Biomedical subjects
B Horio
Publications and source records attributed to B Horio.
Regulation and bioelectrical effects of cyclic adenosine monophosphate production in the ciliary epithelial bilayer.
PURPOSE: To determine whether the intact isolated ciliary epithelial bilayer retains the alpha-2 and beta adrenergic receptor activation and interaction described for whole ciliary processes and whether this pure epithelial bilayer displays bioelectric parameters sensitive to alterations in cyclic adenosine Monophosphate (cAMP) production induced by adrenergic compounds. METHODS: The intact ciliary epithelial bilayer of the rabbit eye isolated by perfusion was mounted in a specially constructed Ussing-type chamber. The transepithelial potential difference and short-circuit current were monitored for effects induced by agents that stimulated or blocked (some did both) caMP production. Using a radioimmunoassay, the latter were studied in the pure epithelial bilayers and in whole ciliary processes. RESULTS: A reproducible increase in cAMP production and an increase in the short-circuit current induced in the bilayer by isoproterenol, a nonspecific beta adrenergic agonist, were both blocked by pretreatment with either timolol, a nonspecific beta adrenergic blocking agent, or with para-aminoclonidine, an alpha-2 agonist. Maximal stimulation of cAMP with forskolin in this pure isolated epithelial preparation yields a response that is 60% of the value found in whole processes, indicating that the latter tissue contains responsive sites that are nonepithelial, probably vascular, or perhaps stromal. The degree of inhibition of the beta adrenergic receptor by alpha-2 agonists was not very different in the two preparations. On the other hand, inhibition of the epithelial vasointestinal peptide receptor by neuropeptide Y or alpha-2 agonism was considerably heightened in the pure bilayered epithelial preparation. CONCLUSIONS: The isolated intact ciliary epithelial bilayer, when stimulated with beta adrenergic receptor agonists, vasointestinal peptide, or forskolin, produces increased cAMP and its transepithelial potential becomes hyperpolarized. These chemical and bioelectrical effects are prevented by pretreatment with either alpha-2 adrenergic agonists or beta adrenergic blocking agents. The results obtained in the isolated intact purely epithelial ciliary bilayer confirm that the ciliary epithelium is the source of adrenergic receptor activation and interaction and support the hypothesis that aqueous humor production is regulated by interactions between epithelial alpha-2 and beta adrenergic receptors.
Regulation of cyclic AMP production in adult human ciliary processes.
Cyclic AMP production in intact ciliary processes from elderly human donors is subject to stimulatory and inhibitory control by various agents. Stimulation of cAMP production is observed with forskolin, vasoactive intestinal peptide, or the beta-adrenergic agonist isoproterenol. Inhibition of forskolin-stimulated cAMP production is observed with endothelin-2 or PAC. The inhibitory effect of PAC is blocked by the specific alpha 2-adrenergic antagonist, yohimbine. Endothelin-2 has no effect on basal cAMP production. These data document the positive and negative regulation of cAMP responses in adult human ciliary processes and support the idea that cAMP is a key intermediate in the regulation of aqueous humor formation.
Human autologous serum for the treatment of full-thickness macular holes. A preliminary study.
BACKGROUND: Recent studies have shown the usefulness of pars plana vitrectomy with/or without the use of transforming growth factor-beta in treating macular holes. The purpose of the present study is to test the efficacy of autologous serum in conjunction with current surgical techniques in the repair of stage 3 or 4 macular holes. METHODS: A total of 11 eyes in nine patients with stage 3 or 4 full-thickness macular holes were treated. The patients ranged in age from 53 to 80 years (mean, 68 years). These patients were followed for 4 to 11 months (mean, 8.4 months). Preoperative best-corrected visual acuity ranged from 2/200 to 20/80 (mean, 20/200). A standardized pars plana vitrectomy was performed with removal of the posterior hyaloid and/or removal of epiretinal membranes, fluid-gas exchange. Autologous serum (0.1 ml) was instilled over the macular hole followed by perfluorocarbon gas tamponade and head positioning for 2 weeks. RESULTS: All 11 (100%) of the eyes in 9 patients had resolution of the surrounding subretinal fluid and flattening of the macular hole. All of the eyes showed an improvement of at least two lines or more (mean, 4.7 lines) in visual acuity. Three eyes (27%) had visual acuities of 20/40 or better, and seven (64%) had visual acuities of 20/60 or better. No exuberant fibrosis proliferation was noted in any eye. CONCLUSION: The results of this preliminary study indicate the possible benefit of autologous serum when used in conjunction with current surgical techniques in treating stage 3 or 4 macular holes.
Characterization of vasoactive intestinal peptide receptors in rabbit ciliary processes.
PURPOSE: To demonstrate a potential role for vasoactive intestinal peptide (VIP) in the regulation of ciliary process function, VIP receptors on rabbit ciliary process membranes were identified and characterized in biochemical and immunochemical studies. METHODS: Membranes were isolated from rabbit ciliary processes, and VIP receptors were characterized by competition binding, affinity cross-linking, and N-glycanase digestion. A site-specific polyclonal antibody directed against the NH2-terminal end of the deduced sequence of the recently cloned rat VIP receptor was generated and used to identify the VIP receptor by immunoblot analysis. RESULTS: Membranes isolated from rabbit ciliary processes exhibited a high-affinity VIP binding site (KD approximately 1 nM). Secretin and glucagon, which possess considerable primary sequence homology with VIP, were ineffective in inhibiting 125I-VIP binding to ciliary process membranes. In conjunction with the chemical cross-linking agent disuccinimidyl suberate, 125I-VIP specifically labeled a 63-kd protein in membranes from ciliary processes. This apparent size was confirmed by immunoblot analysis of ciliary body membranes using a site-specific polyclonal antibody that recognizes residues 92 to 104 of the rat VIP receptor. Digestion of the affinity-labeled receptor with N-glycanase generated an N-linked oligosaccharide free core protein of -50 kd. CONCLUSIONS: These findings demonstrate the presence of specific VIP receptors in rabbit ciliary processes. The differences in ligand specificity and structure of the ciliary process VIP receptor, compared to VIP receptors on peripheral tissues, suggest either a specific role(s) for VIP that may be unique to the anterior segment or the existence of VIP receptor isoforms.
