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.

Endothelins inhibit cyclic AMP production in rabbit and human ciliary processes.

The effects of endothelins on cyclic AMP production have been studied in rabbit and human ciliary processes. Endothelins inhibit basal, isoproterenol-, and VIP-stimulated cyclic AMP production in rabbit ciliary processes and forskolin-stimulated cyclic AMP production in rabbit and human ciliary processes. EC50s for inhibition of forskolin-stimulated cyclic AMP production using rabbit tissue were similar: ET-1, 70 nM; ET-2, 40 nM; ET-3, 70 nM; maximum inhibition was about 76% for each ligand. These data are consistent with the presence of an ETB receptor. Endothelin-like immunoreactivity in rabbit aqueous humor was measured to be 226 pg/ml.

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.

Stimulatory and inhibitory cyclic AMP responses in rabbit ciliary processes after cervical ganglionectomy.

Cyclic AMP production in response to agonists which act at a variety of receptors to either stimulate or inhibit cyclic AMP production has been studied in intact, dissected ciliary processes from rabbit eyes after unilateral surgical removal of the cervical ganglion. Cyclic AMP responses to stimulatory ligands vasoactive intestinal peptide (VIP), isoproterenol, and forskolin and inhibitory agonists neuropeptide Y (NPY), the synthetic somatostatin analogue SMS 201-995, and alpha-adrenergic agents were investigated in tissues from normal eyes and compared to the same responses in tissues from sympathetically denervated eyes. Neither stimulated cyclic AMP production nor inhibition of stimulated cyclic AMP production was significantly different in tissues from denervated vs. normal eyes. Inhibition of VIP-stimulated cyclic AMP production by epinephrine and paraaminoclonidine in tissues from both normal and denervated eyes was blocked by the alpha 2-adrenergic antagonist yohimbine but not by the alpha 1-adrenergic antagonist prazosin. These data indicate that the VIP, NPY, somatostatin, and alpha 2- and beta 2-adrenergic receptors which regulate cyclic AMP production in rabbit ciliary processes are postjunctional and suggest that ligands known to modulate cyclic AMP levels in this tissue may exert effects on aqueous humor formation independently of adrenergic innervation.

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.

Alpha 2-adrenergic and VIP receptors in rabbit ciliary processes interact.

The interaction between alpha 2-adrenergic and VIP receptors has been studied by examining inhibition of VIP-stimulated cyclic AMP production by adrenergic agonists in intact, excised rabbit ciliary processes. Epinephrine, norepinephrine, isoproterenol, dopamine, and the specific alpha 2-adrenergic agonists clonidine and p-aminoclonidine exhibit dose-dependent inhibition of VIP-stimulated cyclic AMP production. I50s, clonidine (0.05 microM) = p-aminoclonidine (0.05 microM) congruent to epinephrine (0.1 microM) less than norepinephrine (2.0 microM) less than isoproterenol (15 microM) = dopamine (15 microM), are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors. Inhibition of VIP-stimulated cyclic AMP production by clonidine, epinephrine, isoproterenol, and dopamine is blocked by yohimbine but not by prazosin. These data establish the alpha 2-adrenergic specificity of the inhibitory effects observed. We have previously shown that beta 2-adrenergic receptor-mediated stimulation of cyclic AMP production in rabbit ciliary processes is also inhibited by postjunctional alpha 2-adrenergic receptors. These studies support the idea that the catecholamines may regulate aqueous humor formation by inhibiting stimulation of cyclic AMP production via postjunctional alpha 2-adrenergic receptors in ciliary processes.

Interaction between alpha 2- and beta 2-adrenergic receptors in rabbit ciliary processes.

The interaction between the alpha 2- and beta 2-adrenergic receptors of ciliary processes has been studied by examining dose-response curves for adrenergic agonist stimulation of cyclic AMP production by intact, excised rabbit ciliary processes. Stimulation of cyclic AMP production by 1-isoproterenol is maximum from 0.1 to 1.0 microM; at higher concentrations stimulation decreases and approaches basal levels. Decreased cyclic AMP production at high concentrations of isoproterenol is blocked by the specific alpha 2-adrenergic antagonist, yohimbine, but not by the alpha 1-adrenergic antagonist, prazosin. Ciliary processes from animals after bilateral cervical ganglionectomy also show reduced cyclic AMP production at high concentrations of isoproterenol and this reduction is blocked by yohimbine, but not prazosin. This experiment suggests that the inhibition at high concentrations of isoproterenol is mediated by postsynaptic alpha 2-adrenergic receptors. Cyclic AMP production is relatively insensitive to epinephrine and norepinephrine, but their responses are potentiated by yohimbine. Catecholamines and clonidine, a specific alpha 2-adrenergic agonist, exhibit dose-dependent inhibition of forskolin-stimulated cyclic AMP production by ciliary processes. I50s from the dose-response curves are consistent with the characteristic binding affinities of these adrenergic agonists for alpha 2-adrenergic receptors: clonidine = epinephrine greater than norepinephrine greater than isoproterenol. Inhibition of forskolin-stimulated cyclic AMP production by clonidine is blocked by yohimbine but not by prazosin.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of hCG on cyclic AMP in rabbit ciliary processes.

