ARTICLE
Received 4 Jun 2015 | Accepted 22 May 2016 | Published 27 Jun 2016

DOI: 10.1038/ncomms12034

OPEN

PACAP suppresses dry eye signs by stimulatingtear secretion
Tomoya Nakamachi1,2, Hirokazu Ohtaki2, Tamotsu Seki2, Sachiko Yofu2, Nobuyuki Kagami2,
Hitoshi Hashimoto3,4,5, Norihito Shintani3, Akemichi Baba6, Laszlo Mark7,8,9,10, Ingela Lanekoff11, Peter Kiss12,
Jozsef Farkas2,12, Dora Reglodi12 & Seiji Shioda13

Dry eye syndrome is caused by a reduction in the volume or quality of tears. Here, we showthat pituitary adenylate cyclase-activating polypeptide (PACAP)-null mice develop dryeye-like symptoms such as corneal keratinization and tear reduction. PACAP
immunoreactivity is co-localized with a neuronal marker, and PACAP receptor (PAC1-R)
immunoreactivity is observed in mouse infraorbital lacrimal gland acinar cells. PACAP eye
drops stimulate tear secretion and increase cAMP and phosphorylated (p)-protein kinase A
levels in the infraorbital lacrimal glands that could be inhibited by pre-treatment with a
PAC1-R antagonist or an adenylate cyclase inhibitor. Moreover, these eye drops suppress
corneal keratinization in PACAP-null mice. PACAP eye drops increase aquaporin 5 (AQP5)
levels in the membrane and pAQP5 levels in the infraorbital lacrimal glands. AQP5 siRNAtreatment of the infraorbital lacrimal gland attenuates PACAP-induced tear secretion. Basedon these results, PACAP might be clinically useful to treat dry eye disorder.

1 Laboratory of Regulatory Biology, Graduate School of Science and Engineering, University of Toyama, 3190-Gofuku, Toyama-shi, Toyama 930-8555, Japan.
2 Department of Anatomy, Showa University School of Medicine, Shinagawa-Ku, Tokyo 142-8555, Japan. 3 Laboratory of Molecular Neuropharmacology,
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan. 4 iPS Cell-based Research Project on Brain
Neuropharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan.
5 Molecular Research Center for Children’s Mental Development, United Graduate School of Child Development, Osaka University, Kanazawa University,
Hamamatsu University School of Medicine, Chiba University and University of Fukui, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. 6 Hyogo University of
Health Sciences, 1-3-6 Minatojima, Chuo-ku, Kobe, Hyogo 650-8530, Japan. 7 Department of Analytical Biochemistry, Institute of Biochemistry and Medical
Chemistry, Medical School, University of Pécs, Szigeti u 12, Pécs 7624, Hungary. 8 Imaging Center for Life and Material Sciences, University of Pécs, Szigeti u
12, Pécs 7624, Hungary. 9 János Szentágothai Research Center, University of Pécs, Szigeti u 12, Pécs 7624, Hungary. 10 PTE-MTA Human Reproduction
Research Group, Szigeti u 12, Pécs 7624, Hungary. 11 Department of Chemistry-BMC, Uppsala University, PO Box 599, Uppsala 751 24, Sweden.
12 Department of Anatomy, MTA-PTE PACAP Lendulet Research Group, Centre for Neuroscience, University of Pécs, Szigeti u 12, Pécs 7624, Hungary.
13 Innovative Drug Discovery, Global Research Center for Innovative Life Science, Hoshi University, 4-41 Ebara 2-chome, Shinagawa-ku, Tokyo 142-8501,
Japan. Correspondence and requests for materials should be addressed to S.S. (email: shioda@hoshi.ac.jp)

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NATURE COMMUNICATIONS | DOI: 10.1038/ncomms12034

D

ry eye syndrome, also known as keratoconjunctivitis sicca,
is a common eye disease caused by a reduction in thevolume or quality of tears. Tear components are
secreted from the main lacrimal gland, accessory lacrimal gland(Krause and Wolfring glands), meibomian gland, and the corneal
and conjunctival epithelia in humans. A thin layer of tear film
containing water, lipid electrolytes and B10 mg ml ! 1 protein
comprising different tear proteins, covers the ocular surface,
thereby maintaining and protecting the eye. The major categories
of dry eye are the aqueous tear-deficient type, in which the
lacrimal glands fail to produce enough of the watery componentof tears to maintain a tear film, and the evaporative type, in which
impaired lipid secretion from the meibomian glands destabilizes
the tear film1. Dry eye syndrome correlates with old age andaffects females to a larger degree2. The number of patientsdiagnosed with the condition has increased in recent years,
which could be due to the popularity of video display use(computer vision syndrome) or the wearing of contact lenses3,4.
The orthodox strategy for the treatment of dry eye syndrome issymptomatic therapy, such as tear replacement using artificial
tears. Although artificial tears provide temporary symptomatic
relief, they do not address the underlying pathophysiology of dryeye syndrome, and the outcome is not always satisfactory5.
Pituitary adenylate cyclase-activating polypeptide (PACAP;
encoded by the gene Adcyap1), which exists in 27- or 38-aminoacid isoforms, was originally discovered in extracts of ovine
hypothalamus6,7. The amino-acid sequence of PACAP—amember of the vasoactive intestinal polypeptide (VIP)/secretin/
growth hormone-releasing hormone family of peptides—shows a
68% sequence homology with VIP. PACAP and VIP share threedifferent receptors: the VPAC1 and VPAC2 receptors (VPAC1-R
and VPAC2-R; gene names: Vipr1 and Vipr2) and the PAC1receptor (PAC1-R; encoded by the gene: Adcyap1r1) withdifferent splice variants8,9. The affinity of PAC1-R for PACAP
is more than 1,000 times higher than its affinity for VIP,
indicating that PAC1-R is a relatively selective receptor for
PACAP (ref. 10). The amino-acid sequences of PACAP27 and
PACAP38 are highly conserved among mammals, and those ofmouse and human are identical. PACAP is distributed mainly inthe central nervous system, but it is also detected in the testis,
adrenal gland, digestive tract and other peripheral organs10. The
amino-acid sequence of PAC1-R is conserved with a 96.6%
homology between mouse and human. Adcyap1r1 mRNA is
widely detected in the central nervous system and peripheralorgans, including the adrenal gland, testis, anterior pituitary
gland, pancreas and placenta10.
PACAP is a multi-functional peptide that can act as aneurotrophic factor, neuroprotectant, neurotransmitter, immunomodulator and vasodilator10–13. During the past 10 years, PACAPnull (Adcyap1 ! / ! ) mice have been generated by several
laboratories and their phenotypes have been analysed14–16.
Recently, we observed that corneal keratinization, associated witha decreased tear volume, frequently occurs in Adcyap1 ! / ! mice. It
has been reported that PACAP immunopositive nerve fibres wereobserved in cat lacrimal gland17, but PACAP and PACAP receptor
expression and function in lacrimal glands has not been wellstudied to date. To address this interesting finding, we investigate
the effects and underlying mechanism of action of PACAP on
lacrimal gland tear secretion in PACAP-null mice, as well as inmice treated with topical eye drops.
Results
Dry eye-like signs in the Adcyap1 ! / ! mouse. During theroutine housing of Adcyap1 ! / ! mice in our animal facility, we
unexpectedly discovered that some mice exhibited cloudiness of2

