Expression of Functional Prostaglandin D (DP) Receptors in Human Corpus Cavernosum Smooth Muscle

Expression of functional prostaglandin D (DP) receptors in human corpus cavernosum smooth muscle

RB Moreland1*, A Nehra2,NNKim1, Kweon-sik Min1, H Albadawi3,4, MT Watkins3,4, I Goldstein1 and AM Traish1,5

1Department of Urology, Boston University School of Medicine, Boston, Massachusetts, USA; 2Department of Urology, Mayo Clinic and Foundation, Rochester, Minnesota, USA; 3Department of Surgery, Boston University School of Medicine, Boston, Massachusetts, USA; 4Department of Pathology, Boston University School of Medicine, Boston, Massachusetts, USA; and 5Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, USA

Prostaglandin D2 (PGD2) binds to speciﬁc G-protein coupled receptors (DP) and induces smooth muscle relaxation by stimulating the synthesis of intracellular cAMP. In this study, we examined the role of PGD2 and DP receptors in regulating human penile smooth muscle contractility. We determined that human corpus cavernosum tissue and smooth muscle cells in culture expressed functional DP receptor and lipocalin-like prostaglandin D synthase by reverse-transcribed polymerase chain reaction (RT-PCR). Functional PGD synthase activity was conﬁrmed by the synthesis of PGD2 in human corpus cavernosum smooth muscle cells upon addition of exogenous arachidonic acid. Organ bath preparations of human corpus cavernosum tissue strips, contracted with phenylephrine, relaxed in a dose-dependent fashion to either PGD2 or the DP selective agonist BW245C. Cultures of human corpus cavernosum smooth muscle cells treated with BW245C showed a two-fold increase in cAMP synthesis. These data are consistent with the expression of functional DP receptors in human corpus cavernosum. This suggests the presence of an intact prostanoid autocrine system that may play a role in regulating penile erectile function. International Journal of Impotence Research (2002) 14, 446–452. doi:10.1038=sj.ijir.3900900

Keywords: DP receptor; PGD2; vascular smooth muscle; lipocalin PGD synthase; corpus cavernosum; erectile dysfunction

Introduction smooth muscle is a major determinant of blood flow within the erectile tissue and regulates tumescence.3,4 In addition to their vasoactive properties, Prostanoids are bioactive lipids derived from prostanoids play key roles in regulating diverse 20-carbon, polyunsaturated fatty acids. The physiological processes. Prostaglandin D modu- 1,2 2 most common precursor is arachidonic acid. lates nociception, body temperature, olfactory func- Prostaglandin G=H synthase (cox 1 and cox 2) tion, sleep, immune response and hormone release converts arachidonic acid to prostaglandin H2, in the central nervous system and in the peri- requiring oxygen as a co-substrate. Prostaglandin phery.2,5 – 7 The DP receptor, which binds PGD, is the H2 is then converted to PGD2, PGE2, PGF2a, PGI2 least characterized with respect to expression and (prostacyclin) and TxA2 (thromboxane A2) by dis- biochemical and physiological properties, relative to tinct synthases. These prostanoids interact with other prostanoid receptors.2,5 Detectable expression specific G-protein-coupled receptors and, depend- of human DP receptor mRNA has been reported only ing on the prostanoid and receptor subtype, mediate in the retina and small intestine.2,5 Cells transfected 1,2 smooth muscle relaxation or contraction. In the with the cloned human DP receptor cDNA or freshly penis, the contractile state of corpus cavernosum isolated nonchromaffin cells from bovine adrenal medulla respond to prostaglandin D2 and other DP agonists with increased cAMP synthesis and calcium *Correspondence: RB Moreland, PhD, Neuroscience mobilization.5,8 Research, Global Pharmaceutical Research and Development, Building AP9, Room 1125, Abbott Laboratories, 100 Abbott Prostaglandin D2 is synthesized from PGH2 by Park Road, Abbott Park, IL 60064-6118, USA. glutathione-independent, lipocalin PGD synthase E-mail: [email protected] or glutathione-dependent, hematopoietic PGD Received 11 January 2002; revised 4 April 2002; synthase; both of which have been cloned.6,9 The accepted 26 April 2002 synthesis of PGE2, PGF2a, PGI2 and TxA2 by human Functional DP receptors in trabecular smooth muscle RB Moreland et al 447 penile corpus cavernosum and the effects of these amplified products were 484 bp for DP receptor and prostanoids on human corpus cavernosum tissue 370 bp for lipocalin PGD synthase. Digestion at and smooth muscle cells have been previously unique restriction endonuclease sites within the 3,10 – 15 reported. However, the synthesis of PGD2 amplified fragments (ScaI for DP receptor, XhoI for and the expression of functional DP receptors in PGD synthase) was utilized to further confirm the human corpus cavernosum smooth muscle are yet identity of the DNA products. cDNA for DP receptor to be determined. The goal of this study was to and PGD synthase were cloned by RT-PCR using the investigate the synthesis of PGD2 in human corpus same primer sets and Pfx-Platinum thermostable cavernosum and the ability of PGD2 to elicit polymerase (Life Sciences, Bethesda, MD), a faithful, biological response in this tissue. proof reading polymerase. Reaction conditions included human corpus cavernosum or small intestine cDNA and amplification at 94C, 65C, 68C for Materials and methods PGD synthase and 94 C, 72 C, 68 C for the DP receptor. Blunt-ended DNA fragments were isolated Chemicals and supplies and cloned into pCR-Script AMP (Stratagene, La Jolla, CA) and DNA sequenced at our institutional core facility. BW245C, PGD1, PGD2 and SQ29,548 were obtained from Cayman Chemical Company (Ann Arbor, MI).