Experiments designed to address the role of cyclic AMP as a mediator of the eye pressure and aqueous flow reducing effects of human chorionic gonadotropin (hCG) in the rabbit have been conducted. No significant stimulatory effect of hCG on particulate preparations of adenylate cyclase from rabbit ciliary processes could be found under several different experimental conditions. Forskolin, an activator of adenylate cyclase can, in some biological systems, reveal a significant hormonal cyclic AMP response which is undetectable in the presence of hormone alone. Forskolin did potentiate the effect of isoproterenol on cyclic AMP production by intact rabbit ciliary processes. Cyclic AMP production by intact ciliary processes was not affected by hCG, either in the presence or absence of forskolin. Significant specific binding of iodinated hCG to particulate preparations of rabbit ciliary processes could not be detected. These results dispute the role of cyclic AMP as a mediator of the flow and pressure reducing effects of hCG; however, theoretical and methodological complications relating to this interpretation are discussed.

Intravitreal injection of purified human chorionic gonadotropin lowers IOP in rabbits.

Human chorionic gonadotropin (hCG) is a glycoprotein hormone composed of two dissimilar subunits alpha and beta held together by noncovalent forces. Each subunit contains about 30% carbohydrate and is extensively crosslinked by disulfide bonds. Previous work from our laboratory with commercial preparations of hCG indicated that intravitreal injection of hCG lowered intraocular pressure (IOP). Our work has been extended by using purified hCG obtained by reverse phase high performance liquid chromatography of a commercial preparation. With a wide pore octyl silica column and a step gradient composed of dilute aqueous trifluoroacetic acid and methanol, several peaks were obtained. The major peak was shown by sodium dodecylsulfate-polyacrylamide gel electrophoresis and amino acid analysis to contain both alpha and beta subunits. That this major peak contained intact hormone rather than a mixture of subunits was revealed by its ability to enhance the fluorescence of 8-anilino-1-naphthalene sulfonate and stimulate the release of cyclic AMP from isolated rat testes; subunits of hCG lack these properties. Physiological doses of hCG from this major peak injected intravitreally in rabbit eyes resulted in significant decreases in IOP without associated irritation when compared with contralateral control eyes.

Forskolin lowers intraocular pressure by reducing aqueous inflow.

Forskolin is a diterpene derivative of the plant Coleus forskohlii that stimulates adenylate cyclase activity without interacting with cell surface receptors. Forskolin lowers the intraocular pressure of rabbits, monkeys, and humans. In rabbits, net aqueous humor inflow decreases, outflow facility remains unchanged, and ciliary blood flow increases. Tolerance to the intraocular pressure lowering effect did not occur in rabbits after topical doses given every 6 hr for 15 days. In vitro forskolin activates adenylate cyclase of crude particulate homogenates prepared from cultured human ciliary epithelia or from dissected ciliary epithelial processes of rabbit or human eyes. This activation is not blocked by timolol. The stimulation of adenylate cyclase by isoproterenol in vitro is potentiated in the presence of forskolin. Forskolin represents a potentially useful class of antiglaucoma agents differing in molecular mechanism of action from previously used drugs.

Enzyme-linked immunosorbent assay of substance P: a study in the eye.

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.

Ultracytochemistry of cholera-toxin binding sites in ciliary processes.

Cholera toxin reduces the rate of aqueous humor in concentrations (10-11M) that do not disturb the morphology of the aqueous-humor forming epithelial cells of the ciliary processes of the rabbit eye. The search for an endogenous mediator of aqueous-humor formation comparable to cholera toxin in its mode of operation prompted us to map the distribution of cell surface receptors for cholera toxin in the ciliary processes of the eyes of rabbits. Cytochemical studies were carried out with the use of conjugates of cholera toxin to fluorescein isothiocyanate (CT-FITC) and to horseradish peroxidase (CT-HRP), and of the B subunit of cholera toxin to horseradish peroxidase (B-HRP). Multiple fluorescent CT-FITC binding sites were observed on the outer nonpigmented epithelial layer near the crests of the processes. Processes incubated with CT-HRP in vitro showed surface staining of 30-40% of the nonpigmented epithelial cells. A prominent reaction product was observed along the basal and lateral plasma membranes of these cells. In vivo studies carried out after arterial infusion of B-HRP showed a reproducible dense reaction product between the apical surfaces of the pigmented epithelium (PE) and of the nonpigmented epithelium (NPE) facing each other. Aggregations of reaction product were observed with the electron microscope in the extracellular space between the apices of PE and NPE. The apical plasma membrane of the endothelium of the blood vessels near the crests of the ciliary processes was stained after either in vivo or in vitro exposure to peroxidase conjugates. These findings indicate that the cell-surface receptors which mediate the action of cholera toxin on aqueous humor formation are very likely localized in the apical plasma membranes of the epithelium of the ciliary processes.

