. Prostaglan- din E can bind to several different subtypes of EP receptors. EP1 (Gaq11-coupled) and EP3 (Gai/o- coupled) receptors can potentially mediate trabecu- *Correspondence: AM Traish, PhD, Director, Urology lar smooth muscle contraction, while EP2 and EP4 Research, Boston University School of Medicine, Room (Gas-coupled) may mediate trabecular smooth mus- W607D, 700 Albany Street, Boston, MA 02118, USA. cle relaxation via different signal transduction E-mail: [email protected] 1–3 4 pathways. Furthermore, the EP3 receptor can Current address: RB Moreland, PhD, Neuroscience 3 Research, Global Pharmaceutical Research and Develop- exist as six different splice variants. In the light of ment, AP9 Room 1125, Abbott Laboratories, 100 Abbott the current use of injectable and intraurethral PGE1 Park Road, Abbott Park, IL 60064-6118, USA. to treat erectile dysfunction and since PGE1 has the Received 18 December 2002; revised 25 March 2003; potential to bind to nine distinct EP receptor accepted 27 March 2003 subtypes, an understanding of the prostaglandin Functional EP receptors in human corpus cavernosum RB Moreland et al 363 receptors in corpus cavernosum is critical for ranged from 45 to 70 y of age and had a variety of designing and targeting effective therapy. etiologies including prostatectomy, pelvic The lack of specific receptor antagonists and trauma, peripheral vascular disease, diabetes melli- limited selective suggests that characteriza- tus and Peyronie’s disease. HCC SMC cultures were tion of EP receptors may require a combination of prepared as previously reported.12 molecular biologic and pharmacologic approaches. The advances over the past decade in paved the way for investigation of the Detection of human EP receptor subtypes by RPA expression and function of these receptors, especially in the light of cDNA cloning and genomic mapping of the specific receptors for PGD, PGE, PGF2a, PGI and Total RNA was prepared from HCC biopsies or from 1–3 TxA2. The presence of EP1 receptors in the penis HCC SMC by homogenization in guanidinium and a potential role for this receptor in the pain isothiocyanate followed by phenol/chloroform ex- 11 13 associated with PGE1 has been proposed. traction and isopropanol precipitation. RPAs were EP1 receptors are Gq/11 coupled and would be performed using Ambion’s commercially available expected to contract corpus cavernosum tissue. The RPA kit (RPA II). Plasmid constructs for the human availability of selective tools to distinguish pharma- EP214 (gift of Dr John Regan, University of Arizona) 15 cologically individual EP receptor families is limited. and human EP3I and EP3II (gift of Dr Mark In this report, we investigated the expression and Abramovitz, Merck-Frost, Canada) have been de- function of EP1, EP2 and EP3 receptors in HCC tissue scribed. For EP2, a 398 bp StuI/HindIII restriction and in HCC SMC. fragment was subcloned into pGEM3zf(À) digested with SmaI/HindIII. Plasmids were linearized with the appropriate restriction endonucleases (Table 1) Methods and transcribed with either T7 RNA polymerase (Ambion) or SP6 RNA polymerase (Ambion) in the presence of nonradioactive nucleotides to prepare 32 Chemicals and supplies sense RNA or in the presence of [ P]CTP (800 Ci/ mmol, NEN Life Sciences, Boston, MA, USA) to prepare antisense RNA. Nonradiolabeled sense RNA PGE1, 17-phenyltrinor PGE2 (17-PTP) and sulpros- was quantitated by measuring absorbance at 260 nM tone were obtained from Cayman Chemical Com- (e ¼ 40 mg/A260). The RNA to be tested was precipi- pany (Ann Arbor, MI, USA). AH-6809 and SC-51322 tated in the presence of 0.5 M ammonium acetate were obtained from Biomol (Plymouth Meeting, PA, (30–40 mg for HCC biopsies and 30–40 mg for HCC USA). RPA II RNase protection assay (RPA) kits as SMC cultures), dried and used immediately for the well as MaxiScript RNA transcription kit were assay. The hybridizations were carried out for 18-h obtained from Ambion (Austin, TX, USA). Cyclic at 451C. RNase digestions after hybridization were AMP radioimmunoassay kits were obtained from allowed to proceed for 90 min at 371C. After Biomedical Technologies (Stoughton, MA, USA). inactivation of RNase and precipitation of the Bretylium and 3-isobutylmethylxanthine (IBMX) protected fragments, samples were electrophoresed were obtained from Sigma Chemical Company (St on 40 cm 5% urea-polyacrylamide denaturing gels, Louis, MO, USA). dried and subjected to autoradiography.

