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

hopes of further illuminating signal transduction pathways leading to trabecular meshwork contractility. All electrophysiological experiments were carried out using cultured trabecular meshwork cells of either human or bovine origin [14, 101, 119±122]. Bovine trabecular meshwork cells were all from the third passage. Human trabecular meshwork cells were obtained from donor eyes and cultivated for up to the eighth passage; for the cell puncture experiments, human cell lines were used.

Resting Membrane Voltage

Classical measurements of membrane voltage using cell puncture demonstrated the existence of three different cell types in cultures of bovine trabecular meshwork cells: a spindle cell type, with a membrane voltage of ±71 B 2 mV (n = 48), an epithelial cell type with a membrane voltage of ±50 B 1 mV (n = 143) and a mixed type with a voltage of ±55 B 1 mV (n = 191) [14]. Five different cell lines of human trabecular meshwork displayed membrane voltages in the range of ±44 to ±63 mV [120], cell puncture experiments on cells from a primary culture of human cells fell within that range.

All of these voltages are more positive than the equilibrium potential for potassium due to the flux of other ions like sodium, calcium or chloride across the cell membrane. This fact was confirmed by changing the concentration of extracellular potassium. This maneuver resulted in a depolarization of the cells which depended on the concentration of potassium applied (fig. 10). Subsequently, the relative K+ permeability could be calculated,

showing that potassium ions only account for 50±70% of the total potassium conductance. No significant differences between human and bovine cells emerged [14, 101, 119, 120].

Excitability

One of the key features of smooth muscle cells is their excitability pattern, the predominant feature of which are spontaneous oscillations of membrane potential called `abortive action potentials' or `spikes' which can either occur spontaneously or can be induced by such maneuvers as applying extracellular Ba2+ [123±125]. In cell puncture experiments, both human and bovine meshwork exhibited such behavior [14, 120] so that electrophysiologically, they react like smooth muscle cells (fig. 11). Smooth muscle cell `spikes' are typically insensitive to tetrodotoxin, an inhibitor of fast Na+ channels; this behavior could also be observed in trabecular meshwork. Instead, the voltage oscillations depended on the presence of extracellular calcium and could be blocked by application of nifedipine, a highly selective Ca2+ channel blocker [101] (fig. 11).

The exact sequence of events leading to the spikes has not been clarified [126]. However, it is known that the application of barium blocks a number of potassium channels and should therefore depolarize the cells. Depolarization in turn opens voltage-operated (nifedipine-sen- sitive) calcium channels; driven by the concentration gradient, Ca2+ ions flow in, leading to further depolarization. The rise in cytosolic calcium, especially in the compart-

ment immediately under the cell membrane, should then activate calcium-dependent potassium channels, leading to repolarization in form of the downward stroke of the spike. This theory is validated by the fact that trabecular meshwork cells do indeed express calcium-dependent potassium channels in the form of maxi-K channels [121]. Interestingly, it could be shown that in trabecular meshwork, these channels are indeed insensitive to the application of external barium and should thus not be blocked by the barium necessary to evoke the spike.

Electrical Characteristics of Ion Channels and Transporters

In a series of experiments, we tried to determine what ionic channels and transporters are involved in regulating the membrane potential of trabecular meshwork cells and what pharmacological agents are able to modify this parameter.

Potassium Channels. We have already indicated that potassium conductance is the major factor contributing to the resting membrane voltage of trabecular meshwork cells. Accordingly, an elevation of external potassium to 135 mmol/l depolarized both human and bovine trabecular meshwork cells by preventing efflux of potassium [14, 119, 120]. The same effect could be achieved by blocking potassium channels with Ba2+. More detailed analysis of the potassium channels involved was obtained using the patch-clamp technique; the results will be discussed below.

Calcium Channels. While removal of calcium did not have an impact on resting membrane voltage, this maneuver lowered the excitability suppressing the formation of spikes (see above). The same effect was observed when L-type channels were blocked by the specific calcium channel blocker nifedipine or by the somewhat less specific verapamil [101, 103].

