Regulation of Sertoli cell tight junction dynamics in the rat testis via the nitric oxide synthase/soluble guanylate cyclase/3',5'-cyclic guanosine monophosphate/protein kinase G signaling pathway: An in vitro study
Lee,Nikki P.Y.; Cheng,Chuen-yan
Endocrinology 144(7): 3114-3129
Publication date: 2003
Nitric oxide (NO) synthase (NOS) catalyzes the oxidation ofL-arginine to NO. NO plays a crucial role in regulating variousphysiological functions, possibly including junction dynamicsvia its effects on cAMP and cGMP, which are known modulatorsof tight junction (TJ) dynamics. Although inducible NOS (iNOS)and endothelial NOS (eNOS) are found in the testis and havebeen implicated in the regulation of spermatogenesis, theirrole(s) in TJ dynamics, if any, is not known. When Sertoli cellswere cultured at 0.5-1.2 x 10 cells/cm on Matrigel-coateddishes or bicameral units, functional TJ barrier was formedwhen the barrier function was assessed by quantifying transepithelialelectrical resistance across the cell epithelium. The assemblyof the TJ barrier was shown to associate with a significantplummeting in the levels of iNOS and eNOS, seemingly suggestingthat their presence by producing NO might perturb TJ assembly.To further confirm the role of NOS on the TJ barrier functionin vitro, zinc (II) protoporphyrin-IX (ZnPP), an NOS inhibitorand a soluble guanylate cyclase inhibitor, was added to theSertoli cell cultures during TJ assembly. Indeed, ZnPP was foundto facilitate the assembly and maintenance of the Sertoli cellTJ barrier, possibly by inducing the production of TJ-associatedproteins, such as occludin. Subsequent studies by immunoprecipitationand immunoblotting have shown that iNOS and eNOS are structurallylinked to TJ-integral membrane proteins, such as occludin, andcytoskeletal proteins, such as actin, vimentin, and -tubulin.When the cAMP and cGMP levels in these ZnPP-treated sampleswere quantified, a ZnPP-induced reduction of intracellular cGMP,but not cAMP, was indeed detected. Furthermore, 8-bromo-cGMP,a cell membrane-permeable analog of cGMP, could also perturbthe TJ barrier dose dependently similar to the effects of 8-bromo-cAMP.KT-5823, a specific inhibitor of protein kinase G, was shownto facilitate the Sertoli cell TJ barrier assembly. Cytokines,such as TGF-ß and TNF-, known to perturb the Sertolicell TJ barrier, were also shown to stimulate Sertoli cell iNOSand eNOS expression dose dependently in vitro. Collectively,these results illustrate NOS is an important physiological regulatorof TJ dynamics in the testis, exerting its effects via the NO/solubleguanylate cyclase/cGMP/protein kinase G signaling pathway.