Karakteristik efek reseptor vasopressin V2 pada berkeringat, keseimbangan cairan,

dan kinerja selama latihan.

Characterization of the effects of the vasopressin V2 receptor on sweating, fluid balance,
and performance during exercise

Penulis
Nama : Lalu Muh. Furqon Ali Hidayat
Nim : 1810301056

DOSEN PENANGGUNG JAWAB MODUL FISIOLOGI LATIHAN

Tyas Sari Ratna Ningrum, SST.Ft.,M.Or

PROGRAM STUDI FISIOTERAPI

FAKULTAS ILMU KESEHATAN
UNIVERSITAS ‘AISYIYAH YOGYAKARTA

Abstrak
Efek konservasi air arginin va- sopressin (AVP) dari keringat telah
dihipotesiskan tapi tidak dievaluasi. penyisipan keringat dan kelenjar ludah
aquaporin-5 (AQP5) saluran air AVP-dimediasi melalui aktivasi jenis vasopressin 2
reseptor (V2R) tetap menarik, namun belum dicari, mekanisme yang bisa
menghasilkan keringat lebih terkonsentrasi dengan yang dihasilkan. Sepuluh pelari
berpartisipasi dalam double-blind acak control trial treadmill di bawah tiga kondisi
farmakologis yang terpisah: plasebo, V2R agonis (0,2 mg desmo- pressin), atau V2R
antagonis (30 mg tolvaptan). Setelah sosialisasi, pelari berlari selama 60 menit pada
60% dari kecepatan puncak diikuti dengan uji coba kinerja kelelahan. variabel hasil
dikumpulkan di tiga titik waktu latihan: dasar, setelah mapan run, dan setelah kinerja
jangka panjang.
 Pendahuluan
Arginine vasopressin ( AVP) adalah regulator neuroendokrin utama dari metabolisme
air, bertindak untuk mempertahankan osmolalitas plasma dalam kisaran fisiologis normal
275-295 mosmol / kg H 2 O (38). Regulasi osmotik tonisitas plasma umumnya terjadi
melalui aktivasi jenis vasopressin 2 reseptor (V2R) di ginjal. Aktivasi V2R memulai
translokasi dan penyisipan berikutnya dar aquaporin-2 (AQP2) saluran air ke membran apikal
mengumpulkan saluran sel pokok. Hal ini memungkinkan untuk reabsorpsi air di sepanjang
OS gradien MOTIC dan antidiuresis bersamaan ketika tonicities plasma.
Tujuan utama dari penelitian ini adalah untuk menilai apakah atau tidak berkeringat
kadar air dan konsentrasi natrium yang akut diatur oleh perubahan dinamis dalam AVP
bertindak atas V2R selama berjalan treadmill. Tujuan sekunder adalah untuk eval- uate
menjalankan kinerja dan suhu inti untuk mencirikan lanjut peran AVP dalam keseimbangan
terkoordinasi cairan dan suhu homeostasis selama latihan. Potensi signifikansi dari temuan
positif dapat: 1) membuat sambungan yang masuk akal antara AVP dan AQP5 saluran air
translokasi tion, dan 2) menghidupkan kembali ilmiah minat kelenjar keringat primitif
sebagai regulator penting dari cairan dan keseimbangan natrium selamalatihan ketahanan.
 Bahan dan Metode
Kriteria Peserta adalah sehat (tidak ada akut atau kronis kondisi medis yang
memerlukan penggunaan obat resep biasa) sepuluh, pelari kebiasaan (50 km mingguan
berjalan jarak untuk 6 minggu sebelum studi partisipasi) be- tween usia 18-60 tahun yang
direkrut untuk berpartisipasi dalam uji coba ini. Kriteria eksklusi adalah 1) sejarah masalah
ginjal; 2) ketidakmampuan untuk merasakan haus; 3) Kesulitan menelan; 4) riwayat
gangguan pencernaan; dan 5) sejarah pingsan terkait dengan venipuncture. Lisan dan tertulis
informed consent diperoleh dari masing-masing peserta sebelum sidang dimulai.
 Hasil

