Larry P. Bausher

Interaction between alpha2-and beta2-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)

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

Abstract: 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 and the absorbance is read in a spectrophotometer 16‐24 h 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.

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.

Alpha2-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.

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.

Ultracytochemistry of cholera-toxin binding sites in ciliary processes.

SummaryCholera toxin reduces the rate of formation of aqueous humor in concentrations (10−11 M) that do not disturb the morphology of the aqueoushumor 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.

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.

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

Delivery of 2 μg 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 μg 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 nonpigmented epithelium with either agent. No hemorrhage, invasion of inflammatory cells or appearance of fibrin exudates in the ciliary processes could be detected.