Fine structural studies of ciliary processes after treatment with cholera toxin or its B subunit.

Delivery of 2 micrograms of cholera toxin (CT), a specific, irreversible activator of adenyl cyclase, via the blood causes dilation of capillaries and stromal edema of the ciliary processes. These morphologic changes occur within 3 h, are maximal at 12 to 24 h, then gradually return to normal by 72 h. In the late phase of hypotony, ultrastructural changes in the ciliary epithelia, similar to Greeff vesicles, are due to a "paracentesis effect" from hypotony, caused by decreased aqueous flow through the eye. Delivery of 2 micrograms of the B subunit of CT (Sub-B) causes very mild capillary dilation and stromal edema of ciliary processes. These changes reach their peak at 3 h, then return to normal at 24 h. No significant damage occurred to the pigmented or non-pigmented epithelium with either agent. No hemorrhage, invasion of inflammatory cells or appearance of fibrin exudates in the ciliary processes could be detected.

Intraocular pressure and aqueous flow are decreased by cholera toxin.

Delivery of 2.1 microgram of cholera toxin, a specific, irreversible activator of adenylate cyclase, via the blood lowers IOP from 17.4 to 11.2 mm Hg in 81/2 hr. decreases net aqueous flow by about 50% in 8 hr, and doubles blood flow to the anterior uvea at 8 to 13 hr. Intravitreal injection of 0.26 microgram of cholera toxin lowered IOP from 15.0 to 9.6 mm Hg, but heat-inactivated toxin had no effect on IOP. The toxin activates adenylate cyclase from ciliary processes 2.2-fold and stimulates cyclic AMP production by ciliary processes 7.4 times. Absence of aqueous flare, normal protein concentrations in the aqueous, and histologic examination all confirmed the functional and structural integrity of the blood-aqueous barrier after cholera toxin infusion. The data point to an important role for ciliary process adenylate cyclase in regulation of aqueous flow and maintenance of IOP.

Potentiation of the effects of topical epinephrine on the pupil and intraocular pressure in the sympathetically denervated rabbit eye by a catechol-O-methyl transferase inhibitor.

Dose-response curves of increase in pupil size and decrease in intraocular pressure with topical epinephrine have been determined in the sympathetically denervated rabbit eye. Topical pretreatment with the catechol-O-methyl transferase inhibitor U-0521 potentiated the effects of epinephrine on both the pupil and pressure. These observations suggest a possible role for catechol-O-methyl transferase in the aqueous humor dynamics of the supersensitive eye. The possible use of the denervated rabbit eye as an experimental model for the glaucomatous eye in evaluating the ocular effects of adrenergic agents is discussed.

Identification of A and B forms of monoamine oxidase in the iris-ciliary body, superior cervical ganglion, and pineal gland of albino rabbits.

The properties of monoamine oxidase activity in homogenates of the iris-ciliary body, superior cervical ganglion, and pineal gland of albino rabbits have been studied. Inhibition curves using specific inhibitors support the concept of A and B enzyme forms, with the following ratios of A/B activity: iris-ciliary body, 40/60; superior cervical ganglion, 90/10; pineal gland, 13/87. Experiments on enzymes from animals with superior cervical ganglionectomy indicate that both A and B forms in the iris-ciliary body have a predominantly extraneuronal location. No significant differences in activity were observed between iris-ciliary body preparations from normal and denervated animals with substrates specific for A or B forms, or with substrates deaminated by both A and B forms. With tryptamine as substrate the iris enzyme can be inhibited by a variety of common monoamine oxidase inhibitors. Topical application of the inhibitor pargyline lowers intraocular pressure in the normal rabbit eye but not in the sympathetically denervated eye. This observation and in vitro data suggest that the mechanism of action of pargyline is through the adrenergic system and not dependent upon intrinsic activity.

The effect of d-isoproterenol on intraocular pressure of the rabbit, monkey, and man.

D-isoproterenol d-bitartrate applied topically lowers intraocular pressure (IOP) in normal albino rabbits and rabbits with alpha-chymotrypsin-induced glaucoma. This effect is independent of any effect on systemic blood pressure or pulse rate. A similar response could not be obtained in monkey or human eyes. Subconjunctival injection of d-isoproterenol d-bitartrate to monkey eyes did not alter IOP.