P2Y(2) receptor elicits PAS-positive glycoprotein secretion from rabbit conjunctival goblet cells in vivo.

We investigated in vivo whether UTP and ATP increased periodic acid and Schiff's reagent (PAS)-positive glycoprotein release from rabbit conjunctival goblet cells. Fifty microL of UTP or ATP at the concentrations of 0.003, 0.03, 0.3, 3.0, 8.5% (54 microM-154 mM) or saline were applied to rabbit eyes. Impression cytology was performed on the upper nasal bulbar conjunctiva and the cells were stained with PAS. To clarify purinergic receptor-mediated involvement in this response, suramin (1%; 7 mM), P2Y(2) antagonist and pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid (PPADS, 0.01%; 167 microM), P2Y(1) antagonist were applied in the rabbit conjunctival sac for 10 min. before UTP or ATP application. Images of the specimens were taken with a digital camera mounted on a microscope and the PAS staining area was measured using an image analyzing system. UTP or ATP eye drop instillation transiently decreased the PAS staining area in a dose-dependent manner, but it gradually recovered after another 30 min. Saline instillation had no effect until 60 min. later. All of the agonists-induced declines were inhibited by pretreatment with 1% (7 mM) suramin but not 0.01% (167 microM) PPADS. UTP and ATP stimulate PAS-positive glycoprotein secretion via P2Y(2) receptor on goblet cells in the rabbit bulbar conjunctiva in vivo.     

INS365 suppresses loss of corneal epithelial integrity by secretion of mucin-like glycoprotein in a rabbit short-term dry eye model.

P2Y2 receptor agonists, like UTP and ATP, stimulate mucin secretion from goblet cells in vitro. Therefore, mucin stimulants could be good candidates for the treatment of dry eye syndrome because mucin increases the tear film stability and protects against desiccation of ocular surface. INS365 is a more stable P2Y2 receptor agonist than UTP. In the present study, we evaluated, in normal rabbit eyes, its effectiveness to release mucin from goblet cells and to protect the corneal damage induced by desiccation. For mucin secretion, impression cytology was performed following the instillation of INS365 solution or saline into the conjunctival sac. The specimens were stained with periodic acid and Schiff (PAS) reagent, and then the staining area was calculated using computer software. INS365 dose-dependently decreased the PAS staining area of conjunctival goblet cells from 2 to 15 min post-application. Furthermore, we utilized the rabbit short-term dry eye model to evaluate if INS365 eyedrops could protect against any of the damage produced by blockage of blinking with ocular speculum. INS365 significantly suppressed corneal damage at concentrations of more than 0.1% w/v. These results suggest that this P2Y2 agonist is a good candidate for the treatment of dry eye disease.     

P2 purinergic receptor-coupled signaling in the rabbit ciliary body epithelium

PURPOSE: To identify and characterize P2 purinergic receptors and their signaling pathways in the epithelial cells of the rabbit ciliary body. METHODS: Real-time fluorescence ratio imaging of the intact fura-2-loaded nonpigmented ciliary body epithelial (NPE) cells of rabbit were used to record changes in the intracellular free calcium concentration ([Ca(2+)](i)), in response to a number of purinergic agonists and antagonists. The effects of some of these drugs on the inositol phosphate (IP) levels in ciliary processes were also examined. RESULTS: Adenosine diphosphate (ADP), adenosine triphosphate (ATP), and uridine triphosphate (UTP) dose dependently increased the [Ca(2+)](i) and IP levels. The [Ca(2+)](i) increases induced by ADP and UTP were distinguishable, both kinetically and pharmacologically. The effect of ADP on [Ca(2+)](i) was mimicked by a number of P2Y(1)-selective agonists, and was blocked by three P2Y(1)-receptor-specific antagonists. The [Ca(2+)](i) increases elicited by ADP (or its analogs) and UTP were additive. CONCLUSIONS: Rabbit ciliary body epithelium possesses both P2Y(1) and P2Y(2) metabotropic purinergic receptor subtypes, which differentially use the IP(3)/Ca(2+) second-messenger pathway.     