Sepuluh peserta berhasil menyelesaikan uji coba studi (8 laki-laki dan 2 perempuan).
Satu perempuan melaporkan menstruasi teratur dan diuji hanya selama fase folikuler dari
siklus sementara haid terhenti perempuan lain dan diuji setiap minggu, seperti subjek laki-
laki. Sejak uji coba sebelumnya menunjukkan tidak ada perbedaan jenis kelamin dalam salah
satu variabel(13), data pria dan wanita digabungkan untuk analisi ses. Usia rata-rata peserta
adalah 35,5 13,1 tahun dengan 8.8 8,7 tahun menjalankan pengalaman dan indeks massa
tubuh (BMI) 22,5 3,3 kg / cm 2. Rata-rata v O2peak dari semua peserta adalah 58,7 6,2 ml ·
kg 1 · min 1, dengan puncak treadmill kecepatan berjalan dari 11,0 1.4 mph dan rata-rata
waktu untuk v O2peak 11,2 2,4 s. Rata-rata kecepatan yang diwakili 60% dari v O2peak
( ditunjuk mapan berjalan kecepatan) adalah 6,6 0,8 mph.
 Diskusi

Hipotesis utama yang kadar air keringat, dilihat dari keringat [Na ], Diatur oleh V2R
vasopressin di tingkat kelenjar keringat tidak didukung oleh hasil penelitian ini. stimulasi
V2R oleh selektif V2R agonis desmopresin itu dibuktikan dengan nilai-nilai osmolalitas urin
yang lebih tinggi dari nilai yang diperoleh baik dalam plasebo dan kondisi antagonis V2R di
semua titik latihan diuji (Gbr. 4 B). Demikian pula, con Penegasan dari V2R antagonisme
dengan selektif V2R antagonis tolvaptan itu dibuktikan dengan nilai-nilai osmolalitas urin
yang secara signifikan lebih rendah dari yang diperoleh baik dalam plasebo dan kondisi
agonis V2 di semua titik latihan diuji. Menghalangi V2R ditambah ekskresi air bebas kemih,
diverifikasi oleh volume produksi urin yang lebih besar tiga kali lipat dari pada plasebo dan
V2R uji agonis. Kurangnya penurunan signifikan dalam volume urin dengan pengobatan
desmopresin kemungkinan merefleksikan dekat-maksimal antidiuresis latihan-induced di
plasebo.
 Perspektif

Penelitian ini menggunakan manipulasi farmakologis dengan reseptor-spesifik agonis

