Michael Wiederholt

The regulation of trabecular meshwork and ciliary muscle contractility.

Current models of aqueous humor outflow no longer treat trabecular meshwork (TM) as an inert tissue passively distended by the ciliary muscle (CM). Instead, ample evidence supports the theory that trabecular meshwork possess smooth muscle-like properties and is actively involved in the regulation of aqueous humor outflow and intraocular pressure. In this model, trabecular meshwork and ciliary muscle appear as functional antagonists, with ciliary muscle contraction leading to a distension of trabecular meshwork with subsequent reduction in outflow. and with trabecular meshwork contraction leading to the opposite effect. Smooth-muscle relaxing substances would therefore appear to be ideal candidates for glaucoma therapy with the dual goal of reducing intraocular pressure via the trabecular meshwork and of improving vascular perfusion of the optic nerve head. However, for such substances to effectively lower intraocular pressure, the effect on the ciliary muscle would have to he minimal. For this reason, more information is needed on the signalling processes involved in regulating trabecular meshwork and ciliary muscle contractility. This review attempts to outline current knowledge of signal transduction pathways leading to relaxation and contraction of ciliary muscle and trabecular meshwork. Pathways can be classified as involving or not involving changes of membrane voltage and of requiring or not requiring external calcium: possibly, other pathways exist. These different pathways involve different ion channels and isoforms of PKC and are expressed to a differing degree in ciliary muscle and trabecular meshwork, leading to differential responses when exposed to relaxing or contracting pharmacological agents. Some of these agents. like tyrosine kinase inhibitors and inhibitors of PKC. have been shown to relax trabecular meshwork while leaving ciliary muscle comparatively unaffected. This profile makes these substances appear as ideal drugs for simultaneously improving ocular outflow and retinal circulation, parameters that determine the time course of visual deterioration in glaucoma.

Endothelin-like immunoreactivity in aqueous humor of patients with primary open-angle glaucoma and cataract

Abstract• Background: Experimental evidence suggests a role of endothelin-1 (ET) in the regulation of intraocular pressure (IOP). • Method: Therefore, inpatients undergoing cataract surgery, ET-like immunoreactivity (STIR) was measured by radioimmunoassay in pooled samples of aqueous humor of eyes with primary open-angle glaucoma (POAG) and normotensive eyes with cataract only. • Results: ETIR was significantly (P < 0.05) higher in patients with cataract and POAG (20.5 ± 1.8 pg/ml,n = 12; preoperative IOP 21.4 ± I.1 mmHg,n = 33) than in patients with cataract only (15.8 ± 1.6 pg/m1,n = 15; preoperative IOP 16.0 ± 0.6 mmHg,n = 77). • Conclusion: This finding may indicate a role of ET in POAG or ocular antihypertensive treatment, and its relevance should be further investigated.

Evidence for Coupled Transport of Bicarbonate and Sodium in Cultured Bovine Corneal Endothelial Cells

SummaryUsin gintracellular microelectrode technique, the response of the voltageV across the plasma membrane of cultured bovine corneal endothelial cells to changes in sodium and bicarbonate concentrations was investigated. (1) The electrical response to changes in [HCO3−]o(depolarization upon lowering and hyperpolarization upon raising [HCO3−]o) was dependent on sodium. Lithium could fairly well be substituted for sodium, whereas potassium or choline were much less effective. (2) Removal of external sodium caused a depolarization, while a readdition led to a hyperpolarization, which increased with time of preincubation in the sodium-depleted medium. (3) The response to changes in [Na+]o was dependent on bicarbonate. In a nominally bicarbonate-free medium, its amplitude was decreased or even reversed in sign. (4) Application of SITS or DIDS (10−3m) had a similar effect on the response to sodium as bicarbonate-depleted medium. (5) At [Na+]o=151mm and [HCO3−]o=46mm, the transients ofV depended, with 39.0±9.0 (sd) mV/decade, on bicarbonate and, with 15.3±5.8 (sd) mV/decade, on sodium. (6) After the preincubation of cells with lithium, replacement of Li by choline led to similar effects as the replacement of sodium by choline, though the response ofV was smaller with Li. This response could be reduced or reversed by the removal of bicarbonate or by the application of SITS. (7) Amiloride (10−3m) caused a reversible hyperpolarization of the steady-state potential by 8.5±2.6 mV (sd). It did not affect the immediate response to changes in [Na+]o or [HCO3−]o, but reduced the speed of regaining the steady-state potential after a change in [HCO3−]o. (8) Ouabain (10−4m) caused a fast depolarization of −6.8±1.1 (sd) mV, which was followed by a continuing slower depolarization. The effect was almost identical at 10−5m. (9) It is suggested, that corneal endothelial cells possess a cotransport for sodium and bicarbonate, which transports net negative charage with these ions. It is inhibitable by stilbenes, but not directly affected by amiloride or ouabain. Lithium is a good substitute for sodium with respect to bicarbonate transport and is transported itself. In addition, the effect of amiloride provides indirect evidence for the existence of a Na+/H+-antiport. A model for the transepithelial transport of bicarbonate across the corneal endothelium is proposed.

