International Journal of Impotence Research (2002) 14, 446–452 ß 2002 Nature Publishing Group All rights reserved 0955-9930/02 $25.00 www.nature.com/ijir 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 tume- scence.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 intes- tine 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 poly- merase, 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