Ординатура / Офтальмология / Английские материалы / Aging and Age Related Ocular Diseases_Lutjen-Drecoll_2000

References

1Lampert PW, Vogel MH, Zimmerman LE: Pathology of the optic nerve in experimental acute glaucoma: Electron microscopic studies. Invest Ophthalmol Vis Sci 1968;7:199±213.

2Gaasterland D, Tanishima T, Kuwabara T: Axoplasmic flow during chronic experimental glaucoma. 1. Light and electron microscopic studies of the monkey optic nerve head during development of glaucomatous cupping. Invest Ophthalmol Vis Sci 1978;17:838±846.

3Hayreh SS, March W, Anderson DR: Pathogenesis of block of rapid orthograde axonal transport by elevated intraocular pressure. Exp Eye Res 1979;28:515±523.

4Radius RL, Pederson JE: Laser-induced primate glaucoma. II. Histopathology. Arch Ophthalmol 1984;102:1693±1698.

5Quigley HA, Addicks EM: Chronic experimental glaucoma in primates. II. Effect of extended intraocular pressure elevation on optic nerve head and axonal transport. Invest Ophthalmol Vis Sci 1980;19:137±152.

6Quigley HA, Addicks EM, Green WR, Maumenee AE: Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol 1981;99:635±649.

7 Quigley HA, Hohman RM, Addicks EM, Green WR: Blood vessels of the glaucomatous optic disc in experimental primate and human eyes. Invest Ophthalmol Vis Sci 1984;25:918± 931.

8Quigley HA, Addicks EM, Green WR: Optic nerve damage in human glaucoma. III. Quantitative correlation of nerve fiber loss and visual field defect in glaucoma, ischemic neuropathy, papilledema, and toxic neuropathy. Arch Ophthalmol 1982;100:135±146.

9May CA, Hayreh SS, Furuyoshi N, Ossoinig K, Kaufman PL, Lütjen-Drecoll E: Choroidal ganglion cell plexus and retinal vasculature in monkeys with laser-induced glaucoma. Ophthalmologica 1997;211:161±171.

10Aguayo AJ, Rasminsky M, Bray GM, Carbonetto S, McKerracher L, Villegas-Perez MP, Vidal-Sanz M, Carter DA: Degenerative and regenerative responses of injured neurons in the central nervous system of adult mammals. Philos Trans R Soc Lond B Biol Sci 1991;331: 337±343.

11Cohen A, Sivron T, Duvdevani R, Schwartz M: Oligodendrocyte cytotoxic factor associated with fish optic nerve regeneration: Implications for mammalian CNS regeneration. Brain Res 1990;537:24±32.

12Eitan S, Schwartz M: A transglutaminase that converts interleukin-2 into a factor cytotoxic to oligodendrocytes. Science 1993;261:106±108.

13Schnell L, Schneider R, Kolbeck R, Barde YA, Schwab ME: Neurotrophin 3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesion. Nature 1994; 367:170±173.

14Hopkins JM, Ford-Holevinski TS, McCoy JP, Agranoff BW: Laminin and optic nerve regeneration in the goldfish. J Neurosci 1985;5: 3030±3038.

15Bastmeyer M, Bähr M, Stuermer CAO: Fish optic nerve oligodendrocytes support axonal regeneration of fish and mammalian retinal ganglion cells. Glia 1993;8:1±11.

16Bastmeyer M, Beckmann M, Schwab ME, Stuermer CAO: Growth of regenerating goldfish axons is inhibited by rat oligodendrocytes and CNS myelin but not by goldfish optic nerve tract oligodendrocyte-like cells and fish CNS myelin. J Neurosci 1991;11:626±650.

17Battisti WP, Shinar Y, Schwartz M, Levitt P, Murray M: Temporal and spatial pattern of expression of laminin, chondroitin sulphate proteoglycan and HNK-1 immunoreactivity during regeneration in the goldfish optic nerve. J Neurocytol 1992;21:557±573.

18Ito S, Karnovsky MJ: Formaldehyde-glutaral- dehyde fixatives containing trinitro com-

pounds. J Cell Biol 1968;39:168a±169a.

19Alm A, Lambrou GN, Mäepea O, Nilsson SFE, Percicot C: Effects on ocular flow and optic nerve morphology of experimental glaucoma in

cynomolgus monkeys. Ophthalmologica 1997; 211:178±182.