the cornea (Fig. 1a). The ocular surface appeared white andsandy, and blood vessels could be seen in the cornea (Fig. 1b).
Based on fluorescein staining, which is commonly used tovisualize corneal injury, strong fluorescence was observed in thecentral part of the cornea in these mice (Fig. 1c). On examinationof this pathology, we discovered that the corneal epithelial cells
were hypertrophied and the surface was keratinized (Fig. 1d). To
quantify the degree of corneal keratinization, corneas wereclassified into four grades with the aid of a dissecting microscope(from Grade 0, denoting normal, to Grade 3 signifying
hypertrophy of the surface and keratinization, as shown in
Supplementary Fig. 1). Wild-type and Adcyap þ / ! male miceover the age of 20 weeks had normal corneas, whereas about 40%
of Adcyap1 ! / ! male mice over 30 weeks of age had Grade 3
corneas (Fig. 1e). In female mice, all groups showed a higher
frequency of keratinization than that observed in male mice
(Fig. 1f). In female Adcyap1 ! / ! mice, the percentage of animals
showing corneal keratinization was o20% in animals younger
than 10 weeks of age, but increased with age to 90% of animals
over 30 weeks of age (Fig. 1f). These data indicate that corneal
keratinization frequently occurs in older Adcyap1 ! / ! mice, andparticularly in female animals.
Because keratinization is a common feature of dry eye disorder,
we postulated that the corneal keratinization was caused by areduction in tear fluid or quality. To test this, the tear secretionlevel in Adcyap1 ! / ! mice was measured using the cotton thread
method. As expected, tear secretion levels of male and female
Adcyap1 ! / ! mice were reduced compared with those ofwild-type mice aged 10 weeks or younger (Fig. 1g,h). The tear
volume in eyes with corneal keratinization was significantly
reduced compared with that of Grade 0 eyes (Fig. 1i,j), while thetear volume and the corneal grade were weakly thoughsignificantly inversely correlated (r ¼ ! 0.242, P ¼ 0.007, twotailed Spearman’s correlation test). On histological examination,
the infraorbital lacrimal gland, conjunctiva and corneal neuralnetwork of Adcyap1 ! / ! mice were found to be morphologicallynormal (Supplementary Fig. 2a–c). Taken together, theseobservations suggest that Adcyap1 ! / ! mice exhibit a dry eyelike phenotype with a reduction in tear volume and corneal
damage, despite the structure of the infraorbital lacrimal gland,
conjunctiva and neural network of the cornea remaining normal.
Distribution and function of PACAP in the lacrimal gland.
Based on the above data, we hypothesized that the PACAP/PAC1-R
system was associated with altered tear secretion by the lacrimal
gland, the major source of tear secretion. To test this, we firstexamined PACAP/PAC1-R expression and distribution in themouse infraorbital lacrimal gland. Using the RT-PCR method,
Adcyap1 and Adcyap1r1 mRNAs were detected in gland extracts,
producing a signal with the same band size as that obtained from aneye ball sample that was used as a positive control (Fig. 2a). PACAP
immunoreactivity was observed around acinar cells, and co-localizedwith immunoreactivity for the neuronal marker NeuN (Fig. 2b,c),and the parasympathetic neuronal marker choline acetyltransferase(ChAT) (Fig. 2d). The PACAP antibody recognized PACAP38 but
not VIP (Supplementary Fig. 3a). PAC1-R immunoreactivity wasobserved on the basal side of acinar cells and ducts, but did notco-localize with smooth muscle actin as a myoepithelial cell marker(Fig. 2e,f). The PACAP and PAC1-R immunoreactivities wereabolished by pre-absorption of the antibody with antigen(Supplementary Fig. 3b,c). PACAP38 was detected in wild-type, but
not Adcyap1 ! / ! mouse tears by matrix-assisted laser desorption/
ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS),and nanospray desorption electrospray ionization (nano-DESI)
Orbitrap MS/MS (Supplementary Figs 4 and 5).

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200

Grade 1

Grade 3

80
60
40
20
0

8
6

*

4
2
0

Grade0 Grade1 Grade2 Grade3
n = 4819

26

26< 10 w 10 w to 20 w > 20 w

Adcyap1+/+
Adcyap1+/–
Adcyap1–/–

20

Female

**

15
10

**

**

**

5
< 10 w 10 w to 20 w > 20 w
24
22
17

n=

j

10

5

23

77
44

44

0
36
1
16
7

25

4
26
27

+/–

**

*

0

–/–

Male

**

10

h 25

Wet cotton thread (mm)Wet cotton thread (mm)

i

12

17

n=

0

+/+

70

–/–

15

14
20
20

28

28

39

54

21

Grade 2

100

20

n=

Wet cotton thread (mm)Grade 0Percentage in female

3
59

10

27

27

39

2

16

5

19

Adcyap1+/+
Adcyap1+/–
Adcyap1–/–

0

–/–

0–
11 10w–
21 20w
–3
0
31 w
w0–
11 10
– w
21 20w
–3
0w
31
w0–
11 10
– w
21 20w
–3
0w
31
w-

d +/+

+/–

25

38
26
28

40

n=

f

g
Wet cotton thread (mm)

60

–/–

–/–

Grade 3

80

+/+

c +/+

Grade 219
36
46

Grade 119
19
38

Grade 0100

0–11 10w
–
21 20
–3 w
0w
31
w0–
11 10w–
21 20w–30w
31
w0–
11 10w
–2
21 0w
–3
0w
31
w-

b +/+

–/–

17
16
27

e
+/+

Percentage in malea7
6

P < 0.005

5
4
3
2
1
0

Grade0n = 48Grade1–3
71

Figure 1 | Corneal keratinization and reduction of tear volume in Adcyap1 ! / ! mice. (a–d) Low- and high-power images of the corneal surface showing

pathological changes. Scale bars, 10 mm in a, 2 mm in b and c, and 50 mm in d. (e,f) Scoring of corneal keratinization in wild-type, Adcyap1 þ / ! andAdcyap1 ! / ! mice as described in Supplementary Fig. 1. The classification was evaluated on both sides, and the highest grade was used for the grading ofmale (e) and female (f) mice at different ages. (g,h) Tear volume measured with the cotton thread method in wild-type, Adcyap1 þ / ! and Adcyap1 ! / !
mice at different ages. The total wet cotton thread length from both eyes is shown for male (g) and female (h) mice. *Po0.05, **Po0.01 versus wild-type
mice. (i,j) Relationship between the wet cotton thread length and the corneal grade in female Adcyap1 ! / ! mice. The dot plot graph shows the wet
thread length for each eye of each grade expressed in terms of the mean±s.e. value (i). *Po0.05 versus Grade 0. The bar graph (j) shows the wet thread
length of Grade 0 and Grades 1–3, based on the data in Fig. 1i.

To investigate the function of PACAP in the lacrimal gland,
PACAP38 was delivered in the form of eye drops to wild-type
mice, and the level of tear secretion was measured using thecotton thread method (Fig. 3a). Eye drops containing 10 ! 10
to 10 ! 8 M PACAP38 significantly increased tear secretion from15 to 45 min after treatment, with levels returning to baseline by
120 min (Fig. 3b). The basal tear secretion level and PACAPinduced tear secretion level did not differ significantly between
males and females (Supplementary Fig. 6). PACAP27-containing
eye drops also stimulated lacrimation, whereas the structurally
related peptide VIP did not (Fig. 3c,d). Given that it has beenreported that PACAP38, rather than PACAP27, is predominantly
expressed in mammalian tissues10, PACAP38 was used in
the following experiments. When PACAP was administeredunilaterally, tear secretion was only induced on the
PACAP-treated side (Supplementary Fig. 7). When corneas
were pre-treated with the topical anaesthetic Benoxil tosuppress the corneal reflex, the basal lacrimation level
decreased, but PACAP still elicited a significant increase in tearsecretion (Supplementary Fig. 8). PACAP can thus induce tearsecretion under topical anaesthesia, showing that it has a direct
effect on the infraorbital lacrimal gland in the absence of thecorneal/conjunctival reflex. Moreover, when the acute tosemi-acute toxicological effect of PACAP38 (10 ! 7 M) eye
drops was evaluated at a concentration 1,000 times higher thanan effective dose of PACAP38 (10 ! 10 M) 48 h after the eye drop
treatment, no morphological changes were observed in thecorneas or in the infraorbital lacrimal glands (Supplementary
Fig. 9). In addition, we examined the effect of PACAP on
angiogenesis in vitro using human endothelial cells andfibroblasts in co-culture systems. No changes were observed ineither the PACAP38 or PACAP6–38 (a PAC1-R and VPAC2-R
antagonist)-treated groups (Supplementary Fig. 10). These datasuggest that PACAP eye drops act locally to stimulate lacrimation
without causing acute to semi-acute toxicity or eliciting a corneal
reflex. Mice have two lacrimal glands (the infraorbital andexorbital glands) (Supplementary Fig. 11a), both of which were
found to express PAC1-R and VPAC1-R (Supplementary

NATURE COMMUNICATIONS | 7:12034 | DOI: 10.1038/ncomms12034 | www.nature.com/naturecommunications

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Lacrimal
gland

Brain

a

Eye ballNATURE COMMUNICATIONS | DOI: 10.1038/ncomms12034

Adcyap1

500 bpAdcyap1r1

800 bp700 bpGapdh

500 bp

b

c

d

PACAP
NeuN
DAPI

PACAP
NeuN
DAPI

PACAP
ChAT
DAPI

e

f

PAC1R
DAPI

PAC1R
SMA
DAPI

Figure 2 | PACAP and PAC1-R expression in wild-type male mouse infraorbital lacrimal gland. (a) Reverse transcriptase PCR analysis to detect Adcyap1
and Adcyap1r1 mRNA in the infraorbital lacrimal gland. The eye ball was used as a positive control. (b–d) PACAP immunoreactivity in the mouse infraorbital
lacrimal gland with NeuN immunoreactivity as a neuronal marker (b,c), and ChAT immunoreactivity as a marker of parasympathetic neurons (d). The whitesquare in b is expanded in c. Scale bar, 20 mm in b and c and 10 mm in d. (e,f) PAC1-R immunoreactivity in the infraorbital lacrimal gland identified withsingle immunostaining in the acinus (arrow head) and in the duct (arrow) (e), and with smooth muscle actin (SMA) immunoreactivity as a myoepithelial
cell marker (f). Scale bar, 20 mm in e and 5 mm in f. DAPI was used as a nuclear marker (e,f).

