Thapsigargin inhibits a potassium conductance and stimulates calcium influx in the intact rat lens

Abstract

1. An increase in lens cell calcium has long been associated with cortical cataract. Recently, it has been shown that thapsigargin induces a rise in lens cell calcium by release from endoplasmic reticulum stores. The effects of this rise on the optical and membrane characteristics of the lens were studied in the isolated rat lens.

2. The electrical characteristics of the isolated, perifused rat lens were measured using a two-internal microelectrode technique that permits measurement of plasma membrane conductance (G_m), membrane potential (V_m) and junctional conductance in the intact lens.

3. Thapsigargin (1 μM) induced a rapid overall depolarization of V_m that was accompanied by first a decrease and then an increase in G_m.

4. Replacing external Na⁺ with tetraethylammonium (TEA) abolished the decrease in G_m. However, a transient increase phase was still observed.

5. The changes in conductance were further characterized by measuring ²²Na⁺ and ⁴⁵Ca²⁺ influxes into the isolated lens. Thapsigargin (1 μM) induced a transient increase in ⁴⁵Ca²⁺, but did not affect Na⁺ influx.

6. The Ca²⁺ channel blocker La³⁺ (10 μM) totally inhibited the thapsigargin-induced Ca²⁺ influx. It also blocked the increase in G_m observed in control and in Na⁺-free-TEA medium. In the absence of external calcium, thapsigargin induced a small depolarization in V_m.

7. These data indicate that thapsigargin induces both a decrease in K⁺ conductance and an increase in Ca²⁺ conductance. These probably result from release of stored Ca²⁺ and subsequent activation of store-operated Ca²⁺ channels (capacitative Ca²⁺ entry).

8. Thapsigargin application over the time course of these experiments (24 h) had no effect on junctional conductance or on the transparency of the lens.

It has been recognized for some time that Ca²⁺ has an important role to play in cataract formation (Duncan & Bushell, 1975; Marcantonio, 1996) probably through the activation of the calpain system. More recently, however, it has been shown that Ca²⁺ may have a more subtle role through calcium cell signalling pathways. For example, it has been shown that lens cells possess a number of G-protein-coupled and tyrosine kinase receptors that are coupled to the release of Ca²⁺ from intracellular stores (Williams et al. 1993; Riach et al. 1995; Duncan et al. 1996). Lens membrane permeability and internal resistance are sensitive to raised calcium levels (Gandolfi et al. 1990) and, interestingly, an increase in external calcium appears to block lens K⁺ conductance (Lucas et al. 1986). We have previously shown that mobilizing lens cell calcium and activating G-protein-coupled muscarinic receptors leads to a depolarization and subsequent oscillation of membrane potential (Thomas et al. 1998). As lens cells are well coupled electrically, these changes in voltage initiated by receptor activation in one part of the lens are transmitted to all cells. Such responses are maintained by a complex interaction of different channels and this study was undertaken to identify the membrane conductances modulated by the calcium mobilization element of the cell-signalling pathway without the complication of possible contributions from direct receptor-coupled channel conductances. Lenses were exposed to the plant alkaloid thapsigargin, which empties calcium stores by inhibiting the Ca²⁺-ATPase-dependent re-uptake mechanism (Thastrup et al. 1990). By monitoring voltage and conductance changes in the whole lens, the effect of store depletion and cytoplasmic Ca²⁺ rise on the membrane conductance and internal resistance of an intact organ could be studied in some detail.

METHODS

Experiments were performed on lenses from 10- to 12-week-old rats (200-250 g) killed by cervical section. The lenses were dissected free using a posterior approach and the vitreous humour, iris and ciliary body were removed. The lenses were placed in a 1 ml Perspex chamber and artificial aqueous humour was perifused at 1 ml min⁻¹ at 35°C.

