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THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
http://dx.doi.org/10.1124/jpet.114.218263
J Pharmacol Exp Ther 352:119–128, January 2015
Melatonin Receptors Trigger cAMP Production and Inhibit
Chloride Movements in Nonpigmented Ciliary Epithelial Cells
Fernando Huete-Toral, Almudena Crooke, Alejandro Martínez-Águila, and Jesús Pintor
Departamento de Bioquímica y Biología Molecular IV, Facultad de Óptica y Optometría, Universidad Complutense de Madrid,
Madrid, Spain
Received July 11, 2014; accepted October 23, 2014
Introduction
Melatonin is a relevant hormone controlling various physiologic actions, many of which are related to the photoperiod
(Pandi-Perumal et al., 2006). It is generally accepted that blood
melatonin levels increase overnight as a consequence of its
production and release from the pineal gland (Caprioli and
Sears, 1984; Alarma-Estrany and Pintor, 2007; Stehle et al.,
2011). This gland is not exclusively responsible for melatonin
production. In the orbital cavity and in the eye, for instance,
some areas, such as the retina (Cardinali and Rosner, 1971a,b;
Alarma-Estrany and Pintor, 2007), ciliary body (Martin et al.,
1992; Alarma-Estrany and Pintor, 2007), and harderian glands
(Djeridane et al., 1998; Alarma-Estrany and Pintor, 2007), have
the ability to synthesize and release melatonin. As occurs in other
tissues, the eye melatonin exerts many of its actions by means
of membrane receptors termed melatonin receptors, divided into
This work was supported by grants from the Spanish Ministry of Economy
and Competition [Grants SAF2010-16024 and SAF-2013-44416-R]; and the
Ministry of Health Social Services and Equality RETICS [Grant RD12/0034/
0001]. Additionally, this work was supported by Spanish Ministry of Economy
and Competition studentship to F.H.-T. and Universidad Complutense de
Madrid studentships to A.M.-Á.
dx.doi.org/10.1124/jpet.114.218263.
reverses the melatonin action by acting as a selective MT3 antagonist. However, at 15 nM it acts as an a-adrenergic receptor
antagonist, enhancing the melatonin effect. Regarding the intracellular pathways triggered by melatonin receptors, neither
phospholipase C/protein kinase C pathway nor the canonical
reduction of intracellular cAMP was responsible for melatonin or
5-MCA-NAT actions. On the contrary, the application of these substances produced a concentration-dependent increase of cAMP,
with pD2 values of 4.6 6 0.2 and 4.9 6 0.7 for melatonin and
5-MCA-NAT, respectively. In summary, melatonin reduces the
release of chloride concomitantly to cAMP generation. The reduction of Cl2 secretion accounts for a decrease in the water
outflow and therefore a decrease in aqueous humor production.
This could be one of the main mechanisms responsible for the
reduction of IOP after application of melatonin and 5-MCA-NAT.
MT1, MT2, and the putative MT3 melatonin receptors (AlarmaEstrany and Pintor, 2007; Dubocovich et al., 2010).
One significant physiologic process in the eye undergoing
circadian control is the regulation of the intraocular pressure
(IOP). IOP is the result of the balance between the production of the aqueous humor by the ciliary body (Civan and
Macknight, 2004; Do and Civan, 2004) and its drainage by the
trabecular meshwork and uvesoscleral pathway (An and Ji,
2011; Pattabiraman et al., 2012). In this sense, the circadian
fluctuation of IOP has been widely studied (Rowland et al., 1981;
Liu et al., 2011) as well as its melatonin levels relationship
(Samples et al., 1988). Interestingly, this circadian pattern can
be modified by applying exogenously melatonin or any of its
analogs (Pintor et al., 2001; Serle et al., 2004). In this sense, the
topical application of melatonin on the ocular surface produces
a transient reduction in IOP that is enhanced by melatonin
analogs, such as 5-MCA-NAT (5-methylcarboxyamino-N-acetyl
tryptamine) or IIK7 [N-butanoyl 2-(9-methoxy-6H-isoindolo
[2,1-a]indol-11-yl)ethanamine]. Melatonin and these two analogs,
acting through MT3 and MT2 melatonin receptors, can modify
IOP by acting on the ciliary body (Pintor et al., 2001, 2003;
Alarma-Estrany et al., 2007).
From a therapeutic point of view, the implications of melatonin and analogs on IOP control are relevant. IOP is elevated
ABBREVIATIONS: DH97, N-pentanoyl-2-benzyltryptamine; IBMX, 3-isobutyl-1-methylxanthine; IIK7, N-butanoyl 2-(9-methoxy-6H-isoindolo[2,1-a]indol11-yl)ethanamine; IOP, intraocular pressure; 5-MCA-NAT, 5-methylcarboxyamino-N-acetyl tryptamine; MQAE, N-(6-methoxyquinolyl) acetoethyl ester; NPE,
nonpigmented epithelial cells; PE, pigment epithelium; PKC, protein kinase C; PLC, phospholipase C; 4-P-P-DOT, 4-phenyl-2-propionamidotetralin; U73122,
1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione; V, velocity.