Human tissue procurement and culture of smooth Assay of cAMP in tissue culture cells muscle cells HCC SMC were prepared as previously reported.12 The protocol to acquire human tissue was approved Cells were grown to confluence in DMEM contain- by the Institutional Review Board for Human ing 25 U/ml penicillin, 250 U/ml streptomycin, Subjects at Boston University Medical Center and 25 U/ml nystatin, 2 mM glutamine and supplemen- the Mayo Clinic and Foundation. HCC tissue was ted with 10% fetal bovine serum. The cells were re- obtained during penile prosthesis insertion. Patients fed with the same media 24 h before the experiment.

Table 1 Details of RNase protection plasmids and protected fragments

Receptor Plasmid backbone cDNA location Codons Size protected (bp)

EP2 pGEM3zf(À) 1111–1509 P320–L358 398 EP3I pBluescriptII 1257–1823 I340–R390 556 EP3II pBluescriptII 1233–1682 I340–A388 449

International Journal of Impotence Research Functional EP receptors in human corpus cavernosum RB Moreland et al 364 Cells were then treated with either 10 mM PGE1, cultured HCC SMC (Figure 1a). In addition, mRNA 10 mM PGE1 plus 10 mM AH6809 or 10 mM forskolin for EP3I and EP3II receptors was detected as 549 and with varying concentrations of sulprostone. All 446 bp protected fragments, respectively (Figure 1b treatments were for 5 min. Assays for cAMP were and c). Of these two different receptor variants, EP3II carried out as previously described.13 is the most interesting as it has been shown to couple to Gas, increasing cAMP synthesis (EP3 receptors normally couple through Gai, decreasing Organ baths cAMP synthesis).2 The other bands at lower mole- cular weights on the EP3 RPA gels suggest the presence of other splice variants present in corpus Organ bath experiments were carried out as pre- cavernosum tissue and cells. Of the receptor mRNAs viously reported.9 Strips of HCC were mounted to examined, the EP2 receptor (Figure 1a) appeared the tension transducers and submerged in thermostated most abundant based on exposure time using equal (371C) 25 ml organ baths containing bicarbonate amounts of total RNA, and was apparent in HCC buffered physiologic salt solution (PSS; 118.3 mM smooth muscle biopsies as well as in cultured cells. NaCl, 4.7 mM KCl, 0.6 mM MgSO4, 1.2 mM KH2PO4, We have previously demonstrated the expression of 2.5 mM CaCl2, 25 mM NaHCO3, 0.026 mM CaNa2ED- EP2 and EP4 receptors in HCC and in cultured SMC TA, 11.1 mM glucose) and aerated with 95% air/5% 6 using RT-PCR. We were not successful in demon- CO2 to attain a pH of 7.4. Optimal isometric tension strating EP1 receptor mRNA expression either by for contraction was attained as described pre- RNase protection or RT-PCR (data not shown) viously.9 All experiments were carried out in the despite pharmacologic functional evidence suggest- presence of 3 mM indomethacin to inhibit produc- ing the presence of these receptors (see below). tion of endogenous arachidonic acid metabolites. Dose–responses to 17-PTP, PGE1 and sulprostone were carried out by cumulative addition. In some experiments, tissue strips were incubated for 20– Effects of EP agonists in HCC 30 min with 10 mM bretylium before beginning the dose–response. Relaxation responses were normal- We utilized the EP1/EP3 