Sodium Channels. Tetrodotoxin, a blocker of fast sodium channels, had no effect on resting voltage [14, 119, 120]. However, this does not rule out a participation of these channels in the response to pharmacological stimulation [127].

Transporters. Ouabain, a blocker of the Na+/K+- ATPase, depolarized trabecular meshwork [14, 120]. Low sodium and low bicarbonate induced a DIDS-sensitive depolarization in human trabecular meshwork cells. These effects can be explained by the presence of an electrogenic Na+-HCO±3 symport, which mediates a flux of bicarbonate coupled to sodium into the cell [14, 120]. This symport should also influence pHi and has been described in bovine corneal endothelium and human cil-

Fig. 11. In human and bovine trabecular meshwork, application of barium resulted in typical spikes of membrane voltage. Spiking could be blocked by application of nifedipine, a specific blocker of L-type channels.

iary muscle [14, 120]. Interestingly, this transporter was absent in trabecular meshwork cells of bovine origin [14, 120].

Muscarinic, Adrenergic and Endothelin Receptors. Trabecular meshwork cells are known to express muscarinic receptors [22, 31, 128]. In measurements of membrane voltage on human trabecular meshwork, the voltage response observed upon application of acetylcholine exhibited the pattern typical of muscarinic receptors coupled to a phospholipase-C-dependent second-messenger system [120, 129] (fig. 12). This cascade generally leads to a release of calcium from cytosolic stores and to an influx of extracellular calcium. This elevation in calcium is thought to activate calcium-activated potassium channels, resulting in a transient hyperpolarization which we were able to observe in our preparation [130, 131]. However, the complete picture is far from clear [132] and also involves suppression of potassium channels. The predominant effect of muscarinergic stimulation, however, is that of a long, sustained depolarization (fig. 12), the exact cause of which has yet to be determined. Possibly, it is due to the opening of nonselective cation channels [32, 132]. The entire voltage response of trabecular meshwork cells to acetylcholine could be blocked by the application of atropine [120].

Fig. 12. Acetylcholine reversibly depolarized human and bovine trabecular meshwork cells. Note the transient hyperpolarization prior to the prolonged depolarization.

Fig. 13. Endothelin, too, depolarized trabecular meshwork cells of both species in measurements of membrane voltage.

Application of isoproterenol, too, caused trabecular meshwork cells to depolarize [120]. The response was sensitive to metipranolol. Other authors, too, have described the existence of ß-adrenergic receptors in trabecular meshwork, mainly of the ß2-subtype.

As in ciliary muscle cells, endothelin dose-dependently depolarized cultured bovine and human trabecular meshwork cells [101] (fig. 13). This indicates the presence of

endothelin A receptors in both tissues. The response to endothelin seems to follow a mechanism similar to that of other receptor-mediated signalling cascades.

In summary, it appears that agents that contract trabecular meshwork depolarize trabecular meshwork cells. This corresponds to the classical, well-described mechanism known for other preparations of smooth-muscle tissue. However, it must be pointed out that contractility experiments using high-potassium solution imply that only part of the contractile response of trabecular meshwork is mediated by changes in membrane potential [29], an important part apparently being due to non-voltage- mediated, pharmacomechanical coupling [36, 133].

Patch-Clamp Measurements

Measurements of membrane voltage have the advantage of leaving cells close to their native state. However, in order to determine if particular channels are indeed involved in determining the resting membrane voltage level of the cell or in its response to agents that alter membrane voltage, more information is needed than provided by a measurement of membrane potential. Using the patch-clamp technique, it is possible to determine both voltage and current during a measurement and to alter the ionic constituents on both sides of the cytosolic membrane. The drawback of this method is that voltage, which by definition is clamped using this technique, cannot fluc-

tuate freely as in physiological situations. The solution used to fill the cell via the pipette allows an exact determination of the reversal potentials to be expected but alters the protein content of the cell, making complex secondmessenger cascades virtually impossible. An alternative to exchanging the entire cytosolic fluid is the perforatedpatch modification of the patch-clamp technique [134], by which the cell membrane is perforated using the ionophore nystatin, allowing potassium (and voltage) to freely equilibrate on both sides of the membrane. This leaves the other ionic and proteinoid constituents of the cytosolic compartment untouched.