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ABSTRACT
Melatonin and its analog 5-MCA-NAT (5-methylcarboxyaminoN-acetyl tryptamine) are active compounds reducing intraocular
pressure (IOP). This action is mediated through MT2 and the
putative MT3 melatonin receptor, producing a transient reduction of IOP that lasts for a few hours and has not yet been
characterized. The use of melatonin and its analog are causing
a decrease in chloride efflux from rabbit nonpigmented epithelial
cells (NPE), possibly explaining the decrease in IOP. Melatonin
and 5-MCA-NAT inhibited rabbit NPE chloride release in a
concentration-dependent manner, whereas the pD2 values were
between 4.5 6 1.2 and 4.4 6 1.0, respectively. Melatonin hypotensive action was enhanced by the presence of MT2 antagonists,
such as DH97 (N-pentanoyl-2-benzyltryptamine) and 4-P-P-DOT
(4-phenyl-2-propionamidotetralin) and by the nonselective melatonin receptor antagonist luzindole. Prazosin (1.5 mM) partially
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Materials and Methods
Cell Culture. Nonpigmented epithelial cells (NPE), an immortalized
cell line of rabbit ciliary nonpigmented epithelium, were kindly supplied
by Dr. Coca-Prados (Yale School of Medicine, New Haven, CT). Cells
were grown in high glucose Dulbecco’s modified Eagle’s medium
(Gibco/Invitrogen, Carlsbad, CA) containing 10% fetal bovine serum
(Sigma-Aldrich, St. Louis, MO) and 0.05 mg/ml Gentamicin (Gibco/
Invitrogen) at 37°C in humidified atmosphere 5% CO2–95% air.
Chloride Efflux Studies. Chloride efflux was measured using
MQAE [N-(6-methoxyquinolyl) acetoethyl ester; Invitrogen, Carlsbad,
CA] as chloride indicator (Lee et al., 1984). Briefly, cells were seeded
in 48-wells plates (Iwaki, Tokyo, Japan) at a density of 104 cells/well
and grown to confluence. Twenty hours before the experiment, the
cells were incubated in Dulbecco’s modified Eagle’s medium containing 1 mM MQAE (West and Molloy, 1996). After incubation, the cells
were washed three times in chloride-containing buffer and incubated
in this buffer, with or without different antagonists, for 10 minutes (at
37°C) to induce chloride channel activation. This buffer consisted of
2.4 mM K2HPO4, 0.6 mM KH2PO4, 1 mM CuSO4, 1 mM MgSO4, 10 mM
Hepes, 10 mM D-glucose, and 130 mM NaCl (Panreac, Barcelona,
Spain). After this new incubation, the buffer was replaced by a chloridefree buffer, with or without the corresponding agonist or antagonist. In
this buffer, NaCl was replaced by an equimolar concentration of NaNO3
(Panreac). Plates were then read on Fluoroskan FL fluorescence plate
reader (Thermo Labsystems Inc., Waltham, MA) following the methodology described by Huete et al. (2011).
Melatonin receptor antagonists luzindole (nonspecific antagonist
melatonin receptor), 4-P-P-DOT (4-phenyl-2-propionamidotetralin),
DH97 (N-pentanoyl-2-benzyltryptamine; MT2 receptor antagonists;
Tocris, Bristol, UK) (100 mM), prazosin (a-1 and MT3 receptor antagonist; Santa Cruz Biotechnology, Dallas, TX) (15 nM, 150 nM,
1.5 mM), specific a-1 antagonist corynanthine (Santa Cruz Biotechnology)
(100 mM), protein kinase C (PKC) inhibitors staurosporine (100 nM) and
bisindolylmaleimide I (1 mM), phospholipase C (PLC) inhibitor U73122
(1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole2,5-dione; 3 mM), and protein phosphatase 1/2A okadaic acid (100 nM;
Tocris) were added in chloride-containing buffer and maintained
in a chloride-free buffer. Forskolin (40 mM) and IBMX (3-isobutyl-1methylxanthine; 50 mM) were added only in a chloride-free buffer.
Data are expressed as mean 6 S.E.M. of relative fluorescence units
normalized to the initial time (Ft 2 F0), where Ft is the fluorescence at
time t and F0 is the initial fluorescence.
Calculation of the Stern-Volmer Constant. To calculate SternVolmer constant (Ksv) in NPE cells, we used the double ionophore technique. As described by West and Molloy (1996), isosmotic buffers with
different concentrations of chloride were used to create a range of chloride
concentrations. The whole range of concentrations was assayed simultaneously in the same plate to avoid MQAE leakage problems during
monitoring of chloride concentrations in the given sample.