UTP and diadenosine tetraphosphate accelerate wound healing in the rabbit cornea

Nucleotides are naturally occurring substances present in tear film that can stimulate tear secretion in animals and humans. We investigated whether certain nucleotides can affect the rate of wound healing in the cornea of white rabbits. In the absence of any added compound, the rate of healing was 72.4 +/- 2.2 microm h(-1). Of all the tested nucleotides, UTP and Ap(4)A were the most active ones, maximally increasing the rate of healing to 121.6 +/- 3.7 and 93.7 +/- 3.2 microm h(-1), respectively. Responses to UTP were dose dependent. UTP had a pD(2) value of 8.9 +/- 0.1 (EC(50): 1.25 nM). P2 purinoceptor antagonists such as suramin and reactive blue-2, inhibited the effect of UTP indicating the involvement of P2Y receptors. Mitogen-activated protein kinase (MAPK) cascade inhibitors also abolished the effects of UTP, suggesting that P2Y receptors are coupled to the MAPK cascade, and that this is involved in controlling the rate of epithelial cell migration.     

P2Y2受体激动剂用于干眼治疗的研究进展

干眼属常见的眼表疾病,是指由于泪液的量和（或）质的异常引起的泪膜不稳定和眼表面的损害,从而导致眼部不适症状的一类疾病。目前我国干眼的治疗包括病因治疗、人工泪液替代治疗、抗炎治疗、免疫抑制剂治疗、手术治疗以及中医治疗等。Ｐ２Ｙ２受体激动剂是一种治疗干眼的新型药物,它能够通过激动位于眼表的Ｐ２Ｙ２受体,促使结膜上皮细胞泪液的分泌以及结膜杯状细胞对黏蛋白的分泌,从而增强泪膜稳定性,改善干眼症状。３０ｇ·Ｌ－１地夸磷索四钠滴眼液是目前唯一上市的Ｐ２Ｙ２受体激动剂。本文就近年来地夸磷索四钠用于干眼治疗的临床疗效、安全性以及相关研究进展作一综述。     

Fibrinolysis in cornea and conjunctiva: Evidence of two types of activators

The nature of corneal and conjunctival plasminogen activators (PAs) from human and rabbit eyes was examined in tissue culture. The fibrinolytic activity of culture fluid from human corneas was low, roughly one-eighth of that of rabbits. The main activity was found to be of the urokinase (UK) type, as demonstrated by crossed immunoelectrophoresis against alpha₂-antiplasmin after incubation with mixed plasma. The fibrinolytic activity of culture fluid from human conjunctival tissue was higher and a mixed secretion of tissue type (tPA) and UK activator was demonstrated. These activators were separated by affinity chromatography against Sepharose-immobilized antibodies against tPA. Fibrinolytic activity in tears was quenched by antibodies against tPA but not by the use of anti-UK antibodies. Thus, in man, tear fibrinolytic activity may depend primarily on a release of PAs from conjunctival tissue.     

去势雄兔泪液分泌及泪膜稳定性的改变

目的观察去势后雄兔眼表及泪膜的变化,评价雄激素对泪液分泌及泪膜稳定性的影响.方法将16只雄兔随机分为去势(实验)及对照两组,每组8只.实验组分别于去势前和去势后1、2、3、4周、2及3个月行Shirmer试验、泪膜破裂时间(break-up time, BUT)及虎红染色检测;利用化学发光法检测实验组手术前、后血清睾酮水平,并行统计学分析处理.3个月后处死两组兔,取其泪腺、Harder腺、结膜、角膜及角膜缘组织行病理组织学观察.结果实验组Shirmer试验、泪膜破裂时间明显低于正常对照组,且随观察时间的延长差异有显著性(t=9.032,P＜0.01;t=4.747,P＜0.01).虎红染色实验组为阳性,对照组为阴性.实验组术后血清睾酮水平(4.52±0.81) nmol/L明显低于术前(19.76±1.53) nmol/L(t=22.290,P＜0.01).病理学观察,实验兔泪腺上皮细胞胞浆萎缩扁平,腺腔扩大,腺泡泡状黏液消失,PAS染色阳性物质减少,结膜杯状细胞数量减少,角膜上皮及角膜缘干细胞形态学上无明显改变.结论去势后雄兔睾酮水平明显低下,泪腺上皮细胞萎缩,腺泡泡状黏液物质消失及结膜杯状细胞减少,导致泪液分泌的质和量改变及泪膜稳定性降低.     