dan antagonis untuk mengaktifkan dan menghambat AVP V2R, sehingga mengesampingkan
efek dari sekresi AVP endogen. Dengan demikian, hasil ini memberikan bukti tidak langsung
bahwa V2R tidak mempengaruhi translokasi saluran AQP5 dalam keringat dan kelenjar
ludah.
Penelitian ini diverifikasi bahwa V2R aktivasi diproduksi secara fisiologis yang tepat,
osmotik diinduksi, perubahan sekunder dalam endogen [AVP] P dan haus stimulasi melalui
aktivasi atau blokade AQP2 saluran penyisipan di saluran pengumpul ginjal. Meskipun
antagonis V2R diproduksi hipernatremia ringan tanpa ditemani oleh kerugian volume plasma
yang lebih besar pada akhir sidang latihan, keseimbangan air secara keseluruhan, perubahan
berat badan, dan latihan perfor- Mance tidak secara signifikan berbeda antara kondisi.
 Am J Physiol Regul Integr Comp Physiol 307: R366 –R375, 2014.
First published June 18, 2014; doi:10.1152/ajpregu.00120.2014.
CALL FOR PAPERS
Central Control of Fluid and Electrolyte Homeostasis
Characterization of the effects of the vasopressin V2 receptor on sweating,
fluid balance, and performance during exercise
Tamara Hew-Butler,1 Jed Hummel,1 Brian C. Rider,1 and Joseph G. Verbalis2
1
Exercise Science Program, Oakland University, Rochester, Michigan; and 2Endocrinology and Metabolism, Georgetown
University Medical Center, Washington, DC
Submitted 18 March 2014; accepted in final form 11 June 2014
Hew-Butler T, Hummel J, Rider BC, Verbalis JG. Character-
ization of the effects of the vasopressin V2 receptor on sweating,
fluid balance, and performance during exercise. Am J Physiol Regul
Integr Comp Physiol 307: R366 –R375, 2014. First published June
18, 2014; doi:10.1152/ajpregu.00120.2014.—A regulatory effect of
arginine va- sopressin (AVP) on sweat water conservation has been
hypothesized but not definitively evaluated. AVP-mediated insertion
of sweat and salivary gland aquaporin-5 (AQP5) water channels
through activation of the vasopressin type 2 receptor (V2R) remains
an attractive, yet unexplored, mechanism that could result in a more
concentrated sweat with resultant decreased water loss. Ten runners
participated in a double-blind randomized control treadmill trial under
three separate pharmacological conditions: a placebo, V2R agonist
(0.2 mg desmo- pressin), or V2R antagonist (30 mg tolvaptan). After
a familiarization trial, runners ran for 60 min at 60% of peak speed
followed by a performance trial to volitional exhaustion. Outcome
variables were collected at three exercise time points: baseline, after
the steady-state run, and after the performance run. Body weight
losses were <2% across all three trials. Significant pharmacological
condition effects were noted for urine osmolality [F = 84.98; P <
0.0001] and urine sodium concentration ([Na+]) [F = 38.9; P <
0.0001], which verified both pharmacological activation and
inhibition of the V2R at the kidney collecting duct. Plasma osmolality
and [Na+] demonstrated significant exercise (F = 26.0 and F = 11.1;
P < 0.0001) and condition (F = 5.1 and F = 3.8; P < 0.05) effects
(osmolality and [Na+], respectively). No significant exercise or
condition effects were noted for either sweat or salivary [Na+].
Significant exercise effects were noted for plasma [AVP] (F = 22.3;
P < 0.0001), peak core temperature (F = 103.3; P < 0.0001),
percent body weight change (F = 6.3; P = 0.02), plasma volume
change (F = 21.8; P < 0.0001), and thirst rating (F = 78.2; P <
0.0001). Performance time was not altered between conditions (P =
0.80). In summary, AVP acting at V2R does not appear to regulate
water losses from body fluids other than renal excretion during
exercise.
arginine vasopressin; sweat sodium; V2 antagonist; running
ARGININE VASOPRESSIN (AVP) is the primary neuroendocrine
regulator of water metabolism, acting to maintain plasma
osmolality within the normal physiological range of 275–295
mosmol/kg H2O (38). Osmotic regulation of plasma tonicity
