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Agents that elevate intracellular cyclic AMP (cAMP) have been found to enhance the synaptic discharge of norepinephrine (NE) from sympathetic nerve terminals in the rabbit iris-ciliary body and other peripheral tissues. We explored the hypothesis that prejunctional alpha 2-adrenergic receptors that mediate feedback inhibition of NE release may be coupled to adenylyl cyclase inhibition. To indirectly monitor cAMP changes in sympathetic axon terminals, we analyzed the cAMP-mediated activation of tyrosine hydroxylase, a sympathetic marker protein that undergoes acute phosphorylation and activation by cAMP-dependent protein kinase A. Tyrosine hydroxylase activity was assayed in situ by incubation of rabbit iris-ciliary body tissue segments in buffered Krebs-Ringer solution containing the substrate tyrosine (100 microM) and the DOPA decarboxylase inhibitor brocresine (30 microM). Intraneuronal DOPA accumulation was quantified by HPLC with electrochemical detection. Tyrosine hydroxylase activity was increased approximately 2 fold by incubation with forskolin (10 microM) plus IBMX (0.5 mM) or with 8-Bromo-cAMP (3 mM). Simultaneous addition of the alpha 2-adrenergic agonist clonidine (1 microM) attenuated the response to forskolin/IBMX, but had no effect on the response to 8-Br-cAMP. Clonidine-mediated inhibition of the forskolin/IBMX response was abolished by treatment of tissues with N-ethylmaleimide (NEM), an alkylating agent that inactivates pertussis toxin-sensitive G proteins (Gi) that couple receptors to adenylyl cyclase inhibition. These findings suggest that prejunctional alpha 2-adrenoceptors in the rabbit iris-ciliary body are negatively coupled to adenylyl cyclase. This mechanism may contribute to autofeedback regulation of NE biosynthesis and release.

PGE2 binding sites or receptors of porcine ciliary nonpigmented epithelial (NPE) and pigmented epithelial (PE) membranes were solubilized with detergents (CHAPS and Triton X100). From the Scatchard plots of PGE2 binding to CHAPS-solubilized proteins, the Kd and Bmax values were calculated to be 35 nM and 470 fmol/mg protein for NPE protein and 65 nM and 430 fmol/mg protein for PE protein, respectively. On the basis of the Kd and Bmax values, the solubilized receptor proteins correspond to PGE2 binding sites of the membranes which have previously been shown to be coupled to adenylate cyclase inhibition. For both NPE and PE proteins, the order of binding potency was PGE2 > PGF2 alpha > PGD2. By gel filtration chromatography of NPE and PE proteins, the molecular mass of the major PGE2 binding peak was estimated to be about 150 KDa when solubilized in CHAPS and 46 KDa for Triton X100 extracts. The Bmax values of membrane-associated binding proteins were increased by GTP, indicating a close association of the PGE2 binding sites with a GTP-binding protein. However, GTP did not affect the Bmax values of detergent-solubilized receptor proteins.

Cyclooxygenase products of arachidonic acid, for example prostaglandins (PGs), prostacyclin, and thromboxane A2, mediate a wide range of physiological actions. Vasodilation, increased vascular permeability, platelet aggregation and its inhibition, and immunomodulation are some of the important biological actions of cyclooxygenase products (1). Depending on type and dose, PGs cause vasodilation, increase or decrease intraocular pressure, and disrupt the blood-aqueous barrier (2, 3). These actions also vary qualitatively and quantitatively with the animal species. Prostaglandins, like any biological molecule, must act by binding with their specific receptors. Coleman and coworkers (4, 5), from a series of studies with PG agonists and antagonists in vascular and non-vascular smooth muscle preparations, classified PG receptors. This classification led to a greater appreciation of the relationship between PG actions and specific PG receptors in various tissues. Ocular actions of PGs linked with specific PG receptors are far from being clear. In this communication we will review our work on PG binding sites in ocular tissues and PG receptors coupled to adenylyl cyclase or phosphoinositidase C signal transduction pathways in ocular tissues of various animal species.

An overview of the ocular hypotensive actions of some alpha 2-agonists with imidazoline structures is presented. These agents inhibit isoproterenol-stimulated adenylate cyclase (AC) activity in ciliary process membrane through a Na+ and GTP-dependent mechanism. Receptor binding studies with the alpha 2-agonist radioligand [125I] p-iodoclonidine ([125I]PIC) in rabbit ciliary body membranes indicate that the alpha 2-receptor subtype is alpha 2A. Gpp(NH)p and NaCl dose-dependently decreased the number of [125I]PIC binding sites by shifting the receptor-G protein complexes from the high affinity state to the low affinity state for agonist binding. This is consistent with the observations that inhibition of AC was Na+ and GTP dependent. The NaCl and Gpp(NH)p effects on binding appeared to be through different mechanisms. The alpha 2-receptor in ciliary process thus appears to be an alpha 2A-receptor that is negatively coupled to the AC-cAMP generating system.