IgG and IgA antibody in tears of rabbits immunized by topical application of ovalbumin

Rabbits were immunized by daily topical conjunctival application of ovalbumin. Control rabbits received ovalbumin intravitreally or intravenously. The direct plaque assay was used to detect IgM antibody-producing cells in lymph node, conjunctival, lacrimal gland and Peyer's patch tissues. A culture ELISA test was used to detect IgG and IgA antibody-producing cells. Serum and tears were tested for IgG antibody by ELISA and passive hemagglutination tests, and for IgA antibody by an ELISA assay. The conjunctival and cervical lymph node cells of topically immunized rabbits produced IgG and IgM antibodies. Neither IgG nor IgM antibody-producing cells were found in the Peyer's patches or lacrimal glands. Serum IgG antibody was detected by both ELISA and HA tests. IgG antibody was found (by the ELISA assay) in tears collected from most topically immunized rabbits, and from some intravitreally immunized rabbits, but not from the intravenously immunized animals. IgA antibody was found in tear samples collected after several weeks of daily topical immunization. IgA antibody-producing cells were detected in the conjunctival tissues, but not in the lymph nodes of those rabbits.     

Novel noninvasive sensitive determination of tear volume changes in normal cats

We describe a novel, high-resolution and noninvasive method for measuring tear volume changes in cats. The method entails photographing at the lid margin the tear meniscus area defined by instillation of 0.1% fluorescein solution into the cul-de-sac. The inferior tear meniscus area was obtained from the digitized images with computer-assisted software. The tear meniscus area increased in proportion to the saline volume applied into the conjunctival sac, which validates the technique. Furthermore, this technique detected with high sensitivity previously described increases in tear fluid secretion induced by the P2Y(2) agonist. We demonstrate in cats that changes in conjunctival sac tear volume can be evaluated by measurement of its inferior tear meniscus area.     

Effect of nucleotide P2Y2 receptor agonists on outward active transport of fluorescein across normal blood-retina barrier in rabbit

BACKGROUND: To evaluate the effect of nucleotide P2Y(2) receptor agonists INS542 and uridine 5'-triphosphate (UTP) on the outward active transport of fluorescein across rabbit blood-retina barrier (BRB) in vivo. METHODS: Injection (0.1 ml) of INS542 (0.1 or 1mM), phosphate buffered solution, or UTP (1 or 10mM) was made in Dutch-belted rabbits. Differential vitreous fluorophotometry (DVF) was performed 3hr later and the fluorescein (F)/fluorescein monoglucuronide (FG) ratio was then calculated. F/FG ratios are inversely proportional to outward active transport of F across BRB at the level of the retinal pigment epithelium (RPE). In another set of experiments, the effect of 0.1 ml vitreous injection of INS542 (1mM) on F/FG ratios was evaluated at different time points ranging from 0.5 to 48hr before conducting DVF. RESULTS: F/FG ratios obtained 3hr after intravitreal injection were as follows (mean+/-standard error): 0.49+/-0.14 (0.1mM INS542), 0.19+/-0.04 (1mM INS542), 0.48+/-0.09 (PBS), 0.40+/-0.08 (1mM UTP) and 0.36+/-0.05 (10mM UTP). The F/FG ratio for 1mM INS542 was significantly lower than in the other groups (P<0.05). In the time course experiments, a significant decrease in the F/FG ratios was observed between 1 and 12hr following administration of INS542 when compared with F/FG ratios obtained in the contralateral (untreated) eye. CONCLUSION: Intravitreal administration of INS542 (but not UTP) enhances outward active transport of F across RPE in intact rabbit eye, indicating that activation of P2Y(2) receptors in vivo directly stimulates RPE active transport.     

Quantitation of rat lacrimal secretion: a novel sandwich ELISA with high sensitivity

Modulation of lacrimal acinar cell tear secretion may involve multiple factors acting both in subtle and pronounced ways. Functional screens of recombinant protein products arising from gene array technologies, or protein fractions derived from lacrimal conditioned media or extracellular matrix, will require a highly sensitive assay capable of monitoring tear protein secretion by small replicate cultures. To improve significantly on current methods, a rat- and mouse-specific sandwich ELISA was developed. For this purpose, chickens and rabbits were immunized with serum-free secretion media from carbachol and VIP-stimulated rat lacrimal acinar cell cultures. Immune sera were characterized by ELISA, Western blotting and immunohistochemistry, and subsequently optimized for use in a sandwich ELISA. Both antisera detected a wide range of different rat tear proteins, and immunostained only the secretory granule-rich juxtalumenal region in sections of rat lacrimal gland. Chicken, but not rabbit, antiserum cross-reacted with rabbit and human tears. In sandwich ELISA, capture with purified chicken immunoglobulin fraction and detection with rabbit antiserum detected as little as 1 ng ml-1 tear protein in 10,000-fold diluted rat secretion medium--a level of sensitivity 8000 times greater than the rat tear peroxidase assay. Such specificity and sensitivity greatly reduce the quantity of media needed for assay, and makes feasible functional screens for scarce factors that may influence lacrimal secretory processes, and in turn possibly play a role in human lacrimal insufficiency syndromes.     