Chemicals and solutions

The composition of the artificial aqueous humour (AAH) was (mM): NaCl, 130; KCl, 5; NaHCO₃, 5; CaCl₂, 1; MgCl₂, 0.5; glucose, 5; and Hepes, 20; pH adjusted to 7.25 with NaOH, 35°C. Na⁺-free-TEA AAH was composed of (mM): TEACl, 135; KCl, 5; Trizma carbonate, 5; CaCl₂, 1; MgCl₂, 0.5; glucose, 5; and Hepes, 20; pH adjusted to 7.25 with Trizma base, 35°C. Thapsigargin was dissolved in DMSO and the final concentrations in AAH were 0.05 % DMSO and 1 μM thapsigargin. All chemicals were purchased from Sigma.

Electrophysiological recordings

Lens membrane potential (V_m) and conductance (G_m) were measured as described by Duncan et al. (1981). The first microelectrode (2 M KCl, 1-6 MΩ) was inserted into superficial posterior fibre cells and provided a measurement of the lens membrane potential with reference to a low resistance bath electrode (Fig. 1). Lens cells are well coupled electrically (Duncan, 1969; Rae et al. 1982) and so V_m gives a measure of the potential across the plasma membrane of the outermost cells. A current-passing microelectrode was placed deeper into the lens (approximately 200 μm). Current pulses (ΔI) were injected at regular intervals between this second microelectrode and a bath electrode. The resulting change in potential (ΔV_m) recorded by the first electrode was the result of the current passing through several resistances dominated by the resistance of the outermost membranes (Duncan, 1969; Duncan et al. 1981; Rae et al. 1982). The cytoplasmic and junctional resistance contributed approximately 10 % of the membrane resistance under these conditions and was responsible for the rapid initial transient in potential in response to a pulse of current (Lucas et al. 1987). Variations in this component were monitored by measuring the amplitude of the initial transient. No variation in junctional resistance was observed in any of the experiments reported here. Therefore, variations in the amplitude of the voltage transient initiated by current pulses were representative of the changes in the G_m of the lens. The electrical measurements were recorded with a two-channel high-impedance amplifier (Firbank Electronics, Norwich, UK), printed on a chart recorder (CR600 recorder, JJ Instruments, Southampton, UK) and digitized (HandyScope, TiePie Engineering, Leeuwarden, The Netherlands) for storage on computer disks and off-line analysis. V_m measurement was continuous while G_m was measured at regular intervals (ΔI/ΔV_m).

Figure 1. Structure of the mammalian lens.

The cells likely to play a role in the thapsigargin response in the lens are those with an intact endoplasmic reticulum (epithelial cells and elongating fibre cells; Bassnett, 1995). Note that the electrodes were placed in accessible mature cortical fibres. For clarity, only the voltage-measuring electrode is shown.

Note that, for all these measurements, the electrodes were inserted through the posterior surface of the lens, near the pole where the capsule is thinnest. The mammalian capsule is extremely robust and thickest on the anterior surface and at the equator and stable measurements are not possible from these areas.

Ca²⁺ and Na⁺ influx

Two protocols were used, the first to monitor the Ca²⁺ and Na⁺ influx over 2 h and the second to measure the kinetics of the Ca²⁺ influx over 24 h.

Ca²⁺ and Na⁺ influx over 2 h

The rat lenses were pre-incubated at 35°C in AAH and then for 2 h in AAH containing 40 kBq ml⁻¹⁴⁵Ca²⁺ and 40 kBq ml⁻¹²²Na⁺. At the end of the incubation period, the lenses were washed in 5 ml non-radioactive medium for 1 min to remove excess external ⁴⁵Ca²⁺ and ²²Na⁺. They were rolled on dry filter paper, weighted and placed in scintillation vials together with 2 ml AAH and 10 ml Optiphase SuperMix scintillation fluid (Wallac Scintillation Products, Turku, Finland). The radioactivity was assayed using a Wallac 1409 liquid scintillation counter. Ca²⁺ and Na⁺ influx were calculated using the following equation:

where cpm_AAH and cpm_lens are the measured activity of the loading solution and of the lens, [ion]_AAH is the Na⁺ or Ca²⁺ concentration in the loading solution, V_AAH the volume of loading solution counted, M_lens the mass of the lens, and T the duration of the incubation in the presence of the isotopes.

