Regional Differences in Functional Receptor Distribution and Calcium Mobilization in the Intact Human Lens

Abstract

purpose. To investigate regional differences in Ca²⁺ mobilization kinetics in the intact human lens produced by exposure to agonists of tyrosine-kinase and G-protein–coupled receptors and to characterize the major receptor subtypes involved in Ca²⁺ signaling in the different regions.

methods. Whole human lenses were placed anterior side down in a plastic chamber and perifused with artificial aqueous humor (AAH) at 30°C. After fura-2 incorporation, cytosolic Ca²⁺ levels were monitored by using epifluorescence techniques in either the equatorial or central anterior epithelial cells of the intact lens. Agonists dissolved in AAH were applied to the lens in successive short pulses.

results. Central anterior lens epithelial cells produced a large response to 10μ M acetylcholine (ACh) and histamine; only a small response to adenosine triphosphate (ATP); and no response to 10 μM adrenalin, 10 ng/ml epithelial growth factor (EGF) or TGFα, or 50 ng/ml platelet-derived growth factor (PDGF)-AB. Conversely, the equatorial cells produced a strong response to 10 μM ATP and histamine, 10 ng/ml EGF (or TGFα), and 50 ng/ml PDGF-AB, but failed to respond to 10 μM ACh or 10 μM adrenalin. The EGF-induced response in the equatorial cells was blocked completely by tyrphostin (AG1478), a specific inhibitor of the EGF receptor tyrosine kinase. Carbachol, a nonhydrolyzable analogue of ACh, and pilocarpine, the M1 muscarinic receptor–specific agonist, both produced the same trend of response amplitude elicited by ACh in each region of the lens. The potency order of purinergic agonist-induced Ca²⁺ mobilization at the equator was consistent with the P2Y₂ receptor subtype. The histamine-induced response was abolished by 10 μM triprolidine, a specific H₁ receptor antagonist, but remained unaffected by the specific H₂ and H₃ antagonists, ranitidine and thioperamide, respectively.

conclusions. There is a spatial heterogeneity in functional receptor activity in different regions of the whole lens. The important growth factor receptors for EGF and PDGF are functionally active only in the equatorial cells of the mature human lens. This study further shows that the ACh, histamine, and ATP-induced responses arise from the activation of M1 muscarinic, H₁ histamine, and P2Y₂ purinergic receptors, respectively.

In many types of epithelial cells, including human lens cells, agonist-induced Ca²⁺ release can occur through activation of either G-protein– or tyrosine-kinase–coupled receptors. In the former case, the β isoform of phospholipase C (PLCβ) is activated, whereas in the latter, the γ isoform of PLC (PLCγ) is activated, ^{1 2} both of which release the second messenger inositol 1,4,5-trisphosphate (IP₃) into the cytoplasm. IP₃ diffuses rapidly within the cytosol and interacts with IP₃ receptors located on the endoplasmic reticulum (ER) membrane that serve as Ca²⁺ channels to release stored Ca²⁺ and initiate the first phase of the Ca²⁺ signal. ^{3 4} In many cell types, it is evident that Ca²⁺ signals originating from either G-protein or tyrosine-kinase receptors control a variety of cellular functions that include cell communication and secretion, as well as growth and proliferation. ^{5 6} In the lens, more is known of the functional effects after tyrosine-kinase receptor activation, and the result is generally a stimulation of cell division or differentiation, or both. ^{7 8 9 10}  

Most studies have been performed in animal model systems, and there is little consensus of opinion concerning which of the possible receptors play the major role in lens growth. ⁷ However, there is evidence to show that alterations in the continuous process of cell division and fiber cell differentiation due to biochemical changes in the ocular environment may lead to the appearance of cataract. ¹¹ It is important to know, therefore, which receptor systems are functionally active in the human lens. There is little doubt, however, that Ca²⁺ signaling itself plays a key role in growth and that both G-protein and tyrosine-kinase receptor systems are involved. ¹²  