Fig. 11b). Although PACAP still significantly stimulated
lacrimation in a mouse model in which the exorbital lacrimal
gland had been removed, it could not stimulate tear secretion in asecond model in which both lacrimal glands had been removed(Fig. 3e,f). These findings indicate that the target organfor PACAP administered in an eye drop formulation is theinfraorbital lacrimal gland.
We next examined the signalling cascade associated with
PACAP-induced lacrimation. Pre-treatment with PACAP6–38
significantly suppressed PACAP-induced tear secretion (Fig. 3g),
whereas VIP6–28 (a VPAC1-R and VPAC2-R antagonist) did not
suppress tear secretion (Fig. 3h). Moreover, a single drop of
PACAP6–38 (10 ! 8 M) to the ocular surface reduced the level ofnormal lacrimation at the 15 and 60 min time points postadministration (Fig. 3g). The intravenous infusion of PACAP also
increased tear secretion in a manner that could be inhibited byco-treatment with PACAP6–38 (Supplementary Fig. 12). These
results indicate that PACAP eye drops stimulate lacrimation via
an action on PAC1-R.
PACAP eye drops to Adcyap1 ! / ! mice. We also used PACAPcontaining eye drops on Adcyap1 ! / ! mice. PACAP38 (10 ! 10 M)
drops increased tear secretion in these mice as well as in their wildtype counterparts, suggesting that PACAP transiently restores tearsecretion in Adcyap1 ! / ! mice (Fig. 4a). We subsequently tested
the effects of repeated administration of PACAP38 on the eyes ofAdcyap1 ! / ! mice (one eye treated with PACAP38, the other withsaline) with a view to preventing corneal keratinization. After 3
weeks of treatment, the injury score had increased in saline-treatedeyes, but was still at the pre-treatment level in PACAP-treated eyes
(Fig. 4b,c). Angiogenesis and ocular hyperaemia were not observedin PACAP-treated eyes (Fig. 4b).
Signalling associated with PACAP-induced tear secretion. To
determine the pathway related to PACAP-induced tear secretion,
we next investigated the signalling pathways downstream of
PAC1-R, focusing on the adenylate cyclase (AC)-cAMP-dependent pathway. As determined by ELISA, the cAMP level in mouse
infraorbital lacrimal glands was increased at 15 min and peaked
30 min after the application of PACAP38-containing eye drops(Fig. 5a). The signal for phosphorylated (p) protein kinase A(PKA), a cAMP-dependent protein kinase, was significantly
increased at 30 min (Fig. 5b), while pre-treatment with the AC
4inhibitor SQ22536 or with PACAP6–38 7.5 min before theadministration of PACAP eye drops significantly suppressed
the PACAP-induced phosphorylation of PKA (Fig. 5c). Pretreatment with the AC inhibitor dramatically suppressed
PACAP-induced tear secretion (Fig. 5d).
Aquaporin expression in wild-type and Adcyap1 ! / ! mice. The
aquaporins (AQPs) are a family of water channel proteins that are
expressed in numerous tissues and organs, with expression of the
AQP4 and AQP5 subtypes being reported in the lacrimal gland18.
To evaluate the relationship between PACAP-induced tearsecretion and AQPs, we examined AQP4 and AQP5
immunoreactivities in the infraorbital lacrimal gland in wild-type
and Adcyap1 ! / ! mice. AQP5 immunoreactivity was identified on
the apical side of acinar cells in wild-type mice, but only weak
immunoreactivity was observed in these cells in Adcyap1 ! / ! mice
(Fig. 6a). AQP4 immunoreactivity was observed on the basal sideof acinar cells in both wild-type and Adcyap1 ! / ! mice, without
any obvious difference between the two (Fig. 6a). The specificity of
the AQP4 and AQP5 immunoreactivities was confirmed using anantigen pre-absorption test for AQP5 antibody or comparison witha primary antibody-free (AQP4 antibody) negative control(Supplementary Fig. 3d,e). On immunoblotting, the AQP5 signal
was found to be significantly lower in the infraorbital
lacrimal glands of Adcyap1 ! / ! mice than in those of wild-type
animals, but the AQP4 signal was almost the same in both cases(Fig. 6b).
The trafficking of AQP5 protein from the cytosol to themembrane contributes to increased water permeability19. After
fractionation and immunoblotting, the AQP5 signal in themembrane fraction was significantly lower in Adcyap1 ! / !
mice than in wild-type mice, but was not significantly different
in the cytosolic fraction (Fig. 6c). In contrast, the APQ4 signals in
the cytosolic and membrane fractions were similar for the twogroups (Fig. 6c).
AQP5 expression and distribution after PACAP eye drops. The
phosphorylation of AQP5 has been postulated to initiate its
trafficking to the membrane20,21. On this basis, the cellular
localization and degree of phosphorylation of AQP5 wasevaluated in PACAP-treated infraorbital lacrimal glands. Thirty
minutes after treatment with 10 ! 10 M PACAP, AQP5
immunoreactivity on the apical side of acinar cells was greater

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ab

PACAP(10 –10M)

30

Wet cotton thread (mm)Saline

25
*
*

20

10
5

20

**

**

15
10
5
0

pre

15

30

45

60

pre

15
30
45
60
Time after eye drop (min)d 25
Wet cotton thread (mm)Wet cotton thread (mm)SalinePACAP27 10–10 M

25

20
15
10
5
pre

Time after eye drops (min)

**

5

0

30

45

0

60

Time after eye drops (min)25
20

SalinePACAP6-38
PACAP
PACAP + PACAP6-38
*

15

*

10

h
**

*

5
0

pre

15
30
45
60
Time after eye drops (min)30

Wet cotton thread (mm)Wet cotton thread (mm)

g 30

45

60

120

5

Pr

Pr

15

10

eop
Pr
e
edr
op
(0
)

**

30

Removal of intraorbital andextraorbital lacrimal gland
SalinePACAP38

15

Wet cotton thread (mm)

**

f

Pr

10

15

Time after eye drops (min)Removal of extraorbital
lacrimal gland
SalinePACAP38

15

eop
e
edr
op
(0
)

Wet cotton thread (mm)e120

SalineVIP 10–10 M
VIP 10–8 M

0120

SalinePACAP38 10–12 M
PACAP38 10–10
M
M
PACAP38 10–8
PACAP38 10–6 M

15

0

c

*
*

*
**

25

15

30

45

60

Time after eye drops (min)SalineVIP6-28PACAP
PACAP + VIP6-28

20
15
10
5
0

pre

15
30
45
60
Time after eye drops (min)Figure 3 | Effect of PACAP eye drops on tear secretion in male mice. (a) Representative images of the cotton thread measurements 30 min afteradministration of 10 ! 10 M PACAP38 or saline eye drops with a 30 s insertion of the thread into both lower eyelids. The blue bar in the lower pictures
shows the length of the tear-absorbed thread that changed colour from yellow to red. Scale bar, 10 mm. (b) Dose-response effect of PACAP38 eye drops ontear secretion in mice. Test solution eye drops were applied to both eyes. The sum of the wet length of cotton from the two eyes is shown on the Y-axis(n¼ 10 per group). *Po0.05, **Po0.01. (c,d) Effect of PACAP27 (c) and VIP (d) eye drops on tear secretion (n¼ 9–10 per group). **Po0.01 versus thesaline-treated group. (e,f) Effect of 10 ! 10 M PACAP38 eye drops on two lacrimal gland-deficient models: removal of the exorbital lacrimal gland (e) andremoval of both the infraorbital and exorbital lacrimal glands (f) (n¼ 10 per group). **Po0.01 versus the saline-treated group. (g,h) Effect of PAC1-R and
VPAC2-R antagonist, PACAP6–38 (g) and VPAC1-R and VPAC2-R antagonist, VIP6–28 (h) treatments on PACAP-induced tear secretion (n¼ 10 pergroup). *Po0.05, **Po0.01.

than that in the saline-treated, SQ22536-pre-treated or PACAP6–
38-pre-treated groups (Fig. 7a,b). The AQP5 levels in the
total lysates of the infraorbital lacrimal gland extracts
showed no difference between the groups at 30 min (Fig. 7c).
However, the AQP5 signal in infraorbital lacrimal gland extracts
immunoprecipitated with a pan-phospho antibody was clearly
detectable in the PACAP-treated group, but was less obvious

in the other groups (Fig. 7c). An AQP4 signal was not detectedin the sample immunoprecipitated with the pan-phospho
antibody (Supplementary Fig. 13). The AQP5 signals in themembrane fractions were increased 30 min after treatment
with PACAP compared with the other groups (Fig. 7d,e). To
elucidate the contribution of AQP5 to PACAP-induced
tear secretion, an AQP5 gene-silencing experiment was

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SalinePACAP3810–10M

*

*

b

c

Saline PACAP38
pre
pre

3.0
2.5

*

1.5
1.0

Saline PACAP38
3w
3w
pre

*

2.0

0.5
0

15
30
45
60
120
Time after eye drops (min)Salin
PA pre e
C
AP
pr 38
e
Sa
lin
PA 3w e
C
AP
3w38

14
12
10
8
6
4
2
0

Score

Wet cotton thread (mm)

a

*

b
*

0
7.5 15 30
Time after treatment (min)
–
–
–

+
–
–

+
+
–

+
–
+

pPKA
37 kDaPKA

37 kDa400

pPKA/PKA(% of control)