ELISA kits for PGD assay were obtained from R&D PGD2 synthesis by human corpus cavernosum Systems (Minneapolis, MN) while cAMP radio- smooth muscle cells immunoassay kits were obtained from Biomedical Technologies (Stoughton, MA). Pfx Platinum polymerase, Taq DNA polymerase and Superscript II Human corpus cavernosum smooth muscle cells Moloney virus reverse transcriptase were obtained were grown from tissue explants17 and cultured from Life Sciences (Rockville, MD). All other on CytodexTM III microcarrier beads (Pharmacia, restriction endonucleases and molecular biological Piscataway, NJ), as previously described for human supplies were obtained from New England Biolabs corpus cavernosum smooth muscle cells12 and (Beverly, MA), unless noted. All chemicals were of human umbilical endothelial cells.19 Cells were reagent grade. grown and maintained under low shear conditions at media PO2 corresponding to blood PO2 during penile erection (100 mm Hg).20 Cells were incubated RT-PCR assay for DP receptor expression with exogenous 20 mM arachidonate for 30 min. Media was collected, treated with methyloxime to prevent decay of PGD2, clarified by centrifugation at Primers for RT-PCR were designed such that they 1000 g for 1 min, frozen in liquid nitrogen and stored flanked one or more exon – intron junctions and were at 770C until assay. A commercially available as follows: human DP receptor 50-CAGAACCGGAG- ELISA kit (Cayman Chemicals, Ann Arbor, MI) was 0 TCTGCGG (nucleotides 614 – 635), 3 -GGGACGCT- used to assess PGD2 production. This protocol was TCCCTCCCGTCC (nucleotides 1092 – 1113)5 and repeated four times, using cells from four different human lipocalin PGD synthase 50-CACACCACTGG- patients. CACCAGGCCC (nucleotides 40 – 79), 30-CCGGT AGCTGTAGGAGCCGAG (nucleotides 382 – 430).9 Total RNA from human kidney and small intestine Organ baths was purchased from Clontech (Palo Alto, CA). Total RNA from human Jurkatt cells and from IMR90 human neonatal lung fibroblasts were the gifts from The Institutional Review Boards at Boston Drs Douglas Faller and Peter Polgar, respectively University Medical Center and the Mayo Clinic (Boston University School of Medicine, Boston, MA). and Foundation approved the use of human tissue. Total RNA was prepared from human corpus caver- Human corpus cavernosum tissue was obtained nosum biopsies and cultured human corpus caverno- during penile prosthesis insertion. Patients ranged sum smooth muscle cells as previously reported.17 from 45 to 70 y of age and had a variety of etiologies Reverse transcription was carried out as previously including radical prostatectomy, pelvic trauma, described18 using random hexamers (Perkin Elmer- peripheral vascular disease, diabetes mellitus and Cetus, Foster City, CA) and Superscript II Moloney Peyronie’s disease. Organ bath experiments were virus reverse transcriptase (Life Sciences, Bethesda, carried out as previously reported.15,16 Human MD). Polymerase chain reaction (PCR) was carried corpus cavernosum tissue strips at optimal iso- out using 40 cycles of 1 min each at 94C, 65C, 72C metric tension were contracted with 1 mM phenyl- in an MJ Research Model 200 thermocycler (MJ ephrine. After stable contraction was attained, Research, Watertown, MA). Expected sizes of the tissue strips were exposed to increasing concentra-