The human lens is a dynamic organ, in that it continues to grow throughout life, and the equatorial region of the lens is the site of cell division and differentiation. In contrast, the central anterior cells represent a mitotically quiescent population. ¹³ An obvious question is whether the different cell populations have different sets of Ca²⁺-signaling receptors. Past studies have been performed largely on tissue-cultured human lens cells, using G-protein–coupled agonists such as acetylcholine (ACh), adenosine triphosphate (ATP), and histamine, ^{14 15} although Duncan et al. ¹² demonstrated that platelet-derived growth factor (PDGF) can elicit Ca²⁺ responses in tissue-cultured rabbit lens cells. These studies have been performed on growing cells rather than the quiescent cells of the anterior epithelium, and further, in a previous study, we have shown that the subset of native anterior epithelial muscarinic receptors can change in culture. ¹⁶ Therefore, it was vitally important to undertake Ca²⁺ mobilization studies in the intact human lens—first, to investigate which receptors are functionally active in native cells, and, second, to identify whether regional differences in receptor distribution exist. 

Materials and Methods

All chemicals were obtained from Sigma Chemical Co., (Poole, UK), unless otherwise stated. All Ca-free medium contained 1 mM EGTA. 

Native Human Lens Preparations

Thirty-two human lenses were used for this study from donors aged between 25 and 80 years. Human globes and lenses were obtained from the East Anglian Eye Bank or Bristol Eye Bank, respectively, usually within 48 hours of enucleation from the donor and after the cornea had been removed for transplantation surgery. As no donor details, apart from age, sex, and cause of death were released, this research followed the tenets of the Declaration of Helsinki. 

Lenses were carefully dissected from the globes, and surrounding ciliary, iris, and vitreous bodies were removed. Lenses were then bathed in 30°C artificial aqueous humor (AAH), with the following composition (mM): 130 NaCl, 5 KCl, 5 NaHCO₃, 1 CaCl₂, 0.5 MgCl₂, 5 glucose, and 20 HEPES, adjusted to pH 7.25 with NaOH. Immediately after removal from the globes, lenses were placed in one of two plastic chambers used for calcium imaging. The first chamber had a depth of 6 mm and could accommodate a whole lens, anterior side down. The lens was secured in place by resting it against pins pushed into the plastic base of the chamber. This arrangement allowed imaging of both the central anterior cells and the equatorial region of the lens (Fig. 1) . Calcium measurements were also performed on cells of the isolated epithelium. ¹⁶ In this case, the lens was placed in a chamber with a depth of 3 mm. The lens capsule with its adherent epithelium was dissected from the fiber mass and secured to the base of the plastic chamber by pinning (see Collison et al. ¹⁶ for further details). Any remaining lens fiber fragments were removed by successive irrigation of the lens capsule with artificial aqueous humor (AAH). 

Measurement of Intracellular Calcium Levels

Both preparations (whole lens and isolated epithelium) were loaded with the acetoxymethylester form of 3 μM fura-2 (fura-2/AM) for 40 minutes at 30°C. The cells were then washed in AAH for 20 minutes to allow complete de-esterification of the dye. Ratiometric imaging of cytosolic Ca²⁺ took place on the stage of an epifluorescence microscope (Nikon, Tokyo, Japan) fitted with a× 20 objective (Fig. 1) . The ratio image of the anterior epithelium (whether attached to the lens or isolated) gives a homogeneous field of view (see Collison et al. ¹⁶ ), and the cells are too small for individual cell analysis. Therefore, data from regions of interest consisting of approximately 10 confluent cells were acquired as a running ratio average. The image of equatorial cells was, however, a sharp, narrow band (approximately 50 μm in width), and a portion of the band was selected as a region of interest—approximately the same area as in the anterior cell preparation. Again, when stimulated with agonists, the region responded in a homogeneous manner across the band, and the data were acquired as a running-ratio average (Fig. 2) . No fluorescence signal was obtained from the lens posterior region or lens nucleus, but stable ratio signals were obtained from anterior and equatorial cells. All preparations were continuously perifused with AAH (30°C). Solutions were administered through a two-way tap, and every effort was made to ensure that solution turnover time in each of the chambers was kept the same (approximately 10 seconds). Cells were excited alternatively with light of 340- and 380-nm wavelengths. Resultant fluorescent emissions at both wavelengths were collected by a charge-coupled device (CCD) camera at 510 nm and sampled every 2 seconds. After background subtraction and calibration, fluorescence ratios (R) were converted into real Ca²⁺ concentrations, by using the formula of Grynkiewicz et al. ¹⁷  