selective agonist 17-PTP- ized as percent of tone caused by 1 mM phenylephr- PGE (see Table 2) in combination with the EP1 ine. Contractile responses were normalized as 2 selective antagonist SC51322 to demonstrate func- percent of tone caused by PSS containing 80 mM tional EP1 receptors in HCC smooth muscle. The K+. Baseline tension was determined at the end of agonist 17-PTP induced potent contractions in each experiment by exposing tissue strips to 10 mM trabecular smooth muscle strips in organ baths and 10 mM nitroprusside. The selectivity (EC B100 nM, Figure 2a). To further confirm the of the various EP agonists/antagonists used in these 50 presence of pharmacologic EP1 receptors, the effects experiments is shown in Table 2.16,17 of the EP1 antagonist SC51322 on 17-PTP contrac- tions were examined. This antagonist attenuated the Results contractile response to 17-PTP at concentrations below 30 mM, as indicated by the shift of the dose– response curve to the right (Figure 2a). Interestingly, Expression of EP2 and EP3 receptor mRNA in HCC at concentrations above 30 mM 17-PTP, EP1 blockade by SC51322 potentiated contraction. It has been reported that EP1/EP3 agonists can either stimulate The EP2 receptor mRNA was detected as a 398 bp or inhibit release of norepinephrine from nerve protected fragment expressed in both HCC and in terminals through prejunctional receptors.18,19 To determine if the contraction to 17-PTP was due in Table 2 Binding characteristics of ligands used in this report part to norepinephrine release, we used the sympa- (taken from Abramovitz et al16 and Kiriyama et al17) thetic nerve inhibitor bretylium. Interestingly, bre- tylium potentiated rather than inhibited the actions Compound EP1 EP2 EP3 EP4 of 17-PTP (Figure 2b). In HCC tissue strips contracted with phenylephr- PGE 9.1 4.9 0.3 0.7 2 ine, PGE caused smooth muscle relaxation (EC PGE1 36.0 10.0 1.0 2.0 1 50 17-PTP 14.0 >100000 4.0 >100000 B20 nM). At concentrations greater than 1 mM, PGE1 Sulprostone 107.0 >100000 0.4 7740.0 induces contraction (Figure 4a), which is attributed AH6809 1217.0 1150 1597.0 >100000 to cross-reactivity with TP () recep- SC-51322 13.8 >100000 698.0 14089 tors since the TP receptor antagonist SQ29548 blocks this contraction.9,10 Since PGE binds to all Data in italics are taken from mouse recombinant receptors 1 expressed in Chinese hamster ovary cells.17 All other data from four EP receptor subtypes, we examined the con- recombinant human receptors expressed in HEK293 cells. Ki (nM) tribution of EP1, EP2 and EP3 receptor subtypes 3 determined by competition with [> H]PGE2. with the antagonist AH6809 (Table 2). Treatment

International Journal of Impotence Research Functional EP receptors in human corpus cavernosum RB Moreland et al 365

Figure 2 Contraction to 17-PTP in HCC tissue strips. Organ bath preparations of human cavernosal tissue strips at optimal isometric tension were contracted with physiological salt solution containing 80 mM K+. After washout, tissues were subjected to cumulative additions of 17-PTP in the absence (control) or presence of 10 mM SC51322 (panel a) or 10 mM bretylium (panel b). Data are expressed as percent of contraction to physiological salt solution containing 80 mM K+ (n ¼ 6 for each protocol).

