application) The long term goal of this application is to investigate the cellular and molecular mechanisms of diabetic impotence. Based on prior publications and preliminary data, we hypothesize that diabetic impotence is associated with alterations in signaling pathways of noradrenergic non-cholinergic (NANC) neurotransmission adenosine 3', 5' cyclic monophosphate (cAMP), guanosine 3', 5' cyclic monophosphate (cGMP), nitric oxide synthase (NOS), adrenoreceptor alpha, and growth factors (TGFa, TGFb1, TGFb2, TGFb3, IGF, and NGF). We will induce diabetes in Fisher rats by streptozotocin (35 mg /kg) and pursue the following experiments to test our hypothesis:
In Specific Aim 1, we will test the hypothesis that streptozotocin-induced diabetes will alter NANC vasodilator system in rats. Under this aim we will analyze intracavernous pressure (functional assessment) of penile erection in streptozotocin-induced diabetic (2, 4, 6, 8 weeks) rats. Immunohistochemical examination (NADPH diaphorase and NOS antibodies staining) of NOS-containing neurons and nerve fibers of the following sites: major pelvic ganglion and intra-penile nerves (dorsal, cavernous and spongiosal) and endothelium in early and late diabetic rats.
In Specific Aim 2, we will test the hypothesis that the cellular and molecular mechanisms of impotence associated with streptozotocin-induced diabetes are due to altered gene and protein expression of NOS, transforming growth factor (TGF), nerve growth factor (NGF), insulin like growth factor (IGF), and adrenoreceptor in rats. Under this specific aim, we will analyze NOS activity by enzymatic assay, NOS gene expression by Norther blot, cAMP, cGMP by radioimmunoassay in diabetic rats. The protein and gene expression for adrenoreceptor, NOS, TGFa, TGFb, NGF, and IGF-I will be done by immunohistochemistry/ Western blotting and Norther blot / reverse transcription/polymerase chain reaction (RT-PCR), respectively.
In Specific Aim 3, we will test the hypothesis that insulin treatment can alter diabetes induced cellular and molecular mechanisms of importance in rats. Under this we will first induced diabetes in rats (streptozotocin injection/35 mg/kg i.p., for 2, 4, 6, and 8 weeks, and then treat them with insulin 915 IU/Kg wt, subcutaneous injection). We will study: (1) functional assessment of penile erection, (2) immunohistochemical parameters (NADPH diaphorase and NOS antibodies staining) of NOS containing neurons and nerve fibers, and (3) protein and gene expression for adrenoreceptor, NOS, TGFa, TGFb, NGF, and IGF-I will be done by immunohistochemistry/ Western blotting and Norther blot / RT-PCR, respectively.