7.5

15

30 (min)
37 kDaPKA

PACAP38
SQ22536
PACAP6-38

*

*

*

300
200
100

0
PACAP38
SQ22536
PACAP6-38

0
pPKA

pPKA/PKA (% of 0 min)

c

160
140
120
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80
60
40
20
0

*
400
200
0

+
–
–

+
+
–

+
–
+

0
7.5
15
30
Time after treatment (min)dSaline+PACAP38
SQ22536+PACAP38

30
25
20
15

**

**

**

15

30

45

10

*

5
0

–
–
–

37 kDa600

Wet cotton thread (mm)acAMP
(f mol mg–1 tissue)

Figure 4 | Effect of PACAP eye drops on female Adcyap1 ! / ! mice. (a) Tear secretion level in Adcyap1 ! / ! mice after application of saline or 10 ! 10 M
PACAP38 eye drops (n¼ 9 per group). *Po0.05 versus saline-treated group. (b,c) Corneal scoring before and after the repeated application of PACAP eye
drops to Adcyap1 ! / ! mice over a 3-week period. (b) Representative images of the corneal surface before and after eye drop application. Scale bar, 2 mm.
(c) Eighteen female Adcyap1 ! / ! mice were given saline or PACAP eye drops unilaterally. The classification of each side of the cornea was evaluatedseparately before, and 3 weeks after eye drop treatment commenced. *Po0.05.

pre

60

120

Time after eye drops (min)Figure 5 | Contribution of the cAMP/PKA pathway to PACAP-induced tear secretion. (a) cAMP content in infraorbital lacrimal gland extracts after
application of 10 ! 10 M PACAP eye drops (n¼ 8 per group). *Po0.05 versus 0 min. (b) Phosphorylated PKA (pPKA) levels in the mouse infraorbital
lacrimal gland after PACAP eye drop application (n¼ 6 per group). *Po0.05 versus 0 min. (c) Pre-treatment with the adenylate cyclase inhibitor SQ22536
or the PACAP receptor antagonist PACAP6–38 suppressed PKA phosphorylation 30 min after PACAP eye drop administration (n¼ 6 per group). *Po0.05.
(d) Effect of SQ22536 pre-treatment on PACAP-induced tear secretion in male mice (n¼ 10 per group). A single drop of test solution was applied to eachocular surface. The sum of the wet lengths of thread from both eyes is shown. *Po0.05, **Po0.01 versus saline þ PACAP group.

designed (Fig. 8a). When infraorbital lacrimal glands weretreated with AQP5 siRNAs, the Aqp5 mRNA level was
significantly decreased by about 70%, whereas the Aqp4 mRNAlevel remained almost the same 24 h after the siRNAtreatment compared with the control group (Fig. 8b,c). AQP5
siRNA treatment reduced AQP5 immunoreactivity in theinfraorbital lacrimal glands, but not AQP4 immunoreactivity(Fig. 8d). The AQP5 siRNA treatment significantly decreased
the basal level of tear secretion, and PACAP-induced
tear secretion was attenuated 24 h after the siRNA treatment(Fig. 8e).
6Taken together, these findings suggest an underlying mechanism whereby PACAP and its receptor are expressed in mouse
infraorbital lacrimal glands. PACAP stimulates tear secretion via
an AC/cAMP/PKA cascade, which in turn stimulates AQP5
translocation from the cytosol to the membrane of lacrimal acinar
cells to bring about an increase in water permeability (Fig. 8f).
Discussion
Dry eye syndrome is more common in women than in men,particularly in older patients2. This study has made use of the

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c

150
100
50
0
–

mem AQP4/pancadherin (%)

0
–

A

–

ya WT
p1
–/

50

A

A

Ad

0

100

ya WT
p1
–/

0

*

50

150

dc

50

100

cyt AQP4/GAPDH (%)

100

150

A

150

dc

–

/–

cy
a
Ad

100 kDa
37 kDaGAPDH

mem AQP5/pancadherin (%)

140
120
100
80
60
40
20
0
W
cy
T
ap
1 –/

*

AQP4/GAPDH (%)

120
100
80
60
40
20
0
W
T
p1 –

AQP5/GAPDH (%)

Adcyap1–/–

Adcyap1–/–

pan-Cadherin

37 kDa

GAPDH

–

AQP4

25 kDa37 kDa

AQP4

ya WT
p1
–/

WT

25 kDa37 kDa

dc

WT

AQP5

AQP5

AQP4

cyt AQP5/GAPDH (%)

AQP5

mem

ya WT
p1
–/

Adcyap1–/–

WT

Adcyap1–/–
cyt
mem

WT
cyt

dc

b

a

Figure 6 | AQP4 and AQP5 levels in the infraorbital lacrimal glands of male wild-type and Adcyap1 ! / ! mice. (a) AQP4 and AQP5 immunoreactivities
in wild-type and Adcyap1 ! / ! mouse infraorbital lacrimal glands. Scale bar, 50 mm. (b) AQP4 and AQP5 signals in infraorbital lacrimal gland total lysatesfrom wild-type and Adcyap1 ! / ! mice detected by western blot analysis and expressed as semi-quantified results (n¼ 6 per group). *Po0.05 versus
wild-type mice. (c) AQP4 and AQP5 signals from fractionated infraorbital lacrimal glands and the semi-quantified results for wild-type and Adcyap1 ! / !
mice (n¼ 6 per group). *Po0.05 versus wild type. GAPDH and pan-cadherin signals were used as internal controls for cytosolic and membrane protein,respectively.

acsalinePACAP
SQ22536
PACAP6-38

PACAP

–
–

+
–

+
+

+
–

–

–

–

+

anti-AQP5

25 kDaanti-GAPDH

37 kDapan-phosphp IP : anti-AQP5

25 kDaNormal IgG IP: anti-AQP5

25 kDaTotal lysate

PACAP+SQ

PACAP+PACAP6-38

d

PACAP
SQ22536
PACAP6-38
Fraction

–
–
+
+
+
+
+
+
+
+
–
–
–
–
–
–
+
–
–
–
–
–
–
+
cyt mem cyt mem cyt mem cyt mem

AQP5

b
**

**

cyt AQP5/GAPDH(% of saline)

100
50

*

300
250
200
150
100
50

PACAP+6–38

PACAP+SQ

PACAP

0Saline0

PACAP+6–38

+
–
+

150

PACAP+SQ

+
+
–

200

PACAP

+
–
–

100 kDa
37 kDa

GAPDH

e

–
–
–

25 kDamem AQP5/panCadherin (% of saline)

Mean density on apicalmembrane (% of saline)**

Saline

180
160
140
120
100
80
60
40
20
0
PACAP38
SQ22536
PACAP6-38

pan-Cadherin

Figure 7 | PACAP eye drop-induced phosphorylation and trafficking of AQP5. (a) Representative images of AQP5 immunoreactivities in infraorbital
lacrimal gland acinar cells after the application of saline, PACAP38, PACAP38 þ SQ22536 (SQ) or PACAP38 þ PACAP6–38 eye drops. Scale bar, 10 mm.
(b) AQP5 signals in fractionated infraorbital lacrimal glands and the semi-quantified results (n¼ 11 per group). **Po0.01. (c) AQP5 signals in total lysate
and immunoprecipitation with a pan-phospho antibody. Immunoprecipitation with a normal IgG antibody was used as a negative control. (d,e) AQP5
signals in fractionated infraorbital lacrimal glands (d) and semi-quantified results (e) (n¼ 11 animals per group). *Po0.05
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–2d

–1d

e

7

PACAP
or saline
Eye drop Cotton thread method

0 15m 30m 45m 60m

c
140
120
100
80
60
40
20
0

*
Control AQP5
siRNA siRNA

d
160
140
120
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40
20
0

Control siRNA

AQP5 siRNA

AQP5-ir

AQP4-ir

Control AQP5
siRNA siRNAf

6

Control siRNA + saline
Control siRNA + PACAP
AQP5 siRNA + saline
AQP5 siRNA + PACAP

**

5

*

4

Parasympathetic neuron

PAC1R

AC

ia

lc

el
l

** **

3

Eyedrop

PACAP

el

cAMP
p

PKA

yo

ep

ith

2
1
0
1day after siRNA
injection

1 day after ex-LG
removal

15

30

45

60

Time after eye drop (min)AQP5

T
locranati sonM

Wet cotton thread (mm)Removal of
exorbital LG

controlor AQP5
siRNA
injection

Correct infraorbital LG
for qPCR and IHC

Aqp5/Gapdh mRNA(% of control)b

Aqp4/Gapdh mRNA(% of control)aAcinar cell

PKA

p
AQP5

p
AQP5

Tear
secretion

Figure 8 | Contribution of AQP5 to PACAP-induced tear secretion in mice. (a) Experimental design of AQP5 siRNA study. (b–d) AQP4 and AQP5 levelsin the infraorbital lacrimal glands of male wild-type mice 1 day after AQP5 siRNA treatment. Aqp4 (b) and Aqp5 (c) mRNA levels were measured by realtime PCR (n¼ 12 per group). *Po0.05 (d) Representative pictures of AQP4 and AQP5 immunoreactivities in the infraorbital lacrimal gland after control
and AQP5 siRNA injection. Scale bar, 20 mm. (e) Effect of AQP5 siRNA treatment of infraorbital lacrimal glands on PACAP-induced tear secretion (n¼ 12).
*Po0.05, **Po0.01. (f) Schema of proposed mechanisms of PACAP-induced tear secretion.