Distinct P2Y receptor subtypes regulate calcium signaling in human retinal pigment epithelial cells

PURPOSE: Nucleotide signaling plays a role in retinal pigment epithelial (RPE) function, and receptors for nucleotides are potential therapeutic targets for various ocular diseases. The purpose of this study was to investigate the expression of P2Y receptor subtypes in native and cultured human RPE cells. METHODS: Intracellular Ca(2+) levels were monitored using real-time fluorescence imaging in cultured human RPE cells loaded with Fura-2. Expression of P2Y receptors in native and cultured RPE cells was determined by quantitative RT-PCR and Western blot analysis. RESULTS: Adenosine triphosphate (ATP), uridine triphosphate (UTP), adenosine diphosphate (ADP), 2-methylthio ATP (2MeSATP), and uridine diphosphate (UDP) produced concentration-related increases in [Ca(2+)](i) in cultured RPE cells. However, differences between the magnitude and shape of agonist responses were observed. ATP and UTP showed similar response characteristics, including a distinct Ca(2+) influx component. ATP and UTP were equipotent (EC(50), 6 muM) and maximum responses were equivalent, suggesting activation of a P2Y(2) receptor. Maximal responses to ADP and 2MeSATP were equivalent with EC(50)s of 1 muM and 0.3 muM. The P2Y(1) antagonist MRS 2179 (10 muM) inhibited these responses, confirming functional expression of P2Y(1) receptors. The presence of a response to UDP suggested P2Y(6) expression. There was no influx component to P2Y(1)- and P2Y(6)-mediated responses. mRNA for P2Y(1), P2Y(2,) P2Y(4), and P2Y(6) receptor subtypes was found in cultured RPE cells, and for P2Y(1), P2Y(2,) P2Y(4,) P2Y(6), and P2Y(12) it was found in native RPE cells. Expression of P2Y(1), P2Y(2), and P2Y(6) protein was found in native and cultured RPE cells. CONCLUSIONS: These data define the expression profile of P2Y receptors in human RPE and show that different P2Y subtypes control distinct calcium responses in these cells.     

P2Y2受体激动剂治疗干眼的研究进展

干眼是一类累及眼表和泪膜的多因素疾病.临床上治疗干眼的传统方法主要是被动性增加泪液,如人工泪液点眼、泪小点栓塞等,但新的关于干眼发病机制的研究表明,理想的干眼治疗方法是促进泪腺分泌泪液.研究已经证实,P2Y2受体激动剂具有促进水分及黏蛋白分泌的作用,可以促进泪腺主动分泌泪液.目前研制了多种人工合成的P2Y2受体激动剂并用于干眼的治疗,临床研究证实,P2Y2受体激动剂能够明显增加水性泪液的分泌和黏蛋白的分泌,改善临床症状,且Ⅰ期临床试验证实其具有较好的安全性.就近年来P2Y2受体激动剂在干眼治疗方面的作用机制、临床疗效和研究进展进行综述.     

Effects of a stable analogue of PGE2 (11-deoxy-13,14-didehydro-16 (S)-methylester methyl PGE2: FCE 20700) on the secretory processes of conjunctival goblet cells of rabbit

The effects of the instillation of a PGE2-analogue (11-deoxy-13,14-didehydro-16 (S)-methyl PGE2 methylester: FCE 20700) in the conjunctival sac of the rabbit were studied by means of two methods. The former is a clinical study (Dohlman test), the latter is a morphological investigation (semithin sections) on specimens of the conjunctival mucosa. From both methods it was possible to demonstrate that the FCE 20700 instillation enhanced the mucous lacrimal secretion in rabbits.     