\[{[}\mathrm{Ca}^{\mathrm{2{+}}}{]}{=}K_{\mathrm{d}}{[}(R{-}R_{\mathrm{min}}){/}(R_{\mathrm{max}}{-}R){]}(S1{/}S2)\]

Calibration involved permeabilizing the cells at the end of the experiment with ionomycin (10 μM) and bathing the cells in Ca-free AAH that contained 1 mM EGTA, 1 μM thapsigargin, 150 mM KCl, and 100μ M of the plasma membrane Ca-ATPase inhibitor, W7. This allowed a measurement of the fluorescence ratio in zero Ca²⁺ (R _min). The same cells were then exposed to a similar solution that had 10 mM Ca²⁺ replacing EGTA to obtain a maximal ratio (R _max). The factor (S1/S2) is the fluorescence intensity at 380 nm when all the fura-2 is in the Ca-free form divided by the fluorescence intensity when the fura-2 is in the bound form. R _min and R _max were determined in calibration experiments. The dissociation constant (K _d) for fura-2 was taken as 224 nM. ¹⁷ Cells in the isolated epithelium and the central anterior region of the lens were successfully calibrated using this procedure. However, calibration of cells in the equatorial region of the lens was unsuccessful, because R _min failed to stabilize throughout the calibration procedure lasting more than 1 hour. 

Agonist Application

The different agonists were applied to the preparations in random order. It should be noted that the order in which the agonists were applied to the whole lens made no difference in the resultant response amplitude elicited by each agonist. Furthermore, 15 minutes was allowed between each agonist application, because successive pulses of test agents gave the same response when applied with this time interval. ¹⁶  

Technical Note

It is important to note that neither securing the lens in the manner described nor loading the lens with fura-2 perturbed the normal membrane characteristics of the lens. Stable resting membrane potentials were recorded from human lenses in parallel studies (unpublished data, 2001) that were of a magnitude similar to those reported from freshly isolated human lenses. ¹⁸ Illuminating the lens with 340- and 380-nm UV light to measure intracellular Ca²⁺ also did not perturb the voltage (Collison et al., unpublished data, 2001). 

Results

Cellular Origins of the Fura-2 Fluorescent Signals

The field of view when the cells on the anterior face of the lens was imaged was very similar to that of the isolated anterior epithelium (see Collison et al. ¹⁶ for further details). A homogeneous ratio image was obtained, and every cell in the field of view responded (e.g., to ACh) in both cases. The cells in the equatorial region of the lens appeared as a bright fluorescent band approximately 50 μm wide, when loaded with fura-2. A 30-second pulse of ATP (10 μM) induced a transient change in cytosolic Ca²⁺ in these cells that lasted approximately 100 seconds (Fig. 2) . There was no fura-2 fluorescence from the posterior face of the lens. Because relatively low-magnification (×20), large-working-distance lenses had to be used, it was not possible to determine whether short equatorial fiber cells and epithelial cells were both imaged. However, the data are consistent with the fluorescent loading pattern obtained in the embryonic chick lens by Bassnett et al., ¹⁹ who demonstrated that it was possible to apply confocal optics to show that only epithelial cells were involved. 