discovery of a new Adcyap1 ! / ! mouse phenotype, namely the
manifestation of corneal keratinization, which was particularly
apparent in female mice as a function of age, the overall
implication being that Adcyap1 ! / ! mice manifest dry eye-likesigns. The tear-secreting response to PACAP eye drops and thebasal level of tear secretion were, nevertheless, similar between
male and female wild-type mice (Supplementary Fig. 6). It has
been suggested that the sex difference in dry eye syndrome inhumans is due to the influence of female sex hormone levels22.
Given that female Adcyap1 ! / ! mice exhibit decreased serum
progesterone levels23, dry eye-like symptoms in these animals
may be due to an imbalance in their sex hormone levels.
Animal models that mimic dry eye disorder have been
established by several groups24,25. One type of dry eye is theaqueous-deficient model, which mimics the dry eye symptoms
caused by autoimmune diseases such as Sjögren’s syndrome,
removal or irradiation of the lacrimal gland, or neuronal pathway
dysfunction. Another type is the evaporative dry eye model, as
seen with meibomian gland dysfunction, environmental stresssuch as exposure to a dry environment, and pharmaceuticallyinduced tear film instability caused by a decrease in oil or mucinsecretion in the tears as evidenced by dry eye symptoms1.
However, a dry eye model arising from a specific endocrine
imbalance has not been reported to date. We have shownthat Adcyap1 ! / ! mice exhibit (1) reduced lacrimation
when left untreated, (2) increased lacrimation upon PACAP
administration and (3) a morphologically normal lacrimal gland.
These findings suggest that the impairment of lacrimal
secretion in Adcyap1 ! / ! mice results from lacrimal gland
dysfunction rather than developmental or structural
8

abnormalities. Moreover, Adcyap1 ! / ! mice spontaneously
develop corneal keratinization with aging, implying that thismouse phenotype could serve as a novel non-Sjögren’s typeaqueous-deficient dry eye model arising from lacrimal gland
dysfunction.
AQP family genes and proteins are expressed in the eye and its
accessory organs26,27. It has been reported that AQP5
immunoreactivity is markedly decreased in the lacrimal acinar
cells of people with Sjögren’s syndrome, a chronic autoimmune
disorder with impairment of the moisture-producing glands28.
The reduced expression of AQP5 suggests that this protein may
be related to the decrease in tear secretion in this disease. On theother hand, Verkman’s group reported that AQP5 KO mice do
not exhibit an altered tear volume29. However, the same group
recently published data showing that the Na þ content of tears
from AQP5 KO mice is significantly higher than that of wild-type
mice30, suggesting that the ion concentration in AQP5 KO mousetears is elevated due to decreased water secretion into thetear fluid. In our study, AQP5 gene silencing-attenuated
PACAP-induced tear secretion, as well as the basal level of tearsecretion. Taken together, we propose that AQP5 is associatedwith water secretion into the tear.
It has been reported that the activation of cAMP/PKA can
induce the translocation of AQP5 from the cytosol to theapical membrane31,32. Moreover, X-ray analysis of thestructure of human AQP5 has revealed that phosphorylation at
the C-terminal is required for the conformational change
for trafficking21. Although the relationship between membranetrafficking and phosphorylation of AQP5 is not yet fully
understood, in the case of AQP2, the closest paralog of AQP5,

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a key event for membrane trafficking is the phosphorylation of a
C-terminal site by PKA (refs 33,34). Here, we demonstrate that
PACAP eye drops induce an elevation of cAMP, pPKA andpAQP5 levels and membrane trafficking of AQP5 in the mouse
infraorbital lacrimal gland, suggesting that PACAP is a regulatorof AQP5 trafficking in this gland.
Gilbard and collaborators reported that topical administrationof a cAMP inducer such as VIP stimulates tear secretion in a
rabbit model of keratoconjuctivitis sicca35 and in patients withdry eye disease36. However, they did not further study the natureof the target organ, or how reagents stimulate tear secretion. Here,
we have demonstrated that PACAP stimulates tear secretion fromthe infraorbital lacrimal gland via a PAC1-R/cAMP/PKA/AQP5
cascade. Moreover, PACAP had a much greater effect on tearsecretion than VIP. It should be noted that the Gilbard study useda high concentration of VIP (2 $ 10 ! 6 M) as eye drops in their
rabbit model, whereas we used 10 ! 10 M and 10 ! 8 M, the sameas our effective doses of PACAP in mice. We also found that
PAC1-R is the main receptor for PACAP-induced tear secretion.
The affinity of PACAP for PAC1-R is 1,000 times higher thanthat of VIP, implying that the higher concentration of VIP may
be able to stimulate tear secretion via PAC1-R. Taken together,
these results suggest that cAMP signalling is an important step fortear secretion, and that PACAP is an endogenous tear regulator in
the lacrimal gland, with much greater potential than VIP tostimulate lacrimation.
Tear fluid includes several antibacterial proteins, growth
factors and secretary mucin for corneal maintenance37,38. As it
has been reported that systemic infusion of PACAP alters thecomposition of tears, especially the keratin family of proteins in
rats39, PACAP may regulate tear protein secretion as well as tearfluid secretion. It is also well known that tear secretion isimportant for corneal healing40, and for this reason we postulated
that a reduction in tear fluid would be an important factor
underlying corneal keratinization in the Adcyap1 ! / ! mouse,
and that PACAP could protect the corneal surface by stimulatingtear secretion. Moreover, we used MALDI-TOF MS andnano-DESI MS/MS to identify the presence of PACAP inmouse tear fluid, a finding that may imply that PACAP issecreted from the lacrimal gland into the tear fluid, thereby
directly affecting the cornea. However, it remains questionablefrom which tissues in tear fluid PACAP is derived. The PACAP
could be coming from corneal and conjunctival nerve endings
released by dromic (parasympathetic or sympathetic nerves) or
anti-dromic (sensory nerves) stimulation, conjunctival goblet cells
or stratified squamous cells, or the meibomian glands. If thecorneal keratinization in Adcyap1 ! / ! mice is due to evaporative
water loss caused by a reduced volume of tears, it could be
postulated that lid closure (for example, reversible closure, using
cyanoacrylate glue) in Adcyap1 ! / ! mice would inhibit thedevelopment of this phenotype. Future studies will be needed toclarify the source of PACAP in tear fluid and the corneal healing
effects of PACAP.
Although our findings indicate the potential of PACAP as a
stimulator of tear production in mice, there are still problems thatwould need to be resolved in relation to drug development. First,
the human and mouse lacrimal apparatuses are structurally
different. In mice, the exorbital lacrimal glands are located near
the ear on the lateral side of the skull, and connect with theexcretory ducts into the eyelid. The infraorbital lacrimal glands
and the lipid-secreting Harderian glands are found in the orbit of
the eye. In humans, one main lacrimal gland is located in the
lacrimal fossa of each orbit next to the eye ball; this connects withexcretory ducts in the upper fornix. However, over 50 accessory
lacrimal glands are scattered over the inner surfaces of the lower
and upper eyelids, and the lipid-secreting meibomian gland islocated in the tarsal plate of the upper and lower eyelids41.
We anticipate that PACAP eye drops will not reach the main
lacrimal gland in humans because of its location, making thetarget of PACAP the accessory lacrimal glands. However, it is still
an open question whether PACAP only affects the lacrimal gland,or whether the corneal and conjunctival epithelia, and theintraorbital lipid-secreting gland are also involved. In any future
clinical trials of PACAP on dry eye patients, it will be important
to clarify the expression of PACAP and PACAP receptors intear-secreting tissue, including the lacrimal glands, as well as theeffect and target of PACAP eye drops in humans. Moreover, we
showed that PACAP eye drops did not cause any adverse reactionin acute to semi-acute phase at a concentration of 10 ! 7 M(Supplementary Fig. 9), or in chronic eye drop treatment at aconcentration of 10 ! 10 M for 3 weeks in Adcyap1 ! / ! mice
(Fig. 4). However, these toxicological evaluations may not be
sufficient for safety trials of PACAP eye drops to proceed onhealthy volunteers. Further suitable toxicological tests will be
required before a clinical trial.
In this study, PACAP38 eye drops at the lower dose of 10 ! 10
M stimulated mouse lacrimation for o1 h. Even if the effects of
PACAP are similar in mice and humans, this short periodof action would pose a problem in a clinical setting. We believethat the short-acting effect of PACAP in our study was due tothe PACAP eye drops being washed from the ocular surface
by lacrimation. One solution could be to formulate an ointment
to provide sustained release. However, low doses of bioactive
peptides have both advantages and disadvantages in terms of drug
development. A low dose of peptide-based medicine offers
good efficacy, safety and high selectivity and potency. In contrast,
there are issues in relation to instability, short half-life andrapid elimination42. These problems would need to be overcome
if PACAP were to be developed as an effective stimulator of
lacrimation.
In conclusion, our results highlight a new function of PACAP
as a stimulator of tear production initiated via the PAC1-R/AC/
cAMP/PKA/AQP5 cascade. We found that PACAP eye drops
induce tear secretion and suppress the progression of corneal
keratinization in Adcyap1 ! / ! mice. Topical administration of
cyclosporine has been developed for dry eye patients to provide
an anti-inflammatory effect; however, eye drops focusing on
tear-stimulating mechanisms are still in the developmental stage.
The findings from our work are encouraging and should provide
the impetus for further preclinical and clinical studies on theefficacy of PACAP eye drops to treat dry eye patients.
Methods
Animals. All experimental procedures involving animals were approved by theInstitutional Animal Care and Use Committee of Showa University (08116, 09110,
50021, 51033, 510043, 52007, 52013, 53020, 53025). The Adcyap1 ! / ! mice on the
C57BL/6 background were established by Dr Akemichi Baba15 and were bred andmaintained under specific pathogen-free conditions in the animal facility of Showa
University. Animals were housed in a facility with a 12-h/12-h light/dark cycle andwere given free access to water and standard rodent chow. Eight to twelve weeks
old mice were used, excepting Figs 1 and 4, and Supplementary Figs 1 and 2 with 8
to over 30 weeks old mice. In this study, both male and female mice were used as
described in the figure legends.
Evaluation of corneal scoring. Keratinization of the cornea was classified into
four grades (Grades 0–3) by visual observation under a dissecting microscope asfollows: Grade 0 (normal) ¼ no observable abnormality; Grade 1 ¼ clouded cornea;
Grade 2 ¼ angiogenesis; and Grade 3 ¼ hypertrophy of the corneal epithelium asshown in Supplementary Fig. 1a–d. The evaluation of corneal score was performed
in a blinded fashion. The corneal keratinization of wild-type, Adcyap1 þ / ! , andAdcyap1 ! / ! mice was evaluated on both sides, and the higher grade was used forthe grading of the animals in Fig. 1e,f.
Corneal fluorescein staining. Corneal injuries were visualized with fluoresceinstaining. Adcyap1 ! / ! and wild-type mice were anaesthetized with an

