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The physiologic mechanism that underlies the epinephrine-induced increase in facility of outflow (C) in glaucomatous human eyes and normal primate eyes is not completely understood. In this study, a recently developed in vitro human eye perfusion model was used to simultaneously monitor facility and cyclic adenosine monophosphate (AMP) changes in response to epinephrine (EPI). In this system, EPI (2.5 x 10(-5) mol/l) resulted in a maximal 44% increase in C, with an ED50 occurring at approximately 8 x 10(-6) mol/l. The C-increasing effect of 10(-5) mol/l EPI was unaffected by 10(-6) mol/l phentolamine. However, it was completely blocked in the presence of 10(-6) mol/l timolol or 2 x 10(-7) mol/l ICI118,551, suggesting the involvement of beta-2 adrenergic receptors. In biochemical studies, 10(-5) mol/l EPI induced a 12- to 14-fold increase in cyclic AMP in the perfusate of treated eyes. This increase was blocked by ICI118,551. In isolated intact human trabecular tissue, a 10 min incubation with 10(-5) EPI stimulated cyclic AMP by a factor of 2.7 over control levels. After 90 min, cyclic AMP levels were increased 4.2 fold over control levels. Collectively, these results show that the intraocular pressure lowering effect of EPI in the human eye is mediated, at least in part, by an increase in facility of outflow. Furthermore, the facility increase appears to be mediated by beta-2 adrenergic receptors and is correlated in time with increased cyclic AMP production.

Cell cultures derived from trabecular meshworks of human and bovine eyes and from bovine vascular endothelia were incubated at 37 degrees C for 1 hr with ethacrynic acid (ECA, 0.1-0.5 mmol/l) dissolved in culture medium. At 2 hr after the initial exposure, ECA at concentrations up to 0.4 mmol/l induced a reversible alteration in cell shape in all three cell types that was coincident with a change in the staining pattern of major cytoskeletal components including actin, alpha-actinin, vinculin, and vimentin. Distinct progressive alterations in beta-tubulin also occurred, with initial changes observed 10 min after ECA exposure. The ECA-induced changes in tubulin were blocked in part by preincubation with taxol (which stabilizes the microtubule structure), but they appeared to differ from those occurring with nocodazole (which interferes with tubulin assembly). These results suggest the possibility that ECA-induced increases in outflow facility may be mediated by alterations in the cytoskeletons of outflow pathway cells.

Twice daily topical administration of echothiophate for 2 weeks to the eyes of living cynomolgus monkeys produced profound subsensitivity of the accommodative response to pilocarpine and an approximately 50% decrease in the number of specific binding sites for 3H-quinuclidinyl benzilate (3H-QNB) in the ciliary muscle without a change in their affinity. When echothiophate treatment was discontinued, functional cholinergic sensitivity and the number of QNB binding sites both returned to normal over a similar 4-8 week period. Most animals had a modest overshoot of both functional sensitivity and number of binding sites for at least several weeks thereafter. The treated to control eye ratios for the number of binding sites and accommodative response to pilocarpine were correlated and the plot of log treated to control binding site ratio versus treated to control accommodation ratio resembled a dose-response curve. Similarly, the treated versus control eye differences for the two parameters were correlated, with the regression line passing through the 0-0 axis intercept. Collectively, these findings suggest that agonist-induced modulation of functional cholinergic sensitivity in the parasympathetically innervated (as opposed to denervated) ciliary muscle occurs by a muscarinic receptor-mediated mechanism. This system appears to provide a useful model to study the regulation of ciliary muscle cholinergic sensitivity.

The effect of the vasoactive peptide, endothelin, on facility of outflow, accommodation, and pupil diameter was measured in the monkey eye in vivo. Endothelin increased the outflow facility 22-71% at approximate anterior chamber concentrations ranging from 10(-10)-10(-7) M. Endothelin-induced accommodation was modest but consistent and statistically significant ranging from 1.61 to a maximum of 2.43 diopters at 10(-9) M and 10(-7) M endothelin, respectively. No change in pupil diameter was noted with any of the administered doses. These data demonstrate an action of endothelin on outflow facility and, together with prior evidence indicating the presence of endothelin receptors on the ciliary muscle, suggest that, like cholinergic agonists, the observed effects of endothelin on outflow facility may be mediated, at least in part, through an action on the ciliary muscle.

During the course of constant-pressure anterior chamber perfusion experiments in eyes from a number of species, measured outflow facility increases progressively with perfusion time. One possible explanation for this phenomenon has been that the technique of perfusion induces a loss of extracellular material from the outflow pathway. Therefore, this general observation has been termed the "wash-out" effect. Data from the authors' laboratories demonstrate that although outflow facility increases in cynomolgus monkey eyes by 26% and 42% from baseline over 90 minutes in vivo and in vitro, respectively, this does not occur in the enucleated human eye. The absence of washout in the human eye probably cannot be explained by postmortem conditions since enucleated monkey eyes undergo a similar magnitude of washout as the monkey eye in vivo. Furthermore, since washout does not occur in the perfused human infant eye, the age of the donor cannot explain the difference in washout properties between human and other primate eyes. The monkey eye in vivo is a valuable model in the study of outflow physiology. However, the difference in washout behavior between human and other primate eyes may point to a potentially important fundamental difference in the biology of the outflow pathways in these two species.