To equilibrate intracellular and extracellular buffer chloride concentration, 10 mM tributyltin (Sigma-Aldrich) and 5 mM nigericin (SigmaAldrich) were added to the buffer. Applying changes in fluorescence
in cells under these different chloride buffers, a Stern-Volmer plot was
obtained, responding to the equation:
F0 =F 5 1 1 Ksv ½Cl– ;
where F0 is the MQAE fluorescence in the absence of chloride, F is the
MQAE fluorescence in presence of chloride, and Ksv (Stern-Volmer
constant) is the slope of linear plot representing the efficiency of collisional quenching.
Concentration-Response Curves. To determine concentrationresponse curves, different concentrations of melatonin (Sigma-Aldrich) and
5-MCA-NAT (Tocris) were tested according to the previous methodology.
Concentrations tested varied in ranges from 1 nM to 150 mM. The Fmax
and the slope of the straight segment of each dose curve were converted into % of fluorescence (Ft 2 F0) versus control (taken as 100%)
and plotted. Data are plotted as percent of mean fluorescence (versus
control) in relative fluorescence units 6 S.E.M. versus logarithm of
agonist concentration.
Cyclic AMP Studies. Cyclic AMP accumulation was measured
using cAMP Enzyme Immune Assay EIA KIT (Cayman Chemical
Company, Ann Harbor, MI). Cells were grown to confluence in 6-well
plates (Iwaki). Then medium was replaced with fresh medium containing different concentration of agents. Antagonists were preincubated over 15 minutes, and after 10 minutes of incubation with
agonists, the medium was removed and immediately each well was
incubated for 20 minutes in medium containing 275 ml HCl 0.1 M. The
cells were then scraped and centrifuged at 1000g for 10 minutes, and
the supernatant was assayed as indicated in the protocol of cAMP
Cayman EIA Kit. The results were expressed as picomoles per milliliter
to avoid errors due to the resuspension and protein quantification in
small volumes. All wells were examined before the assay, and cells were
counted to ensure homogeneity of the study. Results were shown as
a mean 6 S.E.M.
Statistical Analysis. GraphPad Prism (GraphPad Software Inc.,
San Diego, CA) was used to obtain the linear regression (for the straight
lines), nonlinear regression curves, and calculation of slope (for the
straight lines and straight segments of the curves), pD2, and IC50
values. Statistical significance was calculated by analysis of variance
(Bonferroni post tests) and Student’s t test, when needed. Value of
P , 0.05 was taken as significant.
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in primary open angle glaucoma, affecting more than 65 million
patients all over the world (Quigley and Broman, 2006). In most
of the cases, the treatment of the reduction of the abnormally
elevated IOP is by means of adrenergic compounds, carbonic
anhydrase inhibitors, prostaglandins, or parasympathomimetics (Webers et al., 2008; Lee and Goldberg, 2011; Carta et al.,
2012). Because melatonin reduces IOP in experimental models,
it would be of interest to see whether this effect is also feasible
in humans. In this sense, some ophthalmologists have started
to use melatonin to reduce IOP in patients undergoing cataract
surgery, indicating the relevance of this molecule as regulator
of IOP (Ismail and Mowafi, 2009), suggesting its possible use as
a treatment of ocular hypertension.
The actions of melatonin in the reduction of IOP are taking
place mainly on the ciliary body as noted above. Interestingly,
the actions melatonin and analogs can exert on this part of the
eye are not only the short-term IOP reduction (Pintor et al.,
2001, 2003) but they also produce a long-term effect. This
second aspect of melatonin action is due to the modification in
the expression of key genes encoding for proteins relevant in
the control of aqueous humor production, such as adrenergic
receptors (Crooke et al., 2011) and carbonic anhydrases (Crooke
et al., 2012). These proteins indirectly regulate water efflux, therefore controlling aqueous humor production and subsequently IOP.
On the contrary, little is known about the mechanisms of
IOP rapid reduction by melatonin or 5-MCA-NAT. It is supposed that melatonin and analogs may also modify the aqueous
humor water production, but to date there is no evidence of the
possible mechanism involved.
One of the ions driving the water movement from the ciliary
body to the posterior chamber to form aqueous humor is chloride
(Civan and Macknight, 2004; Do and Civan, 2004). Because
there may be a connection between melatonin receptor activation, chloride movement, and aqueous humor production, the
present experimental work studies the ability of melatonin and
5-MCA-NAT to modify the chloride efflux in ciliary body nonpigmented epithelial cells.