Micropuncture study of renal transtubular concentration gradients of sodium and potassium in adrenalectomized rats

SummarySamples of proximal and distal tubular fluid were collected from control rats and from adrenalectomized animals maintained on 0.9% NaCl solution. Plasma and tubular fluid were analyzed for Na22 after adequate isotope equilibration and for Na and K by microflamephotometry. Similar analyses were performed in stationary microperfusion experiments in which the magnitude of the limiting concentration gradients for Na and K was estimated. Transtubular electrical potential differences were measured by means of glass-microelectrodes. A comparison of proximal tubular concentration gradients showed no significant differences between the control and the adrenalectomized rats for either Na or K. In contrast, the ability of the distal tubular epithelium to develop concentration gradients for Na and K was significantly reduced by adrenalectomy. This functional impairment could be reversed by the administration of d-aldosterone. No changes in proximal and distal transtubular electrical potential differences were observed in the various experimental conditions.

Differential smooth muscle-like contractile properties of trabecular meshwork and ciliary muscle

The contractile properties of bovine trabecular meshwork and ciliary muscle strips were investigated using an electromagnetic force-length transducer for isometric force measurements. Acetylcholine, pilocarpine and aceclidine administration resulted in dose-dependent contractions of trabecular meshwork and ciliary muscle. Absolute forces were approximately 10 times larger in ciliary muscle than in trabecular meshwork. Maximal force evoked by aceclidine (5 x 10(-5) M), when compared to the pilocarpine (5 x 10(-5) M) response, was significantly higher in trabecular meshwork than in ciliary muscle. The results were 172.5 +/- 12.6% (n = 7) and 138.9 +/- 4.0% (n = 8, P less than 0.05), respectively. Depolarization induced by raised external potassium (120 mM), when compared to the acetylcholine response (10(-3) M), resulted in a small contraction of 19.3 +/- 4.2% in trabecular meshwork (n = 5), and of 59.0 +/- 13.7% in ciliary muscle (n = 4, P less than 0.01). Both responses were inhibited by atropine (10(-5) M). The differential potassium effect may be explained by the large number of cholinergic nerve endings in ciliary muscle as compared to trabecular meshwork tissue. Recently, a dissociation between the effects of aceclidine on outflow resistance and accommodation has been described. Our data are consistent with these observations and provide evidence for a direct role of trabecular meshwork contractility in aqueous outflow regulation.

K+ Channels and the Intracellular Calcium Signal in Human Melanoma Cell Proliferation

Abstract. K+ channels, membrane voltage, and intracellular free Ca2+ are involved in regulating proliferation in a human melanoma cell line (SK MEL 28). Using patch-clamp techniques, we found an inwardly rectifying K+ channel and a calcium-activated K+ channel. The inwardly rectifying K+ channel was calcium independent, insensitive to charybdotoxin, and carried the major part of the whole-cell current. The K+ channel blockers quinidine, tetraethylammonium chloride and Ba2+ and elevated extracellular K+ caused a dose-dependent membrane depolarization. This depolarization was correlated to an inhibition of cell proliferation. Charybdotoxin affected neither membrane voltage nor proliferation. Basic fibroblast growth factor and fetal calf serum induced a transient peak in intracellular Ca2+ followed by a long-lasting Ca2+ influx. Depolarization by voltage clamp decreased and hyperpolarization increased intracellular Ca2+, illustrating a transmembrane flux of Ca2+ following its electrochemical gradient. We conclude that K+ channel blockers inhibit cell-cycle progression by membrane depolarization. This in turn reduces the driving force for the influx of Ca2+, a messenger in the mitogenic signal cascade of human melanoma cells.

A randomised prospective study of treatment of non-ischaemic central retinal vein occlusion by isovolaemic haemodilution.