Figure 3 Effect of AH6809 on PGE1-mediated relaxation and cAMP accumulation. Organ bath preparations of human caverno- sal tissue strips at optimal isometric tension were contracted with 1 mM phenylephrine and subjected to cumulative additions of PGE1 in the absence (control) or presence of 10 mM AH6809 (panel a; n ¼ 6 for each protocol). Primary cultures of human cavernosal SMC were incubated with increasing concentrations of PGE1 with or without 10 mM AH6809 for 5 min and cAMP was measured by radioimmunoassay in deproteinized cell extracts (panel b; data represent cells from three different patients).

with AH6809 enhanced relaxation to PGE1 (Figure 3a). However, AH6809 attenuated cAMP accumulation in HCC SMC and shifted the dose- response curve to the right (EC50 B200 nM to 2 mM; Figure 3b). The role of EP3 receptors in regulating smooth muscle contractility was examined using the EP3 selective agonist sulprostone. Sulprostone induced very weak contractions in HCC, with significant Figure 1 RPAs for EP receptors in HCC and HCC SMC. For each effects only at the highest concentrations (Figure 4a). lane, 40 mg or RNA was loaded and electrophoresed through 40 cm In HCC SMC, the effects of sulprostone on forskolin- 5% urea-polyacrylamide denaturing gels, dried and subjected to induced cAMP synthesis were examined. Since EP3 autoradiography. (a) EP2 is apparent as an B400 bp band (panel a; receptors are Gai/o coupled, one would expect an 3-day exposure). EP3I is apparent as an B560 bp band (panel b; 7- B inhibition of cAMP synthesis similar to what has day exposure). EP3II is apparent as an 450 bp band (panel c; 5- 20 day exposure). The presence of a doublet (protected band been reported for the a2-. B420 bp) indicates expression of one or more of the other However, no inhibition was noted (Figure 4b) and alternatively spliced EP3 variants. (M ¼ molecular weight mar- at the higher concentrations of sulprostone, a kers; P ¼ untreated probe; R ¼ RNase-treated probe; H1, H2, H3 ¼ RNA from three different patients; C1, C2, C3 ¼ RNA from stimulation of cAMP synthesis was observed, con- HCC SMC derived from three different patients. S ¼ sense RNA sistent with coupling to Gas as has been reported for 11,21 control.) muscarinic receptors HCC SMC.

