Rabbit conjunctival epithelium transports fluid, and P2Y22 receptor agonists stimulate Clminus and fluid secretion

Yansui Li1, Kunyan Kuang1, Benjamin Yerxa2, Quan Wen1, Heinz Rosskothen1, and Jorge Fischbarg1,3

Departments of 1 Ophthalmology and 3 Physiology and Cellular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032; and 2 Inspire Pharmaceuticals, Incorporated, Durham, North Carolina 27703


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Rabbit conjunctival epithelium exhibits UTP-dependent Cl- secretion into the tears. We investigated whether fluid secretion also takes place. Short-circuit current (Isc) was 14.9 ± 1.4 µA/cm2 (n = 16). Four P2Y2 purinergic receptor agonists [UTP and the novel compounds INS365, INS306, and INS440 (Inspire Pharmaceuticals)] added apically (10 µM) resulted in temporary (~30 min) Isc increases (88%, 66%, 57%, and 28%, respectively; n = 4 each). Importantly, the conjunctiva transported fluid from serosa to mucosa at a rate of 6.5 ± 0.7 µl · h-1 · cm-2 (range 2.1-15.3, n = 20). Fluid transport was stimulated by mucosal additions of 10 µM: 1) UTP, from 7.4 ± 2.3 to 10.7 ± 3.3 µl · h-1 · cm-2, n = 5; and 2) INS365, from 6.3 ± 1.0 to 9.8 ± 2.5 µl · h-1 · cm-2, n = 5. Fluid transport was abolished by 1 mM ouabain (n = 5) and was drastically inhibited by 300 µM quinidine (from 6.4 ± 1.2 to 3.6 ± 1.0 µl · h-1 · cm-2, n = 4). We conclude that this epithelium secretes fluid actively and that P2Y2 agonists stimulate both Cl- and fluid secretions.

dry eye; purinergic; adenosine 5'-triphosphate; uridine 5'-triphosphate; INS365; short-circuit current


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE SECRETION OF TEAR FLUID appears to have two main components: 1) baseline secretion, and 2) secretion after a stimulus. It has been proposed that the stimulated secretion arises from the main and accessory (palpebral) lacrimal glands (18), but other sources cannot be excluded. The nature and origin of the baseline secretion is less clear. Some accounts (18) attribute it to the accessory lacrimal glands of Krause and Wolfring, but others even doubt it exists as such (19). These unsolved questions lead naturally to the consideration of all conceivable routes of tear fluid secretion.

A possible source that had not been tested until now is the conjunctival epithelium. Some evidence has appeared to implicate it, since it has been reported (13) that, in the absence of or at low levels of stimulation, the secretion of the cannulated rabbit lacrimal gland is hypertonic (334 mosM) while the tears are isotonic. This led to the suggestion that fluid from the conjunctiva and cornea could dilute the lacrimal gland secretion (13). In keeping with this possibility, it was recently found that the rabbit conjunctival epithelium transports electrolytes (25, 26, 42, 44), expressing both secretory and absorptive electrolyte transport mechanisms (42). In addition, AQP3 water channels have been located to conjunctival epithelium (11). Water channels are conduits for transcellular and translayer water flow in the kidney collecting duct, and they are present in all fluid-transporting epithelia known. Fittingly, the conjunctival apical surface is highly permeable to water, with a transepithelial diffusional water permeability (1.3 µm/s) largely exceeding paracellular water permeability (10-2 µm/s) (5).

Given this background, we have investigated whether the rabbit conjunctival epithelium can transport fluid, and we have found that it does. The direction of fluid transport is from the stromal (basolateral) to the mucosal (apical) side, which is the same direction in which Cl- is secreted by this layer (25, 42). Furthermore, we found that fluid and Cl- transports are stimulated by UTP and by a novel purinergic receptor agonist, P1,P4-di(uridine 5'-)tetraphosphate, or Up4U (INS365), which are, respectively, natural and synthetic agonists of the P2Y2 receptor (34, 38).