20Kettenmann H, Ransom BR: Neuroglia. New York, Oxford University Press, 1995.

21Bignami A, Dahl D: Gliosis; in Kettenmann H, Ransom BR (eds): Neuroglia. New York, Ox-

ford University Press, 1995, pp 843±858.

22 Eng LF, Lee YL: Intermediate filaments in astrocytes; in Kettenmann H, Ransom BR (eds): Neuroglia. New York, Oxford University Press, 1995, pp 650±667.

23Dahl D, Bignami A, Weber K, Osborne M: Filament proteins in rat optic nerves undergoing Wallerian degeneration: Localization of vimentin, the fibroblastic 100 Å filament protein in normal and reactive astrocytes. Exp Neurol 1981;73:496±506.

24Dahl D, Strocchi P, Bignami A: Vimentin in the central nervous system: A study of the mes- enchymal-type intermediate filament-protein in Wallerian degeneration and in postnatal rat development by two-dimensional gel electrophoresis. Differentiation 1982;22:185±190.

25Iwaki T, Wisniewski T, Iwaki A, Corbin E, Tomokane N, Tateishi J, Goldman JE: Accumulation of alphaB-crystallin in central nervous system glia and neuromas in pathologic conditions. Am J Pathol 1992;140:45±356.

26Iwaki T, Kume-Iwaki A, Goldman JE: Cellular distribution of alpha B-crystallin in non-lentic- ular tissues. J Histochem Cytochem 1990;38: 31±39.

27Lowe J, McDermott H, Pike I, Spendlove I, Landon M, Mayer RJ: Alpha B crystallin expression in non-lenticular tissues and selective presence in ubiquinated inclusion bodies in human disease. J Pathol 1992;166:61±68.

28Kato S, Hirano A, Umahara T, Llena JF, Herz F, Ohama E: Ultrastructural and immunohistochemical studies on ballooned cortical neurons in Creutzfeldt-Jacob disease: Expression of alpha B-crystallin, ubiquitin and stress-re- sponse protein 27. Acta Neuropathol 1992;84: 443±448.

29Renkawek K, deJong WW, Merck KB, Frenken CW, van Workum FP, Bosman GJ: Alpha B-crystallin is present in reactive glia in Creutz- feldt-Jakob disease. Acta Neuropathol (Berl) 1992;83:324±327.

30Lütjen-Drecoll E, May CA, Polansky JR, Johnson DH, Bloemendal H, Nguyen TD: Localization of the stress protein ·B-crystallin and trabecular meshwork inducible glucocorticoid response protein in normal and glaucomatous trabecular meshwork. Invest Ophthalmol Vis Sci 1998;39:517±525.

31Duhamel RC, Johnson DA: Use of nonfat dry milk to block nonspecific nuclear and membrane staining by avidin conjugates. J Histochem Cytochem 1985;33:711±714.

32Skoff RP, Vaughn JE: An autoradiographic

study of cellular proliferation in degenerating rat optic nerve. J Comp Neurol 1971;141:133± 156.

33 Mansour H, Asher R, Dahl D, Labkovsky B, Perides G, Bignami A: Permissive and nonpermissive reactive astrocytes: Immunofluorescence study with antibodies to the glial hyal- uronate-binding protein. J Neurosci Res 1990; 25:300±311.

34Oblinger MM, Singh LD: Reactive astrocytes in neonate brain upregulate intermediate filament gene expression in response to axonal injury. Int J Dev Neurosci 1993;11:149±156.

35Varela HJ, Hernandez MR: Astrocyte responses in human optic nerve head with primary open-angle glaucoma. J Glaucoma 1997;6: 303±313.

36Hernandez MR, Pena JDO: The optic nerve head in glaucomatous optic neuropathy. Arch Ophthalmol 1997;115:389±395.

Key Words

Glaucoma W Trabecular meshwork W Smooth muscle W Aqueous humor outflow W Contractility

Abstract

Ample evidence supports the theory that trabecular meshwork possesses smooth-muscle-like properties. Trabecular meshwork cells express a large number of transporters, channels and receptors, many of which are known to regulate smooth-muscle contractility. It has been shown that trabecular meshwork can be induced to contract and relax in response to pharmacological agents. In the model of the bovine eye, confirmed in some cases by experiments on primates, agents that contract trabecular meshwork reduce outflow. On the cellular level, this is coupled with depolarization and a rise in intracellular calcium. Relaxation of trabecular meshwork, on the other hand, appears to be coupled to a stimulation of the maxi-K channel, inducing hyperpolarization and a closure of L-type calcium channels. No significant differences between cells from a human and a bovine source emerged, either in classical measurements of membrane voltage, in measurements of intracellular calcium or patch-clamp experiments. Thus, pharmacological agents that relax trabecular meshwork seem promising candidates for further research ± the ultimate goal being an improvement of glaucoma therapy in humans.