The intent of this work was to examine the actions of allicin on 1) intraocular pressure (IOP) in normal and unilaterally sympathectomized (SX) rabbits; 2) cAMP accumulation in the rabbit iris-ciliary body (ICB) and cultured nonpigmented epithelial (NPE) ciliary body cells; and 3) 3H-norepinephrine (NE) release by calculating fractional tritium overflow in response to electrical field stimulation (EFS, 5 Hz, 12 V/cm) in isolated, perfused rabbit ICBs. Allicin, one of the active compounds produced by garlic, was evaluated on IOP and it was determined that allicin (1, 2.5, or 10 micrograms), topically, but not the precursor, alliin (10 micrograms), lowered the IOP unilaterally in normal rabbits. Allicin (10 micrograms) reduced the IOP by 6 +/- 1 mmHg (n = 4) in normal rabbits at 2 hrs (maximum response) whereas no change occurred in sympathectomized rabbit eyes. Moreover, allicin (0.01, 0.1, or 1 microM) caused 40, 40, or 52% inhibition, respectively, of 3H-NE overflow in response to EFS. Isoproterenol (ISO, 1 microM) stimulated cAMP accumulation by 3.6 and 9 fold in isolated rabbit ICB and cultured NPE cells, respectively. Allicin (1 microM) had no effect on basal cAMP level while it inhibited ISO-stimulated cAMP accumulation by 40% and 23% in ICB and NPE cells, respectively. This study suggests that allicin lowered IOP, in part, by dual actions at the neuroeffector junction.

Treatment with histamine (10(-4) M) of cultured non-pigmented human ciliary epithelial cells led to a biphasic elevation of free intracellular calcium mediated by H1-receptors. The initial transient increase was due to Ca(2+)-release from intracellular calcium stores and could be blocked with a high concentration of verapamil (10(-4) M). The subsequent sustained elevation of cytoplasmic calcium caused by an influx of extracellular calcium was reduced by staurosporine (10(-7) M). We conclude that the sustained increase of cytoplasmic calcium by histamine may be partially mediated by activation of protein kinase C. Since depolarization of the cells had no effect on intracellular calcium, we conclude that typical voltage-operated calcium channels do not significantly influence intracellular calcium.

The activity to synthesize prostaglandins (PGs*) from arachidonic acid [i.e. cyclooxygenase (CO) activity] was compared between nonpigmented epithelial (NPE) cells and pigmented epithelial (PE) cells from porcine ciliary processes. PG synthesizing activities were about the same (0.58 and 0.52 pmol per mg protein per 15 min for NPE and PE, respectively), and inhibited by indomethacin and anti-CO antibody but not by nordihydroguaiaretic acid. By immunogold labeling, CO immunoreactivity was found both in NPE and PE. The present results and the previous observation on higher PG omega/omega-1 hydroxylation activities in NPE than in PE, taken together, suggest that controlled synthesis and inactivation of PGs may have a role in functions of NPE. When PGE2 was incubated with NPE or PE, NPE cells accumulated PGE2 more actively than did PE cells. PG uptake by the NPE cells may prevent aqueous and vitreous PGs from entering the anterior chamber and causing anterior uveitis.

The effect of topical unilateral application of lofexidine, an alpha 2-agonist, on ocular regional blood flow was tested in anesthetized rabbits using radiolabeled microspheres. A significant reduction in blood flow was found only in the ciliary body of the treated eye at 1 hr after lofexidine treatment. However, the IOP of both eyes was decreased significantly at 30 and 60 min post lofexidine treatment. Yohimbine (i.v.) blocked this IOP lowering effect, but only partially prevented the blood flow response. In addition, no significant difference was found in either basal or amphotericin-B stimulated short-circuit current or potential difference of the isolated rabbit iris-ciliary body after 30 minutes of lofexidine treatment. These observations suggest that the IOP lowering effect of lofexidine appears to be mediated by alpha 2-receptors but unrelated to the reduction in the ocular blood flow and net electrogenic ion transport.

