ABSTRACT
Pannexins (Panxs) are channel-forming proteins that have homology to the invertebrate gap junction proteins, the innexins. These proteins form membrane channels implicated in ATP release. To evaluate the role of Panxs in the male reproductive tract, we investigated the distribution and regulation of Panx1 and 3 in the testis, efferent ducts (ED), and epididymis of adult rats. In the testis, Panx1 localized to the basal compartment of the seminiferous epithelium, while Panx3 was expressed in Leydig cells. In the ED, both Panxs were expressed in the apical region of ciliated cells. In the epididymis, Panx1 was detected at the base of the epithelium, at times encompassing basal cells, while Panx3 was restricted to the apical plasma membrane of principal cells. Panx3 immunoreactions were high throughout the entire epididymis while Panx1 was high in all regions except the initial segment. Multiple transcripts for Panx1 were identified, and sequence analysis indicated that alternative splicing might account for them. Orchidectomy resulted in the expression of multiple immunoreactive Panx1 bands, and these appeared to be androgen-repressed throughout the epididymis. Panx3 levels in all epididymal regions were also androgen-repressed. Deglycosylation experiments indicated that some Panx1 species were due to glycosylation, but this did not account for all Panx1 immunoreactive species. In summary, Panxs expressed in the epididymis and regulated by both alternative splicing events and androgens. These proteins may play a role in ATP secretion into the epididymal lumen and basal extracellular spaces for functions involving sperm transport and maturation.
Subject(s)
Androgens/metabolism , Connexins , Nerve Tissue Proteins , Organ Specificity/physiology , Animals , Connexins/genetics , Connexins/metabolism , Ejaculatory Ducts/metabolism , Epididymis/metabolism , Glycosylation , Ion Channels/metabolism , Leydig Cells/metabolism , Male , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Orchiectomy , Rats , Rats, Sprague-Dawley , Reproduction , Seminiferous Epithelium/metabolismABSTRACT
BACKGROUND: The kinin B1 receptor (B1R) is upregulated by pro-inflammatory cytokines, bacterial endotoxins and hyperglycaemia-induced oxidative stress. In animal models of diabetes, it contributes to pain polyneuropathy. This study aims at defining the cellular localization of B1R in thoracic spinal cord of type 1 diabetic rats by confocal microscopy with the use of a fluorescent agonist, [Nalpha-Bodipy]-des-Arg9-BK (BdABK) and selective antibodies. METHODS: Diabetes was induced by streptozotocin (STZ; 65 mg/kg, i.p.). Four days post-STZ treatment, B1R expression was confirmed by quantitative real-time PCR and autoradiography. The B1R selectivity of BdABK was determined by assessing its ability to displace B1R [125I]-HPP-desArg10-Hoe140 and B2R [125I]-HPP-Hoe 140 radioligands. The in vivo activity of BdABK was also evaluated on thermal hyperalgesia. RESULTS: B1R was increased by 18-fold (mRNA) and 2.7-fold (binding sites) in the thoracic spinal cord of STZ-treated rats when compared to control. BdABK failed to displace the B2R radioligand but displaced the B1R radioligand (IC50 = 5.3 nM). In comparison, IC50 values of B1R selective antagonist R-715 and B1R agonist des-Arg9-BK were 4.3 nM and 19 nM, respectively. Intraperitoneal BdABK and des-Arg9-BK elicited dose-dependent thermal hyperalgesia in STZ-treated rats but not in control rats. The B1R fluorescent agonist was co-localized with immunomarkers of microglia, astrocytes and sensory C fibers in the spinal cord of STZ-treated rats. CONCLUSION: The induction and up-regulation of B1R in glial and sensory cells of the spinal cord in STZ-diabetic rats reinforce the idea that kinin B1R is an important target for drug development in pain processes.