Melatonin Reduces the Ciliary Release of Chloride via cAMP
121
Results
Fig. 1. Effect of melatonin and 5-MCA-NAT on chloride efflux. (A)
Stern-Volmer linear regression fit in NPE cells. We can see that there is
a linear relationship between intracellular chloride and fluorescence
intensity. Data represents mean 6 S.E.M. of normalized fluorescence
(Ft 2 F0) in varying ratios of intracellular chloride (n = 6). (B) Traces
represent normalized fluorescence at time 0 (Ft 2 F0) of control and
treated cells with melatonin (s) and 5-MCA-NAT (m) both at 100 mM
versus time. There were extremely significant differences between treated
cells and control in Fmax, t50, and v (slope of straight segment) in all the
cases with a significance of P , 0.001 (n = 6). Values represent the mean 6
S.E.M.
versus changes in the slope (velocity) for melatonin and
5-MCA-NAT, sigmoidal curves were obtained, providing interesting data. As observed in Fig. 2A, melatonin presented
a pD2 value of 4.7 6 0.2, whereas 5-MCA-NAT provided
a pD2 value of 5.0 6 0.1, corresponding to EC50 values of
19.9 and 10 mM for melatonin and 5-MCA-NAT, respectively
(n 5 5). Interestingly, the Hill slopes for both compounds were
different, their values being 0.6 6 0.3 for melatonin and 1.8 6
0.2 for 5-MCA-NAT (Fig. 2B) (n 5 5).
Studies with Antagonists. Although the presence of melatonin receptors, mainly MT2 and MT3, has already been
described in the ciliary body nonpigmented epithelial cells, we
tried to investigate which receptor is involved in the changes in
the intracellular chloride concentrations. In this sense, the MT2
antagonist 4-P-P-DOT was unable to modify the effect of melatonin. Interestingly another MT2 antagonist, DH97, and the
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Effect of Melatonin and 5-MCA-NAT on Ciliary Body
Epithelial Cells. MQAE is a highly sensitive chloride fluorescence probe. This fluorescence is quenched in the presence of
chloride so that changes in fluorescence are inversely proportional to changes in chloride concentration. By using the
protocol specified in Materials and Methods, it was possible to
verify the linear relationship between dye fluorescence and
intracellular chloride concentrations in this cell type (Fig. 1A).
By using this data it was also possible to calculate the SternVolmer constant (Ksv), whose value was 12.18 6 0.89 M21 (n 5 6),
in rabbit NPE cells. This constant permits us to calculate the
intracellular Cl2 concentration, which was 74.37 6 3.8 mM
(n 5 6) in rabbit nonpigmented ciliary epithelial cells when
external Cl2 concentration was 130 mM.
By using this fluorescence probe, we were able to measure
changes in the intracellular chloride concentrations after challenging the cells with melatonin and analogs. In particular,
melatonin and 5-MCA-NAT were able to modify intracellular
chloride. Normalized plots of fluorescence versus time always
presented sigmoid patterns. From these plots, three different
parameters were calculated: the maximal fluorescent signal,
Fmax, which corresponded to the minimal intracellular chloride
concentration ([Cl2]i); t50, which corresponded to the time
necessary to produce 50% of Fmax, an indication of how fast
was the release of chloride; and finally, the slope of the curve
straight segment representing the velocity (V) of the chloride
efflux. We used these parameters to evaluate the treatments
versus the untreated cells that were taken as controls. These
control cells showed the normal release of chloride of this cell
type in chloride-free buffer. As previously indicated, nontreated
cells depicted a sigmoid behavior that was consistent to a
chloride efflux from inside the cells to the extracellular milieu
(Fig. 1B). From this curve it was possible to calculate Fmax, t50,
and V values presented in Table 1.
Melatonin and 5-MCA-NAT (100 mM) clearly and significantly changed the Cl2 efflux as can be seen in Fig. 1B. There
were differences in the Fmax, t0.5, and V when comparing
melatonin and 5-MCA-NAT with control (Fig. 2; Table 1).
Interestingly, both melatonin and 5-MCA-NAT showed a strong
inhibition compared with control. Indeed, melatonin completely
inhibited chloride release for about 602 seconds (10.0 minutes)
and 5-MCA-NAT for roughly 860 seconds (14.3 minutes). After
that, and in the presence of these two compounds, the slope of
their respective curves was not as steep as the control and,
moreover, they did not reach the Fmax the control did (Fig. 1B).
Concentration-Response Curves for Melatonin and
5-MCA-NAT. To fully study the effect of melatonin and 5MCA-NAT on chloride fluxes, cells were challenged with graded
concentrations of both compounds following the protocol described in Materials and Methods. We focused on how these two
compounds were able to diminish cell fluorescence (Fmax) and
its concentration dependency. In this sense, and as can be seen
in Fig. 2A, both compounds depicted concentration-response
curves that were almost identical. From both curves it was
possible to obtain pD2 value of 4.5 6 1.2 for melatonin and
4.4 6 1.0 for 5-MCA-NAT (n 5 5). These values corresponded to
EC50 values of 31.6 and 39.8 mM for melatonin and 5-MCA-NAT,
respectively.
