Nitric Oxide-Mediated Regulation Of Connexin43 Expression In Human And Bovine Ciliary Bodies

Gap junctions (GJ) are clusters of intercellular plasma membrane channels that act as conduits for the direct cell-to-cell communication by allowing intercellular exchange of matter, energy and information via gap junctional intercellular communication (GJIC), which is thought to play an important role in the control of variety cellular functions, maintenance of metabolic homeostasis and neuronal transmission.Connexins (Cxs) are a mulitigene family which have the same basic molecular structure to compose gap junctions between adjacent cells. To date, Cxs have been reported with wild distributions in the eye, for instance in cornea, lens, iris, ciliary body and retina, etc. As the major member of Cxs, Cx43 has been proved localized to the PE-NPE interface of ciliary epitheliums to form gap junction but not found in the ciliary muscle in a number of species. Considering its special location and crucial role, more and more studies were focus on Cx43 and begun to investigate on its phosphorylation and relationship to aqueous humor formation.Nitric oxide (NO) which synthesized by the enzyme nitric oxide synthase (NOS) is an important signaling molecule in the vertebrate annimal's eyes, it can activate the soluble guanylate cyclase (sGC) and adenylate cyclase (AC) to induce the second messenger molecules such as cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) expression, the laters are important regulators in Cx43 channels and some drugs can enhance or reduce their effects. There may exist many signal pathways to influence aqueous humor formation via regulating of Cx43 gap junction.This study aimed to investgate the distribution of Cx43 in human and bovine ciliary body and also the regulation of NO on it. The results indicated that as a effective stimulant, NO can enhance the expression of Cx43 in the two-layers ciliary epithelium, and this effect possiblly depended on the activation of its downstream signal channels such as cGMP and cAMP. Hence our work takes an important role in further studies of aqueous humor generation mechanism and can help to kown more about the action mechanism of some anti-glaucoma medicines,including:PART IExpression of Nitric Oxide Synthase and Guanylate Cyclase in the Human Ciliary Body and Trabecular Meshwork【Purpose】To investigate the expression and distribution of nitric oxide synthase (NOS) isoforms and guanylate cyclase (GC) in human ciliary body and trabecular meshwork.【Methods】Sections of human ocular anterior segments were used. Expression of three NOS isoforms (i.e. neuronal NOS, nNOS; inducible NOS, iNOS and endothelial NOS, eNOS) and GC were assessed by immunohistochemical staining using monoclonal or polyclonal antibody of NOS and GC.【Results】Expression of three NOS isoforms and GC were observed in the ciliary epithelium, ciliary muscle, trabecular meshwork and the endothelium of the Schlemm's canal. Immunoreactivity of nNOS was detected mainly along the apical cytoplasmic junction of the non-pigmented epithelium (NPE) and pigmented epithelial (PE) cells, while iNOS and eNOS were evenly distributed in the cytoplasm of both the PE and NPE. In some ciliary processes, GC was shown to be evenly distributed in-the cytoplasm of the PE and NPE, while in the others it was expressed mainly along the apical cytoplasmic junction of the PE and NPE. NOS and GC were evenly distributed in the ciliary muscle cells, trabecular meshwork and the endothelium of the Schlemm's canal.[Conclusions]The distribution patterns of nNOS and GC in human ciliary epithelium suggest the possible involvement of NO and cyclic guanosine monophosphate (cyclic GMP, cGMP) signalling pathway in the intercellular communication between the PE and NPE cells, and may play a role in both processes of aqueous humor formation and drainage.PART IIExpression of Connexin43 and Connexin40 in Human and Bovine Ciliary Bodies [PURPOSE]To investigate the expression of connexin43 (Cx43) and connexin40 (Cx40) in the dual-layered ciliary epithelium of human and bovine eyes. [METHODS]Sections of human and bovine ocular anterior segments were used. Expression of Cx43 and Cx40 were tested using rabbit polyclonal antibody by immunohistochemistry methods. 【RESULTS】Expression of Cx43 was observed in ciliary epithelium of human and bovine but not in the ciliary muclse. Immunoreactivity of it was detected mainly along the apical cytoplasmic portions of pigment ciliary epithelial (PE) cells and the non-pigmented ciliary epithelial (NPE) cells with high concentration. However, there was no staining signal of Cx40 detected in human ciliary body.【CONCLUSIONS】The distribution patterns of Cx43 in human and bovine ciliary epithelium suggest that Cx43 may be a major component of PE-NPE gap junction channel to mediate intercellular communication between PE and NPE cells, and may play a role in the processes of aqueous humor formation.PARTⅢNitric Oxide-Mediated Regulation of Connexin43 Expression in Human and Bovine Cillary Bodies【PURPOSE】To investigate the potential role of Nitric Oxide (NO) and its downstream signaling molecules cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) in the regulation of Connexin43 expression in human and bovine ciliary bodies.【METHODS】The expression level of Cx43 protein in human and bovine ciliary bodies was measured by Western blot, before or after incubated with different drugs. Tissues were exposed for 18 hours to sodium nitroprusside (SNP, an NO donor, 100μM), 8-Bromo-cGMP(an cGMP analogue,500μM),8-Bromo-cAMP(an cAMP analogue, 250μM) respectively. Some experiments were conducted in the presence of ODQ (an soluble guanylate cyclase inhibitor,50μM), KT5823 (an Protein Kinase G inhibitor, 2μM), H89 (an Protein Kinase A inhibitor, lOμM) for 30 minutes before.[RESULTS]Incubation of ciliary bodies with SNP enhanced the basal expression of gap junction protein Cx43.8-bromo-cGMP and 8-bromo-cAMP exerted effects similar to SNP. Moreover, the synergistic action of SNP on Cx43 expression was significantly prevented by OQD, KT5823 and H89. The up-regulation effects of 8-bromo-cGMP and 8-bromo-cAMP were prevented by KT5823 and H89 respectively.[CONCLUSIONS]In short, these results suggested a possible involvement of NO/cGMP/PKG and NO/cAMP/PKA signal pathway in the control of Cx43 expression in human and bovine ciliary bodies. All these findings will open a new window toward our further understanding of the relationship between NO and aqueous humor production.