The role of NaKCl cotransport in blood-to-aqueous chloride fluxes across rabbit ciliary epithelium.
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PURPOSE: To evaluate the role of NaKCl cotransport in short-circuit current (Isc) and chloride fluxes across rabbit ciliary epithelium mounted in a Ussing-type chamber. METHODS: Bilayered intact ciliary epithelium free of stroma was obtained after perfusion and dissection of rabbit eyes and mounted in an Ussing-type chamber. The effects of bumetanide and other drugs on Isc and transepithelial 36Cl fluxes in bicarbonate-containing Ringer's were determined. Immunoblot analysis was performed by standard techniques. RESULTS: Bumetanide (100 microM) applied to the blood (pigmented epithelium [PE]) side of the ciliary bilayer caused a dose-dependent decrease in Isc from 18.2 +/- 2.2 to 10.4 +/- 1.4 microA/cm2 (43%). Bumetanide applied to the aqueous (nonpigmented epithelium [NPE]) side of the tissue inhibited Isc by only 12%. Immunoblots of dissected NPE and PE tissue probed with an antibody to mammalian NaKCl cotransporter detected approximately 10 times more NaKCl cotransporter protein in PE than in NPE. 36Cl flux studies revealed a PE-to-NPE chloride flux of 180.3 +/- 37.2 microEq/cm2 per hour and an NPE-to-PE flux of 72.3 +/- 22.9 microEq/cm2 per hour, indicating a net PE-to-NPE flux of 108.0 +/- 31.3 microEq/cm2 per hour across rabbit ciliary epithelium. Bumetanide inhibited the PE-to-NPE chloride flux by 52% but did not inhibit the NPE-to-PE flux. Isoproterenol (10 microM) added to the PE side of the bilayer increased Isc by a dose-dependent 53%. Prior addition of bumetanide to the PE side blocked the increase due to isoproterenol by 37%. Isoproterenol (10 microM) stimulated the PE-to-NPE chloride flux by 75% but had no stimulatory effect on the NPE-to-PE chloride flux. 4,4'Diisothiocyanatostilbene-2,2'disulfonic acid (DIDS) inhibited Isc when added to either side of the bilayer but was more potent at low concentrations (<100 microM) when added to the NPE side and more potent at higher concentrations (>100 microM) when added to the PE side. Prior addition of 1 mM DIDS to the NPE side decreased isoproterenol stimulation of Isc by 56%. CONCLUSIONS: NaKCl cotransporters located primarily on the blood side of rabbit ciliary epithelium contribute to aqueous-negative Isc and to blood-to-aqueous chloride transport across the tissue in bicarbonate-containing medium. DIDS-inhibitable mechanisms, possibly including HCO3-Cl exchange and Cl channels, also play a role. Isoproterenol stimulation of Isc involves coordinate upregulation of PE-side NaKCl cotransport and an NPE-side DIDS-inhibitable mechanism(s). (+info)
Neuronal propagation of HSV1 from the oral mucosa to the eye.
(58/1033)
PURPOSE: To identify possible neuronal pathways leading to herpetic ocular disease after primary oral infection in mice. METHODS: The SC16 strain of herpes simplex virus (HSV)-1 (10(6) plaque-forming units) was injected into the mucocutaneous border of the left upper lip. Animals were killed 2 to 10 days postinoculation (DPI). Spread of the virus in neural structures was studied by immunochemistry. RESULTS: HSV1 first replicated at the site of inoculation and then at the superior cervical ganglion (at 2 DPI). The trigeminal ganglion and the facial nerve fibers were infected by 4 DPI. Infection of the ciliary body and iris occurred at 6 DPI, together with several brain stem nuclei belonging to the autonomic or sensory pathways. Between 8 and 10 DPI, the neural infection gradually cleared up, except for the ipsilateral sympathetic ganglion, and ipsilateral keratitis appeared in some animals. CONCLUSIONS: The pattern of viral dissemination in this mouse model suggests that infection of iris and ciliary body results from transfer of virus in the superior cervical ganglion from sympathetic neurons innervating the lip to neighboring neurons innervating the anterior uvea. Later, zosteriform spread of virus from the trigeminal system may have contributed to the clinical and histologic findings. (+info)
Regional differences in the fine structure of the ciliary epithelium related to accommodation.