Regional Differences in Receptor-Induced Ca²⁺ Mobilization in the Whole Lens

It has been previously reported that freshly isolated human lens cells ¹⁶ and tissue-cultured lens cells ^{14 15} maintain a relatively low cytosolic Ca²⁺ concentration that undergoes a large transient increase when the cells are exposed to a range of G-protein–coupled agonists, including ACh, ATP, and histamine. The anterior cells in the intact lens had a similar stable resting Ca²⁺ concentration (approximately 100 nM) and also gave large responses when exposed to ACh, ATP, and histamine (Fig. 3A) . However, anterior cells in the intact lens failed to respond significantly to adrenalin, EGF (Fig. 3A) , or TGFα (Fig. 3B) . It was interesting to note that equatorial cells gave large responses when stimulated by ATP or histamine, but the responses to ACh and adrenalin were extremely small and remained close to the baseline. 

Furthermore, the equatorial cells were also activated by tyrosine-kinase receptor ligands, and EGF (10 ng/ml), for example, elicited a very large, prolonged response (Fig. 4A) that was abolished by the specific EGF receptor tyrosine-kinase inhibitor, tyrphostin AG1478 (100 nM; Fig. 4B ). Furthermore, there was no interaction of this inhibitor with G-protein–coupled receptors, because the response to ATP after tyrphostin was identical with the first ATP response (Fig. 4B) . TGFα is also a ligand for the EGF receptor and, as expected, produced a prolonged response (Fig. 4C) . PDGF-AB (50 ng/ml) also induced a response in the bow region (Fig. 4C) , but failed to produce a response in anterior cells (Fig. 3B) . Tyrphostin had no effect on the equatorial response to PDGF (data not shown). 

The averaged response amplitudes (n = 8 lenses) to ACh, ATP, histamine, adrenalin, and EGF in the anterior and equatorial regions are given in Figures 5A and 5B , respectively, and were normalized to the histamine response, which was always present in both regions of the lens. EGF and adrenalin failed to produce a significant response in anterior epithelial cells in all eight preparations, whereas the responses to ACh and adrenalin in the equatorial cells were either absent or extremely small (Fig. 5A) . Both ATP and histamine consistently induced responses in the two regions, and, at equal concentrations, ATP was always the dominant response at the equator, whereas ACh induced the largest response in the anterior cells. 

Involvement of Lens Capsule Cholinesterase Activity in the Muscarinic Response

The lens capsule has been reported to have an intrinsically high ACh–esterase activity, ²⁰ and in fact the capsule is much thicker at the equator than at the central epithelium. ²¹ To test the possibility that a higher esterase activity could account for the very small ACh response in the equatorial region, the nonhydrolyzable analogue of ACh, carbachol (CCh), was used. In the central anterior lens cells, CCh produced a robust Ca²⁺ transient (Fig. 6A) that was again greatly attenuated in the equatorial region (Fig. 6B) . It was interesting to note that in both regions CCh actually produced a smaller response than ACh. This is not unexpected, because at equal concentrations, CCh is considered to be less potent at activating muscarinic receptors than ACh. ²²  

Pharmacology of the Intact Lens Responses

The pharmacology of the ACh response in the lens is important to establish, because the subtype responsible for Ca²⁺ release has been ascribed to M1, M3, and M5 in different human lens cell preparations. ^{16 23} Furthermore, an ACh response was reported to be absent from tissue-cultured sheep cells that responded well to ATP and histamine. ²⁴ Pilocarpine is reported to be a selective partial agonist for the M1 subtype of muscarinic receptor ²⁵ and is commonly used clinically in the treatment of glaucoma. ²⁶ Because native lens cells have been shown to possess the M1 muscarinic receptor subtype, ¹⁶ it was not surprising to find that pilocarpine produced a large response in the central anterior lens cells and isolated epithelium (Figs. 7A 7B , respectively).The pilocarpine and ACh responses were biphasic, and the second (slower) phase probably arose from activation of the store-operated Ca²⁺ entry pathway. ^{3 27} Previous results have shown that this pathway has extremely slow activation kinetics in human lens cells. ^{15 28} Pilocarpine failed, however, to produce a response in cells of the human lens cell line HLE-B3 (data not shown), and in fact these cells express the M3 rather than the M1 subtype. ¹⁶ Pilocarpine also failed to elicit a response in the bow region of the intact lens (Fig. 7C) . It should be noted that the spectrum of the responses to the different G-protein–coupled agonists was the same for the isolated epithelial cells compared with that in the central anterior cells of the intact human lens (Figs. 7A 7B) . 