MK-927 (dl-5,6-dihydro-4-(2-methylpropylamino)-4H-thieno(2,3b)thiopyra n-2-sulfonamide-7,7-dioxide hydrochloride) is a water soluble, carbonic anhydrase inhibitor (CAI) possessing a Ki of 12.0 nM against purified human carbonic anhydrase II in vitro. The acute instillation of one drop (50 microliters) of 0.5%, 1% and 2% solutions of MK-927 maximally decreased the intraocular pressure (IOP) of ocular hypertensive, cynomolgus monkeys by 4.7, 5.9 and 9.6 mm Hg, respectively. The decline of 9.6 mm Hg represented a reduction of 27% from the corresponding vehicle-treated value of 35.3 mm Hg. Peak reductions in IOP were present at 2 to 4 hr after the instillation of the three doses and the ocular hypotensive effect was waning at 6 hr. The IOP of normotensive, monkey eyes was significantly lowered by 1% and 2% solutions of MK-927 with the effect being more transient in these eyes than in hypertensive eyes. The elevated IOP of alpha-chymotrypsinized rabbits was dose-dependently decreased by 0.01%, 0.1% and 0.5% solutions of MK-927. MK-927 modestly bound to rabbit ocular pigment in vitro and the concentrations of MK-927 in the iris + ciliary body of pigmented rabbits were higher than those in the same tissue of albino rabbits after dosing with 0.5% MK-927. The ocular hypotensive effect of 2% MK-927 was greater in magnitude and longer in duration in normal pigmented than in albino rabbits. The IOP lowering action of MK-927 was local as evidenced by results of ocular distribution studies and the observation that the unilateral instillation of 0.5% MK-927 into the contralateral eye was devoid of effect on the untreated, hypertensive eye of alpha-chymotrypsinized rabbits. MK-927 has been selected for topical evaluation in glaucoma patients.

Ocular tissues have the capacity to metabolize arachidonate to prostaglandins and related materials, such as hydroxy arachidonate derivatives (HETEs) which are potent mediators of the inflammatory response. Reactive oxygen species are also present during the inflammatory response, resulting in an altered oxidative environment within the eye. This study was designed to evaluate the possible impact of the oxidant hydrogen peroxide and anti-oxidants, ascorbic acid and glutathione, upon arachidonate metabolism. It was found that hydrogen peroxide was observed to potently inhibit arachidonate metabolism in the cornea, but not in the iris-ciliary body. This might be related to a more efficient detoxification of hydrogen peroxide by iris-ciliary body. Ascorbate appeared to have a general stimulatory influence upon arachidonate metabolism in the iris-ciliary body. In the cornea, ascorbate selectively reduced metabolism to HETE while enhancing the products generated by the cyclo-oxygenase pathway. In both cornea and iris-ciliary body reduced glutathione suppressed conversion of arachidonate to its active metabolites. These observations indicate that arachidonate metabolism in ocular tissues is sensitive to the oxidative environment.

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.

The ocular distribution of the carbonic anhydrase inhibitors 6-hydroxybenzothiazide-2-sulphonamide (L-643,799) and 6-hydroxybenzothiophene-2-sulphonamide (L-650,719) has been investigated in albino rabbits after conjunctival administration of these compounds or related alkyl prodrugs. The ocular penetration of L-650,719 has been compared in ocular normotensive rabbits and in animals whose intraocular pressure has been experimentally elevated.

3H-Prazosin and 3H-rauwolscine binding sites were identified in a membrane suspension prepared from albino rabbit iris + ciliary body. Scatchard analysis of saturation binding experiments demonstrated that both 3H-prazosin and 3H-rauwolscine bind to a single population of binding sites with KD values of 0.87 nM and 5.33 nM, respectively. Bmax values of 65.7 and 198 fmol/mg protein were obtained for 3H-prazosin and 3H-rauwolscine, respectively. Displacement studies by several adrenergic agonists and antagonists indicated that 3H-prazosin and 3H-rauwolscine labelled alpha 1- and alpha 2-adrenoceptors, respectively, in the iris + ciliary body. Epinephrine, norepinephrine and phenylephrine were able to stimulate the synthesis of 3H-inositol phosphates in ciliary processes labelled with 3H-inositol, with EC50 values of 2.4, 12 and 10 microM, respectively. The corresponding maximum stimulations of basal activity were 433, 430 and 283%, respectively. Phenylephrine behaved like a partial agonist in this assay. The norepinephrine response could be potently antagonized by prazosin (Ki = 27 nM), with rauwolscine being 285-fold less potent. An epithelial cell suspension was prepared from the ciliary process. Stimulation of phosphatidylinositol turnover by norepinephrine (0.1 mM) was observed, and this could be blocked by prazosin (10 microM), thus, indicating the presence of alpha 1-adrenoceptors, coupled to phosphatidylinositol turnover, in epithelial cells of the rabbit ciliary process.