Copyright © 2000 S. Karger AG, Basel

Introduction

The major route for the outflow of aqueous humor is via the trabecular meshwork into Schlemm's canal [1±4]. The form and area of the spaces which are enclosed by the trabecular beams are important for aqueous drainage. The composition of the extracellular material in these spaces seems to be mainly responsible for outflow resistance [5]. In glaucoma, outflow is reduced due to an increase in different forms of extracellular material deposited within the cribriform layer of the trabecular meshwork, correlating with the loss of axons in the optic nerve [5].

In the traditional concept, trabecular meshwork is an inert tissue, with no regulatory properties of its own. In this concept, regulation of outflow resistance is determined by the ciliary muscle. Connected to the trabecular meshwork by fibers called zonules, contraction of the ciliary muscle passively distends the trabecular meshwork, increasing intratrabecular spaces [3, 4]. The same mechanism determines the accommodative state of the lens. Recent evidence speaks for an additional involvement of the scleral spur, a contractile structure containing myofibroblasts and projecting like a shelf into the trabecular meshwork from its posterior margin [4, 6, 7]. Physiologically, the regulation of two such massively different parameters as accommodation and intraocular pressure by the tone of the same muscle seems surprising.

ABC

Fax + 41 61 306 12 34 E-Mail karger@karger.ch www.karger.com

Work done during the last decade has established that, in addition to being passively distended by the ciliary muscle, the trabecular meshwork has contractile properties of its own [1], and that the contraction and relaxation of this structure may influence ocular outflow in the sense that relaxation reduces intraocular pressure. It seems possible that administration of smooth-muscle-relaxing substances might lower intraocular pressure, via relaxation of trabecular meshwork, while simultaneously improving retinal circulation by vasodilation of retinal capillaries. Trabecular meshwork thus appears as an interesting target tissue for new approaches in glaucoma therapy.

Ba´ra´ny [8] was the first to advance a hypothesis that trabecular meshwork possesses contractile properties of its own. Subsequently, a number of studies have been performed to investigate this theory. Histologically, extensive innervation of the trabecular meshwork has been shown [4, 9]. Using electron microscopy, Ringvold [10] observed cytoplasmic filaments in meshwork cells of monkeys. Using a histochemical technique, he demonstrated that these filaments consist of actin material. Various scientists have shown that the microfilaments present in trabecular meshwork are smooth-muscle ·- actin [11±15] and that there may be smooth-muscle myosin in the human trabecular meshwork [16, 17]. Morphologically, data from various authors show that muscarinergic agonists such as pilocarpine directly affect the trabecular meshwork of the eye of humans and various animal species and vary their form in culture [8, 18±21].

Measurements of intracellular calcium underscore the notion that trabecular meshwork possesses characteristics of smooth-muscle cells. Shade et al. [22] and Llobet et al. [23] have shown that, as in smooth muscle, substances that contract trabecular meshwork elevate cytosolic calcium.

Thus, it can by now be seen as evident that trabecular meshwork resembles smooth muscle, with the potential for regulating intraocular pressure. The search for specific pharmacological agents that interact with channels, transporters, receptors or other proteins in the signalling cascade leading to changes in trabecular meshwork contractility is thus a worthwhile undertaking, potentially leading to a better and more specific pharmacological control of intraocular pressure.

Contractility Measurements

While the contractility of ciliary muscle has been studied in various mammalian species including man [24±27], measurements of trabecular meshwork contractility had

not been performed before we attempted to obtain direct measurements of isolated trabecular meshwork strips. While the ciliary muscle extends into the trabecular meshwork of higher primates making it hard to isolate strips of trabecular meshwork, the bovine eye is well suited for contractility experiments. In this species, the ciliary muscle is more posteriorly located and can easily be removed from the trabecular meshwork.

For the contractility measurements, bovine eyes were dissected and strips of trabecular meshwork and ciliary muscle prepared according to established methods [29]. Using a purpose-built force-length transducer similar to that described by Brutsaert et al. [30], the isometric force of the strips could be monitored for a period of several hours after an equilibration period of approximately 1 h [29, 31±34].