Although the expression of the muscarinic receptor subtype changes during culture, ¹⁶ this does not appear to be the case for either the purinergic or histamine receptors. The potency sequence uridine triphosphate (UTP) ≥ ATP ≫ uridine diphosphate (UDP) = adenosine diphosphate (ADP) obtained from the intact lens (Fig. 8A) is precisely that obtained for tissue-cultured human ¹⁵ and sheep lens cells ²⁴ and indicates that the P2Y₂ (previously called P2U) receptor subtype is responsible. There appears to be no contribution from P2X₁ or P2X₂ ionotropic receptors in the human lens, because high concentrations of the specific agonists α,β-methylene ATP (α,β-meATP) andβ ,γ-methylene ATP (β,γ-meATP) failed to stimulate a change in Ca²⁺ concentration (Fig. 8B) . Similarly, there appeared to be no adenosine receptors coupled to Ca²⁺ mobilization in the intact human lens (Fig. 8A) . The fact that the histamine response at both the equatorial and anterior regions is totally inhibited by 10 μM of the H₁ antagonist, triprolidine (Fig. 9 and data not shown) indicates that the H1 receptor subtype was responsible for Ca²⁺ mobilization. The histamine-induced response was unaffected by the specific H₂ receptor antagonist ranitidine and the H₃ receptor antagonist thioperamide. Again, a similar sensitivity has been reported in human tissue-cultured cells. ¹⁵  

Discussion

There is some controversy at present concerning which receptor systems (both tyrosine-kinase and G-protein–coupled) are of prime importance in the human lens. ^{7 9} Such controversies exist partly because most experimental work is performed in a range of animal models, ^{29 30} but also because, when human lens cells alone are compared, different tissue-culture models produce a different spectrum of receptors. ¹⁶ One unequivocal way of finding out which receptor systems are functionally active is to measure downstream signaling events in the intact organ. The present experiments have clearly demonstrated the presence of functional tyrosine-kinase and G-protein–coupled receptors that mobilize intracellular Ca²⁺ in the whole human lens. Furthermore, there is evidence of heterogeneity with respect to which regions of the lens preserve functionally active receptors. For example, ACh known to activate receptors in tissue-cultured lens cells, ¹⁴ elicited large changes in cytosolic Ca²⁺, only in the central anterior epithelium, but not in the equatorial region (Figs. 3 4) . In contrast, the tyrosine-kinase–linked growth factors EGF, TGFα, and PDGF-AB produced large responses in the equatorial cells, but did not induce responses in central epithelial cells. 

It is interesting that EGF, TGFα, and PDGF-AB produce responses only in the equatorial region of the intact lens, because this region is solely responsible for lens cell growth and differentiation. Previous studies have shown that PDGF contributes to lens cell growth and transparency in the intact chick lens, by initiating cell proliferation and cell division. ³⁰ PDGF has also been reported to induce lens cell proliferation and some aspects of the fiber cell differentiation pathway in transgenic mice. ³¹ Potts et al. ³² found that PDGFα receptors were present in the peripheral lens epithelium of the embryonic chick lens during development. Furthermore, they confirmed the mitogenic effect of PDGF on tissue-cultured chick lens epithelial cells. Tyrosine-kinase–linked growth factors PDGF-AB and EGF have both been shown to mobilize Ca²⁺ from internal stores and to influence growth of lens cells in culture ^{12 33} (Duncan and Wormstone, unpublished data), whereas Ibaraki et al. ¹⁰ found that EGF (10 ng/ml) not only greatly increased cell proliferation but also stimulated fiber cell differentiation in human lens cell cultures. More detailed analysis of the morphologic changes in cultured human lens cells induced by EGF revealed the presence of multilayered cells, a proportion of which possessed ball-and-socket junctions, characteristic of differentiated lens fiber cells. ³⁴ Although EGF has been shown to be a potent mitogen for primate and rabbit epithelial lens cells in culture, similar proliferative responses to TGFα have also been obtained. ³⁵ Because a relatively low concentration of the specific EGF receptor inhibitor tyrphostin AG1478 (100 nM) abolished the EGF-induced Ca²⁺ response (Fig. 4B) , it is likely that the EGF receptor plays a critical role in cell division and differentiation in the equatorial region of the human lens. 