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The ciliary bodies of five monkey eyes and one human eye were subdivided into five zones. The ciliary epithelium with its bordering stroma was investigated electron microscopically. The number of cell organelles of the nonpigmented (NPE) and pigmented (PE) epithelium (mitochondria, rough endoplasmic reticulum, Golgi complexes); intercellular junctions between NPE and NPE, PE and PE, and NPE and PE (desmosomes, puncta adhaerentia, gap junctions, tight junctions); and fenestrations of the capillary endothelium were quantitatively evaluated. All these types of cell organelles, fenestrations of the capillary endothelium, and gap junctions in the NPE were found in greater numbers at the crests of the ciliary processes than in the valleys between processes. On the other hand, the number of puncta adhaerentia is significantly higher in the valleys than at the crests. In the valleys, the internal limiting membrane performs an elaborate network of electron-dense strands in which many fine zonular fibers terminate. These fibers are believed to belong to the "tension fiber system." Their firm attachment to the ciliary epithelium and the great number of intercellular junctions known as mechanical structures lend further support to our concept that these structures function as a fulcrum in the process of accommodation. (+info)
Hyperopia and loss of accommodation following ciliary muscle disinsertion in the cynomolgus monkey: physiologic and scanning electron microscopic studies.
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Twenty-three cynomolgus monkeys underwent 360-degree disinsertion and retrodisplacement of the ciliary muscle in one eye. Ten to 12 weeks after unilateral disinsertion, resting refraction in the "disinserted" eyes was more hyperopic than in the opposite eyes by 1.12 +/- 0.21 (mean +/- S.E.M.) diopters (p less than 0.001). Accomodative responses to intramuscular pilocarpine (2 or 3 mg/kg) were 0.90 +/- 0.14 (mean +/- S.E.M.) diopters in the disinserted eyes and 13.88 +/- 0.79 diopters in the opposite eyes. The induced hyperopia and loss of accommodation in the disinserted eyes seemed permanent, persisting for at least 14 months in one monkey and 29 months in three monkeys tested periodically after disinsertion. By light microscopy, the ciliary muscle in the disinserted eyes appeared normal and was contracted by pilocarpine. Scanning electron microscopy of the accommodative apparatus revealed retrodisplacement of the ciliary muscle, ciliary processes, and zonular plexus in the disinserted eyes. Structural alterations in the zonular apparatus seemed insufficient to account for the physiological findings. Hyperopia and loss of accommodation following ciliary muscle retrodisplacement are consistent with a new theory of zonular action during accommodation. (+info)
Multiple receptor activation elicits synergistic IP formation in nonpigmented ciliary body epithelial cells.
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We have examined the interaction between muscarinic and alpha(2)-adrenergic receptor activation on inositol phosphate (IP) formation in the nonpigmented cells of the ciliary body epithelium (NPE cells) of the rabbit. We have compared these changes with those previously observed in the intracellular free Ca(2+) concentration. Whereas muscarinic receptor activation causes an increase in intracellular Ca(2+) and IP formation, activation of alpha(2)-receptors does not significantly increase either intracellular Ca(2+) or IPs over basal levels. However, simultaneous activation of muscarinic and alpha(2)-adrenergic receptors with the specific agonists carbachol and UK-14304 produces massive Ca(2+) increases and results in a synergistic increase in IP formation. This synergistic IP formation is inhibited by both muscarinic and alpha(2)-adrenergic receptor antagonists as well as by pertussis toxin and an inhibitor of phospholipase C. IP formation is predominantly independent of intracellular Ca(2+), because it is decreased but not prevented by blocking the entry of Ca(2+) with LaCl(3) or chelating intracellular Ca(2+) with 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Thus synergistic IP formation underlies, at least in part, the synergistic increase in intracellular Ca(2+) resulting from simultaneous activation of muscarinic and alpha(2)-adrenergic receptors. (+info)
The flightless I protein localizes to actin-based structures during embryonic development.