Kinetics of the Ca²⁺ influx over 24 h

The rat lenses were pre-incubated at 35°C in AAH and then incubated in AAH containing 1 μM thapsigargin. Ca²⁺ influx was monitored at different times during the experiment (0, 1, 3, 7 and 23 h) by adding 40 kBq ml⁻¹⁴⁵Ca²⁺ to the AAH. After 60 min in the presence of ⁴⁵Ca²⁺, the lenses were washed in 5 ml of non-radioactive medium for 1 min. They were then rolled on dry filter paper, weighted and placed in scintillation vials together with 2 ml AAH and 10 ml Optiphase SuperMix scintillation fluid. The radioactivity was assayed as described above.

Lens opacity

Lens opacity was assessed using the method described by Sanderson & Duncan (1993). The lenses were incubated in AAH (35°C) in the presence or absence of 1 μM thapsigargin. At the end of the experiment, they were placed in front of a black background and illuminated from above. The photographic negative was then digitized (LKB Ultrascan XL, Pharmacia, Uppsala, Sweden) and a light-scattering index calculated.

RESULTS

The rat lens has a relatively high and stable membrane potential (V_m = -63.4 ± 0.6 mV, n = 36) that depolarizes upon exposure to thapsigargin (Fig. 2A). Perifusing the lens with thapsigargin for 4 min induced a response similar to that induced by a 20 min stimulation (data not shown). This is not surprising, as thapsigargin is known to have a relatively long-lasting effect (Thastrup et al. 1990). Therefore, 4 min-long exposures to thapsigargin were generally employed. Although in each case the overall response was characterized by an initial fast depolarization followed by a relative stabilization of V_m, the initial phase comprised more than one component. In 29 cases out of 36 (81 %), the rate of the initial depolarization was clearly biphasic. The start of the second phase of depolarization is indicated by an arrow in Fig. 2A. The membrane conductance (G_m) changes were in fact more obviously multiphasic, and successive, distinct stages in the response were observed. Firstly, there was a fast decrease in G_m, followed by a rapid increase and a final slow and long-lasting decrease. Changes in membrane voltage and conductance during the different phases were measured as described in Fig. 2 and the means ± s.e.m. from 35 lenses are shown in Fig. 2A. It appears that the different phases of the V_m response coincided with the phases of the G_m response. Therefore, both the decrease and the increase in G_m were associated with a membrane potential depolarization.

Figure 2. Thapsigargin induced multiphasic changes of G_m and V_m in the intact lens.

A, the intact rat lenses were stimulated by perifusing with AAH supplemented with thapsigargin (1 μM) and DMSO (0.05 %) for 4 min. The results, characteristic of 35 independent experiments, indicate that thapsigargin induces complex variations in G_m (dashed line) and a multiphasic depolarization of V_m (continuous line). B (expanded time scale of A, centred on the region indicated by the arrow), the depolarization was multiphasic with a rapid depolarization first, followed by a relative stabilization (arrow, also in A) and a second rapid depolarization. The dashed lines indicate the rate of the rapid depolarizations. C, discrete measurements of G_m and V_m were performed in order to determine the mean (±s.e.m.) amplitude and kinetics of the response to thapsigargin. For G_m (dashed line), the first measurement was taken at the beginning of the response (time = 0, baseline), the second at the peak of the G_m decrease, the third at the peak of the G_m increase and the last 20 min after the beginning of the response. For V_m (continuous line), the first measurement was taken at the beginning of the response (time = 0, baseline), the second at the break in V_m depolarization (see A and B, arrow), the third at the peak of the depolarization and the last 20 min after the beginning of the response.