The uveal tract is responsible for the nutritional supply of many intraocular structures, especially the avascular lens, through the production of aqueous humor. Several growth factors, including EGF and PDGF, have been detected in the uveal tract of human eyes. ³⁶ In vivo, a breakdown of the blood–aqueous barrier arising from an injury is likely to release growth factors from neighboring ocular structures, therefore increasing the amount of growth factors in the aqueous humor. ³⁶ ELISA assays and sensitive radioimmunoassays, however, have in fact failed to detect significant amounts of either EGF or TGFα, the main ligands of the EGF receptor, in aqueous humor from human eyes, ^{37 38} and although EGF is largely absent from normal lenses, it appears to be present in certain cataractous lenses. ³⁹  

It should also be noted that the EGF receptor can be transactivated by a wide range of agonists, including those activating G-protein–coupled systems. ^{40 41 42 43} Furthermore, the mitogen-activated protein (MAP) kinases represent a point at which cell surface signals for either G-protein– or tyrosine-kinase–coupled receptors converge to regulate cell growth and division. ⁴⁴ Additional signaling systems may therefore be required that synergistically enhance the downstream effects of EGF receptor-ligand interactions. ¹¹  

Previous work from this laboratory has shown that ATP can modulate the PDGF-driven growth of lens cells, ¹² and it has been suggested that this modulation arises through cell-signaling“ cross-talk” between G-protein–coupled P2U and tyrosine-kinase–coupled receptors. It is interesting that ATP produces a much larger response, relative to histamine, in the equatorial region of the intact lens than it does in the anterior epithelium. In fact, the prime role of Ca²⁺ cell signaling in driving the growth of lens cells can be seen by exposing cells to the selective ER membrane Ca²⁺-ATPase inhibitor, thapsigargin (100 nM), which induces total cell growth arrest. ¹²  

It should be noted that histamine produced relatively large responses in both the equatorial and central anterior cells by activation of H₁ receptors, which suggests that the human lens may in some way be able to receive information concerning the ocular inflammatory response. Human ocular allergic pathophysiology is mediated by many cellular and molecular mechanisms, ^{45 46} invariably requiring conjunctival mast cell activation and release of histamine, along with other agents, into the aqueous humor. In the light of present findings, it is reasonable to assume that certain signal-transduction pathways may be stimulated in the human lens, after release of such inflammatory agents into the bathing medium from surrounding ocular tissues. 

It was possible to obtain quantitative values for the Ca²⁺ levels in anterior epithelial cells, both when they resided in the intact lens and when freshly isolated from the lens (Figs. 3 7 , and Collison et al. ¹⁶ ). Not only were they similar, but both preparations responded to the same range of agonists. The equatorial cells, however, responded differently in most respects, and the reason that these cells do not calibrate (see the Methods section) probably lies in the different coupling characteristics of anterior and equatorial cells. Several studies have shown that the anterior cells, while coupled to one another, do not appear to be functionally coupled to the fibers beneath, nor do they possess typical gap junction structures on the apical membranes facing the fibers. ^{19 47} The equatorial epithelial cells, however, possess junctional plaques and appear to be coupled to some extent to underlying bow fibers. ^{47 48} This functional coupling would make it very difficult to calibrate the equatorial cells, because, after exposure to Ca-free conditions in the external medium, Ca²⁺ could still enter these cells from the bulk of the rest of the lens. The anterior epithelial cells, on the other hand, would be much more easily drained of Ca²⁺ if they were not functionally coupled to the underlying fibers. 