International Journal of Impotence Research Functional EP receptors in human corpus cavernosum RB Moreland et al 366 norepinephrine from nerve terminals.18,19 However, in mammalian tissues, prejunctional EP3 receptors inhibit norepinephrine release.18 This inhibitory action of prejunctional EP3 receptors is consistent with our data. Assuming that there is basal release of norepinephrine in isolated cavernosal tissue strips, it is possible that higher concentrations of 17-PTP bind to prejunctional EP3 receptors and inhibit basal release of norepinephrine. In the presence of 10 mM SC51322, there may be partial blockade of EP3 receptors in addition to antagonism Figure 4 Effects of EP3 agonist sulprostone on HCC tissue strips and HCC SMC. Organ bath preparations of human cavernosal of EP1, facilitating basal norepinephrine tissue strips at optimal isometric tension were contracted with release. This could result in a slightly enhanced physiological salt solution containing 80 mM K+. After washout, contractile response despite the antagonism of tissues were subjected to cumulative additions of sulprostone. EP1/EP3 receptors on the trabecular smooth Data are expressed as percent of contraction to physiological salt muscle. solution containing 80 mM K+ (panel a; n ¼ 6). Primary cultures of human cavernosal SMC were incubated with 10 mM forskolin To further examine potential regulation of the and increasing concentrations of sulprostone for 5 min and cAMP EP1/EP3-mediated contractile response by sympa- was measured by radioimmunoassay in deproteinized cell thetic nerves, we carried out dose responses to 17- extracts (panel b; data represent cells from three different PTP in the presence of bretylium to inhibit norepi- patients). nephrine release. Interestingly, we observed that bretylium potentiated contraction to 17-PTP. Brety- lium is known to inhibit norepinephrine reuptake Discussion and consequently increase synaptic concentrations of the neurotransmitter in a transient manner. However, it is unlikely that this mechanism is PGE1 has been used to diagnose and treat erectile responsible for the large enhancement in 17-PTP- dysfunction for almost 20 y.8,22,23 However, the exact induced contraction since all tissue strips were mechanisms by which PGE1 relaxes smooth muscle incubated with bretylium for 20–30 min before and which receptors are present on HCC smooth beginning the dose response. We have previously muscle remain to be elucidated. The pharmacology demonstrated that this time period is sufficient to of PGE1 is complicated by the presence of multiple inhibit neurogenic contraction completely in caver- G-protein-coupled receptors, which signal through nosal tissue strips.28 varying pathways. EP2 and EP4 receptors are Alternatively, bretylium, a quaternary ammonium thought to be involved in mediating smooth muscle compound, has been shown to block K+ channels relaxation, whereas EP1 and EP3 may be involved in and inhibit hyperpolarization and relaxation of smooth muscle contraction. The EP1 receptor sub- various smooth muscle tissues.29,30 Although the type has been implicated in pain and allodynia, specific receptor subtypes have not been identified, 24–27 which may be attenuated by nitric oxide. This is PGE1 has been shown to activate the large-conduc- of special interest since one of the major complaints tance KCa channels in penile cavernosal tissue via a 8 31 with intracavernosal PGE1 is pain. In organ bath -mediated mechanism. Thus, + studies, PGE1 produces dose-dependent relaxation bretylium may block K channel-mediated hyper- in HCC tissue strips, and at higher concentrations, polarization to enhance contraction substantially to crossreacts with thromboxane A2 (TP) receptors, 17-PTP. However, this would suggest that 17-PTP, in producing contraction.9,10 part, stimulates cAMP production. The EP3 receptor In this report, we have demonstrated expression is the most diverse of the four subtypes of PGE of mRNA for EP2, EP3I and EP3II receptors as well as receptors. There are six different alternatively functional demonstration of EP1, EP2 and EP3 spliced isoforms differing in the third intracellular receptors. The EP1/EP3 selective agonist 17-PTP loop after the seventh transmembrane domain.1–3 As caused dose-dependent contraction in HCC tissue. a result, most EP3 isoforms are coupled to Gai/o and The EP1 selective antagonist SC51322 inhibited inhibit cAMP. However, the EP3II isoform (EP3D) 2,12,33 contraction to 17-PTP in HCC tissue strips at can also couple to Gas and can increase cAMP. concentrations below 3 mM and enhanced contrac- Thus, it is possible that 17-PTP may stimulate EP3II tion at concentrations greater than 3 mM. While the receptors to increase cAMP. inhibition of 17-PTP-induced contraction suggests Further evidence for the complex nature of EP3 the presence of functional EP1 receptors on HCC receptors can be seen in the functional responses to smooth muscle, the potentiation at higher concen- the EP3 agonist sulprostone. While sulprostone at trations suggests the involvement of other regulatory high concentrations (1 mM) caused weak contraction mechanisms. EP1/EP3 agonists have previously in isolated HCC tissue strips, it actually enhanced been shown to either stimulate or inhibit release of forskolin-induced cAMP accumulation at identical