Depolarization by High External K+

As the membrane potential of the vast majority of cells depends to a large extent on the potassium gradient, raising external potassium has a profound influence on this potential, leading to depolarization. Application of highpotassium external solution is a standard procedure for inducing smooth-muscle contraction [35, 36]. In trabecular meshwork, application of 120 mmol/l KCl evoked a contraction that was biphasic but corresponded to only 19% of the response attainable by acetylcholine [29]. Part of the KCl response was blockable by atropine. This can be explained by the fact that in many tissues, depolarization induces a release of acetylcholine from cholinergic nerve terminals. This indicates that only a fraction of the entire contractile response of trabecular meshwork can be attributed to depolarization of the cell membrane per se. However, this result has to be taken with a grain of salt as the application of such a high-potassium solution has to be seen as a very unphysiological maneuver. We would also like to point out that this experiment does not allow conclusions on the voltage dependence of the signalling pathway leading to relaxation or about the response of trabecular meshwork to hyperpolarization.

Cholinergic Agents

Cholinergic agents induced strong, reproducible contractions in trabecular meshwork cells (fig. 1), which were inhibitable by atropine, demonstrating the presence of muscarinic receptors [29, 31]. The induction of force could be increased by application of physostigmin, a reversible anticholinesterase inhibitor, demonstrating the presence of acetylcholinesterase in trabecular meshwork cells. Experiments using muscarinic antagonists of the M1

and M3 type [37] suggest that both in human and in bovine trabecular meshwork, functional receptors are of the M3 type [31, 38] (fig. 1). However, other muscarinic subtypes have not been excluded. In addition, it has been shown that, in primates, receptor subtype antagonists are modulators of aqueous humor outflow [39].

Adrenergic Agents

Human trabecular meshwork cells have been shown to express ·- and ß-adrenergic receptors, especially of the ß2- adrenergic subtype [40±43]. In addition, agonists like epinephrine have been shown to reduce outflow resistance and to increase outflow facility through direct actions on trabecular meshwork and via the uveoscleral route in a primate model [2, 44, 45]. The interpretation of this fact is complicated, however, due to the fact that epinephrine is nonspecific and dose-dependently acts on both ·- and ß-receptors.

In contractility experiments, both ·1- and ·2-adrener- gic agonists contracted the trabecular meshwork strips with approximately 20% of the potency of carbachol [31]. The effect of ·2-agonists was greater than that of ·1-ago- nists and the effects could be blocked by specific antagonists.

In contrast, ß-agonists such as isoproterenol significantly relaxed the tissue precontracted by carbachol, an effect that could be blocked by metipranolol. Application of a blocker on the unstimulated tissue had no effect [31].

In the concentration used to reduce intraocular pressure (10±4 to 10±3 mol/l), epinephrine contracted strips of

trabecular meshwork. Additional application of metipranolol, a ß-blocker, further increased trabecular meshwork tone, indicating blockage of the relaxing effect of the ß- component of epinephrine. It seems that the net effect of epinephrine on trabecular meshwork contractility should depend on the balance of ·- and ß-adrenergic receptors in the tissue of the species observed.

Low External Ca2+

While relaxation of precontracted ciliary muscle was total when external calcium was removed, only a part of the contractile response of trabecular meshwork depended on the presence of external calcium, with 42% of the response to carbachol and 23% of the response to endothelin remaining after removal of this ion [46]. It seems that in trabecular meshwork, both a calciumdependent and a calcium-independent mechanism of contractile response exists. Similar effects have been reported for other tissues, pointing towards a regulation of myosin activity via protein kinases [47, 48].

Blockers of Ca2+ Channels

Blockage of calcium channels is one of the new therapeutic approaches in glaucoma therapy, aimed at reducing intraocular pressure and, simultaneously, improving retinal circulation. While verapamil has been reported to enhance ocular outflow in humans [49], other calcium blockers have been reported to affect the rate of inflow [50].

Different blockers of calcium channels had varying effects on trabecular meshwork contractility [1, 32, 46].