Since the first phase of the response involved a decrease in G_m and a depolarization in V_m, it is likely that it arose from a blockade of K⁺ conductance (Thomas et al. 1997). In order to investigate the second phase it was necessary to block any contribution from K⁺ conductances and hence the protocol of Farahbakhsh et al. (1994) was employed. Replacing external Na⁺ with TEA not only blocks K⁺ conductances, but also conveniently allows differentiation between changes in Na⁺ conductance and changes in Ca²⁺ conductance by reversing the Nernst potential for Na⁺. Exchange of Na⁺ for TEA would be expected to inhibit Na⁺-Ca²⁺ exchange in the lens, but it is likely that this does not have a major effect on internal Ca²⁺ at least in the short term. Indeed, experiments on tissue cultured lens cells show that although removal of external Na⁺ does cause a small initial increase in internal calcium, this change is transient and [Ca²⁺]_i returns to its baseline level within minutes (Duncan et al. 1993). Perifusion of the rat lens with Na⁺-free-TEA AAH produced a decrease in G_m and a depolarization of V_m. G_m and V_m stabilized at 250.0 ± 8.6 μS and -39.3 ± 1.0 mV, respectively (n = 15). The rat lens is known to have relatively high K⁺ and low Na⁺ permeabilities (Lucas et al. 1987). Therefore, the effect of Na⁺-free-TEA AAH was probably due in a large part to the blockade of K⁺ channels. When G_m and V_m had stabilized, thapsigargin was applied. After an initial delay, a transient depolarization and a transient increase in G_m were observed (Fig. 3A). The changes in conductance and voltage were computed as described in Fig. 3 showing that there was a significant delay of 5.2 ± 0.8 min before thapsigargin induced a significant change in membrane conductance. These data show that V_m and G_m do not recover fully after the initial transient change and stabilize at an elevated value (Fig. 3A).

Figure 3. When external Na⁺ was replaced by TEA, thapsigargin induced an increase in G_m associated with a depolarization of V_m.

A, the lens was perifused with Na⁺-free-TEA AAH and stimulated with thapsigargin (1 μM) for 4 min. The result, representative of 11 independent experiments, indicates that thapsigargin induces a transient increase in G_m (dashed line) and a transient depolarization of V_m (continuous line). B, discrete measurements of G_m and V_m were performed in order to determine the mean (±s.e.m.) amplitude and kinetics of the response to thapsigargin. For G_m (dashed line), the first measurement was taken at the beginning of the stimulation by thapsigargin (time = 0, baseline), the second at the beginning of the G_m increase, the third at the peak of the G_m increase and the last 20 min after the beginning of the stimulation. For V_m (continuous line), the first measurement was taken at the beginning of the stimulation by thapsigargin (time = 0, baseline), the second at the beginning of the depolarization, the third at the peak of the depolarization and the last 20 min after the beginning of the stimulation.

Store-operated Ca²⁺ channels (SOCs) are activated by thapsigargin (due to store depletion) in a range of tissues including the lens and are strongly inhibited by La³⁺ (Hoth & Penner, 1992; Riach et al. 1995). When applied in control solution, La³⁺ (10 μM) induced a very small hyperpolarization of the lens membrane potential (ΔV_m = 0.35 ± 0.20 mV, n = 11; Student's t test, P < 0.05) but no significant change in G_m (Fig. 4A). In Na⁺-free-TEA AAH, La³⁺ (10 μM) did not induce any change in V_m or G_m (data not shown). In control AAH and in the presence of La³⁺, thapsigargin induced a decrease in G_m and a small V_m depolarization (Fig. 4). Both G_m and V_m stabilized within 4 min and then partially recovered. When the lens was perifused with Na⁺-free-TEA AAH and La³⁺ (10 μM), thapsigargin failed to produce an electrical response (n = 4, data not shown).

Figure 4. In the presence of La³⁺ (10 μM), thapsigargin induced a decrease in G_m associated with a depolarization of V_m.