In a randomised study 25 patients over 50 years of age with non-ischaemic central retinal vein occlusion (CRVO) were assigned to either a treatment (14 patients, isovolaemic haemodilution) or a control group (11 patients, no treatment). After three months eight eyes in patients with haemodilution improved, whereas none of the untreated eyes had better visual acuity (p less than 0.01). Thirteen haemodiluted and 11 control patients could be observed for one year. Six eyes of the haemodiluted patients retained a better visual acuity, whereas no improvements had occurred in the control group (p less than 0.025). In fluorescein angiography the lowering of the packed cell volume to 35-32% accelerated the time of maximal venous filling (tmvf) from 17.4 (SEM 1.4) s to 11.4 (SEM 0.9) s (p less than 0.005). In patients with non-ischaemic CRVO without treatment the passage time did not change. The shortened tmvf reflects a higher blood velocity. Thus isovolaemic haemodilution improves the visual prognosis in non-ischaemic CRVO probably by inducing a higher blood fluidity, which results in higher blood velocity, at least in areas of compromised retinal microcirculation.

Regulation of outflow rate and resistance in the perfused anterior segment of the bovine eye

Contractile properties of isolated trabecular meshwork strips have recently been described. In the present paper we characterize the regulation of the outflow pathway in the isolated perfused anterior segment of the bovine eye. Anterior segments of bovine eyes with detached iris, ciliary body and ciliary muscle were perfused at constant pressure of 8.8 mmHg. A constant outflow of approximately 6-8 microliters min-1 could be obtained for at least 3 hr. The calculated outflow resistance was in the range 1.1-1.4 mmHg min microliter-1. The relative outflow was significantly reduced after application of carbachol, reaching a maximal inhibition of 30%. EC50 for carbachol was 3 x 10(-8) mol l-1. Atropin completely blocked the effect of carbachol on outflow. Morphological examination of perfused anterior segments which were perfused with carbachol revealed an intact fine structure of the meshwork cells. Pilocarpine at 10(-5) mol l-1 reduced outflow by 15%. Epinephrine at 10(-5) mol l-1 reduced outflow, while epinephrine at 10(-6) mol l-1 slightly increased the outflow rate. This effect could be blocked by metipranolol. Endothelin-1 in concentrations of 2 x 10(-9) and 2 x 10(-8) mol l-1 inhibited relative outflow by > 30%. Carbachol, pilocarpine, endothelin and a high dose of epinephrine, which have been shown to induce contractions in isolated bovine trabecular meshwork and ciliary muscle strips, induced a reduction of outflow rate and an increase of outflow resistance of the anterior segment. Thus, at least in the bovine eye, the trabecular meshwork per se is directly involved in the regulation of aqueous humor outflow.

A randomised prospective study on treatment of central retinal vein occlusion by isovolaemic haemodilution and photocoagulation.

Thirty eight patients with ischaemic and non-ischaemic central retinal vein occlusion were evaluated for the effect of isovolaemic haemodilution. They were allocated at random to a haemodilution group (19 patients, panretinal photocoagulation and isovolaemic haemodilution) and a control group (19 patients, panretinal photocoagulation). Haematocrit was lowered in steps to 30 to 35% in the haemodilution group by repeated exchanges of whole blood for plasma and dextran (MW 40 000) and kept at this level for a period of six weeks. The haemodilution did not lead to serious complications. Three months after starting the treatment eight of 19 patients with haemodilution showed a better visual acuity, whereas only one of 19 control patients had improved. Seven of 17 patients with haemodilution, but only one of 17 control patients, retained a better visual acuity after one year. In the haemodilution group there were fewer patients with macular fibrosis and more with only minor foveal changes. The haemodilution seems to be more effective in patients with ischaemic than with non-ischaemic central retinal vein occlusion. It is concluded that isovolaemic haemodilution improves the visual outcome of patients with central retinal vein occlusion, probably mediated by enhanced retinal blood flow.

Endothelin-like immunoreactivity in the aqueous humour and in conditioned medium from cultured ciliary epithelial cells

Endothelin-like immunoreactivity was detected in human (15.6 +/- 2.7 pg/ml) and bovine (11.1 +/- 0.98 pg/ml) aqueous humour of the eye. These concentrations are 2-3 times higher than the corresponding plasma levels. Cultured human nonpigmented ciliary epithelial cells released endothelin-like immunoreactivity with a maximum of 2.1 +/- 0.32 pg/(cm2* 48 h). The release was stimulated by fetal calf serum, thrombin, carbachol and phorbol ester and blocked by cycloheximide. Immunocytochemistry showed cytoplasmic staining of cultured human nonpigmented ciliary epithelial cells for endothelin-1. Endothelin-1 was shown to induce contractions in isolated human ciliary muscle by isometric force measurements. Endothelin in the aqueous humour may play a role in the regulation of intraocular pressure.