While the highly specific calcium antagonist nifedipine in the low dosage of 10±5 mol/l only had a relaxing effect on about 10% of the total contractile response, verapamil, which is reported to block a number of other channels including potassium channels and chloride channels [51± 53], blocked more than 70% of the response. Ni2+, an inorganic calcium blocker which has been shown to inhibit low-threshold (T-type) Ca2+ channels [54] and Na+/Ca2+ exchange [55], had a relaxing effect both on the contractile response to endothelin (86%) and carbachol (41%). In summary, it appears that external calcium is needed for a part but not all of the contractile response of trabecular meshwork to carbachol and endothelin.

Endothelin

Endothelin-like immunoreactivity is 2±3 times higher in aqueous humor of human and bovine eyes than in the corresponding plasma [32, 56], and an elevation of endo- thelin-like immunoreactivity in the aqueous humor of glaucoma patients has been reported [57]. It seems that endothelin, the release of which has been shown to be stimulated by stretch and fluid flow rate [58], might be an important hormone regulating aqueous humor production and/or outflow. The finding of higher endothelin levels in glaucoma patients indicates a possible dysfunction involving an altered production of endothelin [57].

This theory is underscored by the fact that endothelin evokes a strong contractile response in isolated trabecular meshwork strips which is in the range of the carbachol effect if equal concentrations are applied (fig. 2). 77% of this contractile response was dependent on extracellular calcium [32, 46].

Nitric Oxide

Nitric oxide has been implicated in the physiology of aqueous humor dynamics [59±61], and the use of nitrovasodilatators in the therapy of glaucoma is being discussed. Thus, NO synthase could be detected in the outflow pathway of the bovine and human eye, and NO synthase immunoreactivity was reduced in patients with primary open-angle glaucoma [59].

NO, nitrovasodilatators and nonnitrates like sodium nitroprusside have been shown to increase cyclic GMP [62, 63]. Application of membrane-permeable cGMP (8- bromo-cGMP) relaxed precontracted strips of trabecular meshwork to 41% of the tone under carbachol [64] (fig. 3). The organic nitrovasodilatators like ISDN (isosorbide dinitrate) and 5-isosorbide mononitrate, too, were able to relax trabecular meshwork. The most potent relaxants were nonnitrates like SNP (sodium nitroprusside) and

Fig. 2. Recording showing the contractile response of bovine trabecular meshwork to endothelin. Again, the contractile force depends on the dose of endothelin applied.

SNAP (S-nitroso-N-acetylpenicillamine), reducing trabecular meshwork tone in response to carbachol by over 60%. Interestingly, inhibition of NO synthase by L-nitroarginine (L-NAG) increased carbachol-induced contraction significantly, while ISDN and SNP also significantly relaxed uncontracted trabecular meshwork. These findings indicate a continuous release of NO by trabecular meshwork both under contraction and under resting conditions.

Prostaglandins

Prostaglandins (PGs) represent a new class of topically effective ocular antihypertensive drugs [65, 66]. PGF2· and its analogues [e.g. PhXA34 (latanoprost)] have been shown to enhance outflow facility [67, 68], although most of the effect seems to have concerned the uveoscleral route. However, the existence of PGF2· receptors in human trabecular meshwork has been demonstrated using the RT-PCR technique [69].

Mediated by a number of different prostanoid receptor subtypes, PGs have been shown to have differing effects on various types of smooth-muscle tissue. Trabecular meshwork, too, responded to the various PGs in different ways [34]. Sulprostone and the thromboxane mimetic U- 46619 caused contraction (fig. 4). These effects could be blocked by the TP thromboxane activated receptor antagonist SQ-29548. In contrast, PGF2· and 17-phenyl PGF2· had no effect, while the nonselective EP (E prostanoid)

agonist 11-deoxy PGE1 and the specific EP2 (E2 prostanoid) agonist AH-13205 significantly relaxed precontracted trabecular meshwork strips. We conclude that trabecular meshwork possesses both TP and EP2 receptors the activation of which causes opposite effects.

Cyclooxygenase Inhibitors

It has been shown that in smooth-muscle tissue, muscarinic stimulation initiates a well-described cascade of second-messenger signalling involving, among other things, the release of PGs. Cyclooxygenase is the enzyme responsible for the production of prostanoids. In contraction experiments [70], application of the cyclooxygenase inhibitor indomethacin (5W10±6 mol/l) on trabecular meshwork and ciliary muscle strips precontracted by carbachol resulted in an additional contractile response to almost 140% of the uninfluenced contraction by carbachol. These results suggest that carbachol induces the production of relaxing PG in trabecular meshwork and ciliary muscle, the inhibition of which results in contraction.