A, the lens was perifused with control AAH and stimulated with thapsigargin (1 μM) for 4 min. The result, representative of 3 independent experiments, indicates that thapsigargin induces a sustained decrease in G_m (dashed line) and a transient depolarization of V_m (continuous line). B, discrete measurements of G_m and V_m were performed in order to determine the mean (±s.e.m.) amplitude and kinetics of the response to thapsigargin. For G_m (dashed line), the first measurement was taken at the beginning of the stimulation by thapsigargin (time = 0, baseline), the second at the peak of the G_m decrease and the last 20 min after the beginning of the stimulation. For V_m (continuous line), the first measurement was taken at the beginning of the stimulation by thapsigargin (time = 0, baseline), the second at the peak of the depolarization and the last 20 min after the beginning of the stimulation.

As well as an increase in Ca²⁺ conductance, an increase in Na⁺ conductance could play a role in the thapsigargin-induced decrease in membrane conductance observed when the lens was perifused with control AAH. In order to determine whether this was the case, Na⁺ and Ca²⁺ influx were determined in the presence and absence of thapsigargin. Over a 2 h period, thapsigargin induced a significant rise in ⁴⁵Ca²⁺ influx, but was without effect on the ²²Na⁺ influx (Fig. 5). La³⁺ (10 μM) abolished the thapsigargin-stimulated Ca²⁺ influx, while having little effect on the resting Ca²⁺ level. When the kinetics of the ⁴⁵Ca²⁺ influx were investigated in more detail, the influx increased within the first 60 min of the response and then declined down to the control level after 24 h (Fig. 5A).

Figure 5. Thapsigargin-induced Ca²⁺ influx into the lens is inhibited by La³⁺.

Lenses were incubated with ⁴⁵Ca²⁺ and ²²Na⁺ for 2 h in the presence or absence of thapsigargin (Tg, 1 μM) and La³⁺ (10 μM). A, thapsigargin strongly stimulated Ca²⁺ influx into the lens. This response was inhibited by La³⁺. B, Na⁺ influx was also monitored during these experiments and it was not affected by thapsigargin. * Not significantly different from control (n = 4, Student's t test, P > 0.05). The experiments are representative of 3 independent experiments. C, kinetics (means ±s.e.m.) of ⁴⁵Ca²⁺ influx. In this experiment, lenses were incubated with ⁴⁵Ca²⁺ for 1 h at 0, 1, 3, 7 and 23 h after the beginning of the application of thapsigargin (1 μM). Thapsigargin was present throughout the experiment. A control was also performed by incubating the lenses for 1 h at times 0 and 23 h in the absence of thapsigargin (dashed line).

To investigate further the possibility that the activation of Ca²⁺ channels could play a role in the response to thapsigargin, the electrical response to thapsigargin was studied in the absence of extracellular calcium. As previously reported, removal of calcium from the AAH induced a large G_m increase and a V_m depolarization (Jacob & Duncan, 1983). Under these conditions, thapsigargin still induced a response characterized by a transient V_m depolarization (Fig. 6). No significant change in G_m could be measured. This is probably due to the brevity and the small amplitude of the response.

Figure 6. In the absence of external Ca²⁺, thapsigargin induced a transient depolarization of V_m.

The lens was perifused with Ca²⁺-free AAH (1 mM EGTA, Ca²⁺ free). In these conditions, thapsigargin (1 μM) induced a transient V_m depolarization. No significant change in G_m could be observed. The second stimulation of the lens with thapsigargin induced a hyperpolarization that is characteristic of the effect that the vehicle alone (0.05 % DMSO) has in some cases.

Since opacification of the lens is associated with an increase in Ca²⁺ content (Duncan & Bushell, 1975; Sanderson et al. 1994), the effect of increasing Ca²⁺ influx with thapsigargin on the lens transparency was monitored. No significant opacification of the rat lens could be observed after a 24 h incubation with 1 μM thapsigargin (n = 4, data not shown).

DISCUSSION

Not all cells in the lens are expected to be sensitive to thapsigargin as only the epithelial cells and nucleated fibre cells appear to possess an intact endoplasmic reticulum (Bassnett, 1995). The voltage-measuring electrode in the present experiments was placed in a terminally differentiated fibre cell that does not have endoplasmic reticulum Ca²⁺ stores and so the thapsigargin-induced signals must be relayed throughout the lens by junctional communication (Fig. 1). A similar conclusion was reached for acetylcholine-induced depolarization (Thomas et al. 1997) and this is not surprising as the lens appears to be particularly well endowed with ionic cell-to-cell communication pathways (Duncan, 1969; Rae & Kuszak, 1983).