International Journal of Impotence Research Functional EP receptors in human corpus cavernosum RB Moreland et al 367 concentrations. Thus, cAMP accumulation does not 3 Pierce KL, Gil DW, Woodward DF, Regan JW. Cloning of necessarily predict the tonic response. human receptors. Trends Pharmacol Sci 1995; 16: A dissociation between relaxation and cAMP 1 – 22. 4 Roy AC, Tan SM, Kottegoda SR, Ratnam SS. Ability of human accumulation may also be seen with AH6809, a corpus cavernosum to generate and thrombox- nonselective antagonist for EP1, EP2 and EP3 anes in vitro. IRCS Med Sci 1984; 12: 608. receptors. AH6809 potentiated PGE1-induced re- 5 Daley JT et al. Prostanoid production in rabbit corpus laxation of HCC tissue but reduced PGE -induced cavernosum. I. Regulation by oxygen tension. J Urol 1996; 1 155: 1482 – 1487. cAMP synthesis in cultured SMC. Since this agent 6 Moreland RB et al. Oxygen-dependent prostanoid synthesis has significantly lower affinity for EP4 receptors activates functional PGE receptors on human corpus caverno- (Table 2), it is likely that the enhanced relaxation is sum smooth muscle cells. Am J Physiol 2001; 281: H552 – H557. due to inhibition of EP1 and EP3I receptors, while the attenuation in cAMP synthesis is due to 7 Hedlund H, Andersson KE. Contraction and relaxation induced by some prostanoids in isolated human penile inhibition of EP2 and EP3II receptors. We have erectile tissue and cavernous artery. J Urol 1985; 134: 1245 – previously shown that AH6809 inhibited PGE1- 1250. induced cAMP synthesis in cultured SMC at high 8 Porst H. The rationale for in erectile failure: oxygen tension.6 In addition, the EP2 selective a survey of worldwide experience. J Urol 1996; 155: agonist butaprost effectively relaxed HCC smooth 802 – 815. 10 9 Moreland RB et al. induces relaxation of human muscle strips in organ baths. These observations corpus cavernosum smooth muscle: comparison to prosta- confirm that EP2 receptors are involved in HCC glandin E1. Int J Impot Res 2000; 12: 107 – 110. 10 Angulo J et al. Regulation of human penile smooth muscle relaxation while EP1 and EP3I receptors may mediate contraction. tone by prostanoid receptors. Br J Pharmacol 2002; 136: 23 – 30. In summary, we have demonstrated the expres- 11 Lue TF, Levine JD. Impotence. J Urol 1995; 155: 85. sion of EP2, EP3I and EP3II receptors at the mRNA 12 Moreland RB et al. PGE1 suppresses the induction of collagen level and EP1, EP2 and EP3 receptors by functional synthesis by transforming growth factor-b1 in human corpus studies. These data together with our previous work cavernosum smooth muscle. J Urol 1995; 153: 826 – 834. suggest that HCC expresses all four known subtypes 13 Traish AM, Palmer MA, Goldstein I, Moreland RB. Expression 6 of functional muscarinic subtypes in of EP receptors. We suggest that EP2, EP3II and EP4 cultured human corpus cavernosum and in cultured smooth receptors predominate in mediating smooth muscle muscle cells. Receptors 1996; 5: 159 – 176. relaxation and EP1 and EP3I receptors modulate 14 Regan JW et al. Cloning of a novel human prostaglandin contraction. It remains to be determined what role receptor with characteristics of the pharmacologically defined EP2 subtype. Mol Pharmacol 1994; 46: 213 – 220. these receptors may play in the physiology and 15 Adam M et al. Cloning and expression of three isoforms of the pathophysiology of erectile function. The availabil- human EP3 prostanoid receptor. FEBS Lett 1994; 338: 170 – ity of receptor knockout mice as well as the reports 174. of selective agonists and antagonists for these 16 Abramovitz M et al. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of receptor subtypes should enable further investiga- prostaglandins and related analogs. Biochim Biophys Acta tion of the pharmacology of EP receptors as well as 2000; 1483: 285 – 293. potential development of improved therapeutics for 17 Kiriyama M et al. Ligand binding specificities of the eight erectile dysfunction.32,33 types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br J Pharmacol 1997; 122: 217 – 224. 18 Jensen TJ, Nedergaard OA. Modulation of norepinephrine release from sympathetic neurons of the rabbit aorta by Acknowledgments prejunctional prostanoid receptors. J Pharmacol Exp Ther 1999; 291: 7 – 11. 19 Allgaier C, Meder W. Cultured chick sympathetic neurons: We acknowledge the excellent technical assistance prostanoid EP1 receptor-mediated facilitation of noradrena- of Yue-hua Huang, Cynthia Gallant and Erika Line´ line release. Naunyn Schmiedebergs Arch Pharmacol 1995; 352: 447 – 450. as well as the administrative assistance of Jerie 20 Traish AM, Moreland RB, Huang YH, Goldstein I. Expression McGrath-Cerqua. This work was supported by of functional alpha-2-adrenergic receptor subtypes in human Grants R29-DK47950, R01-DK39080, R01-DK40025 corpus cavernosum and in human corpus cavernosum smooth and K01-DK02696 from the National Institutes of muscle cells. Recept Signal Transduct 1997; 7: 55 – 67. Health. 21 Traish AM et al. G-protein-coupled receptor agonists augment activity induced by forskolin in human corpus cavernosum smooth muscle cells. Recept Signal Transduct 1997; 7: 123 – 134. 22 Andersson KE. Pharmacology of penile erection. Pharmacol References Rev 2001; 53: 417 – 450. 23 Moreland RB, Hsieh GC, Nakane M, Brioni JD. The biochem- ical and neurologic basis for the treatment of erectile 1 Narumiya S, Sugimoto Y, Ushikubi F. Prostanoid receptors. dysfunction. J Pharmacol Exp Ther 2001; 296: 225 – 234. Structure and function. Physiol Rev 1999; 79: 1193 – 1226. 24 Minami T et al. L-NAME, an inhibitor of nitric oxide synthase, 2 Breyer RM, Bagdassarian CK, Myers SA, Breyer MD. Prosta- blocks the established allodynia induced by intrathecal noid receptors: subtypes and signaling. Ann Rev Pharmacol administration of . Neurosci Lett 1995; 201: Toxicol 2001; 41: 661 – 690. 239 – 242.