Interestingly, in the monkey eye, the effect of decreasing outflow resistance is partly inhibited by indomethacin [71].

Diuretics

Recent research indicates that the function of the Na+- 2Cl±-K+ cotransporter is altered in glaucomatous eyes and that this should affect the cell volume of trabecular meshwork cells [72, 73]. This transporter is sensitive to the loop

Fig. 4. Prostaglandins had varying effects on trabecular meshwork, ranging from relaxation to contraction. This recording shows the strong, contractile response observable after application of the thromboxane mimetic U-46619.

diuretics bumetanide and furosemide, and to the rather unspecific agent ethacrynic acid, which is known to inhibit not only this transporter, but also sodium-dependent anion transporters and to modulate the cytoskeleton. However, contractility of trabecular meshwork was not altered when this transporter was blocked by bumetanide [32, 74]. The diuretic hydrochloride, which blocks the Na+Cl± cotransporter [32], had no effect on contractility.

Fig. 5. Trace demonstrating the relaxation of precontracted trabecular meshwork by flufenamic acid and ethacrynic acid. The effects were additive.

Fig. 6. Trabecular meshwork was relaxed by application of genistein, a tyrosine kinase inhibitor. Tyrphostin 51, a synthetic tyrosine kinase inhibitor, had similar effects.

On the other hand, ethacrynic acid relaxed trabecular meshwork. Studies on primates have shown that systemic or local application of various diuretics including bumetanide had no effect on aqueous humor dynamics and intraocular pressure [74], while local application of ethacrynic acid increased outflow facility in the human eye [75].

Flufenamic Acid

Flufenamic acid, a therapeutically used antirheumatic agent [76±78], relaxed trabecular meshwork strips precontracted by carbachol or endothelin 1 [32] (fig. 5). These relaxing effects were independent of the relaxing effects of ethacrynic acid and isosorbide dinitrate. While in many tissues, the blockage of nonselective cation channels by flufenamic acid has been reported [79, 80], it appears that in trabecular meshwork, the maxi-K channel is stimulated.

Modifiers of the Cytoskeleton

Both ethacrynic acid and cytochalasin D relaxed trabecular meshwork [32] (fig. 5). Both agents are thought to disrupt microtubules which help constitute the cytoskeleton [81±83].

Protein Kinase Inhibitors

Phosphorylation of cellular proteins controls various cellular functions such as mitogenesis but also contractili-

ty [84±87]. Recently, it has been demonstrated that protein tyrosine kinase pathways are involved in the regulation of smooth muscle contractility [88, 89].

In trabecular meshwork, stimulation of the EGF (epidermal growth factor) receptor with tyrosine kinase activity by EGF (100 Ìg/l) caused relaxation of tissue strips precontracted by carbachol (10±6 mol/l) [33]. Application of tyrosine kinase inhibitors like tyrphostin 51 and genistein in concentrations of 5W10±5 mol/l produced relaxation (fig. 6). Inhibition of PKA-PKG by H-8 (5W10±6 mol/l) and of the serine threonine kinase PKC by chelerythrine or NPC-15437 (both at 10±6 mol/l) was also able to relax trabecular meshwork; these effects were additive to the effects of the inhibition of tyrosine kinase [33]. Recently, it has been shown that trabecular facility is indeed increased after application of the nonselective serine threonine protein kinase inhibitors H-7 and staurosporine [27, 90, 91].

Interestingly, there were marked differences in the response of trabecular meshwork and ciliary muscle [33]. In contrast to the relaxing effect of EGF on trabecular meshwork, ciliary muscle was contracted by application of EGF. The effect of inhibiting tyrosine kinase was more pronounced in trabecular meshwork. Inhibiting PKC and PKA-PKG had no effect on the contractility of ciliary muscle.

Fig. 7. Summary of 10 experiments measuring ocular outflow in the model of the perfused anterior segment of the bovine eye in which the ciliary muscle has been removed. The muscarinergic agonist pilocarpine, which contracts trabecular meshwork, reduced ocular outflow.

Cellular Contractions

On a cellular level using cultivated trabecular meshwork cells, changes in shape indicating contractile processes have also been observed. Agents that have been shown to decrease the area of trabecular meshwork cells include the vasoactive compounds bradykinin [92] and acetylcholine [21], as well as ethacrynic acid, colchicine and vinblastine [81, 93, 94], all of which are known to disrupt the cytoskeleton.