Application of thapsigargin induced complex changes in lens membrane conductance and at least two distinct phases were distinguishable (Fig. 2). Firstly, the data presented here indicate that thapsigargin activates a Ca²⁺ conductance. When external Na⁺ was replaced by 135 mM TEA, thapsigargin induced a transient increase in G_m and a depolarization of V_m (Fig. 3). Since these conditions caused a reversal of the Nernst potential for Na⁺, the electrical changes observed were probably due to an increase in Ca²⁺ conductance. This was confirmed by the finding that the Ca²⁺-channel blocker La³⁺ totally inhibited this response. The ⁴⁵Ca²⁺ influx studies confirmed that thapsigargin increases Ca²⁺ conductance in the intact lens (Fig. 5). In the presence of La³⁺ and normal AAH, thapsigargin failed either to increase ⁴⁵Ca²⁺ influx (Fig. 5A) or to induce the late increase in G_m (Fig. 4). Furthermore, in the absence of external Ca²⁺, thapsigargin induced a small and transient electrical response. Taken together, these data indicate that thapsigargin activates a Ca²⁺ influx. Thapsigargin did not alter Na⁺ influx and therefore Ca²⁺ influx occurred via a Ca²⁺-specific pathway. Interestingly, SOCs, which are known to be activated by thapsigargin, have similar kinetics characteristics to the Ca²⁺ conductance observed here. Indeed, Shuttleworth (1994) has shown that, after Ca²⁺ release from the stores, there is a significant delay in the activation of SOCs in exocrine cells. Similar delays have been observed with agonist-induced Ca²⁺ influx in human lens cells (Riach et al. 1995). It is therefore probable that the thapsigargin-induced increase in G_m is due to the activation of SOCs.

The electrical response to thapsigargin in Na⁺-free-TEA AAH indicates that part of the Ca²⁺ influx rapidly inactivated (Fig. 3). SOCs are known to inactivate within minutes after a rise in [Ca²⁺]_i (Zweifach & Lewis, 1995). The inactivation of such channels could explain the rapid inactivation of the current observed in the intact lens (Fig. 3). However, in the lens, the inactivation was partial (Fig. 3B, V_m at 20 min) which suggests that a second sustained Ca²⁺ influx remained. This is supported by the flux data that show a slowly inactivating thapsigargin-induced Ca²⁺ influx (Fig. 5A). Madge et al. (1997) have observed similar kinetics in pulmonary artery endothelial cells where depletion of the Ca²⁺ stores induces both a transient and a more sustained Ca²⁺ influx. In the lens, the Ca²⁺ flux data indicate that the more sustained phase also inactivates.

This transient rise in calcium influx does not appear to be sufficient either to electrically uncouple lens cells or to alter lens transparency. A sustained increase in calcium induced, for example, by oxidative stress can lead to a measurable uncoupling and loss of transparency when monitored with precisely the same techniques as used here (Duncan et al. 1988; Sanderson & Duncan, 1993). This indicates that even a strong activation of the lens calcium signalling pathway does not have a short-term traumatic effect on the lens. It is interesting that Spray et al. (1986) have shown that mammalian epithelial cells, in contrast to insect salivary glands (Rose et al. 1977), are relatively insensitive to uncoupling by calcium and non-physiological levels of calcium (> 10 μM) have to be reached before uncoupling occurs. It is also likely that, in the lens, calcium-induced cell uncoupling and opacification are attained only under conditions associated more with cell death than cell signalling.