Changes in aqueous norepinephrine and cyclic adenosine monophosphate during the circadian cycle in rabbits.
The concentrations of catecholamines and cyclic adenosine monophosphate (AMP) in the aqueous of rabbits entrained to 12-hr light:12-hr dark were measured at six times during the circadian cycle. Aqueous norepinephrine was higher in the dark than in the light; the minimum concentration at 1.5 hr after lights on, and maximum concentration at 6 hr after lights off, were 1.21 +/- 0.17 and 4.60 +/- 0.79 ng/ml, respectively. The concentrations of epinephrine and dopamine were less than the sensitivity of the assay (0.1 and 0.4 ng/ml, respectively) at all times. Aqueous cyclic AMP was also higher in the dark than in the light; the minimum concentration at 6 hr after lights on, and maximum concentration at 10.5 hr after lights off, were 11.5 +/- 1.2 and 29.4 +/- 2.7 pmol/ml, respectively. Superior cervical ganglionectomy reduced aqueous norepinephrine during light and dark to less than the sensitivity of the assay (0.1 ng/ml); preganglionic section of the cervical sympathetic trunk (decentralization) did not reduce aqueous norepinephrine as much as ganglionectomy but markedly reduced its concentration during the light and dark. Superior cervical ganglionectomy or pretreatment with 0.1% timolol virtually eliminated, and decentralization markedly reduced, the dark-phase increase of aqueous cyclic AMP. Most of the daily changes in norepinephrine and cyclic AMP persisted in animals housed in constant dark; this is consistent with the fraction of both changes which persisted in constant dark being circadian. These results support the idea that increased adrenergic activity during the dark increases the rate of aqueous flow and intraocular pressure in rabbits.
Neuropeptide Y and somatostatin inhibit stimulated cyclic AMP production in rabbit ciliary processes.
The interaction of adrenergic and peptide receptors linked to adenylate cyclase and the inhibition by bioactive peptides of stimulated cyclic AMP production has been investigated in intact, excised rabbit ciliary processes. Cyclic AMP production stimulated by isoproterenol, vasoactive intestinal peptide, or forskolin was inhibited by the biologically active peptides neuropeptide Y, somatostatin, and the synthetic somatostatin analogue SMS 201-995. IC50s determined from dose-response curves of inhibition are consistent with the known abilities of these ligands to modulate cyclic AMP and physiological responses in other tissues. Inhibition by neuropeptide Y or SMS 201-995 was unaffected by the specific alpha 2-adrenergic antagonist yohimbine, which shows that peptide inhibition is not occurring via peptide binding to the inhibitory alpha 2-adrenergic receptor. These results suggest that endogenous peptides may participate in modulation of cyclic AMP production and subsequent physiological events influenced by cyclic AMP levels in rabbit ciliary processes by inhibiting stimulated cyclic AMP synthesis.
Isoproterenol-induced amylase release in rabbit parotid acini: relation of protein phosphorylation, cyclic AMP and related kinase activity to changes in secretory rate.
Isoproterenol-induced amylase release from rabbit parotid acini was examined in relation to cyclic AMP (cAMP) concentrations, cAMP-dependent protein kinase (cAMP-PK) activity ratios and protein phosphorylation. Initial stimulation of amylase release by isoproterenol was preceded by increases in cAMP, cAMP-PK activity ratios and phosphorylation of a 34,000 MW (major) and a 30,000 MW (minor) protein in the microsomal fraction. When propranolol was added, decreases in cAMP concentrations and cAMP-PK activity ratios preceded the reduction in amylase release. Detailed analysis was performed on the 34,000 MW protein. The relation of dephosphorylation of protein 34 and reduction in amylase release was complex. Slight dephosphorylation occurred before or concurrently with the decrease in amylase release; however, maximal dephosphorylation was preceded by maximal inhibition of amylase release. When secretion of amylase was reinstituted by isoproterenol or forskolin, increases in cAMP and cAMP-PK activity ratios occurred before or in concert with amylase release but rephosphorylation of protein 34 occurred after the start of amylase release. Photoaffinity labeling studies using [32P]-8-azidoadenosine-3',5'-cyclic monophosphate indicated that proteins 34 and 30 were not regulatory subunits of cAMP-PK or their breakdown products. Although these data are consistent with phosphorylation of proteins 34 or 30 being required for triggering initial secretion, maximum dephosphorylation was not essential for inhibition of secretion. Furthermore, initiation of amylase release by the gland after a short period of quiescence did not depend on prior phosphorylation of protein 34. These data may indicate the absence of a requirement of amylase release for phosphorylation of protein 34.
Phosphorylation of rabbit parotid microsomal protein occurs only with beta-adrenergic stimulation.
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