Because the anterior epithelium did not appear to be coupled to the underlying fibers, it is perhaps therefore not unexpected that the freshly isolated anterior epithelium behaved very similarly to the central epithelium in the intact lens (Figs. 7A 7B) . Significantly, the M1 muscarinic subtype appeared to be activated by ACh in both preparations, as there were large, robust responses from pilocarpine, an M1-selective agonist. ²⁵ The lens capsule contains very high cholinesterase activity, ²⁰ and because the capsule is much thicker at the equatorial region than at the anterior, ²¹ it is possible that a greater activity of the enzyme in the equatorial region could be blunting the ACh response from these cells. However, this is unlikely to be the case, because both CCh and pilocarpine did not give enhanced responses in the equatorial region (Figs. 6B 7C) . In the intact human lens, the time courses of the G-protein–coupled Ca²⁺ responses are significantly faster than those initiated by the tyrosine kinase agonists. This has been observed in other tissues and has been ascribed to the fact that signaling through G-protein–coupled receptors is faster than that arising from tyrosine phosphorylation. ⁴⁹  

It is notable that, although adrenalin produces a large Ca²⁺ mobilization response in sheep cells, ²⁴ there appeared to be no significant response in the intact human lens, either from anterior epithelial cells or from equatorial cells. There also appeared to be significant differences in G-protein signaling mechanisms between human and rat, in that P2Y₂ receptors were identified in the anterior epithelial cells of the former in the present study. However, in the rat lens, in situ hybridization techniques have revealed that P2Y₂ transcripts are present only in elongating fiber cells and not in any epithelial cells, either anterior or equatorial. ⁵⁰ The isolated perifused human lens has thus been shown to be a reliable system with which to investigate the functional activity of different receptor pathways. Because it has recently been shown that the human lens maintains transparency and viability over prolonged culture, ⁵¹ this raises the possibility of applying pharmacologic and molecular techniques to alter receptor expression and hence of assessing directly the relative contribution of each of them to maintaining growth, differentiation, and transparency. 

 Supported by the Biotechnology and Biological Sciences Research Council (BBSRC), The Humane Research Trust, The Sir Halley Stewart Trust, and National Institutes of Health Grant EY10558.

 Submitted for publication March 14, 2001; revised May 18, 2001; accepted May 31, 2001.

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 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked“ advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Schematic diagram of the chamber used in Ca²⁺ imaging. The lens is oriented posterior surface uppermost, whereas the two spatially distinct regions of the lens used for Ca²⁺ imaging are shown as (A) central anterior epithelium and (B) equatorial region. Only these two regions incorporated fura-2. There was no fluorescence signal from the bulk of the lens, shown in yellow (see also Bassnett et al. ¹⁹ ) The diameter of the mature human lens is approximately 10 mm.

Figure 2.

 

Time-lapse fluorometric ratio images of ATP-induced mobilization of intracellular Ca²⁺ in human lens cells loaded with fura-2. The fluorescent band corresponding to resting levels of intracellular Ca²⁺ in the equatorial epithelial cells at t = 0 seconds is superimposed on the white-light image (top left). ATP (10 μM) was perifused for 30 seconds and stimulated a Ca²⁺ transient (averaged running values from the ratio images) that lasted approximately 100 seconds. The fluorescent band corresponding to the equatorial epithelial cells (EE) was 50 μm wide and lay adjacent to the capsule (C). The remainder of the lens appeared as a shade of light gray against the dark background of the chamber. Any residual fluorescence did not change with time.

Figure 3.