A smooth muscle cell line (H7CM) was established from the ciliary muscle of a 1-day-old human infant. The cultured cells had a normal female karyotype (46 XX) and could be maintained in cell culture for at least 11 generations. A common feature of confluent cultures was the presence of abundant bundles of 6-7 nm microfilaments associated with dense bodies. Both the ultrastructural appearance and the presence of smooth muscle-specific alpha-isoactin (also present in the human ciliary muscle in situ) support the smooth muscle origin of the H7CM cell line. Continuous membrane voltage (Vm) recordings were obtained in confluent monolayers of H7CM cells using glass microelectrodes. Resting Vm in 105 impalements averaged -66.2 +/- 0.7 mV (mean +/- standard error of the mean). In this system, rapid membrane transients induced by changing of the superfusing test solutions were detectable. Relative K+ conductance was characterized, and the contribution of electrogenic sodium/potassium adenosine triphosphatase to Vm was investigated. Under control conditions, H7CM cells were electrically quiescent. However, action potentials could be induced by application of 10 mM barium. Barium-induced action potentials were not abolished by removal of extracellular Na+ nor were they inhibited by the presence of tetrodotoxin. However, they were blocked by verapamil, fulfilling criteria believed to be typical for smooth muscle cells. Acetylcholine, carbachol, and to a lesser extent pilocarpine induced a reversible Vm depolarization. The effect of acetylcholine was blocked by atropine, implying muscarinic receptor involvement in the Vm response. Collectively, these findings show the potential usefulness of cultured ciliary muscle cells in understanding further the cellular mechanisms underlying drug-induced contraction of the human ciliary muscle.

Ciliary ganglionectomy in the cynomolgus monkey produced loss of the accommodative response to electrical stimulation of the Edinger-Westphal nucleus and to topical eserine, concurrent with enhanced responsiveness to topical and systemic pilocarpine. This supersensitivity produced by parasympathetic denervation was not associated with an increase in muscarinic receptor number or affinity measured using specific QNB binding sites in ciliary muscle homogenates. In seven of ten cases, ciliary muscle muscarinic receptors were decreased by 60-84%, while in the remaining three cases, the decrease ranged from 12-37%. In no case was supersensitivity accompanied by an increased number of receptors. Six or more months after ganglionectomy, accommodative responses to central electrical stimulation and topical eserine returned to normal in most animals, indicating parasympathetic reinnervation of the ciliary muscle. Ciliary muscle sensitivity to pilocarpine returned to normal with reinnervation and was associated with recovery of normal receptor number and with normal binding affinity.

In cynomolgus monkeys, resting total outflow facility was unaltered 1 and 6 or more months after ciliary ganglionectomy (CG) or postganglionic ciliary neurectomy (PCN). Intraocular pressure (IOP) was decreased in the denervated eye 1 week and 1 month after surgery, but returned to normal after 6 or more months. Although baseline facilities were comparable in CG/PCN and fellow control eyes 6 or more months after surgery, even maximal intracameral doses of pilocarpine did not increase outflow facility in previously denervated eyes, while a normal facility increase occurred in fellow control eyes. However, both previously denervated and fellow control eyes exhibited a large facility increase to both submaximal and greater than maximal intracameral doses of eserine.

Topical echothiophate administration to the cynomolgus monkey eye for 5-6.5 months produced marked subsensitivity of the accommodative response to pilocarpine and a 65% decrease in specific high affinity 3H-QNB binding sites (ostensibly indicating muscarinic receptors) in the ciliary muscle. The decrease in 3H-QNB binding sites was quantitatively similar in surgically untouched, totally iridectomized, and ciliary muscle disinserted eyes. Following a 5-month off treatment recovery period, 3H-QNB binding sites increased to more than twice the number in untreated control eyes. In echothiophate-treated eyes whose contralateral eyes had previously received atropine+echothiophate, 3H-QNB binding sites were three to six times more numerous than in other long-term echothiophate-treated eyes, and one to two times more numerous than in untreated controls. Topical pilocarpine administration for 1 day to 7 months reduced ciliary muscle 3H-QNB binding sites by approximately 25%. Alterations in muscarinic receptors during and following cholinergic drug therapy may in part explain the observed subsensitization and recovery of ciliary muscle physiological responses.

Ciliary ganglionectomy and/or postganglionic ciliary neurectomy in the cynomolgus monkey was followed by supersensitivity to intramuscular (i.m.) pilocarpine and lack of response to topical eserine and to electrical stimulation of the Edinger-Westphal nucleus. Normal responsiveness to pilocarpine and eserine returned in most instances after about 6 months. An accommodative response to stimulus of the Edinger-Westphal nucleus was also present and, as in control eyes, could be blocked by hexamethonium. Collectively, these findings demonstrate that parasympathetic innervation to the ciliary muscle mediating accommodation traverses a typical peripheral autonomic synapse, almost certainly located predominantly in the ciliary ganglion; by 6 months after denervation, the ciliary muscles have reinnervated; and the parasympathetic pathway to the eye exhibits plasticity and capacity for regeneration.

No differences were found between fluorophotometrically determined aqueous flow rates in cynomolgus monkeys having vs not having undergone prior repeated anterior chamber perfusion. This suggests that: repeated perfusion does not produce a progressive decline in the rate of aqueous formation; and the repeatedly perfused monkey eye can be validly used in studies of aqueous formation.