When, instead of studying the relationship between concentration and cell fluorescence, we analyzed concentration
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TABLE 1
Effect of melatonin and 5-MCA-NAT on chloride efflux
Data of Fmax, t50, and slope are expressed as mean 6 S.E.M. Fmax was estimated by sigmoidal allosteric curve regression
(R2 = 0.94, 0.93 and 0.86 for control, melatonin, and 5-MCA-NAT, respectively).
Control
Melatonin
5-MCA-NAT
Fluorescence max Fmax
t50
Slope
RFU
s
RFU/s
3.283 6 0.088
3.151 6 0.02515##
1.756 6 0.1054***,##
2748.08
5242.95***
4010.27***
6.661024 6 3.80 1026
3.231024 6 1.34 1025***
2.701024 6 3.94 1026***
RFU, relative fluorescence units.
***P , 0.001, highly significant differences were found in Fmax, t50, and slope between treated cells and controls; ##P ,
0.05, no significant difference between treatments of melatonin and 5-MCA-NAT except Fmax (n = 6).
nonselective melatonin receptor antagonist luzindole, enhanced
the effect triggered by melatonin and 5-MCA-NAT. Melatonin
effect in the presence of DH97 reduced Cl2 efflux from 47.2 6 5.3
to 21.1 6 1.4% and luzindole to 19.8 6 1.1% (n 5 6).
5-MCA-NAT effect changed from 43.4 6 3.4% (alone) to
33.3 6 1.3% when 4-P-P-DOT was present, to 17.9 6 2.3% in
the presence of DH97, and to 17.5 6 2.1% when luzindole
was present (n 5 6) (Fig. 3). In the same sense, prazosin
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Fig. 2. Concentration-response curves of melatonin and
5-MCA-NAT analyzing Fmax and changes in the slope.
(A) Concentration-response curves for melatonin (s)
and 5-MCA-NAT (m) assayed at concentrations ranging
from 1029 to 1024 M. No significant differences were
found between melatonin and 5-MCA-NAT. Values are
the mean 6 S.E.M. (n = 5). (B) Concentration-response
curves for melatonin (s) and 5-MCA-NAT (m) assayed
at concentrations ranging from 1029 to 1024 M. Points
on the graph represents the variation of the slope (V) in
the straight segment of the fluorescence curve at the
mentioned concentrations. Nonlinear regression asymmetric (five parameter) was plotted. R2 = 0.96 and 0.98
for melatonin and 5-MCA-NAT, respectively. Values are
the mean 6 S.E.M. (n = 5).
Melatonin Reduces the Ciliary Release of Chloride via cAMP
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(MT3 antagonist receptor) 15 nM surprisingly enhanced the
effect of 5-MCA-NAT to 29.5 6 4.3%. Nevertheless, the values
were very close to those of 5-MCA-NAT alone (44.8 63 .5%) when
we used 150 nM prazosin and partially reverted 5-MCA-NAT
effect (78.3 61 6.5%) when it was used at 1.5 mM concentration
(n 5 6) (Fig. 4A). Similar results were obtained using melatonin
(n 5 6) (Fig. 4B); however, no statistically significant differences
were reached between melatonin and melatonin 1 15 nM
prazosin. This lack of statistical significance difference probably
is due to a relatively high S.E.M regarding “n” used and not to
a difference in the behavior of both substances.
Adrenoreceptor a-1 Implications in Chloride Regulation.
The results obtained with prazosin at a low concentration produced an enhancement of 5-MCA-NAT hypotensive effect, whereas at high concentration, it produced the partial inhibition of
5-MCA-NAT effect, suggesting the involvement of an adrenoreceptor
a-1 in the regulation of chloride secretion.
As is shown in Fig. 5, A and B (n 5 6), corynanthine (a-1
antagonist) was able to enhance the effect of 5-MCA-NAT on
chloride efflux from 43.36 6 5.53 to 28.18 6 3.46%, confirming
the role of a-1 receptors in the regulation of chloride flux.
Corynanthine alone has no effect on the chloride secretion
(data not shown).
Second Messengers Triggered by Melatonin and 5MCA-NAT. It has been claimed that MT3 melatonin receptors
are coupled to the PLC/PKC pathway (Huang et al., 2001). To
see whether the effect of melatonin and 5-MCA-NAT was
triggering this intracellular pathway, different blocking agents
of this route were tested in their ability to modify the Cl2 effluxes
triggered by melatonin and 5-MCA-NAT.