While this paper was undergoing review, another paper appeared (45), reporting net fluid secretion across pigmented rabbit conjunctiva in the serosal-to-mucosal direction. Such secretion was increased by mucosal addition of 1 mM 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) and 10 µM UTP and was abolished by 0.5 mM ouabain, which is similar to the results we present. The two laboratories had previously reported some of these findings in abstract form (29, 43).


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Solutions. The physiological medium used to bathe the conjunctiva for short-circuit current (Isc) measurements was Tyrode solution (42), containing (in mM) 113 NaCl, 30 NaHCO3, 4 KCl, 0.8 NaH2PO4, 1.2 MgCl2, 1.8 calcium gluconate, and 5.6 D-glucose, pH 7.4-7.5 (maintained by gassing with air-5% CO2), osmolarity 280 mosM. For fluid transport experiments, since gassing was impractical, we used modified Tyrode solution by adding HEPES (20 mM) while leaving the rest of the components unchanged; pH remained the same but the osmolarity was 300 mosM.

Rabbit conjunctival preparation. New Zealand albino rabbits, each weighing 2.0-2.5 kg, were euthanized with pentobarbital sodium solution injected into the marginal ear vein. The eyes including eyelids were enucleated immediately along the superior and inferior orbit. Only freshly dissected preparations were used, and that entailed that only one of the eyes of a given rabbit was used for each experiment. The eyeball was placed on a semispherical Lucite holder, where it was secured in place by vacuum. The eyelids were then pulled up, which resulted in extending the conjunctiva, forming a cylinder. At that point, the eyelids were sutured to a circular holder held on a manipulator. The holder was adjusted to maintain the conjunctiva in cylindrical shape, with the epithelium covering the internal surface. The inside of the conjunctival cylinder was filled with prewarmed (37°C) Tyrode solution, and the subconjunctival tissues and extraocular muscles were trimmed off the outside. The conjunctival cylinder was then cut open from one of the canthi to the corneal limbus vertically, followed by cutting all along the limbus. At that point, the entire conjunctiva thus obtained, including the free bulbar and fornix sections plus the palpebral conjunctiva still attached to the eyelids, was placed into a dish with Tyrode solution at 37°C and gassed with air-5% CO2. With the help of a dissection microscope, the palpebral conjunctiva was dissected off the eyelids, which yielded the whole conjunctiva isolated in one piece.

Isc. These measurements were done with an Ussing system [World Precision Instruments (WPI), Sarasota, FL]. The isolated rabbit conjunctival epithelium preparation was clamped in a model CHM2 WPI chamber. The solutions were bubbled with air-5% CO2 and maintained at 37°C, using the thermally jacketed glass lifts of the system. The Isc across the tissue was measured using a four-electrode WPI DVC 1000 voltage-current clamp.

Fluid transport. Isolated rabbit conjunctival epithelial preparations were mounted in an insert made of two flat Lucite rings, one of them having a stainless steel mesh (Fig. 1) to support the tissue. The insert was clamped between two thermally jacketed fluid-filled chambers (37°C). The mucosal side (top) chamber was stoppered and was in contact with the outside through a narrow piece of tubing (to minimize evaporation); the serosal (bottom) chamber was closed except for the detector, also enclosed to minimize evaporation. The nanoinjector-driven volume-clamp instrument (see Fig. 1) has been described previously (10, 35). The rate of fluid traversing the preparation was determined by keeping constant the volume of the stromal side chamber. The microelectrode contact detector used is triggered by volume variations of 1-3 nl; to avoid its blockage, voltage to it was limited to ~100 mV. The nanoinjector voltage output was proportional to the volume injected or (withdrawn) by the syringe in each cycle; such output went to a pen-chart recorder and to a computer in oscillograph mode. To function, the method requires the tissue to be applied against its support by a pressure head. In addition, if that pressure head is <1.5 cmH2O, capillarity artifacts at the detector are possible. Because the tissue does not normally have a pressure difference across it, for practical considerations we chose a compromise value. The relative positions of chamber and detector were therefore such that the hydrostatic pressure difference (mucosa minus stroma) across the conjunctival preparation was 3.0 cmH2O.