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intraperitoneal (i.p.) injection of pentobarbital, and 2 ml of fluorescein solution(1 mg ml ! 1) dissolved in saline were dropped on the cornea. After letting the animal
blink five times, excess drops were wiped off with a cotton swab. Photographs weretaken with a digital camera (CAMEDIA C5050, Olympus, Tokyo, Japan) connected
to a stereoscopic microscope.
PACAP-containing eye drops and cotton thread tear test. The rate of lacrimal
secretion was determined with the cotton thread test using standardized phenol
red-impregnated cotton threads (Zone-Quick; Menicon Co. Ltd, Nagoya, Japan).
Mice were anaesthetized with an i.p. injection of pentobarbital to suppress
autonomic nervous system reflexes that could have affected tear secretion. The
basal level of tear secretion was evaluated by insertion of the thread under the lower
eyelid for 30 s, after which the bilateral length of the colour-changed thread that
had absorbed the tear fluid was measured in millimeters. The test solution,
PACAP38 (10 ! 6–10 ! 12 M (Peptide Institute, Osaka, Japan), PACAP27(10 ! 10 M, Peptide Institute), VIP (10 ! 8 M or 10 ! 10 M, Peptide Institute) or
saline was then applied to both ocular surfaces. The mice were forced to blink theireyes five times, and their eyes were then kept closed during all subsequent steps toprevent drying of the surface of the cornea.
Excess drops were wiped off with a swab 7.5 min after application of the eye
drops, and tear secretion was checked at 15-min intervals from 15 to 120 min after
the eye drops had been administered. At each measurement time, the thread was
kept on the lower conjunctival sac of each eye for 30 s. The tear volume in the no eye
drop study (Fig. 1; Supplementary Fig. 12) was performed without anaesthesia. The
total length of wet cotton thread was measured for both sides (Figs 1d,e, 3, 4 and 5;;
Supplementary Figs 8 and 12), and in other specific experiments (Figs 1f,g and 8;
Supplementary Fig. 7), the length was evaluated on each side. The PACAP receptor
antagonist PACAP6–38 (Peptide Institute), VIP receptor antagonist VIP6–28(Sigma, St Louis, MO, USA), adenylate cyclase inhibitor SQ22536 (Sigma-Aldrich)or topical anaesthetic 0.4% oxybuprocaine hydrochloride (Benoxil; Santen
Pharmaceutical Co., Ltd., Osaka, Japan) was applied 7.5 min before theadministration of eye drops containing 10 ! 10 M PACAP38 or saline. When Benoxil
was pre-administered, the loss of the corneal reflex was confirmed by stimulating thecorneal surface with a blunt plastic tip before PACAP or saline treatment.
Histology. Adult C57BL/6 mice (Charles River Japan, Kanagawa, Japan) wereanaesthetized with sodium pentobarbital (50 mg kg ! 1, i.p.) and perfused withphosphate-buffered saline (PBS) followed by 2% paraformaldehyde in PBS.
Immediately afterwards, the infraorbital lacrimal glands were removed and fixed in
the same fixative for 24 h at 4 !C. Fixed tissues were embedded in paraffin forhistopathological studies with hematoxylin and eosin staining, or frozen sections
were used for double-labelling immunofluorescence staining.
Toxicology of the PACAP eye drops. In acute to semi-acute toxicology experiments, 2 ml of 10 ! 7 M PACAP or saline were applied as eye drops, and the eye balland accessory organs of treated animals were fixed with formaldehyde 48 h later. The
acute to semi-acute toxicological effects of PACAP on the cornea and infraorbital
lacrimal gland were evaluated based on hematoxylin and eosin staining.
Double-labelling immunofluorescent staining. The following primary antibodies
were used according to standard protocols43. Eight-mm-thick frozen sections were
blocked in 5% normal horse serum for 1 h and incubated overnight at 4 !C in
mixtures of the following primary antibodies: rabbit anti-PACAP antibody (1:2000;
Peninsula Laboratories, Inc., Belmont, CA, USA), rabbit anti- PAC1-R antibody(1:200; raised by using the N-terminal residue as an antigen44), mouse anti-smoothmuscle actin antibody (1:200; R&D Systems, Inc. Minneapolis, MN, USA), mouseanti-NeuN antibody (1:1000; Millipore, Billerica, MA, USA), goat anti-ChAT
antibody (1:100; Millipore), rabbit anti-AQP4 antibody (1:250; Millipore) andrabbit anti-AQP5 antibody (1:100; Millipore). Immunoreactivity was detectedusing an Alexa 488- or 546-labelled goat anti-mouse IgG, goat anti-rabbit IgG or
donkey anti-goat IgG antibodies (1:800; Invitrogen) following a 90-min incubation
at room temperature. After washing with PBS, the sections were incubated for
5 min with 4’,6-diamidine-2-phenylindole dihydrochloride (DAPI, 1:10,000; Roche
Diagnostics, Indianapolis, IN, USA) to stain cell nuclei. Labelling was imaged witha fluorescence microscope (Axio Imager Z1 with Apotome, Carl Zeiss,
Oberkochen, Germany). In the antigen absorption test, PACAP, PAC1-R or AQP5
antibody solution was incubated with a final concentration of 1 mM of antigens in
5% normal horse serum in PBS overnight at 4 !C. After centrifugation at 10,000g
for 20 min, the supernatant was used instead of the primary antibody.
Reverse transcriptase PCR. Total RNA was isolated from mouse infraorbital
lacrimal glands with an RNAeasy kit (Qiagen, Hilden, Germany). Reverse transcription was performed with random primers. Mouse Adcyap1 primers, forward;
50 -ATG ACC ATG TGT AGC GGA GCA AGG CTG G-30 , reverse; 50 -CTA CAAGTA TGC TAT TCG GCG TCC-30 (product size 525 bp), and mouse Adcyap1r1
primers, forward; 50 -CCT GTC GGT GAA GGC CCT CTA CAC A-30 , reverse;
50 - CCC AGC CCA AGC TCA AAC ACA AGT C-30 (798–801 bp product for the
hip or hop form, and 717 bp product for the short form), mouse Vpacr1 primers,
10