International Journal of Impotence Research Functional EP receptors in human corpus cavernosum RB Moreland et al 368 25 Minami T et al. Characterization of EP receptor subtypes intestinal and uterine smooth muscle. Br J Pharmacol 1992; responsible for prostaglandin E2-induced pain responses by 107: 288 – 295. use of EP1 and EP3 receptor knockout mice. Br J Pharmacol 30 Berry JL, Small RC, Foster RW. Tracheal relaxation induced by 2001; 133: 438 – 444. potassium channel opening drugs: its antagonism by adrener- 26 Stock JL et al. The prostaglandin E2 EP1 receptor mediates gic neurone blocking agents. Br J Pharmacol 1992; 106: pain perception and regulates blood pressure. J Clin Invest 813 – 818. 2001; 107: 325 – 331. 31 Lee SW et al. Prostaglandin E1 activates the large-conductance 27 Malmberg AB, Rafferty MF, Yaksh TL. Antinociceptive KCa channel in human corporal smooth muscle cells. Int effect of spinally delivered prostaglandin E receptor antago- J Impot Res 1999; 11: 189 – 199. nists in the formalin test on the rat. Neurosci Lett 1994; 173: 32 Ungrin MD et al. Key structural features of prostaglandin E2 193 – 196. and prostanoid analogs involved in binding and activation of 28 Kim N, Azadzoi KM, Goldstein I, Saenz de Tejada I. A nitric the human EP1 prostanoid receptor. Mol Pharmacol 2001; 59: oxide-like factor mediates nonadrenergic–noncholinergic neu- 1446 – 1456. rogenic relaxation of penile corpus cavernosum smooth 33 Kawahara H et al. A prostaglandin E2 receptor subtype EP1 muscle. J Clin Invest 1991; 88: 112 – 118. receptor antagonist (ONO-8711) reduces hyperalgesia, allody- 29 Berry JL et al. Inhibition by adrenergic neurone blocking nia, and c-fos expression in rats with chronic nerve agents of the relaxation induced by BRL 38227 in vascular, constriction. Anesth Analg 2001; 93: 1012 – 1017.

International Journal of Impotence Research