 

Examples of Ca²⁺ transients elicited by G-protein–and tyrosine-kinase–coupled agonists. An intact human lens was imaged in the central anterior epithelial region (as shown in Fig. 1 ). (A) The central region of anterior epithelial cells responded well to ACh (10 μM) and histamine (10 μM), less well to ATP (10 μM), and not at all to adrenalin (Adren; 10 μM) or EGF (10 ng/ml). (B) Anterior epithelial cells did not produce a significant response to TGFα (10 ng/ml) or PDGF (50 ng/ml). Trace (A) was obtained from one lens but was repeated using at least eight lenses from four independent donors, and trace (B) was repeated using three different lenses.

Figure 4.

 

Monitoring Ca²⁺ transients in the equatorial region. (A) Cells in the equatorial region responded markedly to ATP, histamine, and EGF (10 ng/ml), but very little to ACh or adrenalin. (B) The EGF-induced response was totally inhibited by tyrphostin AG1478 (100 nM), and subsequent application of EGF after this specific tyrosine kinase inhibitor was removed failed to elicit any further change in intracellular Ca²⁺. The ATP-induced response was unaffected by tyrphostin AG1478. (C) The equatorial cells respond to TGFα (10 ng/ml) and PDGF (50 ng/ml). In both regions of the lens, changes in cytosolic Ca²⁺ levels were taken from regions of interest containing approximately 10 cells. Trace (A) was obtained from one lens and was repeated using at least eight lenses from four independent donors, and traces (B) and (C) were repeated using three different lenses.

Figure 5.

 

Comparison of response amplitudes in both regions induced by each agonist relative to histamine. (A) Central anterior epithelial and (B) equatorial cells. Histamine was chosen as the standard for normalizing, because it elicited significant responses in both regions of the lens. ACh, ATP, and histamine were applied at 10μ M for 30 seconds, whereas EGF was applied for 3 minutes at 10 ng/ml. In both cases, the resting ratio value defines 0% response, and the average peak ratio amplitude of the responses to histamine defines 100%. The data were obtained from a total of eight lenses from four different donors.

Figure 6.

 

Comparison of carbachol-induced responses in anterior and equatorial cells. (A) The nonhydrolyzable analogue of ACh, CCh (10μ M), produced a strong response in the central anterior epithelium, but failed to produce a significant response in the equatorial cells (B). CCh produced a smaller response than ACh in both regions. For both lens regions, n = 3 lenses.

Figure 7.

 

Pilocarpine-induced Ca²⁺ mobilization in human lens cells. The M1 muscarinic-receptor–selective agonist pilocarpine (10 μM) produced a robust Ca²⁺ mobilization response in the central anterior lens epithelial cells (A) and the isolated epithelium (B). However, concentrations up to 1 mM failed to elicit responses in equatorial cells of the intact lens (C). All concentrations are 10 μM, unless otherwise stated. For each lens preparation, n = 3.

Figure 8.

 

Characterization of the ATP-induced Ca²⁺ mobilization response in the equatorial region of the lens. (A) ATP and UTP produced similar responses in equatorial cells, whereas application of either ADP, UDP, or adenosine (Aden) caused little or no change in lens cell Ca²⁺. (B) The P2X₁- and P2X₂-specific agonists, α, β-meATP, and β,γ-meATP, failed to elicit any change in lens cell Ca²⁺. All agonist concentrations are 10 μM, unless otherwise stated. For each lens preparation, n = 3.

Figure 9.

 

Characterization of histamine-induced changes in intracellular Ca²⁺ in the equatorial region of the lens. Triprolidine (Triprol; 10 μM), but not ranitidine (Ranit; 10 μM) or thioperamide (Thiop; 10 μM), abolished the 10-μM histamine-induced Ca²⁺ response. The effect of triprolidine was reversible only after the antagonist had been removed for a long period.

The authors thank Julia Marcantonio for helpful discussions and Rebecca Torguson for technical assistance.