forward; 50 - GTA TGG ATG AGC AGC AAC AGA CTG-30 , reverse; 50 - TTG ATG
ATG GTG TCC CAG CAC-30 (product size 487 bp), mouse Vpacr2 primers,
forward; 50 -CAT CTC TGT GCT GGT CAA GGA C-30 , reverse; 50 - CGC CAT
CTT CTT TTC AGT TCA C-30 (product size 654 bp), mouse Gapdh primers,
forward; 50 - GCC AAG GTC ATC CAT GAC AAC-30 , reverse; 50 -GTC CAC CAC
CCT GTT GCT GTA-30 (product size 498 bp), and mouse Actb primers, forward;
50 -GTG GGC CGC TCT AGG CAC CAA-30 , reverse; 50 -CTC TTT GAT GTC
ACG CAC GAT TTC-30 (product size 540 bp) were mixed with the EX taq kit(TAKARA Bio, Otsu, Japan) with the PCR conditions of penetration for 15 s at
95 !C, annealing at 50 !C for 30 s, elongation at 72 !C for 1 min with a final
extension at 72 !C for 10 min. The PCR products were separated by gelelectrophoresis on an agarose gel and visualized with an illuminometer. Image ofthe gel have been cropped for presentation. The uncropped images in Fig. 2a areshown in Supplementary Fig. 14.
Real-time PCR. RNA isolation was followed by the reverse transcriptase PCR
method. Reverse transcription into complementary DNA was achieved using thereagents and protocol of the PrimeScript RT reagent kit (TaKaRa BIO, Kyoto, Japan).
The PCR primer set was as follows: mouse Aqp4 primers, forward; 50 -CTT TCT
GGA AGG CAG TCT CAG-30 , reverse; 50 -CCA CAC CGA GCA AAA CAA AGA
T-30 , mouse Aqp5 primers, forward; 50 -GGG TAA CCT GGC CGT CAA TG-30 ,reverse; 50 -TGA CCG ACA AGC CAA TGG ATA A-30 , and mouse Gapdh primers,
forward; 50 -TGT GTC CGT CGT GGA TCT GA-30 , reverse; 50 -TTG CTG TTG
AAG TCG CAG GAG-30 . Real-time PCR was performed using SYBR Premix Ex Taq
II reagent (TaKaRa BIO Inc.) and an ABI PRISM 7900 instrument (Applied
Biosystems, Lincoln, CA, USA). Relative gene expression was calculated using thecomparative delta Ct method with the Ct values of the housekeeping gene, Gapdh.
mRNA levels were normalized, with the percentage for control groups taken as 100%.
Repeated eye drop study. Adcyap1 ! / ! female mice were given eye drops
containing 10 ! 10 M PACAP or saline, 2 ml per eye (unilaterally), 2 times per day,
6 days per week for 3 weeks. We selected 18 female Adcyap1 ! / ! mice based on
similar average pre-treatment corneal keratinization scores on both sides. The
scoring method was the same as the previous evaluation for each side separately.
cAMP enzyme immunoassay. Enzyme-linked immunoassays for cAMP were
performed using a cAMP enzyme immunoassay (EIA) kit (Cayman Chemicals,
Grand Rapids, MI, USA) following the manufacturer’s instructions. In brief,
C57/BL6 mice were anaesthetized, and 2 ml of 10 ! 10 M PACAP was applied toboth ocular surfaces. Both infraorbital lacrimal glands were removed at 0, 7.5, 15 or
30 min after the application of the eye drops (n ¼ 8). They were then homogenizedin 200 ml of 5% trifluoroacetic acid on ice. After centrifugation at 1,500g for 10 min,the lysate was mixed with 1 ml of ether for 10 s. After removal of the ether, theaqueous layer was acetylated according to the EIA kit manufacturer’s instructions
and used for the EIA assay. The absorption in each well was measured with a plate
reader (POLARstar Omega; BMG LABTECH GmbH, Offenburg, Germany).
Immunoblotting. For western blot analysis, mice were euthanized by decapitationand their infraorbital lacrimal glands were immediately removed. The infraorbital
lacrimal glands were then homogenized in cold lysis buffer (10 mM Tris-HCl,
0.15 M NaCl and 1% Triton X-100) with a protease inhibitor cocktail (Sigma).
Homogenates were centrifuged at 12,000g for 30 min at 4 !C and the resultant
supernatant was collected as the total cell lysate, which was subsequently diluted
with SDS sample buffer (250 mM Tris-HCl (pH 6.8), 4% SDS, 40% glycerol, 4%2-mercaptoethanol and 0.002% bromophenol blue) and incubated for 12 h at 4 !C.
This lysate sample (30 mg) was electrophoresed on a 7.5% polyacrylamide gel
containing 0.1% SDS at 100 V. The protein bands were then transferred from thegel to polyvinylidinene fluoride membranes (Bio-Rad Laboratories, Inc. Hercules,
CA, USA) for 3 h at 100 mA. The membrane was initially blocked with 2%
Blockace (Dainihon Pharmaceutics, Osaka, Japan) in Tris- buffered saline with
Tween 20 (TBS-T) for 1 h at room temperature and probed overnight with a mousemonoclonal antibody for PKA (1:1,000, Cell Signaling Technology, Danvers, MA),
pPKA (1:1,000, Cell Signaling Technology), AQP4 (1:2,000, Millipore), AQP5
(1:4,000, Millipore), GAPDH (1:2,000, Millipore), or pan-cadherin (1:4,000,
Abcam, Cambridge, UK) at 4 !C. After incubation with a sheep anti-mouse IgG
HRP-conjugated antibody (1:2,000; GE Healthcare Bioscience, Little Chalfont, UK)
for 1 h at room temperature, protein bands were revealed using a SuperSignal West
Dura Extended Duration Substrate (Thermo Fisher Scientific Pierce Biotechnology,
Rockford, IL, USA) and exposed onto X-ray Film. Subcellular protein fractionation(cytosolic and membrane fractions) was performed using the ProteoExtract
Subcellular Proteome Extraction Kit according to the manufacturer’s instructions(Calbiochem, Hessen, Darmstadt, Germany). The fractions were precipitated using
a ProteoExtract Protein Precipitation Kit (Calbiochem). Precipitated protein wasthen dissolved in lysis buffer and the protein content was measured. Equivalentamounts of proteins (5 mg) for each fraction of subcellular fractionation were used
for immunoblotting. GAPDH and pan-cadherin signals were used as the internal
controls for cytosolic and membrane proteins, respectively. Image of the membrane
have been cropped for presentation. The uncropped images in Figs 5–7 are shownin Supplementary Fig. 14.

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Immunoprecipitation. All immunoprecipitations were carried out using theimmunoprecipitation kit Catch and Release v2.0 (Millipore) following the
manufacturer’s instructions. In brief, mouse infraorbital lacrimal gland lysate wasobtained 30 min after the application of eye drops. Four-hundred microgramprotein from the cell lysate, 1 mg of a mouse anti-pThy, -pSer, or -pThr(pan-phospho) IgG antibody (AnaSpec Inc, San Jose, CA, USA) for the capture
antibody or a normal mouse IgG (Millipore) as the control antibody, and 10 ml of
the antibody capture affinity ligand (total 500 ml) were mixed and placed in a Catchand Release v2.0 spin column containing prepacked immunprecipitation capture
resin. After end-over-end shaking for 16 h at 4 !C, the column was centrifuged,
washed 3 times and then eluted with 70 ml of the elution buffer. The eluent was
analysed by immunoblotting.
Removal of lacrimal glands. Under inhalation anaesthesia with sevoflurane, thehair from under the ear to the outer canthus of the eyes was shaved, and a 15 mm
incision was made. In the exorbital lacrimal gland removal model, the exorbital
lacrimal gland, which is located under the ear, was exposed and removed. In theinfraorbital and exorbital lacrimal gland removal model, the exorbital lacrimal
gland and the lacrimal duct were first isolated. The infraorbital lacrimal gland was
exposed by pulling the lacrimal duct, after which both lacrimal glands were
removed. The skin was then sutured and the animals were kept warm during theirrecovery from anaesthesia. The tear secretion level was checked before and after
surgery.
Semi-quantification of AQP5 immunoreactivity in lacrimal acini. The densities
of AQP5 immunoreactivity in the mouse infraorbital lacrimal gland 30 min afteradministration of eye drops containing saline, PACAP38, PACAP38 þ SQ22536, or
PACAP38 þ PACAP6–38 was evaluated. AQP5 immunostaining was performed
following the above method. One hundred pictures of acini from 10 infraorbital
lacrimal glands (10 acinus pictures/lacrimal gland) from five wild-type mice ineach group were cut out with grey scale. Using Image J software (ver. 1.44p), theaverage density in the apical membrane, and in four spots of 1 mm2 in thecytosolic area were measured. The value of the apical membrane density wasdetermined using the cytosolic density value as background. The quantification
was performed with a blinded test, masking sample data for another person
who used image J.
AQP5 siRNA treatment in vivo. An HPLC grade of non-target negative control
and three types of mouse AQP5 siRNAs (sequence was shown in Supplementary
Table 1) were designed and purchased from BONAC Corporation (Fukuoka,
Japan). Mouse AQP5 siRNAs (10 mM $ 3 siRNAs) or negative control siRNA(30 mM) were mixed with an equal volume of atelogene local use (KOKEN, Tokyo,
Japan) and gently incubated for 1 h at 4 !C. One day after the removal of the
exorbital lacrimal gland, the siRNA was applied to surround the infraorbital
lacrimal gland. AQP5 siRNA and control siRNA were used on opposite sides. The
next day, mice were anaesthetized with pentobarbital, after which PACAP38(10 ! 10 M) or saline eye drops were administered. At the 15, 30, 45 and 60 min
time points, the tear secretion level was measured using the cotton thread method.
The infraorbital lacrimal gland was then removed and the AQP4 and AQP5 levels
were checked by real-time PCR and immunostaining.
Whole-cornea immunostaining. After euthanasia, the epithelial layer of thecornea was scraped off under a stereoscopic microscope. The eye ball was excised,
then fixed in 4% paraformaldehyde for 2 h at room temperature. The cornea wasdissected and washed 3 times with phosphate-buffered saline (PBS), after which it
was incubated with blocking buffer (1% Triton X-100 and 10% normal horse serum
in PBS) for 1 h. The cornea was then incubated with primary antibody(Neurofilament 200, 1:500, Sigma, St Louis, MO) diluted in blocking buffer for 3
days at 4 !C, washed four times in PBS, and incubated with a secondary antibody(alexa546-labelled anti-rabbit IgG, 1:500) diluted in the blocking buffer for 4 h atroom temperature. After final washing in PBS, the cornea was mounted surface
side up on a glass slide with aqueous mounting medium. Labelling was imaged witha fluorescence microscope (Axio Imager Z1 with Apotome, Carl Zeiss,
Oberkochen, Germany).
Dot blotting. PACAP38 or VIP (1 ml of 0.2 to 25 pmol ml ! 1) was dropped on anitrocellulose membrane. After drying, the membrane was washed in Tris bufferedsaline with Tween 20 and blocking buffer, followed by immunoblotting as
described above. A primary anti-PACAP antibody (1:4,000; Peninsula Laboratories,
Belmont, CA, USA) was used in the dot blotting study.
MALDI-TOF mass spectrometry of intact PACAP38 in tear samples. Tear
samples were collected from mice by application of sterile filter paper strips (n ¼ 5,Schirmer paper), and PACAP38 was measured using MALDI-TOF mass spectrometry. Aqueous solutions of the standard and tear samples were loaded onto thetarget plate (MTP 384 massive target T, Bruker Daltonics, Bremen, Germany) by
mixing 1.0 ml of each solution with the same volume of a saturated matrix solution,