Trabecular meshwork cells of both human and bovine origin were investigated using the patch-clamp technique.

Inward Rectifier Potassium Channel

Bovine trabecular meshwork cells exhibited an inward current at hyperpolarizing voltages that could be totally blocked by barium, tetraethylammonium chloride and withdrawal of potassium. As with the chloride currents, various pharmacological maneuvers had no impact on this conductance so that, until further evidence emerges, we are inclined to see the function of this channel in the domain of cell homeostasis [unpubl. observation].

Sodium Conductance

An Na+-selective current has been described in human trabecular meshwork cells by Rich et al. [127], which we were also able to detect [103] (fig. 14). This current can be stimulated by melatonin (5W10±8 to 10±4 mol/l) [127]. Interestingly, both intraocular pressure and physiological

melatonin levels show a circadian rhythm [135]. However, more research is needed before a definite link between these two observations can be established.

Outwardly Rectifying Potassium Conductance

Trabecular meshwork cells of both human and bovine origin display a strong, outwardly rectifying current upon depolarization [121] (fig. 15). The outward current level was greatly reduced from 0.5 B 0.1 nA (n = 9) in solutions containing potassium to 0.05 B 0.01 nA (n = 4) in potas- sium-free solutions and blocked almost completely by application of tetraethylammonium chloride (n = 4). To assess precisely which potassium conductances are involved, various specific potassium channel blockers were applied.

ATP-Dependent Potassium Channels

ATP-dependent potassium channels, which shut down when levels of cytosolic ATP rise, have been identified in a number of tissues and are believed to play an important role in regulating smooth-muscle contractility [130]. Derivatives of sulfonylurea, like glibenclamide, are generally believed to be highly specific blockers of this channel. Bovine trabecular meshwork cells did not show a significant alteration in current when glibenclamide (10±5 mol/l, n = 7) was applied [122]. Although it cannot be ruled out that ATP-dependent potassium channels are activated in trabecular meshwork by some pharmacological maneuver, resting current shows no contribution of these channels.

Small-Conductance Calcium-Activated Potassium Channels

Another channel often described in smooth muscle tissue is the small-conductance calcium-activated potassium channel [130]. This channel is activated by elevation of cytosolic calcium, has a single channel conductance of below 100 pS and is specifically blocked by a compound from bee venom, apamin. In bovine preparations of trabecular meshwork, apamin (10±6 mol/l, n = 4) had no effect on resting current [122]. Again, this does not rule out measurable participation of these channels when the cells are in an activated state.

Large-Conductance Calcium-Activated Potassium Channels

Large-conductance calcium-activated potassium channels, also known as maxi-K channels, feature three properties: large conductance of over 200 pS, activation by elevation of cytosolic calcium (fig. 16A, B) and activation by an increase in resting voltage [136]. Charybdotoxin and, especially, iberiotoxin, both won from scorpion venom, are highly specific blockers of this channel [137] (fig. 16C, 17). Given in a dosage of 10±7 mol/l, iberiotoxin blocked current to 22 B 6% (n = 4, p ! 0.001) of current to outward current at 80 mV in bovine trabecular meshwork cells, while charybdotoxin blocked initial 71 B 16% (n = 4, p ! 0.001) of outward current [121], reflecting the greater potency of iberiotoxin for blocking this channel [138]. In human trabecular meshwork, the values were 42 B 8% (n = 5) for charybdotoxin (10±7 mol/l), while iberiotoxin (10±7 mol/l) blocked 44 B 6% (n = 9) of the total outward current. Statistically, no significant differences emerged between the species, either in the absolute levels of out-

