Ethacrynic acid induces reversible shape and cytoskeletal changes in cultured cells.

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.

Epinephrine increases facility of outflow and cyclic AMP content in the human eye in vitro.

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.

Ethacrynic acid increases facility of outflow in the human eye in vitro.

Anterior segments of human donor eyes were perfused with culture medium at a perfusion pressure of 15 mm Hg in a 5% carbon dioxide environment at 37 degrees C. After determination of a baseline facility of outflow, the perfusion chamber contents were exchanged with either drug vehicle or ethacrynic acid, at concentrations ranging from 0.01 to 0.

Accommodation and ciliary muscle muscarinic receptors after echothiophate.

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.

Outflow facility studies in the perfused human ocular anterior segment.

We have recently developed a tissue model of the human aqueous outflow pathway involving placement of the eviscerated anterior corneoscleral shell, [with lens and uveal tissue removed but trabecular meshwork (TM) attached] onto a specialized perfusion apparatus. The TM and associated outflow tissues are perfused with culture medium at a physiologically-relevant perfusion pressure in a 5% CO2 environment at 37 degrees C. Under these conditions, the perfused outflow tissues are similar for several days, to the human and/or subhuman primate outflow system in vivo with regard to morphology as well as several functional parameters.

Effect of endothelin on outflow facility and accommodation in the monkey eye in vivo.

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.

Absence of time-dependent facility increase ("washout") in the perfused enucleated human eye.

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.

Membrane voltage recordings in a cell line derived from human ciliary muscle.

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.

Ciliary muscle choline acetyltransferase and acetylcholinesterase after ciliary ganglionectomy.

Choline acetyltransferase (ChAT; EC 2.3.1.

Dissociation between the effect of aceclidine on outflow facility and accommodation.

The dose/effect relationships for intracameral doses of aceclidine HCl and pilocarpine HCl on total facility of outflow and accommodative amplitude were compared in the cynomolgus monkey eye in vivo. The largest dose of aceclidine tested (20 micrograms), resulted in an average increase of 270% in outflow facility, whereas accommodation was limited to an average of 5 diopters. In contrast, 20 micrograms of intracameral pilocarpine (which is submaximal in increasing facility) resulted in an average of 19 diopters of accommodation.

Ciliary neuromuscular morphology in cynomolgus monkeys after ciliary ganglionectomy.

Cynomolgus monkeys underwent unilateral ciliary ganglionectomy (CG) and/or posterior ciliary neurectomy (PCN). The ciliary muscle was functionally denervated, as evidenced by loss of choline acetyltransferase activity, loss of the accommodative response to topical eserine and electrical stimulation of the Edinger-Westphal nucleus, and supersensitivity of the accommodative response to pilocarpine. Light and electron microscopy carried out 3-28 days after CG/PCN revealed degeneration of myelinated and unmyelinated nerve fibers as evidenced by axonal swelling and shrinkage, mitochondrial degeneration, axoplasmic condensation and vacuolization, and activated, phagocytosing Schwann cells.

Endothelin depolarizes membrane voltage and increases intracellular calcium concentration in human ciliary muscle cells.

The ciliary muscle which is involved in accommodation and regulation of aqueous humour outflow resistance resembles smooth muscle in other parts of the body. In the present investigation we used an established primary cell line (H7CM) to study the effects of endothelin, a novel vasoconstrictor peptide, on membrane voltage (V) and intracellular calcium in cultured human ciliary muscle cells. Membrane voltage was measured in confluent monolayers of H7CM cells using conventional microelectrodes.

Outflow facility studies in the perfused bovine aqueous outflow pathways.

We have recently developed a technique for constant pressure perfusion of the aqueous outflow pathway of the eye. Our preliminary studies, conducted in the calf eye, show surprisingly that the manipulations necessary for preparing the outflow pathways and attached corneoscleral shell for perfusion do not greatly disrupt normal aqueous outflow physiology and anatomy according to the following criteria: 1. facility of outflow is similar before and during outflow pathway perfusion 2.

Time-dependence of perfusion outflow facility in the cynomolgus monkey.

Surgically virgin (N), aniridic (I), and ciliary muscle disinserted (D) cynomolgus monkey eyes had total outflow facility determined in vivo for 88-136 minutes by two-level constant pressure perfusion of the anterior chamber. In all three types of eyes, facility increased during the first hour but stabilized thereafter. Average facility for the second half hour was 15% higher than for the first half hour in N and I eyes, and 10% higher in D eyes.

Dissociation of cholinergic supersensitivity from receptor number in 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%.

Effect of cholinergic drugs on outflow facility after ciliary ganglionectomy.

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.

Reinnervation of primate ciliary muscle following ciliary ganglionectomy.

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.

Cholinergic drugs alter ciliary muscle response and receptor content.

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.

Effect of repeated anterior chamber perfusion on aqueous flow in the cynomolgus monkey.

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.

Comparative anesthetic effects on aqueous humor dynamics in the cynomolgus monkey.

Cynomolgus monkeys were anesthetized with either intravenous (IV) ketamine hydrochloride, IV ketamine and intramuscular (IM) diazepam, IM methohexital sodium and IM pentobarbital sodium, or endotracheal halothane. Intraocular pressure, aqueous humor flow rate (F), anterior chamber volume, corneal endothelial transfer coefficient (Ka), and anterior chamber elimination coefficient (Ke) were determined noninvasively, using applanation tonometry, keratometry, pachymetry, and fluorophotometry. Arterial blood gases (ABG), acid-base balance, and mean arterial pressure (MAP) were monitored.