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Fig. 1.   Scheme for conjunctival fluid transport measurements. A conjunctival preparation was placed on the insert, which in turn was clamped between two thermally jacketed fluid-filled hemichambers. For clarity, jackets are shown on left side only. The height of the chamber assembly can be adjusted to obtain a hydrostatic pressure difference (Delta P) of 3 cmH2O between the two sides of the tissue; bott, bottom.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isc. Unless specified, after mounting, the Isc was allowed to stabilize for 30 min, at which point the average Isc across the rabbit conjunctival epithelium was 14.9 ± 1.4 µA/cm2 (range 5-24 µA/cm2, n = 16). At that time, typically one of four different P2Y2 purinergic receptor agonists utilized was added to the apical side funnel. Each agonist at each given concentration was tested in an individual preparation and only once during a given experiment. After such addition, the Isc initially increased rapidly in all cases. Given that all experiments were done with the same protocol, the behavior of Isc can be expressed as a percent of its value immediately before the solution exchange. With 10 µM (final concentration) of UTP, INS365, INS306, or INS440, the respective increases above the control levels were 88%, 66%, 57%, and 28% (n = 4 in all cases, Fig. 2). The stimulated Isc gradually decreased to its control value except for INS365 and INS440, for which Isc remained 24% and 13% higher than the control level after 30 min, respectively. The effects of these four compounds were concentration dependent (Figs. 3 and 4). Maximal increases in Isc (Emax) for UTP, INS365, INS306, and INS440 at 10-3 M were 104 ± 19, 91 ± 2, 78 ± 14, and 58 ± 2% of the control level, respectively (Fig. 3). The EC50 values calculated from these data are also given in Fig. 4; they were 2.5 ± 1.4, 3.6 ± 1.1, 3.5 ± 2.1, and 9.1 ± 1.5 µM, respectively. Comparing the four compounds, UTP gave the highest change in Isc but could keep Isc stimulated for >30 min only at the highest concentration tested (10-3 M). INS365 not only stimulated the Isc to reach a similar level, but also could keep the Isc stimulated for a longer time at 10-3 and 10-5 M (Fig. 3B). INS440 had the least effect on Isc.


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Fig. 2.   Transconjunctival short-circuit current (Isc) and its stimulation by adding 4 different P2Y2 purinergic receptor agonists at a concentration of 10 µM to the apical side after Isc being stable for 30 min. Points represent means ± SE of 4 preparations. Here and elsewhere, as mentioned in MATERIALS AND METHODS, each preparation corresponds to a different eye and a different rabbit.



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Fig. 3.   Stimulation of Isc by adding 4 different P2Y2 purinergic receptor agonists at 4 different concentrations to the apical side of the rabbit conjunctiva after Isc was stable for 30 min. Points represent means ± SE of 4-5 preparations; n = 4.



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Fig. 4.   Effects of 4 different P2Y2 purinergic receptor agonists at 4 different concentrations on the peak change (%) of Isc. The curves shown were obtained with a dose-response nonlinear fitting algorithm contained in the program Origin (OriginLab, Northampton, MA). Emax, maximum Isc.

