tear lysozyme is a high molecular weight , long chain glycolytic enzyme secreted by the lachrymal gland . among the tear proteins
identified , lysozyme constitutes around 20%40% of the total tear protein ( farris 1985 ) and its concentration in the tear film is higher than in any other fluid of the body ( fleming 1922 ) .
this protein has the capacity to dissolve gram - negative bacteria walls by the enzymatic digestion of mucopolysaccharides ( milder 1987 ) . due to this bactericidal action
, lysozyme has been considered as one of the essential elements of the protective tear film barrier against ocular infection ( mackie and seal 1976 ) .
the importance of this lachrymal component has contributed to develop methods for its detection and measurement , as well as to correlate its concentration with ocular pathologies .
several studies have shown a decrease in the concentration of lysozyme in patients with keratoconjunctivitis sicca ( van bijsterveld 1969 ; mackie 1984 ; montero 1990 ) , suggesting that a drop off in tear lysozyme levels may constitute an important parameter to detect a malfunctioning lachrymal gland ( klaeger 1999 ) .
recently , the presence of a new family of compounds in the tear film have been described : the diadenosine polyphosphates ( pintor , carracedo et al 2002 ) . these naturally occurring dinucleotide compounds exhibit both intracellular and extracellular physiological actions , these including vasoactive properties , neuromodulatory regulation of neurotransmitter release or intracellular modulation of ion channels ( mclennan 2000 ; hoyle 2002 ) . formed by two adenosine molecules joined by a variable phosphate chain , they are abbreviated as apna ( n = 27 , where n describes the number of phosphates ) .
the activity of these nucleotides on ocular tissues is being investigated , and it is known that they act through p2 receptors to modulate intraocular pressure in rabbits ( pintor , peral , pelez et al 2002 ) ; ap4a and utp improve the rate of wound healing in the cornea of new zealand white rabbits ( pintor , bautista et al 2004 ) and also , ap4a , ap5a , and ap6a , can stimulate tear secretion after single - dose topical application in rabbits ( pintor , peral , hoyle et al 2002 ) . in order to investigate the physiological role of nucleotides onto the tear film , lysozyme levels in new zealand white rabbits tears have been measured after a topical application of the mentioned substances . the methodological approach used to get the lysozyme levels has been the agar diffusion method described by van bijsterveld ( 1974 ) .
the analysis of the data suggests an increase in tear lysozyme levels with the application of the tested nucleotides .
twelve male new zealand white rabbits from granja cunicula san bernardo ( navarra , spain ) weighing 2.02.5 kg were kept in individual cages with free access to food and water and subjected to regular cycles of light and dark ( 12 hours each ) .
the new zealand white rabbits were 6 months old and the slit lamp exam evidenced no ocular pathology or alteration that could affect the tear secretion .
all the experiments were performed according to association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research and in accordance with the european communities council directive ( 86/609/eec ) .
three nucleotides were tested along this work : utp from amersham biosciences , inc . , ( piscataway , nj ) ; ap4a from sigma chemical ( st louis , mo ) ; up4u ( diquafosol or formerly ins365 ) kindly provided by inspire pharmaceuticals ( durham , nc , usa ) .
the p2 antagonists employed were pyridoxalphosphate-6-azophenyl-2 , 4-disulfonic acid ( ppads ) , suramin and reactive blue-2 ( rb-2 ) were purchased from sigma / rbi ( natick ma ) . for single dose experiments , all the nucleotides were applied in a concentration of 100 m in a volume of 10 l in the tested eye .
the p2 receptor antagonists ppads , suramin and rb-2 were instilled in concentrations of 100 m ( 10 l ) , 30 min before the application of any of the nucleotides .
the utp , ap4a and up4u nucleotides were tested twice in the whole sample ( n = 24 each agonist ) .
the p2 receptor antagonists were experienced once for each agonist in the whole sample ( n = 12 for each agonist ) .
the diffusion in agar method was employed to measure the lysozyme levels in rabbit tears .
callibration curves as well as all the lysozyme measurements were performed as indicated by van bijsterveld ( 1974 ) and mackie and seal ( 1976 ) .
briefly , the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter .
the paper discs were placed with clamps in the upper bulbar conjunctiva to avoid mucus strands .
when the tears soaked the discs ; they were removed and put on petri dishes with agar medium where micrococus lisodeikticus was grown .
the petri dishes were then incubated at 37 c for 24 hours and finally the zones of lysis of micrococus lisodeikticus were measured .
lysozyme standard curves for its quantification was performed with known concentrations of hel ( hen egg lysozyme ) . for the mentioned curve ,
the 5 mm paper discs were weighed before and after being soaked by rabbit tears in order to estimate the equivalent volume of lysozyme solution to apply on the calibration paper discs .
the difference in weight was the equivalent to 5 l of lysozyme solution , so that volume was applied to estimate the standard curve .
concentrations between 0.1 mg / ml and 1.0 mg / ml of standard hen lysozyme were applied on the discs in a final volume of 5 l .