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The product of the flightless I gene is predicted to provide a link between molecules of an as yet unidentified signal transduction pathway and the actin cytoskeleton. Previous work has shown that weak and severe mutations of the flightless I locus in Drosophila melanogaster cause disruption in the indirect flight muscles and in embryonic cellularization events, respectively, indicative of a regulatory role for the flightless I protein in cytoskeletal rearrangements. A C-terminal domain within flightless I with significant homology to the gelsolin-like family of actin-binding proteins has been identified, but evidence of a direct interaction between endogenous flightless I and actin remains to be shown. In the present study, chick, mouse and Drosophila melanogaster embryos have been examined and the localization of flightless I investigated in relation to the actin cytoskeleton. It is shown that flightless I localization is coincident with actin-rich regions in parasympathetic neurons harvested from chicks, in mouse blastocysts and in structures associated with cellularization in Drosophila melanogaster. (+info)
Effect of cataract extraction and posterior chamber lens implantation on outflow facility and its response to pilocarpine in Korean subjects.
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AIM: To investigate the effect of the lens on outflow facility in Korean patients with cataracts. METHODS: Intraocular pressure was measured by Goldmann applanation tonometry in 42 patients with cataracts and outflow facility was determined by tonography preoperatively, before and after instillation of pilocarpine. All patients received clear corneal phacoemulsification and silicone foldable intraocular lens implantation within the capsular bag by one surgeon. Two months after surgery, slit lamp examination and gonioscopy were performed and intraocular pressure and outflow facility were again determined. Statistical analysis was carried out using the Student's paired t test. RESULTS: There were no anterior chamber reactions and no visible trabecular meshwork damage 2 months after surgery. Intraocular pressure 2 months after lens extraction decreased by a mean of 2.4 (SE 0.4) mm Hg (p<0.001) compared with the preoperative value; postoperative outflow facility with and without pilocarpine increased by 0.080 (0.019) microl/min/mm Hg (p<0.001) and 0.045 (0.014) microl/min/mm Hg (p<0.001), respectively, at 2 months compared with preoperative values. The facility response to pilocarpine after lens extraction, relative to the baseline value and preoperative response, increased by 10.7 (7.1)% which was not statistically significant (p>0.1). CONCLUSION: Lens extraction causes a reduction in intraocular pressure and an increase in outflow facility in Korean subjects. Pilocarpine causes an increase in outflow facility which persists after cataract extraction and posterior chamber lens implantation. (+info)
Expression of human beta-defensins in intraocular tissues.
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PURPOSE: Defensins are naturally occurring antimicrobial peptides. Recently the authors published evidence of defensin production by the human ocular surface. A study was undertaken to look for intraocular defensins that may account for unexplained antimicrobial activity of intraocular fluids. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed on human postmortem ciliary body samples for beta defensins-1 (HBD-1) and beta defensin-2 (HBD-2), and alpha defensins 5 and 6. Induction of defensins by cytokines was analyzed in cultured human ciliary body epithelial (CBE) and retinal pigment epithelial (RPE) cells. Polyclonal antibodies were used to immunoblot aqueous and vitreous to detect HBD-1 and HBD-2 and to estimate their concentration. RESULTS: RT-PCR revealed constitutive HBD-1 message in ciliary body. HBD-2 and alpha defensin 5 and 6 messages were absent. HBD-2 message was induced by cytokine stimulation of both CBE and RPE cells. Immunoblots of vitreous and aqueous stained positively for HBD-1 but not HBD-2. The estimated aqueous concentration of HBD-1 was less than 16 ng/ml. CONCLUSIONS: This study demonstrates that HBD-1 is constitutively present in the aqueous and vitreous, probably at sub-bacteriocidal concentrations. HBD-2 was absent from aqueous, but cytokine stimulation studies suggest that it may be generated in response to inflammatory cytokines during infections. HBD-2 has a wider antibacterial spectrum, is 10-fold more potent, and may play a more significant role in antimicrobial defense than HBD-1. The use of defensins therapeutically may be indicated; however, caution is required because defensins also promote cell proliferation and fibrin formation, which are 2 key elements in ocular scarring processes such as proliferative vitreoretinopathy. (+info)