generally occurs through activation of the vasopressin type 2
receptor (V2R) in the kidney. Activation of V2R initiates the
translocation and subsequent insertion of aquaporin-2 (AQP2)
water channels into the apical membrane of the collecting duct
principal cells. This allows for water reabsorption along os-
Address for reprint requests and other correspondence: T. Hew-Butler
School of Health Science, 3157HHB, Oakland Univ., Rochester, MI 48309-
4482 (e-mail: hew@oakland.edu).
motic gradients and concomitant antidiuresis when plasma
tonicities are high (46).
Unlike this well-characterized interaction of V2R-
stimulated translocation of AQP2 channels in the kidney, a
regulatory effect of AVP on sweat water conservation has yet
to be determined. The capability of water reabsorption within
an estimated 1.6 to 4.0 million sweat glands (41) is a
teleological appealing possibility, which could maximize water
conserva- tion during prolonged periods of thermoregulatory
stress. Ex-
ercise performed at intensities above 50% of V˙ O2
maximum
can shift up to 92% of all water (and 87% of all sodium losses)
away from urine production toward requisite thermoregulatory
sweat production (6, 29, 32). This primary shift in water and
sodium excretion routes would be of increasing homeostatic
importance during endurance exercise, particularly when per-
formed in hot environments. Thus the capacity for osmotically
mediated water conservation to occur in sweat glands would
be an attractive effector mechanism aimed at maintaining fluid
homeostasis during sustained periods of thermoregulatory
stress.
Sweat glands appear to have acute fluid and sodium regula-
tory elements such as aquaporin-5 (AQP5) water channels (4),
Na+/H+ exchangers (10), adrenergic nerve terminals, and þ-
adrenergic receptors (35), cystic fibrosis transmembrane
conductance regulator chloride channels (CFTR-Cl), and epi-
thelial sodium channels (ENaC) (9, 35). Much of the available
evidence support changes in sweat sodium concentration
([Na+]) to reflect changes in sweat rate (11). However, two
studies recently reported statistically significant positive asso-
ciations between sweat [Na+] and plasma AVP concentrations
([AVP]P) (5, 13). These independent investigations hypothe-
size that increases in sweat [Na+] might possibly be mediated
by an AVP-dependent mechanism, perhaps by increasing
water reabsorption either through vasopressin receptor type 1a
(V1aR) or V2R stimulation. The presence of AVP receptors
within human sweat glands is only weakly supported by
indirect evidence (37). Prior investigations looking specifically
at the effects of vasoconstriction on sweat rate and
composition do not support activation of the V1aR as a
plausible mecha- nism for any association between sweat
[Na+] and [AVP]P (7, 33, 34, 45). Therefore, AVP-mediated
insertion of AQP5 water channels through activation of the
V2R within the apical membrane of the sweat gland remains
an enticing, yet unex- plored, mechanism.
AQP5 water channels have been identified within the apical
membrane of secretory cells located in salivary, sweat, lacri- mal, and airway submucosal glands as well as in corneal,
R366 0363-6119/14 Copyright © 2014 the American Physiological Society http://www.ajpregu.org
 AVP EFFECTS ON SWEATING AND FLUID REGULATION R367
nasopharyngeal, uterus when
bronchial, and exposed to
uterus progesterone in
epithelium (4, vivo (20).
20, 28, 43). Whether or not
AQP5 channels the principle
in sweat duct hormone of
epithelium have water
been found in homeostasis,
the distal apical AVP, stimu-
tubule segments lates synthesis or
(4), a location membrane
mirroring that of insertion of
the collecting AQP5 water
duct principle channels has yet
cells within the to be explored,
kidney (17). In although the
renal epithelium, mechanism
collecting duct underlying AVP
aquaporin activation of
activity is the AQP2 channels
key step in the via V2R