A second membrane conductance appears to be modulated by thapsigargin since a decrease in G_m associated with a depolarization of V_m was observed. This is probably due to the inhibition of a K⁺ conductance (Thomas et al. 1997). Since thapsigargin induces an increase in [Ca²⁺]_i, the electrical response presumably occurs by the inhibition of K⁺ channels by Ca²⁺. Evidence for the presence in the lens of such channels has already been reported (Lucas et al. 1986; Alvarez et al. 1996). In response to thapsigargin, [Ca²⁺]_i increases partly due to release from endoplasmic reticulum stores and partly from the resulting influx of Ca²⁺ (Berridge, 1997) and hence at least two phases of electrical response would be expected. In the presence of La³⁺, the thapsigargin-induced Ca²⁺ influx was blocked (Fig. 5A): the increase in [Ca²⁺]_i was then only due to Ca²⁺ release and would therefore be predicted to have a reduced amplitude. In fact, thapsigargin still induced a decrease in G_m in the presence of La³⁺ (Fig. 4) but the amplitude of this transient was smaller than that observed in the absence of La³⁺ (Fig. 2). This supports the idea that the thapsigargin-sensitive K⁺ conductance is modulated by Ca²⁺ and that Ca²⁺ release is sufficient to modulate this conductance. The small amplitude of the thapsigargin-induced response in the absence of external Ca²⁺ (Fig. 6) can be explained by the absence of Ca²⁺ influx and supports the results obtained with La³⁺.

Ca²⁺-sensitive K⁺ channels play an important role in modulating electrical activity in a range of tissues and Selyanko & Brown (1996) have recently reported that the potassium M channel in inside-out patches of neuronal cell membranes is reversibly inhibited by an increase in [Ca²⁺]_i. This channel also appears to be modulated by the calcium-calmodulin-dependent phosphatase calcineurin (Marrion, 1996). It is interesting that acetylcholine has been shown to inactivate this channel directly through the muscarinic receptor system (Brown & Adams, 1980). Although best characterized in neurones and neurone cell lines, it appears to be present in other cell types (Marrion, 1997).

The presence of such Ca²⁺-sensitive K⁺ channels in the lens suggests that they have a potentially important role in cell signalling processes in the lens. It is now well established that lens cells express muscarinic acetylcholine receptors that, upon activation, can induce both [Ca²⁺]_i and V_m oscillations (Williams et al. 1993; Thomas et al. 1998). It is probable that the two mechanisms are closely linked as it has been shown in a range of cell types that simultaneous Ca²⁺ and V_m oscillations take place involving various voltage- and Ca²⁺-sensitive channels (López et al. 1995; Verheugen & Vijverberg, 1995). The most compelling evidence is provided by the fact that thapsigargin sensitizes the lens to acetylcholine-induced V_m oscillations, rather than inhibiting them (Thomas et al. 1998). This could occur if thapsigargin simply raised [Ca²⁺]_i and hence the threshold at which oscillations could occur. Acetylcholine also induces a depolarization of V_m that cannot be prevented by thapsigargin (Thomas et al. 1998). This could be due to the modulation of K⁺ channels via a Ca²⁺-independent cell signalling pathway such as the diacylglycerol/protein kinase C pathway. The presence of both a Ca²⁺-dependent and a Ca²⁺-independent signalling pathway is further shown by the fact that nifedipine, a specific inhibitor of L-type Ca²⁺ channels, blocks acetylcholine-induced oscillatory activity but fails to inhibit the initial acetylcholine-induced V_m depolarization (Thomas et al. 1998). Interestingly, the Ca²⁺ channel blocker verapamil has previously been shown to inhibit a calcium-overload cataract produced in the diabetic rat in vivo indicating a possible role of these mechanisms in cataract formation (Fleckenstein, 1983).

The studies carried out to date indicate that lens cells possess a range of receptors linked with signalling pathways that involve the complex modulation of different ion channels (Ca²⁺ and K⁺ channels). The lens appears to be an interesting model as it allows the characterization of the mechanisms involved over a much longer time course than is possible in isolated c ells. Furthermore, because of the symmetry and basic simplicity of structure of the lens, it is also possible to derive information concerning the transmission of signals between cells and the impact of calcium-induced changes on this transfer.