Fluid transport. Rabbit conjunctival preparations were mounted in the insert with their mucosal (apical) side facing the top chamber. We found that the conjunctival epithelial preparations transported fluid from the bottom to the top chamber (from stroma to mucosa) against a hydrostatic pressure difference of 3 cmH2O. We calculated average rates of fluid transport 30 min before and after adding agonists or inhibitors. The baseline rate of fluid transport observed was 6.5 ± 0.7 µl · h-1 · cm-2 (range 2.1-15.3, n = 20). Figure 5 shows a typical experiment in which the conjunctival preparation transports fluid spontaneously and continuously at an average rate of 8.6 µl · h-1 · cm-2 for a period of 90 min after mounting. We also calculated average rates of fluid transport every 15 min within this entire 90-min period (bar diagram, Fig. 5). As can be seen, fluid transport was reasonably stable except for the first 15 min after mounting. Because the current technique does not allow gassing of the serosal side, we only ran relatively short experiments, all of them in freshly isolated preparations. Because 10 µM UTP and INS365 had significant effects on Isc, we also explored their effects on fluid transport. With our procedure, to keep the volume of the bottom chamber constant, it meant that the bottom chamber was inaccessible during an experiment, and therefore test agents could only be added to the top chamber (mucosal side). We waited for the conjunctiva to transport fluid in stable fashion for about 0.5 h, and then we added UTP or INS365 (10 µM each, final concentration). Both analogs induced a significant increase in fluid transport that could last up to 60 min. Figure 6 shows representative experiments. Agonists induced changes in rates of fluid transport as follows: for UTP, from 7.4 ± 2.3 µl · h-1 · cm-2 (range 2.8-15.3) to 10.7 ± 3.3 µl · h-1 · cm-2 (range 3.1-18.4; n = 5), and for INS365 from 6.3 ± 1.0 µl · h-1 · cm-2 (range 3.2-8.4) to 9.8 ± 2.5 µl · h-1 · cm-2 (range 3.7-20.3; n = 5). Pairing the control and stimulated flow rates within each experiment yielded increases of 45.6 ± 29.5% with UTP and of 50.2 ± 16.2% with INS365.


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Fig. 5.   Typical fluid transport by an excised rabbit conjunctival preparation from its serosal to its mucosal side against a hydrostatic pressure difference (Delta P) of 3 cmH2O. Vertical deflections represent volume accumulated during 5-s recording intervals, as detailed at a magnified time scale in the inset. Bars denote rates of fluid transport averaged for 15-min periods.



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Fig. 6.   Addition of 10-5 M INS365 (A) and UTP (B) to the top chamber (mucosal side) stimulates rabbit conjunctival fluid transport in 2 representative experiments. After the fluid transport was stable for ~30 min, UTP or INS365 were added (each 10 µM final concentration). Both agonists induced a significant increase in fluid transport that could last up to 60 min. A and B, top: accumulation (integral) of the volume of fluid transported, and the slopes summarize the average rates of fluid transport before and after stimulation. A and B, bottom: reproduction of original chart recordings of fluid transport measurements. A and B, middle: bar diagrams denote rates averaged for 15-min periods; fl. tr., fluid transport.

Furthermore, we characterized the effects of inhibitors of electrolyte transporters and channels on transconjunctival fluid movements. We added ouabain or quinidine to the apical side after fluid secretion was stable for ~0.5 h. Fluid transport was abolished by 1 mM ouabain after 30 min of exposure (n = 5) and was drastically inhibited by 300 µM quinidine [from 6.4 ± 1.2 µl · h-1 · cm-2 (range 3.0-8.6) to 3.6 ± 1.0 µl · h-1 · cm-2 (range 1.7-6.4)] during the first 30 min after exposure (n = 4). Figure 7 shows representative experiments for the effects of ouabain and quinidine.


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Fig. 7.   Addition of 1 mM ouabain (A) and 300 µM quinidine (B) to the top chamber (mucosal side) inhibits rabbit conjunctival fluid transport in 2 representative experiments. After fluid transport was stable for ~30 min, ouabain or quinidine were added at the times denoted by the arrows at the final concentrations shown.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Isc. From prior Isc measurements, active Cl- secretion at the apical surface of the freshly excised rabbit conjunctiva accounted for 75% (25) and 60% (42) of the Isc, respectively. The Isc values determined in our study are in agreement with the values found previously (25, 42, 44).