兔角膜上皮的CFTR分泌通路与细胞内钙信号释放的关联研究

背景 研究表明角膜上皮中的囊性纤维跨膜电导调节因子(CFTR)是重要的阴离子和水分泌通道,CFTR激动剂可促进泪液分泌,对干眼的治疗具有一定的意义.曾有研究认为CFTR通路是cAMP-PKA依赖通路而没有钙信号参与.然而,升高cAMP并不能促进角膜上皮CFTR通路的分泌,钙信号在角膜上皮CFTR分泌通道中是否发挥作用尚不清楚. 目的 从生理学角度探讨CFTR在兔角膜上皮分泌功能的实现与钙信号之间的关系. 方法 采用计算机随机数字分配法将16只健康新西兰白兔随机分为2个组,每组各8只.动物全身麻醉下取双眼角膜,然后处死.角膜上皮面朝向正向电流方向置于尤氏小室,奇数组兔右眼角膜仅给予单纯ATP刺激(ATP刺激组),左眼角膜用CFTR特异性抑制剂CFTRinh-172预处理后给予ATP刺激(CFTRinh-172预处理组);偶数组兔右眼角膜用于原代角膜上皮细胞培养并采用激光扫描共焦显微镜进行单个细胞胞质内钙离子荧光强度测定,左眼角膜组织用细胞内钙离子螯合剂BMPTA/AM预处理后给予ATP刺激(BMPTA/AM预处理组),采用短路电流装置记录各组兔角膜上皮的短路电流变化. 结果 ATP刺激组、CFTRinh-172预处理组和BMPTA/AM预处理组角膜上皮电流值分别为(5.73±1.36)、(1.30±0.95)和(2.47±0.55) μA/cm2,CFTRinh-172预处理组和BMPTA/AM预处理组角膜上皮电流值均低于单纯ATP刺激组,差异均有统计学意义(t=11.201、5.508,均P＜0.001).单细胞的细胞内钙离子检测发现,ATP刺激后细胞内钙离子荧光强度迅速升高至ATP刺激前的3.25倍.结论 ATP可引起兔角膜上皮细胞分泌增加,CFTRinh-172抑制整个CFTR通路中ATP促发的角膜短路电流,而BMPTA/AM消除了细胞内游离的钙离子,提示兔角膜上皮细胞分泌的CFTR通道功能的实现与细胞内钙信号释放有关.     

Denervation of rabbit lacrimal gland increases levels of transferrin and unidentified tear proteins of 44 and 36 kDa.

PURPOSE: The main lacrimal gland secretes proteins and fluid that make up the aqueous component of the tears. Previous reports indicate that the parasympathetic innervation of the gland influences the secretion of protein from the lacrimal gland. We investigated the effect of lacrimal nerve transection on the levels of individual proteins and overall protein concentration in the tear fluid. METHODS: The main lacrimal gland was unilaterally denervated in adult rabbits at the site of nerve entry to the gland. The contralateral gland (sham-operated) had identical surgical manipulations, excluding nerve transection. Tears were collected daily from both eyes for up to 9 days, after which lacrimal glands were collected. SDS-PAGE, densitometric and image analysis, and Western blot were performed. RESULTS: Consistently measurable tear protein bands ranged from 6 kDa to 85 kDa, using densitometric analysis. Lacrimal gland denervation produced a sustained increase in proteins of 85, 44, and 36 kDa in tears and lacrimal gland tissue from the denervated side, compared with the sham-operated side (0.025 > p > 0.001). The band at 85 kDa was identified as transferrin by Western blot. Tears from the denervated glands also showed transient decreases in low molecular weight tear proteins (18, 12/10, and 6 kDa), as well as a decrease in overall protein concentration, compared with tears from sham-operated glands and non-operated glands (p < 0.001). CONCLUSIONS: These results demonstrate that, in rabbit tears, the quantities of transferrin and two unidentified tear proteins, as well as overall protein concentration, are influenced by the sensory and/or autonomic innervation to the lacrimal gland. The decrease in overall tear protein concentration after lacrimal gland denervation may be related to a loss of nerve-regulated secretagogue-induced protein secretion.     

Extracellular ATP opposes thrombin-induced myosin light chain phosphorylation and loss of barrier integrity in corneal endothelial cells