final stimulation in
manipulation of response to
urine water loss hypertonic stress
(17), which has been clearly
further supports delineated within
the the kidney (17).
physiological This primary
plausibility of a aim of this study
par- allel was to critically
relationship assess whether
between AVP or not sweat
and AQP water content
activity within and sodium
sweat gland concentration
epithelium. are acutely
AQP5 water regulated by
channels appear dynamic
to be regulated changes in AVP
by factors such acting on the
as tumor V2R during
necrosis factor- treadmill
a, hypertonic running.
stress, and/or Secondary aims
activation of the were to eval-
cyclic adenosine uate running
monophosphate performance and
sec- ond core temperature
messenger to further
system (43). characterize the
Progesterone is role of AVP in
currently the the coordinated
only known balance of fluid
hormonal and temperature
mediator of homeostasis
AQP5 channel during exercise.
protein expres- The potential
sion, as was significance of
demonstrated in positive findings
the epithelial could: 1)
cells of the rat establish a
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 plausible connection between AVP and according to exercise laboratory
AQP5 water channel transloca- tion, and 2) hour (mph) every 60 s until the participant thirst. Thirty protocol for trials
could no longer keep pace with the treadmill minutes before 2, 3, and 4 (B).
revive scientific interest in the primitive (volitional exhaustion).
sweat gland as an important regulator of the trial began
Trials 2, 3, and 4 were then conducted (time 0), each
fluid and sodium balance during endurance utilizing the randomized exercise test lab participant
exercise. protocol (Fig. 1A). These three trials utilized arrived to the
the same exact protocol, differing only in the exercise lab
MATERIALS AND METHODS pharmacological intervention used to assess the where he or she
role of the AVP V2R on sweat sodium was prepped with
Participants concentra- tion and performance. In side-by-side
randomized, double-blind order (both par- sweat patches on
Ten healthy (no acute or chronic medical ticipant and investigator blinded to the
conditions requiring regular prescription the upper back
intervention), either a placebo pill, the V2R and a temperature
medication use), habitual runners (>50 km antagonist tolvaptan (Samsca, 30 mg tablet), or sensor reading
weekly running distance for >6 wk before the V2R agonist desmopressin (DDAVP, 0.2 con- firmed. The
study participation) be- tween the ages of 18 – mg tablet) was ingested along with a CorTemp CorTemp data
60 yr were recruited to participate in this trial. Core Temperature Sensor 2 h before logger was then
Exclusion criteria were 1) history of kidney commencement of the exercise trial (time 0) attached to each
problems; 2) inability to sense thirst; 3) with 240 ml of bottled water. All tablets were partici- pant’s
difficulty swallowing; 4) history of placed within identical colored capsules, with waist, enclosed
gastrointestinal disorders; and 5) history of the investigator from Georgetown University
fainting associated with venipuncture. Oral and within a zippered
(J. G. Verbalis) creating the randomiza- tion running belt for
written informed consent was obtained from key for each participant. Ten coded packets
each participant before the trial began. This continuous
(one per study participant, each containing a monitoring.
study was approved by both the Oakland placebo, V2R agonist, and V2R antag- onist
University and Georgetown University Medical Immediately
tablet labeled in separate envelopes as trial 2, preceding the
Center Institutional Review Boards. The study 3, or 4) were then sent to Oakland University
protocol was registered in ClinicalTrials. gov as exercise test, all
where the lead investigator (T. D. Hew- Butler) participants
identifier NCT02084797. distributed one packet to each participant after