ward current or in the contribution of the maxi-K channel to this current. Current voltage relationships were also identical. In human trabecular meshwork cells, elevation of cytosolic calcium by applying the calcium ionophore (10±5 mmol/l) led to a dramatic increase in the total outward current to 645 B 45% (ppaired ! 0.005, n = 3, V = 80 mV) of the original value; charybdotoxin blocked 37 B 6% (ppaired ! 0.001, n = 3, V = 80 mV) of this current. Subsequent single-channel measurements demonstrated the existence of high-conductance potassium channels with a conductance level of 326 B 4 pS (n = 10; bovine) and 302 B 13 pS (n = 4; human) in symmetrical potassium solution (135 mmol/l); again, significant differences between the bovine and the human species did not emerge. Both in human and in bovine cells, elevating cytosolic calcium from 10±7 to 10±6 mol/l had no significant effect on open probability; however, further elevation to 10±5 mol/l and higher greatly increased channel activity in excised inside-out patches. These values are in good accordance with values reported by other groups for other tissues.

Cytosolic application of ATP (1 mmol/l) also enhanced open probability, a first indication of the fact that while maxi-K channels in trabecular meshwork are indeed stimulated by calcium, calcium is not the only and perhaps not even the physiologically most important stimulant. The effect of barium depended on the side to which it was applied: given from the cytosolic side, a block of outward current through maxi-K channels could be observed, while outside application of barium showed no effect on conductance through these channels. Possibly, blockage of maxi-K channels by barium occurs via the ball-and- chain model.

Stimulation of Outward Current by cGMP

In contractility experiments, substances that elevate cyclic GMP, like the organic nitrovasodilatators ISDN and 5-ISMN or the nonnitrates SNP and SNAP, were shown to relax trabecular meshwork [64]. Direct application of the membrane-permeable cGMP analogue 8-bro- mo-cGMP to strips of trabecular meshwork evoked the same response [64]. In patch-clamp experiments, superfusion of bovine trabecular meshwork cells with a solution containing 8-bromo-cGMP (10±3 mol/l) evoked a stimulation of outward current to 290 B 57% (n = 4, p ! 0.05) that was sensitive to charybdotoxin (10±7 mol/l). These data indicate that cyclic GMP stimulates maxi-K chan-

nels. Extrusion of potassium is thus enhanced, leading to hyperpolarization. In the classical concept of the regulation of smooth-muscle contractility, this hyperpolarization should lead to a reduction in cytosolic calcium through a shutdown of L-type calcium channels and a reduced emission of calcium from stores [103]. Thus, relaxation of trabecular meshwork by substances that elevate cGMP involves stimulation of maxi-K channels.

Stimulation of Outward Current by Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors have been shown to relax trabecular meshwork cells [33]. Application of the tyro-

sine kinase inhibitor genistein (5W10±5 mol/l) on bovine trabecular meshwork cells stimulated with acetylcholine resulted in a reversible increase in outward current to 578 B 154% (n = 16) of the initial current level [122]. In human trabecular meshwork, the effect was comparable (fig. 17). The effect of genistein was dosage dependent. Reversal potential was hyperpolarized by 15 B 3 mV (n = 9). Tyrphostin 51, a synthetic inhibitor of tyrosine kinases, had the same effect (433 B 46%; n = 7). Daidzein, a nonactive structural analogue of genistein, had no effect (n = 4). The stimulation of outward current by tyrosine kinase inhibitors could be blocked by substitution of potassium by tetraethylammonium ions, while the potassium channel blockers glibenclamide (K-ATP) and apamin (small-conductance calcium-activated potassium channel) had no effect. Blockage of the large-conductance cal- cium-activated potassium channel (maxi-K) by charybdotoxin or iberiotoxin (10±7 mol/l) suppressed 86 B 18% (n = 4) of the response. Depleting the cells of calcium did not have an effect on the current stimulated by genistein. In the excised inside-out configuration, open probability increased to 417 B 39% (n = 3) after exposure to genistein. It appears that both in human and in bovine trabecular meshwork, inhibition of tyrosine kinase stimulates maxi-K channels through a mechanism not involving changes in cytosolic calcium. As discussed above, this leads to hyperpolarization due to efflux of potassium and explains the relaxation observed in the contractility experiments.