Increased contractility of the actin cytoskeleton by phosphorylation of the regulatory myosin light chain (MLC) results in a loss of barrier integrity in corneal endothelial cells. This study has investigated the effect of extracellular ATP, which may influence both Ca2+ and cAMP signalling, on MLC phosphorylation and barrier integrity in cultured bovine corneal endothelial cells (BCEC) known to express A2B and P2Y purinergic receptors, and ecto-nucleotidases. Extracellular ATP (100 microM) promoted MLC dephosphorylation (pMLC=61.8% at 18 min; n=9). Pre-exposure to ARL-67156, an ecto-nucleotidase inhibitor, prevented ATP-induced dephosphorylation. Other P2Y agonists, UTP and ATPgammaS, also induced MLC dephosphorylation but to a lesser degree compared to ATP. Thrombin (2 U/ml), which activate Rho kinase through PAR-1 receptors in the endothelium, induced MLC phosphorylation (pMLC=129.2%; n=14). This phosphorylation was completely abolished by concomitant exposure to ATP. When cells were pretreated with adenosine (100 microM; A2B agonist) or forskolin (10 microM), thrombin-induced phosphorylation was suppressed. ATP also led to a significant increase in cAMP (> 3-fold compared to 10 microM adenosine). Thrombin-induced increase in trans-endothelial flux of horseradish peroxidase (44 kDa) and disruption of the cortical actin were suppressed by ATP. These findings indicate that in BCEC (1) ATP induces elevated cAMP through its metabolite adenosine leading to MLC dephosphorylation, (2) Stimulation of P2Y2 receptors also leads to activation of MLCP since UTP- and ATPgammaS caused MLC dephosphorylation, and (3) ATP is antagonistic to thrombin since the latter inhibits MLCP through increased activity of Rho kinase. These findings further emphasize the role of contractility of the actin cytoskeleton in regulating the barrier integrity of corneal endothelium.     

A rabbit dry eye model induced by topical medication of a preservative benzalkonium chloride

PURPOSE: To establish a rabbit dry eye model with topical medication of the ocular preparation preservative benzalkonium chloride (BAC). METHODS: Sixteen white rabbits were used. One eye of each rabbit was chosen randomly for topical administration of 0.1% BAC twice daily for 14 days. The other untreated eyes served as controls. Schirmer test, fluorescein, and rose bengal staining were performed before and after BAC treatment on days 3, 5, 7, and 14. Conjunctiva impression cytology specimens were collected on days 0, 7, and 14. The rabbits were killed after day 14. Immunofluorescence staining was performed to detect mucin-5 subtype AC (MUC5AC) on conjunctival cryosections. Cornea and conjunctiva structures were evaluated by light and electron microscopy. RESULTS: Compared with untreated controls, BAC-treated eyes showed significant decreases in Schirmer scores (P = 0.01) and increases in fluorescein scores (P < 0.001) on days 5, 7, and 14. A significant increase in rose bengal scores was noticed as early as day 3 (P = 0.001). Decreases in goblet cell density occurred on days 7 and 14 (P = 0.001). Decreased MUC5AC and histopathologic and ultrastructural disorders of the cornea and conjunctiva were also observed in the BAC group. CONCLUSIONS: These findings demonstrated that an ophthalmic preservative, benzalkonium chloride, induced a dry eye syndrome in rabbits with damage to the cornea and conjunctiva, decreased aqueous tear basal secretion, goblet cell loss, and MUC5AC deficiency. This rabbit model was consistent with human dry eye syndrome in both aqueous tear and mucin deficiency and may be appropriate for studying dry eye syndrome.     

Toxizität neuer Benetzungs- und Konservierungsmittel in vitro

PURPOSE: The use of preservatives such as benzalkonium chloride (BAC) usually increases the toxicity of pharmaceutical tear substitutes. HP-guar has been recently introduced as a new artificial tear substitute and includes the preservative Polyquad (0.001%), which is considered to be non-toxic. We therefore examined the effect of preserved (cetrimide 0.01%) and unpreserved HPMC (hydroxypropylmethyl cellulose) and HP-guar in dose and time-response experiments in a human corneal and conjunctival epithelial cell culture model. METHODS: Immortalized human conjunctival and corneal epithelial cells were cultured in 96-well plates at 37 degrees C with 5% CO(2) and exposed to the test solutions. The ATP content was quantified by means of a luminescence-based ATP assay, intracellular esterase activity by double fluorescent viability staining (calcein AM/ethidium homodimer D-1) and cell migration by a colony dispersion assay. All experiments were performed in triplicate and repeated at least once. The significance of differences was determined with an unpaired two-sided t-test. RESULTS: HPMC with preservative severely reduced the ATP content at all concentrations tested. Unpreserved HPMC, however, showed an inhibition of ATP production only at 100% and good esterase activity. HP-guar with and without preservative were found to reduce ATP activity more than unpreserved HPMC, but the unpreserved solution was found to reduce cellular ATP levels significantly more than the preserved solution. CONCLUSIONS: The new preservative Polyquad induced significantly less cytotoxicity than cetrimide. However, even unpreserved HP-guar can induce cytotoxicity in vitro, while unpreserved HPMC remains a good alternative tear substitute with low cytotoxicity.