Experimental Protocol
All consenting participants presented to the
exercise lab on four separate occasions, at the
same time of day, every other week. Each
participant was instructed to refrain from
exercise 24 h immediately preceding and
competitive events of >21 km within 3 days of
each lab trial. The first lab visit (trial 1) was a
treadmill familiarization trial
during which an incremental test to volitional
exhaustion was per- formed to determine each
participant’s V˙ O2peak. This V˙ O2peak test was
then utilized in trials 2, 3, and 4 as the
performance test. Specifically,
after a 5-min warmup, each participant ran for 1
min at a self-selected comfortable speed,
equivalent to the average speed of an easy run.
Thereafter, the speed on the treadmill was
increased by 0.5 miles per

completion of the familiarization trial (trial 1). Fig. 1. Diagrams of

The randomization key was unlocked only after the double-blind,
all data collection was completed. randomized
No food was allowed following ingestion of control, study
the capsule and sensor, design (A) and
but ad libitum water intake was allowed randomized
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 R368 AVP EFFECTS ON SWEATING AND FLUID REGULATION

were asked to completely void their bladder. The urine was collected measurement of body weight, plasma volume, [AVP]P, plasma
into a prelabeled plastic container that was later measured for volume oxytocin concentration ([OT])P, and thirst rating.
(ml). After the urine void, 1 ml of saliva was collected via the passive
drool technique. Then 5 ml of venous blood were withdrawn from an
antecubital vein with the participant in a seated position. Each par-
ticipant was weighed in running clothes without shoes on a WW42D
impedance scale (Weight Watchers, New York City, NY) immedi-
ately before mounting the treadmill. All of the measures collected
before treadmill running began have been designated as “baseline”
measures.
Each randomized exercise test (Fig. 1B) consisted of 60 min of
“steady-state” running at a speed corresponding to 60% of each
participant’s V˙ O2peak running speed. After completion of the 60-min
steady-state run segment, the left upper back sweat patch was quickly
removed, 1 ml of saliva was collected, and 5 ml of blood withdrawn
via venipuncture from an antecubital vein with the participant seated.
The participant was then asked to completely void his or her bladder
and all urine was collected in a prelabeled plastic container. After
voiding was completed, each participant was again weighed before
remounting the treadmill for the performance segment of the trial. All
of the measures collected immediately following steady-state running
were designated as “steady-state” measures. Data collection
following the steady-state run was performed within 5 min of
commencing the performance segment.
Each participant then immediately underwent a performance trial:
running for 1 min at a speed according to 60% V˙ O2peak, then
increas-
ing the treadmill speed 0.5 mph every 60 s until volitional exhaustion.
Each participant was blinded to their performance time and speed
between the trials. Blood, sweat (right upper back patch), saliva, and
urine were then collected immediately upon completion of the per-
formance segment of the trial. A treadmill-modified whole body
washdown was performed at the end of the randomized exercise test
to determine whole body sweat sodium loss (1, 42). All of the
measures collected after completion of the performance segment of
the trial treadmill have been designated as “performance” measures.
Thirst rating was assessed before the trial started (baseline) and
immediately poststeady state and performance testing using a 10-point
anchored scale. On the scale, “0” was the lowest possible rating (not
thirsty at all) while “10” was the highest possible rating (extremely
thirsty).
If any research participant needed to urinate during the 60-min
steady-state run (which was possible using the V2R antagonist), those
individuals were allowed to dismount the treadmill and empty their
bladder (into a container for further analysis and volume measure-
ment). The time lost was then subsequently added to the overall
running time so that all participants ran for a total of 60 min on the
treadmill during the steady-state portion of the run. Since the average
time to volitional exhaustion during the performance test (V˙
O2peak
test) was ~10 min (10.4 ± 0.9 min in the pilot trial) it was deemed
unlikely the participants needed to urinate during the performance
stage of the trial. Bottled water was freely available for each partic-
ipant throughout the trial. All core temperature data were downloaded
from the data logger after the trial was completed, with the peak
temperature for each stage of the trial recorded.