Stimulation of Outward Current by Flufenamic Acid

Flufenamic acid relaxes trabecular meshwork [32], reportedly by blocking nonselective cation channels. We tested the impact of flufenamic acid (10±5 mol/l) on membrane currents of trabecular meshwork cells. No impact on inward current was observed, as would have been the case if nonselective cation channels had been affected by flufenamic acid in a major way. Instead, we observed a strong rise in outward current, the major part of which was attributable to maxi-K channels [139] (fig. 18). It appears that in trabecular meshwork, flufenamic acid hyperpolarizes the cell by stimulating potassium efflux. Subsequently, calcium influx through L-type channels and other voltage-dependent influx pathways is reduced, intracellular calcium levels decline and the cell relaxes [32].

Calcium Channels

Both human and bovine trabecular meshwork cells expressed L-type channels with an inactivation time constant of 157 B 76 ms (n = 8; bovine trabecular meshwork) and 194 B 167 ms (n = 9; human trabecular meshwork) in a solution containing 10 mmol/l Ca2+ [103]. No significant differences between the two types of tissue emerged. When calcium was substituted by barium, the inactivation constant typically increased to much larger values (1,582 B 440 and 1,449 B 396 ms, respectively; fig. 19A, B). Application of Bay K 8644 (10±5 mol/l) resulted in a significant increase in inward Ba2+ current to 141 B 10%

Transporters

sIP3

Channels

sKIr (Inward rectifier)

sMaxi-K (KCa)

Ca2+ (L-type)

K+

Na+ Nonselective Ca2+ cation channel

s H2O (aquaporin)

Na2+

(n = 13) of control value in bovine trabecular meshwork cells and to 150 B 2% (n = 7) of control value in human trabecular meshwork cells (fig. 19C). Application of nifedipine (10±3 mol/l) led to a significant decrease in inward Ba2+ current in bovine trabecular meshwork cells to 48 B 13% of control value with a recovery to 96 B 5% (n = 5). In human trabecular meshwork cells, the values were 64 B 4 and 106 B 24% (n = 4), respectively. The existence of voltage-activated L-type calcium channels in trabecular meshwork is further evidence of the smooth-muscle-like characteristics that allow trabecular meshwork to respond to agents that alter membrane voltage by contraction and relaxation.

Functional Antagonism between Trabecular Meshwork and Ciliary Muscle

From the above, it can be concluded that trabecular meshwork is indeed contractile and that trabecular meshwork contractility regulates ocular outflow in the sense that relaxation of trabecular meshwork enhances ocular outflow. Contractility experiments have demonstrated that differences exist between the regulation of the contractility of ciliary muscle and trabecular meshwork. Ideally, it should be possible to find a substance with a maximal relaxing effect on trabecular meshwork but only a minimal effect on ciliary muscle. Such a compound might lead to a new approach in glaucoma therapy.

References

Much work still needs to be done on clarifying the signalling cascade that eventually leads to the contractile response of trabecular meshwork. However, a large number of transporters, channels and receptors have been identified in trabecular meshwork, many of which are known to regulate smooth-muscle contractility (fig. 20). In particular, we have outlined how substances that relax trabecular meshwork stimulate the maxi-K channel, leading to hyperpolarization and ultimately affecting cytosolic calcium. Measurements of contractility demonstrate that a second, voltageand calcium-independent pathway also leads to an alteration of trabecular meshwork tone. It is still not clear how these processes lead to a change in the shape of trabecular meshwork cells, but it appears that ultimately, these pathways would tend to alter the conformation of the cytoskeletal proteins actin, myosin and tubulin [27, 90, 140, 141].

Finally, we should point out that recent research indicates that a third smooth-muscle-like structure seems to be involved in regulating ocular outflow, namely the scleral spur [5±7, 16, 17].

Acknowledgements

The authors want to thank M. Boxberger for expert technical assistance and O. Strauss, K. Steinhausen, H. Thieme, Y. Que and R. Rosenthal for helpful discussions. This work was supported by DFG grant Wi 328/19 and Biomed 2 grant BMH4-CT96-1593.

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