The Perfused Anterior Segment

The isolated, perfused anterior segment of primate and bovine eyes is an established method for studying aqueous humor outflow [28, 92, 95±97]. Perfusion of the anterior segment of bovine eyes with detached iris, ciliary body and ciliary muscle at a pressure of 8.8 mm Hg yielded a constant outflow rate of 6±8 Ìl/min with an outflow facility of 0.87 ÌlWmm Hg/min and an outflow resistance of 1.15 mm HgWmin/Ìl. The perfusion rate remained constant for up to 3 h; no washout occurred. Elevation of the pressure in the outflow chamber increased outflow resistance in a linear fashion.

Relative outflow in this model could be influenced by a variety of drugs [28]. Carbachol reduced outflow by a maximum of 37% with a half-maximal effective concentration of 3W10±8 mol/l; the effect of the drug could be completely blocked by atropine. Pilocarpine was some-

what less effective with a reduction in outflow of 15% (fig. 7). Endothelin 1, a potent vasoactive agent that contracts trabecular meshwork strips [46], also reduced ocular outflow. Bradykinin is generated by the action of the kallikrein-kinin system and known for its vasoactive properties. Intracameral administration of bradykinin is known to increase intraocular pressure [98] and to contract smooth-muscle tissue; in accordance with this, a drop in outflow facility in the model of the perfused anterior segment was observed [92].

As in the contractility experiments, the net effect of epinephrine depended on the concentration used [28]. At 10±5 mol/l, outflow was reduced, while at a lower concentration (10±6 mol/l), outflow in the bovine eye increased, an effect also reported for the human eye. This increase in the perfusion rate could be blocked by application of the ß-blocker metipranolol. While in the higher concentration range the effect of epinephrine on ·-adrenergic receptors predominates, it seems that in the lower concentration range, the net effect can be explained by the stimulation of ß-adrenergic receptors.

Disruption of the cytoskeleton by cytochalasin D, which led to a relaxation of trabecular meshwork cells in contractility experiments, also increased outflow facility [32].

In conclusion, it is possible to say that substances that contract trabecular meshwork strips reduce outflow in the model of the perfused anterior segment, while substances that relax trabecular meshwork induce an increase in the outflow rate.

Intracellular Calcium

It is a well-established fact that smooth-muscle contractility is regulated by the concentration of intracellular calcium [35, 99, 100]. Using fura-2-loaded bovine trabecular meshwork cells, we measured the intracellular calcium concentration by recording the fluorescence ratio of calcium-bound to calcium-free dye [101]. In accordance with findings by Shade et al. [22], we found a basal resting calcium concentration of 40±80 nmol/l. Cytosolic calcium levels could be elevated both by depolarizing the cells using high-potassium solution and by application of the opener of L-type calcium channels, Bay K 8644 (5 Ìmol/l). Application of endothelin 1, which contracts trabecular meshwork [46] and induces a reduction in the outflow rate [28], caused a biphasic increase in the level of cytosolic calcium [28, 102] (fig. 8); the same could be observed when acetylcholine was applied. This biphasic response is well known from other preparations of smooth muscle tissue [35, 99, 100]. The initial peak is thought to be due to a release of calcium from cytosolic stores via an established second-messenger pathway involving G proteins and inositol triphosphate. The subsequent plateau phase is due to calcium influx from the outside through various, tissue-dependent influx pathways [26]. In trabecular meshwork, these include voltage-dependent calcium channels [101, 103]. Thus, an increase in cytosolic calcium could be obtained by depolarization with highpotassium solution and by application of a specific opener of L-type calcium channels, Bay K 8644 [103]. The participation of nonselective cation channels [32] and of cal-

cium-release-activated calcium currents [104, 105] is also discussed.

Measurements on cultured human trabecular meshwork cells have shown that the response to muscarinergic agents is almost identical in the human and the bovine species both in terms of absolute concentrations of cytosolic calcium reached, and in terms of the biphasic profile observed [22, 101]. The use of different muscarinic receptor subtype antagonists has revealed that the M3 receptor subtype is the most important receptor involved [22]. In addition, it was possible to show that phosphoinositide production was stimulated as a result of the stimulation of muscarinic receptors, with subsequent activation of the phospholipase C system. Subsequent research demonstrates a rise in calcium in response to the application of various neuropeptides (neuropeptide Y, substance P, bombesin, calcitonin gene-related peptide, vasoactive intestinal peptide) in trabecular meshwork cells [106]. Neuropeptide Y proved to be a particularly potent elevator of cytosolic calcium levels and phosphoinositide turnover. Bradykinin also elevated cytosolic calcium levels [92], as did the application of PGF2· [69]. Both atrial natriuretic peptide and C type natriuretic peptide increased the accumulation of cGMP, leading to a suppression of carbacholinduced calcium mobilization [107]. Elevation of hydraulic pressure also induced a rise in cytosolic calcium [108], in accordance with the observation that trabecular facility decreases with rising intraocular pressure [1, 96]. Endothelin 1 and histamine [38, 101, 102, 109] also elevated internal calcium in trabecular meshwork, while angiotensin II had no effect [102].