International Journal of Impotence Research Functional DP receptors in trabecular smooth muscle RB Moreland et al 448 711 76 tions of BW245C (10 – 10 M), PGD1 or PGD2 treated with increasing concentrations of BW245C (10710 – 1075 M). In some experiments, tissue strips (10711 – 1076 M) for 5 min. The incubations were were incubated with the TxA2 receptor antagonist terminated by quickly aspirating the medium and 15 SQ29,548 (10 mM) before exposure to PGD1 or adding ice-cold 1 N perchloric acid. Assays to PGD2. quantify cAMP were performed, as previously described.16

Measurement of cAMP synthesis Results

Human corpus cavernosum smooth muscle cells PGD synthesis in human corpus cavernosum were prepared as previously reported.17 Cells were grown to conﬂuence in DMEM containing 25 U=ml penicillin, 250 U=ml streptomycin, 25 U=ml nystatin, In order to determine if the lipocalin-like PGD and 2 mM glutamine and supplemented with 10% synthase mRNA was expressed, RT-PCR was carried fetal bovine serum. The cells were re-fed with the out utilizing total RNA from human corpus same media 24 h before the experiment. Cells were cavernosum biopsy specimens and cultured smooth incubated with 10 mM of 3-isobutyl-1-methyl- muscle cells. Total RNA from human kidney, xanthine (IBMX) for 15 min and were then human small intestine, IMR90 cells and Jurkatt cells

Figure 1 RT-PCR analysis of lipocalin PGD synthase expression in human tissues and cells. RT-PCR was carried out as described in the methods and products separated on a non-denaturing 10% acrylamide gel. (A) Lane 1, DNA control; Lane 2, human kidney total RNA; Lane 3, human small intestines total RNA; Lane 4, human IMR90 cell total RNA; Lane 5, human Jurkatt cell total RNA; Lanes 6 – 9, total RNA derived from human corpus cavernosum biopsies; Lanes 10 – 12, total RNA derived from human corpus cavernosum smooth muscle cell cultures. (B) Identical samples digested with the restriction endonuclease XhoI.

International Journal of Impotence Research Functional DP receptors in trabecular smooth muscle RB Moreland et al 449 were used as positive controls. Figure 1A shows the Expression of DP receptor mRNA in human corpus amplification of a specific 370 bp band. The identity cavernosum of this DNA product was established by digestion of the amplification products with the restriction To determine if DP receptor mRNA was expressed in endonuclease XhoI, which resulted in the expected human corpus cavernosum, RT-PCR was carried out 199 bp and 171 bp DNA fragments (Figure 1B). utilizing RNA from human corpus cavernosum whole Subcloning and DNA sequencing of the amplifica- tissue and cultured cavernosal smooth muscle cells. tion products further confirmed the identity of the Total RNA from Jurkatt cells was used as a negative product as lipocalin PGD synthase. The functional control, while human kidney, small intestine and activity of PGD synthase in cultured human caver- IMR90 cells were used as positive controls. Figure 2A nosal smooth muscle cells was assessed by the shows that RT-PCR of these samples resulted in a measurement of PGD2 in conditioned media. Under 484 bp amplified band in all the positive controls. The basal conditions, PGD2 production was 0.23 Æ amplified band was detected in all four human corpus 0.01 pmol=106 cells. When cells were exposed to cavernosum biopsy specimens and in four of the five 20 mM arachidonate in media with oxygen tension of human cavernosal smooth muscle cell cultures, albeit 100 mm Hg (identical to blood PO2 during penile to different degrees. The identity of this cDNA erection), PGD2 production increased to 4.03 Æ product was confirmed by restriction endonuclease 1.58 pmol=106 cells (n ¼ 4). digestion of the amplification products with ScaI,

Figure 2 RT-PCR analysis of DP receptor expression in human tissues and cells. RT-PCR was carried out as described in the Methods section and products were separated on a non-denaturing 10% acrylamide gel. (A) Lane 1, DNA control; Lane 2, human kidney total RNA; Lane 3, human small intestines total RNA; Lane 4, human IMR90 cell total RNA; Lane 5, human Jurkatt cell total RNA; Lanes 6 – 9, total RNA derived from human corpus cavernosum biopsies; Lanes 10 – 13, total RNA derived from human corpus cavernosum smooth muscle cell cultures. (B) Identical samples digested with the restriction endonuclease ScaI.