In the present study we have measured the oxygen consumption and lactic acid production, under aerobic and anaerobic conditions, in the bovine ciliary process epithelium (CPE) in the presence and absence of transport modifiers. Basal oxygen consumption was 8-15 microliters O2 consumed/mg protein/hr and decreased by 35% when sodium was removed or ouabain was added to the media. Anaerobic metabolism as measured by lactate production was also attenuated by sodium-free or ouabain treatment. When O2 consumption was severely limited by cyanide, lactic acid production increased significantly ("Pasteur effect"), whereas 2-deoxyglucose reduced lactate formation. Both chloride-free and acetazolamide treated CPE increased their dependency on aerobic glycolysis, and this response was also observed under anaerobic conditions, suggesting the presence of an anion transport mechanism. A net lactate production was also found to occur across the aqueous epithelium under aerobic and anaerobic conditions. These results are consistent with the presence of a bicarbonate-sensitive anion transport system located in the ciliary process epithelium.

Relative enzyme activities of the mercapturate pathway were determined in bovine ciliary pigmented (PE) and nonpigmented (NPE) epithelial cells in culture. Glutathione S-transferase activity was greater in PE cells than in NPE cells. Gamma-glutamyl transpeptidase activity appeared to be exclusively associated with NPE cells; the enzyme activity was virtually absent in PE cells. The level of cystine aminopeptidase activity was about the same in NPE and PE cells. N-acetyl transferase was predominantly found in NPE cells. If a similar enzyme distribution should exist in vivo in the bicellular layer of ciliary epithelium, the present result suggests that both NPE and PE cells are involved in the detoxification of xenobiotics via the mercapturate pathway, glutathione conjugation occurring primarily in the PE cells and the reactions catalyzed by the membrane-bound enzymes gamma-glutamyl transpeptidase and N-acetyl transferase taking place mainly in the NPE cells.

Ocular absorption of timolol in rabbits was studied after topical ocular administration of 3H-timolol in an eyedrop or in silicone cylindrical devices that released timolol at 7.2 micrograms/h. The devices were applied in either the inferior or superior conjunctival sac. Timolol concentrations were nearly equal in the inferior and superior portions of ocular tissues when the drug was administered in an eyedrop. Administration in the devices resulted in unequal timolol distribution in the cornea, conjunctiva, sclera, and iris-ciliary body. Timolol concentrations were higher in the part of each tissue that was closer to the site of the device application. Unequal concentrations of timolol in the superior and inferior part of the eye and very low timolol concentrations in the aqueous humor indicated that timolol was absorbed mainly via a noncorneal route from the device placed in the inferior conjunctival sac. Induced blinking at one minute intervals did not change ocular absorption of timolol. Compared with inferior conjunctival sac applications, placement of the devices in the superior conjunctival sac resulted in increased corneal and total ocular absorption of timolol as indicated by higher timolol concentrations in the aqueous humor and by a smaller difference between concentrations in the superior and inferior portions of the examined tissues. The application site dependent ocular absorption indicated that controlled release of timolol in the tear fluid did not result in a uniform timolol distribution in the preocular tear fluid of rabbit eyes.

The effect of t-butyl hydroperoxide (TBHP) on the oxidation of C1-radiolabeled glucose and C6-radiolabeled glucose by bovine ciliary body was investigated in the presence and absence of the glutathione reductase inhibitors nitrofurantoin (NF) and 1,3-bis(2-chloroethyl)-1-nitrosourea [BCNU]. 14CO2 production from 1-14C-glucose was markedly stimulated by TBHP and inhibited by BCNU. Using tissue extracts, BCNU was shown to inhibit both glutathione reductase and glucose-6-phosphate dehydrogenase. These results support the hypothesis that bovine ciliary body has high NADPH regenerating capacity by the hexose monophosphate shunt.

Topical beta blockers are used to treat glaucoma patients. These drugs inhibit aqueous production for prolonged periods of time. The purpose of this study was to determine whether timolol maleate (a non-specific beta blocker) binds to human iris-ciliary body (CB) melanin and to elucidate the binding characteristics of the drug to melanin. Timolol bound to bovine iris and ciliary body by two possible mechanisms. The binding kinetics indicate that the binding is probably of a nonspecific nature. There was no statistically significant differences between the melanotic tissues (CB, iris) and the nonmelanotic tissues (lens, cornea, liver, kidney) regarding the amount of timolol bound. However significantly more timolol was bound to the isolated melanins than the whole tissues. Timolol was released from the nonmelanotic tissues at a much faster rate than from the melanotic tissues. The amount of timolol bound to iris-CB from albino and pigmented rabbits showed that the amount of timolol bound to these tissues diminished in the following order: black or gray greater than brown greater than albino. It was also found that the rate of timolol release decreased in the following order: albino greater than gray greater than brown or black. Our results demonstrate the binding of beta blocker to human, bovine and rabbit iris-CB and consequent slow release of timolol from these tissues.