|Budget Amount *help
¥13,100,000 (Direct Cost : ¥13,100,000)
Fiscal Year 1998 : ¥3,700,000 (Direct Cost : ¥3,700,000)
Fiscal Year 1997 : ¥9,400,000 (Direct Cost : ¥9,400,000)
1) Research on the expression of constitutive and inducible heme-oxygenases (HOs) in the smooth muscle of the urogenital organs.
Methods 1 : Immunohistochemical study ; Immunohistochemical examination of the cryostat sections of the rat penile tissue using antiserum against rat HO1 and HO2 by ABC method.
Methods 2 : Molecular biological study ; Total RNA was extracted from the rat penile tissue, and the total RNA was reverse-transcribed into cDNA.Subsequently, polymerase chain reaction (PCR) was performed using primer pairs specific for either rat HO]. or H02. Amplified PCR products were electrophoreses in agarose gel, and were size-fractionated.
Results : Nerve-like structures which could be stained by NADPH-diaphorase staining and HO-2 immunohistochemistry, but not by HO-1, were clerly confirmed. The expression of HO-2 mRNA, but not HO-1 mRNA was coafirmed by RT/PCR.
2) Research on the presence and function of P2 purinergic receptors in the smooth muscle of the urogenital organs.
1 : Functional examination ; Isometric contraction studies of rat bladder tissues using electrical stimulation was performed, to determine the presence of non-adrenergic, non-cholinergic activity, and to confirm the effect of antagonists against P2X or P2Y receotor.
Methods 2 : Molecular biological study ; Total RNA was extracted from the rat penile, bladder, and seminal vesicle tissues, and the total RNAs were reverse-transcribed into cDNAs. Subsequently, polymerase chain reaction (POR) was performed using primer pairs specific for either rat P2X or P2Y receptor. Amplified POP.products were electrophoreses in agarose gel, and were size-fractionated. In situ hybridization was performed using cyostat sections of above tissues and probes speciflc for either rat P2Xor P2Y receptor.
Results : In the rat penis. P2YI purinoceptor mRNA was expressed, In the ratseminal vesicle, both P2X2 and P2YI purinoceptors were expressed. In the rat urinary bladder, both P2X2 and P2Y 1. purinoceptors were expressed, and P2X-mediated contraction and P2Y-mediatect relaxation were pharmacologically confirmed.
2) 分子生物学的解析:ラット膀胱、陰茎、精嚢から全RNAを抽出し、RT/PCRと直接塩基配列決定を行った.ラット膀胱、陰茎、精嚢から凍結切片を作成し、P2X及びP2Yに対するブローブを用いてin situ hybridization(ISH)を行い、mRNAの局在を調べた.