International Journal of Impotence Research Functional DP receptors in trabecular smooth muscle RB Moreland et al 450 Effects of DP agonists in human corpus cavernosum

The structures of the DP agonists used in this study are shown in Figure 3. In human corpus cavernosum tissue strips contracted with phenylephrine, both PGD1 and the naturally occurring prostanoid PGD2 caused smooth muscle relaxation to varying extents (Figure 4). The estimated EC50 for PGD2 was 200 nM. At concentrations greater than 3 mM, PGD2 induced contraction of corpus cavernosum muscle strips. Pre-incubation of muscle strips with 10 mM SQ29,548 inhibited this contraction, suggesting cross-reactivity with thromboxane A2 receptors (data not shown). Of the three compounds tested, the DP-selective agonist BW245C was the Figure 3 Compounds used in this study. most potent in relaxing human corpus cavernosum (EC50 10 nM). The effects of BW245C were also examined in cultured smooth muscle cells isolated from human corpus cavernosum. In the presence of the non- which resulted in the predicted 248 and 236 bp DNA selective, phosphodiesterase inhibitor 3-isobutyl-1- fragments (Figure 2B). Subcloning and DNA sequen- methylxanthine (IBMX), cAMP synthesis increased cing of ampliﬁcation products from both small in a dose-dependent fashion, up to 2-fold over basal intestine and human corpus cavernosum further levels (Figure 5). conﬁrmed the identity of the product as the DP receptor. Discussion

In this study, we have demonstrated the expression of the lipocalin PGD synthase, as well as functional activity evidenced by the synthesis of PGD2 in human corpus cavernosum smooth muscle cell

Figure 5 Cyclic AMP synthesis in response to BW245C in cultured human corpus cavernosum smooth muscle cells. Conﬂuent Figure 4 Relaxatory dose responses of DP agonists in human cultures of cells were incubated with varying concentrations of corpus cavernosum. Organ bath preparations of human corpus BW245C for 5 min, in the presence of IBMX. Incubations were cavernosum tissue were contracted with 1 mM phenylephrine and terminated by aspiration of media and addition of ice-cold 1 N per- exposed to increasing concentrations of PGD1 (squares), PGD2 chloric acid. Cyclic AMP levels were measured in de-proteinized (circles) or BW245C (triangles). All values are expressed as mean Æ cell extracts by radioimmunoassay. Data are expressed as mean Æ s.e.m. (n ¼ 4 patients for PGD1 and PGD2; n ¼ 6 for BW245C). s.e.m. (n ¼ cells derived from six different patients).