after two control measurements , 10 l of the studied compound was topically instilled in one eye ( taking the contralateral eye as a control ) , and the tear samples were collected every hour for five hours . for measuring the diameter of the halos , petri dishes were scanned and analyzed by the computer program imagej ( v.1.37 , nih , usa ) .
briefly , scanned images of the inhibition halos were transformed into 8-bit black and white images and further transformed into a binary image prior to the corresponding calculation . with this standardized method ,
data were analyzed using the paired t - test and significance was set at p < 0.05 .
twelve male new zealand white rabbits from granja cunicula san bernardo ( navarra , spain ) weighing 2.02.5 kg were kept in individual cages with free access to food and water and subjected to regular cycles of light and dark ( 12 hours each ) .
the new zealand white rabbits were 6 months old and the slit lamp exam evidenced no ocular pathology or alteration that could affect the tear secretion .
all the experiments were performed according to association for research in vision and ophthalmology ( arvo ) statement for the use of animals in ophthalmic and vision research and in accordance with the european communities council directive ( 86/609/eec ) .
three nucleotides were tested along this work : utp from amersham biosciences , inc . , ( piscataway , nj ) ; ap4a from sigma chemical ( st louis , mo ) ; up4u ( diquafosol or formerly ins365 ) kindly provided by inspire pharmaceuticals ( durham , nc , usa ) .
the p2 antagonists employed were pyridoxalphosphate-6-azophenyl-2 , 4-disulfonic acid ( ppads ) , suramin and reactive blue-2 ( rb-2 ) were purchased from sigma / rbi ( natick ma ) . for single dose experiments , all the nucleotides were applied in a concentration of 100 m in a volume of 10 l in the tested eye .
the p2 receptor antagonists ppads , suramin and rb-2 were instilled in concentrations of 100 m ( 10 l ) , 30 min before the application of any of the nucleotides .
the utp , ap4a and up4u nucleotides were tested twice in the whole sample ( n = 24 each agonist ) .
the p2 receptor antagonists were experienced once for each agonist in the whole sample ( n = 12 for each agonist ) .
the diffusion in agar method was employed to measure the lysozyme levels in rabbit tears .
callibration curves as well as all the lysozyme measurements were performed as indicated by van bijsterveld ( 1974 ) and mackie and seal ( 1976 ) .
briefly , the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter .
the paper discs were placed with clamps in the upper bulbar conjunctiva to avoid mucus strands .
when the tears soaked the discs ; they were removed and put on petri dishes with agar medium where micrococus lisodeikticus was grown .
the petri dishes were then incubated at 37 c for 24 hours and finally the zones of lysis of micrococus lisodeikticus were measured .
lysozyme standard curves for its quantification was performed with known concentrations of hel ( hen egg lysozyme ) . for the mentioned curve ,
the 5 mm paper discs were weighed before and after being soaked by rabbit tears in order to estimate the equivalent volume of lysozyme solution to apply on the calibration paper discs .
the difference in weight was the equivalent to 5 l of lysozyme solution , so that volume was applied to estimate the standard curve .
concentrations between 0.1 mg / ml and 1.0 mg / ml of standard hen lysozyme were applied on the discs in a final volume of 5 l .
after two control measurements , 10 l of the studied compound was topically instilled in one eye ( taking the contralateral eye as a control ) , and the tear samples were collected every hour for five hours . for measuring the diameter of the halos , petri dishes were scanned and analyzed by the computer program imagej ( v.1.37 , nih , usa ) .
briefly , scanned images of the inhibition halos were transformed into 8-bit black and white images and further transformed into a binary image prior to the corresponding calculation . with this standardized method ,
data were analyzed using the paired t - test and significance was set at p < 0.05 .
to determine whether these nucleotides were able to modify rabbit tear lysozyme levels at intervals after the nucleotide topical application , single doses of utp , ap4a , up4u were applied at 100 m ( at a final volume of 10 l ) ( figure 1 ) .
the results for the time - course of the single - dose application compared with the basal curve for lysozyme is represented in figure 2 .
it is possible to see that all the three substances increase the concentration of lysozyme in the rabbit tears , and that the maximal effect is obtained two hours after the instillation of the corresponding nucleotide .
this effect remained for at least 3 more hours , then returned to basal levels ( figure 2 ) .
a representative experiment showing the inhibitory halos around whatman n1 paper discs in the moment of maximal effect is presented in figure 3 .
these halos represent the zones of lysis of micrococcus lysodeikticus due to the action of lysozyme .
the first disc shows the basal diameter of lysis for a control rabbit ( basal lysozyme concentration present in tears , figure 3a ) .
the others showed a maximal increased diameter of lysis due to the action of the different nucleotides on the tear lysozyme levels after their topical application ( figure 3a ) .
a representative value to compare effects among nucleotides can be obtained if we represent the mean of lysozyme concentration obtained for the period between 2 and 5 hours  plateau  for any of the substances versus basal concentration . as it is shown in figure 3b , it is possible to see that the mean values for all the nucleotides showed a significant increase in lysozyme concentrations over the basal level . the increase in lysozyme concentration after the nucleotides application was 93 % for ap4a , 119% for up4u and 67% for utp ( figure 3b ) .
