Douglas S. Gregory

Cervical ganglionectomy alters the circadian rhythm of intraocular pressure in New Zealand White rabbits.

A circadian rhythm of intraocular pressure in rabbits could provide a useful model for understanding the daily rhythm of intraocular pressure in humans and for studying mechanisms which regulate intraocular pressure. Our results confirm earlier work showing that New Zealand White rabbits housed in an environment with a lighting cycle of 12 hours light and 12 hours dark have a rhythm of intraocular pressure, and that this rhythm persists in constant dark. We show further that the cycle of light and dark is the zeitgeber for entrainment of the rhythm of intraocular pressure, and therefore persistence of this rhythm in constant dark establishes it as a circadian rhythm. Cervical ganglionectomy demonstrated that intact sympathetic innervation to the eye is required for maintenance of the normal circadian rhythm of intraocular pressure in rabbits. Intraocular pressure in sympathectomized eyes is no different from control eyes during the light, but is significantly reduced during the dark.

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)

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.

LIGHT EXPOSURE DECREASES IOP IN RABBITS DURING THE NIGHT

Elevated IOP observed in rabbits during the dark phase of the circadian cycle decreased rapidly and reversibly when rabbits were exposed to light during the dark phase. The decrease of IOP does not result from decreased aqueous flow and only part of the decrease requires intact ocular sympathetic innervation.

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.

Melatonin does not increase IOP significantly in rabbits.

New Zealand white rabbits have a circadian rhythm of intraocular pressure; pressure is higher during the dark than during the light. We explored the possibility that the dark phase increase of serum and/or ocular melatonin plays a role in regulating the rhythm of intraocular pressure. Exogenous melatonin was delivered by four routes: topical application (0.1 and 10 micrograms), intravenous injection (1 microgram), intravitreal injection (9 micrograms) and intra-arterial infusion (50 ng and 1 microgram). Melatonin delivered by these routes did not increase intraocular pressure. We also measured the concentration of melatonin in the aqueous after unilateral intra-arterial infusion to confirm that melatonin delivered by this route reached the eye. The concentration was 419 +/- 99 and 109 +/- 17 pg/ml (n = 8, p < 0.025) in the ipsilateral and contralateral eyes, respectively. The concentration of melatonin in the aqueous after intra-arterial infusion of saline was less than the sensitivity of the assay (< 16 pg/ml, n = 6). The results of these experiments indicate that melatonin does not increase IOP significantly under the experimental conditions employed in this study.

Rabbits have a circadian rhythm of aqueous humor cyclic AMP

Rabbits have circadian rhythms of intraocular pressure (IOP) and aqueous flow. The dark phase increases of IOP and flow are associated with increased aqueous cyclic AMP. If the daily rhythm of aqueous cyclic AMP reflects changes of tissue levels of cyclic AMP which mediate one of the mechanisms which control the rhythms of IOP and aqueous flow, then it must be a circadian rhythm as are the rhythms of IOP and flow. A recent report showed no change in aqueous cyclic AMP from 2 hrs before to 2 hrs after the beginning of subjective dark in animals housed in constant dark. Because the increase of intraocular pressure at the same times did persist in constant dark, the authors concluded that a significant portion of the circadian rhythm of IOP is unrelated to cyclic AMP mediated ocular mechanisms. However, if an inhibitor of cyclic nucleotide phosphodiesterase is added to aqueous samples after harvesting by paracentesis, it is possible to show that most of the increase of aqueous cyclic AMP from mid-light phase to mid-dark phase persists in constant dark and is therefore likely to reflect a circadian rhythm of tissue cyclic AMP.

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.

Circadian change of adenylate cyclase activity in rabbit ciliary processes.

Purpose. To characterize in vivo changes of adenylate cyclase activity in rabbit ciliary processes during the circadian cycle. Methods. After removal of vitreous, lens, retina and choroid from freshly enucleated rabbit eyes, the anterior segment and attached sclera was chilled in cold buffer containing the non-selective phosphodiesterase (PDE) inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Then ciliary processes were excised and homogenized in ice cold trichloro-acetic acid (TCA) 2.5 min after IBMX treatment. Increased cyclic AMP in response to PDE inhibition was measured in ciliary processes at six times during the circadian cycle, after preganglionic section of the cervical sympathetic trunk (decentralization, DX), and after topical instillation of hydroxyamphetamine, timolol, brimonidine, rauwolscine or a soluble derivative of forskolin. Results. The increase of rabbit ciliary process cyclic AMP levels in response to PDE inhibition with IBMX was enhanced at night. Much of the enhanced nocturnal increase persisted in constant dark and was blunted by DX. Topical instillation of hydroxyamphetamine enhanced the increase during the day; whereas, timolol, a ß-adrenergic antagonist, or brimonidine, an a 2 -adrenergic agonist, blunted the IBMX induced increase. Neither instillation of rauwolscine, an a 2 -adrenergic antagonist, nor the soluble forskolin derivative enhanced the increase of cyclic AMP after IBMX. Conclusions. The technique reported here can be used to estimate ciliary process adenylate cyclase activity in vivo. There is a circadian rhythm of adenylate cyclase activity in rabbit ciliary processes that is driven in part by ocular sympathetic input and stimulation of ß-adrenergic receptors.

Response of rabbit ciliary process cyclic nucleotides to specific phosphodiesterase inhibitors.

Purpose. To determine which cyclic nucleotide phosphodiesterase family activities can be identified in rabbit ciliary processes. Methods. Freshly excised rabbit ciliary processes were incubated in vitro with family selective phosphodiesterase inhibitors in the absence or presence of activators of soluble or membrane bound guanylate cyclase. The resulting increases of cyclic AMP and cyclic GMP were measured by RIA. Results. Rabbit ciliary process cyclic AMP levels were increased by the phosphodiesterase 1 and 4 selective inhibitors, 8-methoxymethyl-IBMX and rolipram, respectively. Cyclic GMP levels were increased by 8-methoxymethyl-IBMX; whereas the phosphodiesterase 5 and 6 selective inhibitor, zaprinast, increased cyclic GMP little. Nitric oxide donors increased cyclic AMP in addition to cyclic GMP, and inhibition of soluble guanylate cyclase eliminated the increase of cyclic AMP. The effects of sodium nitroprusside and the phosphodiesterase 3 selective inhibitor, cilostamide, on cyclic AMP were not additive suggesting that the sodium nitroprusside mediated increase of cyclic GMP, like cilostamide, inhibited phosphodiesterase 3. Conclusions. Cyclic AMP in rabbit ciliary processes is hydrolyzed primarily by phosphodiesterases 1 and 4, and, when cyclic GMP levels are low, by phosphodiesterase 3; cyclic GMP is hydrolyzed primarily by phosphodiesterase 1.