International Journal of Impotence Research Functional DP receptors in trabecular smooth muscle RB Moreland et al 451 cultures. Initially, it was proposed that the lipocalin a difference in signal transduction pathways inher- PGD synthase was localized in the central nervous ent to the HEK cell line. In contrast, Chinese system and spinal fluid while the hematopoietic, hamster ovary cells transfected with the recombi- glutathione-requiring PGD synthase was expressed nant mouse DP receptor, PGD2 and BW245C in the peripheral tissues.6 Recently, the presence of competitively inhibited binding of radiolabeled the hematopoietic PGD synthase, but not the PGD2 with Ki values of 21 nM and 250 nM, respec- lipocalin PGD synthase, was demonstrated by tively.23 This data is consistent with the differences Northern blotting in the rat penis.22 We were unable observed for DP receptor binding of BW245C in mice to detect the 23 kDa hematopoietic PGD synthase in versus humans.2 human corpus cavernosum smooth muscle cell Wehave previouslydemonstrated that PGE1 causes extracts by Western blotting, despite detection of cAMP levels to increase to 750 – 1000 pmol=mg the enzyme in other tissues (unpublished observa- protein in human cavernosal smooth muscle cells tions). It is interesting to note that expression of the through either the EP2 and=or EP4 receptors.12,16 lipocalin PGD synthase has been shown in human This PGE1-induced increase in cAMP synthesis was prostate, epididymis, and testis and PGD synthase observed to be proportional to the relaxation resp- activity was detected in seminal plasma.23 However, onse. In contrast, the induction of cAMP synthesis by this is the first report of lipocalin PGD synthase in BW245C in the present study appears much less the human corpus cavernosum penis. In addition robust than the efficacy of DP agonists on smooth to the synthesis of PGD2, we demonstrated the muscle relaxation. There was only a two-fold increase presence of DP receptors by RT-PCR of DP mRNA in in cAMP synthesis (up to 23 pmol=mg protein) at both human corpus cavernosum whole tissue and 1 mM of BW245C, which caused relaxation in pheny- cultured smooth muscle cells. Restriction enzyme lephrine-contracted human cavernosal tissue strips. analysis, cloning and sequencing of the amplified Since the major increase in intracellular cAMP occurs fragment from human corpus cavernosum and small at much higher concentrations of BW245C, this may intestine confirmed the identity of the DP receptor. be due to crossover with the EP2 receptor as has been We have also demonstrated functional DP recep- suggested in other systems.26,27 tors in human corpus cavernosum. Both the natural One of the mechanisms of smooth muscle relaxa- receptor ligand, PGD2 and the DP receptor agonist tion in response to DP agonists may involve activ- BW245C caused relaxation of human cavernosal ation of calcium channels. A transient increase in tissue strips. At the highest concentration of PGD2 intracellular calcium in response to DP agonists has examined (10 mM), human corpus cavernosum tis- been demonstrated in transfected as well as primary 5,8 sue strips contracted, similar to what has been cell cultures. Gas activation of L-type calcium reported with dose responses to PGE1 or miso- channels has been shown with recombinant L-type prostol.15 Since this contractile response could be calcium channels reconstituted into bilayers,28 in prevented by the addition of the thromboxane A2 cardiac myocytes via b-adrenergic receptor signaling 29 antagonist SQ29,548, higher doses of PGD2 probably as a down-regulation of receptor activity and in 30 cross-react with TP (thromboxane) receptors. A transgenic mice overexpressing cardiac Gas. In the similar phenomenon has been reported in non- later report, activation of L-type calcium channels 30 vascular smooth muscle in the lung, where PGD2 proceeded through a cAMP-independent pathway. plays a role in bronchoconstriction via TP recep- Thus, while cAMP may play a role in DP receptor tors.23 The pharmacological results are consistent signaling in human corpus cavernosum smooth with RT-PCR data demonstrating DP receptor mRNA muscle, the exact signal transduction pathways in both corpus cavernosum smooth muscle biopsy involved in smooth muscle relaxation remain to be specimens and cultured smooth muscle cells. The determined. tissue distribution of the rat DP receptor has been Despite the reported discreet and limited expres- characterized and found predominantly in the brain sion of DP receptor mRNA in human tissues,5 the in the leptomeninges and peripherally in the eye presence of mRNA for DP receptor and PGD and in the oviduct. Interestingly, no DP receptor synthase and functional responses to DP agonists mRNA was detected by Northern blot analysis of in human corpus cavernosum suggests a regulatory 21 polyA RNA in the rat penis. role for PGD2 in erectile physiology. Penile erection The estimated EC50 values for PGD2 and BW245C- is a complex hemodynamic physiological process induced relaxation in our study are comparable to involving central nervous system, peripheral ner- those reported in embryonic bovine tracheal cells vous system and vascular regulatory components. assayed for cAMP production (101 nM for PGD2 and Perhaps most interesting with regard to erectile 59 nM for BW245C).24 When cAMP production is function is the elegant work of Hayaishi and assessed in transfected HEK293 cells expressing the colleagues who have established a role for PGD2 in human DP receptor, the effective doses of PGD2 the induction of both slow wave (SWS) and rapid 7,31,32 (EC50 ¼ 6 nM) and BW245C (EC50 ¼ 0.7 nM) are sig- eye movement (REM) sleep. During REM sleep, nificantly lower.5 This may be due to much greater most human males have three to five episodes of numbers of receptors present on the HEK293 cells or nocturnal penile tumescence. These erections have

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