As we expected, the mucosal application of UTP increased Isc in a dose-dependent fashion, confirming that UTP stimulates Cl- secretion (26). Our results also show that the other three P2Y2 purinergic receptor agonists we used (INS365, INS306, and INS440) could also stimulate Isc in a concentration-dependent manner. As mentioned in the results, they induced maximal stimulation of Isc rapidly after application, and their effects decreased gradually within 30 min. UTP (10 µM) simulated Isc more than INS365, but the effects of the latter remained much longer.

UTP and ATP have previously been demonstrated to be potent mediators of goblet cell mucin secretion and Cl- secretion in airway epithelia (22, 24, 27, 31), as well as in the conjunctiva (20, 26). They act via the P2Y2 nucleotide receptors in the epithelial cells of these tissues and regulate ion transport and secretory functions. The physiological importance of the P2Y2 receptor, a G protein-coupled receptor, has been well established. Signal transduction by P2 receptors has been thoroughly reviewed (1, 16, 39). Studies have indicated that ATP, acting on P2 receptors in the cell membrane, plays a pivotal role in various tissues as an extracellular transmitter (4, 23); ATP is known to affect intracellular Ca2+ concentration in various tissues, including ocular tissues such as corneal epithelium (23), endothelium (7, 46), and lacrimal acinar cells (15). The P2Y2 receptor found on the apical surface of airway epithelia is believed to be the major coordinator of mucociliary clearance mechanisms in the lung. P2Y2 agonists (UTP, ATP) lead to increases in mucin release (22, 27), protein secretion (32), Cl- and ion transport (24, 31), water transport into the airway surface (2, 17), and surfactant release (14). Their role in regulation of mucosal hydration and defense may provide clinical benefit for lung diseases such as chronic bronchitis in cystic fibrosis. It also appears that a whole class of uridine nucleotide-responsive receptors exists in a broad range of tissues, since different receptors that are activated by uridine nucleotides have been identified (36). This poses the possibility of selective drug development for each of these receptors.

Fluid transport. The main finding of this paper is that the conjunctival epithelium transports fluid from its blood side outward into the tear side. Fluid transport takes place against a pressure head of 3 cmH2O, is abolished by 1 mM ouabain (a Na+ pump inhibitor), and is drastically inhibited by 300 µM quinidine (a K+ channel blocker), all of which suggest that the fluid movements observed arise from active transport.

The average Isc we report (14.5 µA/cm2) corresponds to a monovalent ion flux of solute flux (Js) = Isc/zF = 0.54 µeq · h-1 · cm-2 (where F = Faraday's constant and z is signed charge), which in turn would correspond to an isotonic fluid flow of Jv = Js/Ciso = 3.6 µl · h-1 · cm-2 (Ciso = 150 mM where Ciso is the concentration of an isotonic solution). This is less than the average rate of fluid transport we determined (6.5 µl · h-1 · cm-2). However, the ranges for Isc and Jv measurements are relatively large (5-29 µA/cm2 and 2.1-15.3 µl · h-1 · cm-2). Therefore, it is unclear whether the relative current deficit is meaningful or is simply determined by the large relative deviations. As mentioned above, the average Isc values determined by us are consistent with values earlier determined by other laboratories (25, 42, 44), although Kompella et al. (25) report a suggestively wide range (5-44 µA/cm2). It may be that these preparations are especially susceptible to edge damage; however, to determine that will require further work.

One question the findings pose is whether such secretion can account for part or all of the basal rate of tear fluid secretion. It would appear that a large conjunctival contribution is entirely possible; the total area of rabbit conjunctiva is ~7.2 cm2, and, at the average fluid transport rate we determined (6.6 µl · h-1 · cm-2), the total addition to tear fluid of conjunctival origin would amount to 0.79 µl/min or 47.5 µl/h. For comparison, the physiological basal (or minimal) flow rate for rabbit conjunctival tear fluid is reportedly (3) 0.72 µl/min or 43.2 µl/h. All other prior reports summarized in Fig. 8A (3, 6, 8, 9, 13, 19, 33, 37, 40, 41) are also consistent with the possibility that the basal tear secretion may arise in good part (if not mostly) from the conjunctiva. Some supportive indications can be obtained from the data in Fig. 8B. The basal secretion observed in the absence of the main lacrimal gland of squirrel monkeys (~0.4 µl/min in our estimate, Fig. 8B) might correspond to secretion by accessory glands, as interpreted before (30), but might also include a component of basal conjunctival secretion (~0.6 µl/min in the rabbit; cf. Fig. 8A).