As is shown in Fig. 6, none of the compounds tested to inhibit the PLC/PKC pathway was able to produce a change in
Fig. 4. Effect of prazosin antagonism on the secretory response triggered
by melatonin and 5-MCA-NAT. (A) Effect of prazosin at 15 nM, 150 nM,
and 1.5 mM on chloride efflux elicited by 5-MCA-NAT 100 mM. Prazosin at
the lowest concentration enhanced the action of 5-MCA-NAT; however, at
the highest concentration partially antagonized the effect of 5-MCA-NAT
(*P , 0.05; n = 6). (B) Effect of prazosin at different concentration (see A),
on chloride efflux elicited by melatonin 100 mM. The results were very
close to those obtained with 5-MCA-NAT (*P , 0.05; n = 6).
Fig. 3. Effect of MT2 and nonselective MT receptor antagonists on the
melatonin and 5-MCA-NAT secretory responses. Antagonists for melatonin
receptors at the concentrations described in Materials and Methods were
tested in their ability to block melatonin and 5-MCA-NAT effects. All data
are expressed as % of the normal (control) chloride intracellular concentration at Fmax. Antagonists did not reverse melatonin and 5-MCA-NAT effect,
and on the contrary, they were able to increase melatonin and 5-MCA-NAT
effect (***P , 0.001; n = 6). Values are the mean 6 S.E.M.
the fluorescence signal, either alone or in the presence of
melatonin or 5-MCA-NAT.
Involvement of cAMP Pathway. We decided to investigate the canonical cAMP pathway that has been described to
be negatively coupled to both MT1 and MT2 receptors. When
adenylate cyclase activity was increased by means of a forskolin
and IBMX mixture (see Materials and Methods), we could
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To investigate the involvement of the MT3 receptor in this
signaling pathway we blocked this receptor using 1.5 mM
prazosin (n 5 6), measuring the accumulation of intracellular
cAMP. The results were presented in Fig. 8. Data showed a
partial and significant reversion of intracellular cAMP from
92.34 6 6.36 (5-MCA-NAT) to 74.36 6 4.32 pmol/ml (5-MCA-NAT 1
prazosin).
Fig. 5. Action of corynanthine (a-1 antagonist receptor) on the secretory
effect of 5-MCA-NAT. (A) Normalized fluorescence versus time in NPE
cells using 5-MCA-NAT 100 mM, and corynanthine 100 mM (n = 6). (B)
Normalized fluoresce in Fmax at different treatments. Corynanthine
clearly enhances the action of 5-MCA-NAT alone (P , 0.05; n = 6).
Corynanthine alone did not differ from controls.
notice a reduction in Cl2 efflux as observed in Fig. 7A. Because
the chloride efflux triggered by adenylate cyclase activation resembled the behavior of melatonin and 5-MCA-NAT, we studied
the ability of these two substances to increase cAMP concentrations. The results showed that 100 mM melatonin was able to
increase intracellular cAMP levels of 58.05 6 4.22 to 84.59 6
6.78 pmol/ml (n 5 6). In the case of 5-MCA-NAT, 100 mM results
were similar, obtaining an intracellular cAMP concentration of
90.55 6 5.53 pmol/ml (n 5 6) in the presence of the melatonin
analog.
Different concentrations of melatonin and 5-MCA-NAT were
tested to characterize the dose-dependent behavior of these
substances. As shown in Fig. 7B, graded concentrations of melatonin and 5-MCA-NAT evoked the accumulation of concomitant
amounts of cAMP in rabbit NPE cells. The concentrationresponse curve provided a pD2 value of 4.6 6 0.2 for melatonin
and 4.9 6 0.7 for 5-MCA-NAT, which were equivalent to EC50
values of 22.0 and 19.4 mM (n 5 6) for melatonin and 5-MCANAT, respectively.
Discussion
The present manuscript describes the effect of melatonin
and its analog 5-MCA-NAT acting on melatonin receptors of
ciliary body nonpigmented epithelial cells. The main action of
melatonin and its analog is the modulation of intracellular
chloride concentrations, which is important because chloride
rules water movement and therefore is the key ion driving the
production of the aqueous humor (Civan and Macknight, 2004).
The aqueous humor is responsible for the correct eye shape and
acts as a nutritional fluid for avascular structures such as the
lens or the cornea (Civan, 1998). Under certain circumstances,
a lack of drainage of the aqueous humor produces an elevation
of IOP possibly responsible for the pathology termed glaucoma.
There are different reports indicating that melatonin application reduces or increases IOP (Caprioli and Sears, 1984).
We claim that melatonin and analogs reduce IOP in New
Zealand white rabbits, glaucomatous monkeys (Serle et al.,
2004), and even humans (Ismail and Mowafi, 2009).