the activity of the substances on tear lysozyme levels suggests the activation of p2 nucleotide receptors . to confirm this ,
the effects of three non - selective nucleotide receptor antagonists on lysozyme levels were studied : ppads , suramin and rb-2 .
diadenosine tetraphosphate effect on lysozyme production was significantly reversed by the antagonist ppads , returning the lysozyme concentration to control values .
neither of the other two , rb-2 or suramin was able to modify the effect triggered by ap4a .
up4u was antagonized by ppads and suramin , the reduction being 42% and 52% for ppads and suramin respectively .
moreover , in the case of rb-2 , there was a potentiation of utp effect when compared with the effect of this nucleotide alone , showing an increase of almost 25% above the lysozyme concentration obtained when utp is applied alone .
this study shows for the first time the physiological effect of ap4a , up4u and utp on the tear lysozyme levels in new zealand white rabbits .
all the tested nucleotides showed an increase in the lysozyme concentrations for a single - dose application , which was measurable for several hours after the topical application of the compounds .
since lysozyme is one of the main proteins involved in the protective tear film barrier against ocular infection , together with lactoferrin , an increase in their levels may , in general , enhance the bactericidal action of tears , preserving the health of the ocular surface .
the presence of nucleotides and dinucleotides in the tear is suggesting their involvement in several relevant physiological processes of the ocular surface .
we have added to the list of biological actions of nucleotides and dinucleotides the ability of these compounds to increase the tear concentrations of lysozyme .
it is moreover relevant the fact of having important changes in the concentrations of this enzyme when up4u or utp are topically applied .
these two nucleotides have not been described in tears but they behave similarly to ap4a , suggesting that all may activate the same p2y purinoceptor subtype . in this sense ,
the three tested nucleotides have been claimed to be quite selective purinergic receptors ( lazarowski et al 1995 ; for the p2y2 mundasad et al 2001 ) .
nonetheless , the results obtained by means of the non - selective p2 receptor antagonists depict a different pattern depending on the tested nucleotide . in the case of ap4a
the only effective antagonist was ppads while in the case of up4u apart from ppads , suramin was also able to partially reverse the dinucleotide action .
one may expect both nucleotides to stimulate the same receptor or group of receptors ( see the similarities in the structure showed in figure 1 ) .
taking into account our results , it seems that ap4a and up4u share some p2y receptor activation but in the case of up4u it may activate more than one , since its increase in lysozyme is partially blocked by suramin while ap4a effect is not
. this may be at least in part justified by the fact of the presence of several p2y receptor in the lachrymal gland and cornea ( cowlen et al 2003 ; pintor , bautista et al 2004 ; pintor , sanchez - nogueiro et al 2004 ) , and also by the fact that up4u is known to activate at least p2y2 and p2y4 receptors ( brunschweiger and muller 2006 ) .
it is noteworthy the effect the antagonists of p2 receptors display on the utp effect .
although one should expect that rb-2 might block the effect of utp , the observed behavior of this antagonist shows the opposite effect .
reactive blue 2 has been described as a very potent inhibitor of all plasma membrane - bound ntpdases as reported by many authors ( mateo et al 1996 , 1997 ) . since rb-2 inhibits ecto - enzymes and does not antagonize p2y receptors it is reasonable to see a potentiation of the utp effect since it will not be degraded as fast as when it is applied alone .
, it has been described a decrease in lysozyme levels with age ( mackie and seal 1976 ) as well as in other pathological states running with a drop off in tear secretion like keratoconjunctivitis sicca or sicca syndrome ( mackie and seal 1984 ) .
therefore , in these cases , it may be good to incorporate any of the indicated nucleotides as a new compound as a pharmacological treatment to reinforce the barrier against opportunistic infections .
recently , apart from the bactericidal role , tragoulias et al ( 2005 ) and millar et al ( 2006 ) have suggested that lysozyme contributes to lower the surface tension of the tear film . in this case ,
an increase in lysozyme levels could contribute to stablizing the tear film , thus avoiding an excessive evaporation and the resulting problems on the ocular surface . in summary
, we have shown that the p2y receptor agonists , ap4a , up4u and utp , are able to increase the tear concentrations of lysozyme . due to the importance of this protein ,
these nucleotides may be used to fortify the tear film barrier by increasing a naturally occurring defender against some ocular surface infections .
 the present work studies the effects of topical application of nucleotides on rabbit tear lysozyme levels . 
 lysozyme values were determined by the diffusion in agar method described by van bijsterveld in 1974 , and the protein amount was obtained by measuring the inhibitory halos around a whatman n1 paper disc of 5 mm in diameter . 
 the tested nucleotides were utp , ap4a and up4u . 
 these compounds were topically instilled in a single - dose in one eye ( with the contralateral eye as a control ) and the lysozyme halos were measured along 5 hours . 
 the obtained results showed an increase in the lysozyme concentrations of 67% , 93% , and 119% for utp , ap4a , and up4u , respectively , over the basal levels of lysozyme . 
 for this reason , we suggest these molecules as a potential treatment for the reinforcement of the tear film barrier against ocular infection . 
Introduction
Experimental procedures
Animals
Compounds
Diffusion in agar
Statistical analysis
Results
Discussion