Regulation of Intracellular pH

Intracellular pH (pHi) is a key factor in determining the activity of many cellular enzymes [110, 111]. Transporters involved in pHi regulation have been identified in many cell types and have been found to participate in cell homeostasis, transmembrane transport, transepithelial transport, growth factor activation and cell proliferation.

In order to demonstrate the existence of transporters that regulate pHi in trabecular meshwork, bovine trabecular meshwork cells were loaded with 5(6)carboxy-4),5)- dimethylfluorescein and pHi was monitored by measuring the pH-dependent absorbance of this dye [1, 112]. In physiological, bicarbonate-containing Ringer's solution, pHi averaged 7.02. When the CO2/HCO±3 buffer was replaced (ensuring that external pH remained at a constant value of 7.4 by addition of HEPES), pHi dropped significantly, a first indication of bicarbonate-dependent transporters regulating trabecular meshwork pHi. We were able to demonstrate the existence of three independent pH-regulating transporters. In bicarbonate-free medium, both maintaining steady-state pHi and recovery after an acid load was Na+ dependent (fig. 9). The underlying mechanism could be totally blocked by amiloride. These data point to the existence of an Na+/H+ exchanger in trabecular meshwork, a transport process responsible for eliminating acid equivalents from the cytosolic compartment that can be found in a number of cells. The existence of this exchanger was confirmed by another study [113].

When cells were acidified in bicarbonate-containing medium, pHi recovery continued even after the Na+/H+ exchanger had been blocked with amiloride. Additional blockage with DIDS (4,4)-diisothiocyanatostilbene-2,2)- disulfonate) or pyridoxal 5)-phosphate, both blockers of bicarbonate-dependent anion exchangers, eliminated the recovery process. Replacing Cl± in the extracellular solution was also able to eliminate recovery from an acid load and led to alkalinization instead. The recovery was sodium dependent and blockable by ethacrynic acid, a blocker of the sodium-dependent chloride-bicarbonate exchanger. In a further series of experiments, recovery after an alkaline load was tested. This recovery was sensitive to removal of external bicarbonate. Removal of external chloride reversed the direction of regulation. Removal of sodium had no impact, while DIDS blocked alkaline extrusal. These experiments point to the existence of two additional pHi-regulating transporters in trabecular meshwork ± the sodium-dependent and the sodium-independent chlo- ride-bicarbonate exchangers.

Fig. 9. Measurement of pHi using bovine trabecular meshwork. After an acidifying prepulse with NH4Cl, the pHi returns to the resting level. This process is dependent on the presence of extracellular sodium, pointing towards the Na+/H+ exchanger as the underlying transporter.

The pHi-regulating transporters of other ocular tissues

± such as the cornea and ciliary epithelium ± have been described in more detail [110, 111, 114, 115]. In these tissues, the Na+/H+ exchanger is the dominant force after acidification of the cell, the sodium-independent chlo- ride-bicarbonate exchanger is activated by alkalinization, while the sodium-dependent chloride-bicarbonate exchanger maintains pH at the basal level. Further work is needed to determine if these exchangers have housekeeping function only or if they influence other aspects of cellular functioning. Interestingly, trabecular meshwork cells express active receptors for growth factors, the stimulation of which involves changes of cytosolic pH [116]. In addition, endothelin has been shown to increase pHi in trabecular meshwork cells [102]. Considering that endothelin contracts trabecular meshwork cells, it is possible that pHi is involved in the signalling cascade leading to contraction of trabecular meshwork, in parallel to observations on other smooth muscle cells [117, 118].

Electrophysiology of Trabecular Meshwork

The electrophysiology of trabecular meshwork has been investigated using both traditional measurement of membrane voltage and the patch-clamp technique in the