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Fig. 8.   A: comparison of our present data with those in reports of tear and lacrimal gland basal rates of secretion. B: secretion of tears in eyes with and without lacimal glands.

We also report that, importantly, the rabbit conjunctival secretion is subject to purinergic stimulus (Figs. 6 and 8A and see discussion below). Whether the conjunctival secretion is also subject to neural stimulus (as is tear secretion, Fig. 8B) remains to be determined. We find that fluid transport across the rabbit conjunctiva is stimulated by mucosal additions of the P2Y2 receptor agonists UTP and INS365. Figure 6 depicts experiments in which the stimulation caused by these agonists at 10-5 M is clearly visible. However, on average, the extent of stimulation was somewhat less than shown there, and amounted to ~50% (see RESULTS and Fig. 8A). A similar trend is visible in the stimulation of Isc by 10-5 M UTP and INS365 (Fig. 2): integrating the area under the respective curves yields stimulations of 26% and 35%, respectively. Within experimental error, the secretion of fluid is stimulated about the same as Isc; whether electrolyte movements other than apical Cl- secretion are involved in fluid transport remains to be explored.

These results might conceivably account for a role of conjunctival fluid in hydrating the surface of the cornea. In this connection, it has been recently reported that topical instillation of INS365 in vivo consistently increased tear secretion compared with untreated or saline-treated rabbit eyes (47). The increase in tear secretion was statistically significant at 5-15 min after the application, and, consistent with the in vitro results reported here, an 8.5% solution of INS365 caused an approximately twofold increase in tear secretion. It is known that P2Y2 receptors play an important role in regulation of conjunctival mucin secretion. In addition, UTP and ATP induce an increase in mucin release on topical application on the conjunctiva (20), so activation by UTP or its agonists is believed to trigger the coordinated release of mucin as well as fluid by the conjunctiva.

Given these and prior observations, under some conditions, the conjunctival epithelium might serve as a conduit for osmotic flow and contribute to dilute tear gland secretion. In addition, since we have observed that the conjunctival secretion can be modulated, it is also possible that the conjunctival epithelium might serve as part of a mechanism to sense and correct the osmolarity of the fluid bathing it. As we mentioned above, both ATP and UTP are not only highly effective Cl- secretagogues when applied to the apical surface of the airway epithelia but also can induce fluid secretion across airway epithelia (2, 17). A similar role of P2Y2 receptor mediated by UTP and ATP has been explored in the conjunctiva. The results indicate that the rabbit and human conjunctival cells contain functional P2Y2 nucleotide receptors (20), which have been confirmed in human conjunctiva by RT-PCR techniques (21).

A common, vexing condition in ocular pathology known as "dry eye syndrome" is produced by abnormalities in precorneal tear film components (aqueous, mucin, or lipid), which may lead to loss of tear film stability (12). The resulting dry spots on the corneal and conjunctival epithelia have adverse repercussions on the ocular epithelial surface cells. The results from this and other laboratories discussed above indicate that, by stimulating secretions of fluid and mucin, P2Y2 agonists may prove useful to treat dry eye disease.


    ACKNOWLEDGEMENTS

This work was supported by Inspire Pharmaceuticals (Grant CU512848) and in part by Research to Prevent Blindness.


    FOOTNOTES

Address for reprint requests and other correspondence: J. Fischbarg, Dept. of Physiology, College of Physicians and Surgeons, Columbia Univ., 630 West 168th St., New York, NY 10032 (E-mail: jf20{at}columbia.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 28 March 2000; accepted in final form 27 March 2001.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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