prepared fresh every day by dissolving a-cyano-4-hydroxycinnamic acid in
acetonitrile/0.1% trifluoroacetic acid (1/2, v/v). The mass spectrometer used in thiswork was an Autoflex II TOF/TOF (Bruker Daltonics) operated in the linear mode.
Ions were accelerated under delayed extraction conditions (140 ns) in the positive
ion mode with an acceleration voltage of 20.00 kV. The instrument uses a 337 nm
pulsed nitrogen laser, model MNL-205MC (LTB Lasertechnik Berlin GmbH,
Berlin, Germany). External calibration was performed in each case using the
average masses of the Bruker Peptide Calibration Standard (#206195, Bruker
Daltonics). Protein masses were acquired within a range of 1,000–8,000 m/z. Eachspectrum was produced by accumulating data from 800 consecutive laser shots.
Bruker FlexControl 2.4 software was used for control of the instrument and Bruker
FlexAnalysis 2.4 software for spectrum evaluation.
Nanospray desorption electrospray ionization Orbitrap MS/MS analyses of
PACAP38 in tear samples. Nanospray desorption electrospray ionization(nano-DESI) was used to acquire MS/MS spectra directly from Schirmer paper
containing tears (n ¼ 6). The nano-DESI probe consisted of two fused silica
capillaries (ID 50 mm, OD 150 mm, Polymicro Technologies, Molex, Lisle, IL)
positioned at an angle to each other45. A solvent, consisting of methanol/water(9/1, v/v) with 2% formic acid, was propelled, at 0.5 ml min ! 1, through the primarycapillary, forming a liquid bridge to the secondary capillary. The secondary
capillary transported the solvent to the mass spectrometer inlet for nanosprayionization. The filter paper was soaked with 10 ml 0.1% trifluoroacetic acid
(99%, Sigma-Aldrich) on a regular glass slide. The glass slide was placed on a
motorized x,y,z-stage (Newport Corporation, Irvine, CA, USA) to position thesample under the nano-DESI probe46,47. Material was extracted from the wet
surface of the Schirmer paper by the nano-DESI probe and analysed using a
QExactive Plus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany). The instrument mass resolving power was set to 70,000 (m/Dm) and ahigh voltage of 3 kV was applied to the primary capillary. Selective ion monitoring
was set to m/z 648.5±2, corresponding to PACAP38 (z ¼ 7) and tandem mass
spectrometry was performed at m/z 648.5±1 Da, using higher energy
collision-induced dissociation with a normalized collision energy of 20 applied.
The same settings were used for the PACAP38 standard, wild-type mouse samples
and PACAP ! / ! mouse samples.
Effect of PACAP on angiogenesis. The experiments on endothelial cell tube
formation were conducted in 24-well dishes using an angiogenesis kit (Kurabo,
Okayama, Japan), according to the manufacturer’s instructions. Human umbilicalvein endothelial cells and fibroblasts were co-cultured in medium containing
vascular endothelial growth factor (final 10 mg l ! 1) with various concentrations of
PACAP38 (10 ! 9, 10 ! 6 M) and PACAP6–38 (10 ! 8, 10 ! 6 M), with the medium
exchanged on days 4, 7 and 9. On day 11, the cells were washed and directly fixedin the wells with 70% ice-cold ethanol for 30 min. The fixed cells were serially
incubated with 1% bovine serum albumin (BSA) in the buffer, a mousemonoclonal antibody against human CD31, an alkaline phosphatase-conjugatedgoat anti-mouse IgG, and a nitro-blue tetrazolium chloride (NBT)/5-bromo-4chloro-30 -indolylphosphatase p-toluidine salt from the kit, and then washed andphotographed. The images were analysed using Angiogenesis Image Analyzer
software (Kurabo) to measure the gross area of the CD31-positive tubes(the area of endothelial tubes) and the length of CD31-positive tubes in culture.
Data are shown as a percentage of the area of the endothelial cell tubes in theuntreated cultures.
Systemic infusion of PACAP38. Systemic infusion of PACAP was done as in ourprevious study48. Briefly, PACAP38 (5 nmol kg ! 1) or PACAP38 plus PACAP6–38(50 nmol kg ! 1) was injected into the jugular vein with vehicle (0.1% BSA in saline)
under inhalation anaesthesia with sevoflurane. A PE10 polyethylene tube
connected to an Alzet osmotic pump (0.5 ml h ! 1; DURECT Corporation,
Cupertino, CA, USA) that was filled with the vehicle, PACAP38 (32 pmol ml ! 1), or
PACAP plus PACAP6–38 (320 pmol ml ! 1) was then inserted for continuous
administration. Tear volume was measured with the cotton thread method fourdays after the infusion commenced.
Statistical analyses. Data are presented as the mean±s.e. (n ¼ sample size). The
effects of treatments were analysed with the Mann–Whitney U-test (Figs 1i,j and4c), the two-tailed Student’s t-test (Figs 6b,c and 8b,c), one-way ANOVA followedby the Dunnett test (Figs 3b, 5a–c and Fig. 7e) or a one-way ANOVA followed by
the Tukey test (Figs 1g,h, 3c–h, Figs 5d and7b and Fig. 8e and Supplementary
Figs 6–8, 10 and 12). The two-tailed Spearman’s correlation test was used to
identify correlations between corneal grade and tear volume in Adcyap1 ! / ! mice.
Differences due to the treatments were considered as significant for values ofPo0.05.
Data availability. The authors declare that the data supporting the findings of thisstudy are available within the article and its Supplementary Information Files.

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Acknowledgements
We thank Dr Jerome L. Maderdrut for editorial assistance. In memory of
Dr Akira Arimura, for his many helpful suggestions. This work was supported
by JSPS KAKENHI Grant Numbers 24592680, 24592681, 23249079, 15K15670,
26293020, 26670122, 16H02684 and 15H01288); the JSPS Program for Advancing
Strategic International Networks to Accelerate the Circulation of Talented Researchers,
Grant no. S2603 (H.H.). Support was also received from the Hungarian Scientific
Research Fund (OTKA, K104984, D.R., P.K., J.F., L.M.), the Lendulet Program, theArimura Foundation, KTIA_NAP_13-1-2013-0001 (D.R.). PTE AOK KA 34039-2009,2011, 2012-2013 13-18 (L.M.), VR 621-2013-4231 and SSF ICA-6 (I.L.).

Author contributions
T.N. designed and performed the experiments, evaluated the results, and wrote
part of the manuscript. H.O. scored the corneal keratinization and performed themolecular biochemistry. T.S. provided valuable expert advice. N.K. performed theeye drop experiment. L.M., I.L., J.F., P.K. and D.R. performed the mass
spectrometry experiment and wrote part of the manuscript. S.Y. performed theimmunohistochemical experiment. H.H., N.S. and A.B. supplied the experimental
material and edited the manuscript. S.S. designed and supervised the experiments andwrote the manuscript.

Additional information
Supplementary Information accompanies this paper at http://www.nature.com/
naturecommunications
Competing financial interests: The authors declare no competing financial interests.

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How to cite this article: Nakamachi, T. et al. PACAP suppresses dry eye signs
by stimulating tear secretion. Nat. Commun. 7:12034 doi: 10.1038/ncomms12034(2016).

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