Aqueous humor formation relies on the ability of the ciliary
body cells [pigment epithelium (PE) and NPE] to mobilize
chloride ions from the stromal part of the ciliary body to the
posterior chamber of the eye (Do and Civan, 2004). The results
presented in this manuscript suggest that melatonin and
5-MCA-NAT may reduce IOP because they decrease the efflux
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Fig. 6. Effect of PKC/PLC inhibitors on the secretory response triggered
by melatonin and 5-MCA-NAT. The responses of melatonin or 5-MCANAT in the presence of a variety of PLC/PKC pathway (see Materials and
Methods) were completely unaffected when treated with these agents (n =
8). Values are the mean 6 S.E.M.
Melatonin Reduces the Ciliary Release of Chloride via cAMP
125
of chloride from the cytoplasm of NPE toward the extracellular
space. The effect of both melatonin and 5-MCA-NAT inhibiting
this ion movement was strong during 10 and 14 minutes for
melatonin and 5-MCA-NAT (100 mM), respectively. After this
interval of inhibition, the rate of efflux increases, although slope
and Fmax were always below the control values. This fact indicates
that the inhibitory effect does not only affect initial chloride efflux
but inhibition was also present when the equilibrium was reached
(see Fig. 1). The substantial inhibition of the chloride release and
the presumable inhibition of the aqueous humor formation seem
to be higher than the IOP reduction observed in vivo (Pintor et al.,
2003). In this sense, it is important to notice that the in vitro
model we are using, although mimicking the conditions present
in the ciliary body, has certain limitations. One of the main in
vitro restrictions is that we are measuring the secretory layer
NPE cells, producers of the aqueous humor, and not the drainage system, possibly the reason for such differences. Another
limitation to take into consideration is that the measurements
we performed involve only Cl2 efflux processes. This implies
that there is a Cl2 efflux after a concentration gradient (as
occurs in the in vivo model) and, because of the lack of this ion
in the extracellular buffer, mechanisms transporting this ion
from the extracellular space to NPE cells cytoplasm are not
fully activated. Consequently, the data obtained are dependent
solely on the state of chloride channels and transporters. Also,
we should emphasize the absence of the PE, which takes
chloride from stromal and transfers it to NPE layer cells. The
importance of PE and its function has been extensively described
in the literature (McLaughlin et al., 1998; Do and To, 2000; Do
et al., 2004a; Ni et al., 2006). However, the simultaneous study of
both layers complicates precise conclusions concerning the contribution of each structure involved.
Although there are some limitations in the model we are
using, it is important to emphasize that an analysis of the
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
Fig. 7. Effect of melatonin and 5-MCA-NAT cAMP
production on forskolin (Forsk) + IBMX on chloride
efflux. (A) Effect of forskolin + IBMX on chloride efflux.
The plot represents normalized fluorescence at time
0 (Ft 2 F0) of control and Forsk-IBMX treated cells.
There was a statistically significant reduction in the rate
of increase of fluorescence in Forsk-IBMX group compared with control and therefore a decrease in chloride
efflux (P , 0.01; n = 15). (B) Concentration-response
curves for melatonin (s) and 5-MCA-NAT (m) assayed at
concentrations ranging from 1026 to 1023 M on the
generation of cAMP. Values represent the mean 6 S.E.M.
(n = 6).
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Huete-Toral et al.
Fig. 8. Effect of prazosin on cAMP production promoted by 5-MCA-NAT.
Addition of 1.5 mM prazosin with 100 mM 5-MCA-NAT as described in
Materials and Methods. Prazosin was able to partially revert the cAMP
accumulation produced by 5-MCA-NAT (*P , 0.05, n = 6).
Downloaded from jpet.aspetjournals.org at ASPET Journals on June 18, 2017
concentration-curves demonstrates that the effects depicted
by melatonin and 5-MCA-NAT were highly similar but not
identical (especially in Fmax), as we can see in Fig. 1. This
matches the results obtained for both substances on IOP in
New Zealand white rabbits, where the effect of melatonin as
an hypotensive agent is less robust than that of 5-MCA-NAT
(Pintor et al., 2003).
An interesting and unexpected observation was the effect of
the classic melatonin antagonists. The MT2 antagonist DH97
and the MT1/MT2 nonselective antagonist luzindole potentiated the inhibitory effect of both melatonin and 5-MCA-NAT
on chloride movement. This may suggest that melatonin and
5-MCA-NAT are acting through a different MT1/MT2 or luzindolesensitive receptor. Moreover, when MT1/MT2 or luzindolesensitive receptors were blocked, the effect of this putative
receptor was enhanced, suggesting a different signaling pathway
from the canonical described for melatonin receptors. This indirectly implies that the action of MT1/MT2 may have an opposite effect, increasing the efflux of Cl2. Because MT1 and MT2
melatonin receptors are negatively coupled to adenylate cyclase
and there is a concomitant reduction in the concentrations of
cAMP, the blockade of these two receptors might involve
PLC/PKC pathway as happens in some models (Bowler et al.,
1996; Godson and Reppert, 1997; Dortch-Carnes and Tosini,
2013). Interestingly, new studies are appearing indicating that
new second messenger systems can be coupled to melatonin
receptors. For instance, melatonin receptors produce a reduction in sodium nitroprusside–released nitric oxide and cGMP
levels in human nonpigmented ciliary epithelial cells (DortchCarnes and Tosini, 2013). These authors indicate that, at least
in part, melatonin and analog effects can use this pathway in
human NPE cells and that this second messenger system
might be responsible for the melatoninergic compound hypotensive effect. These results are perfectly compatible with those
presented here and with previous ones in which we suggested
that MT2 melatonin receptor agonists reduce IOP (AlarmaEstrany et al., 2008).
Coming back to the second messengers being activated by
melatonin and 5-MCA-NAT in our model, we were unable to
detect any involvement of the PLC/PKC pathway. Some
authors claimed that 5-MCA-NAT is not a selective MT3 receptor agonist but it could activate MT1 or MT2 melatonin
receptors (Vincent et al., 2010). If this were so, we would
expect reductions in the cytosolic concentration of cAMP,
because, as already mentioned above, MT1 or MT2 melatonin
receptors are negatively coupled to adenylate cyclase (Vanecek,
1998). The study on the ability of melatonin and 5-MCA-NAT to
inhibit cAMP formation pointed in the opposite direction,
because it was impossible to see a reduction in cAMP production. On the contrary, we could observe that melatonin and its
analog increased cAMP concentrations in a concentrationdependent manner. This is not a common mechanism of signal amplification, but it has been described in some models
(Raviola, 1974; Beraldo and Garcia, 2005; Schuster et al.,
2005). Indeed it modified chloride efflux, and this effect was
mimicked when we applied forskolin and IBMX. The relationship between the increase of cAMP and the decrease of IOP is
widely studied in the eye. In rabbits and monkeys, it is known
that this decrease in IOP is associated with a significant decrease in AH secretion. Interestingly, it was reported that
5-MCA-NAT is able to produce important increases in cAMP in
chick retinas by a mechanism that may involve an MT3 (Mel1c)
binding site (Sampaio, 2009).
When the involvement of the putative MT3 melatonin receptor was studied, the use of the only available antagonist
prazosin was tested. Low concentrations of this antagonist
produced an unexpected potentiation of the action triggered
by 5-MCA-NAT. Nevertheless, at higher concentrations (1.5 mM),
prazosin had its antagonistic effect blocking melatonin and 5MCA-NAT actions, indicating that at low micromolar concentrations it acts as a MT3 receptor antagonist as previously
described elsewhere (Dubocovich et al., 2003; Pintor et al.,
2003; Alarma-Estrany et al., 2011).
Concerning the results obtained with prazosin at nanomolar
concentrations, the observed contradictory effect potentiating
5-MCA-NAT action can be explained as an effect performed on
a-1 adrenoceptors. This point was confirmed when the same
effect was obtained with the selective a-1 adrenoceptor antagonist corynanthine, as was also described regarding the modulation of IOP in rabbits by other authors (Chidlow et al., 2001).
In the same way, we tested the action of prazosin, in micromolar range, on the cAMP accumulation induced by 5-MCA-NAT.
Prazosin was able to partially revert the increased intracellular cAMP. This fact relates the cAMP increase with the MT3
melatonin receptor.
It was demonstrated that the chloride efflux from NPE cells
is one of the most, if not the most important, ion controlling
aqueous humor secretion as previously commented (Jacob
and Civan, 1996; Forrester, 2002). However, the nature of the
proteins ruling the movement of Cl2 (Ritch et al., 1989;
Paulmichl et al., 1992; Chen et al., 1999; Do et al., 2006) and
the transmitters regulating chloride secretion and secondarily
IOP is not clear. For example, traditionally it has been accepted
that the blockade of b-adrenergic receptors is a pharmacologic
approach to decrease IOP (Freddo, 1987). b-Adrenergic receptors are positively coupled to adenylate cyclase but, surprisingly,
cAMP activates some chloride channels. The explanation seems
to be that the effects of cAMP itself and b-adrenergic receptors
may reflect a complex regulation evolving different mechanisms
(McLaughlin et al., 2001) and presumably several channels and
transporters as suggested by Do and Civan (2004). Recently, it
was demonstrated that the increase in cAMP levels does not only
activate protein kinase A but also may have a direct effect on ion
channels. Fleishauer and coworkers (2001) demonstrated that
cAMP action is conducted via a direct action on chloride channels
Melatonin Reduces the Ciliary Release of Chloride via cAMP
Acknowledgments
The authors thank Penny Rollinson for help with the English edition
of the manuscript.
Authorship contributions
Participated in research design: Pintor.
Conducted experiments: Huete-Toral.
Performed data analysis: Martínez-Águila.
Wrote or contributed to the writing of the manuscript: Crooke.
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