Outcomes
The primary outcome variables included biochemical
measurement of sodium concentration in sweat, blood, and urine
during each randomized trial (placebo, V2R agonist and V2R
antagonist). Sec- ondary outcome variables included measurement of
exercise perfor- mance (time during the incremental test to
exhaustion) and peak core temperature. Tertiary variables included
 Biochemical and Fluid Balance Analyses
Plasma samples. All venous blood samples were collected in
chilled lithium heparin tubes. All blood samples were immediately
centrifuged at 3,000 rpm for 10 min. The separated plasma was then
placed into two 1.5-ml Eppendorf tubes and immediately stored at
—80°C until batch analysis could be performed at Georgetown Uni-
versity Medical Center. Plasma [Na+] and potassium concentration
([K+]) was measured using ion-selective electrodes (Beckman Syn-
chron EI-ISE, Fullerton, CA). Osmolality was measured using a
vapor pressure osmometer (VAPRO 5520, Wescor, Logan, UT).
Changes in plasma volume (PV) were estimated by comparing pre-
and post- intervention measurements of plasma protein (PP) using a
clinical refractometer (Schuco Clinical Refractometer 5711-020,
Japan)(44). Plasma [AVP]P and oxytocin ([OT]P) were measured by
specific RIAs following acetone-ether extraction as described
previously (47). The standard curve for AVP is linear between 0.5
and 10.0 pg/tube with the use of a synthetic AVP standard
(PerkinElmer Life Sciences, Boston, MA). The minimum detectable
concentration of AVP in extracted plasma was 0.5 pg/ml. The AVP
antiserum (R-4) displayed 1% cross-reactivity with OT. The
standard curve of the OT assay was linear between 0.25 and 5.0
pg/tube with the use of a synthetic OT standard (PerkinElmer Life
Sciences). The minimum detectable con- centration of OT in
extracted plasma was 0.25 pg/ml. The OT antiserum (Pitt-Ab2)
displayed 1% cross-reactivity with AVP.
Sweat samples. Sweat was collected via two different techniques:
1) sweat patch collection, and 2) whole body washdown technique
(WBW). For the sweat patch collection, the upper back was cleansed
with alcohol, rinsed with distilled water, and allowed to air dry.
After the area was clean and dry, two sweat patches were applied
side-by- side to the upper back (to the left and right of the thoracic
spine), just below the shoulder blades. Each patch consisted of one
sheet of 8-ply 5 cm × 5 cm sterile gauze covered with one sterile
sheet of 10 cm × 12 cm Tegaderm. For sample collection, after the
AVP EFFECTS ON SWEATING AND FLUID REGULATION
+ +
electrolyte analyses could be performed. Urine [Na ], [K ], and
osmolality were measured as in the plasma samples. After these
sample aliquots were removed and stored, all remaining urine was
measured for volume (in ml) before being discarded.
Fluid balance parameters. Upon entering the laboratory, all par-
ticipants were given free access to bottled water throughout the trial
and asked to drink according to thirst. The volume of water was
quantified by measuring the remaining contents and subtracting this
volume from the known volume of water ingested by counting bottles
emptied during the trial. An estimate of total fluid losses during the
trial was calculated immediately before and after treadmill running by
the following balance equation: fluid balance = water ingested (ml)
— urine produced (ml) — body weight losses (kg losses were used to
approximate sweat losses in ml).
Statistical Analyses
A two-way repeated measures ANOVA was used to examine
potential differences in exercise (baseline, steady-state, and perfor-
mance segments) versus condition (placebo, V2R agonist, and V2R
antagonist) for plasma, urine, sweat, and saliva [Na+], [K+], and
osmolality values. A one-way repeated measures ANOVA was used
to examine potential differences in fluid balance and performance
across conditions (placebo, V2R agonist, and V2R antagonist). When
necessary, a Bonferroni correction with unweighted means was per-
formed post hoc to further identify exercise (row) and/or condition
(column) differences between mean values. Linear regression analy-
ses (Pearson’s) were performed to assess relationships between vari-
ables. Statistical significance a level was set a priori at P < 0.05. All
data were presented as means ± SD unless otherwise noted. It was
steady-state exer- cise, the left sweat patch was carefully removed
and the contents of the gauze poured directly into an Eppendorf tube
that was immedi- ately closed and frozen at —80°C until further
analyses could be performed. After the performance stage of the test,
the right patch was similarly removed with the sweat immediately
collected, frozen, and stored. All assessments of [Na+] and [K+] were
measured as in the plasma samples. For the WBW technique (1, 42),
after completion of the performance stage of the trial, each participant
was placed in a plastic kiddie pool within a fully enclosed walk-in
tent. With the participant seated in the middle of the pool, one
researcher poured
~1.5 liters of distilled water over the participant’s head and body.
The participant was then asked to remove all clothing in the privacy
of the tent and then pour the remaining amount of distilled water
(3.78 liters total) over his or her body, leaving the clothes and the
water jug within the pool. The participant was provided a clean towel
to dry him or herself off and asked to leave the towel within the
kiddie pool. After the participant redressed, the contents of the kiddie
pool were swirled around vigorously for 60 s and two 1.5-ml
Eppendorf tubes were filled with samples of water obtained from the
contents of the kiddie pool. These whole body washdown samples
were then stored at —80°C until electrolyte analyses could be
performed. Sweat [Na+] and [K+] were measured as in the plasma
samples.
Saliva samples. All salivary samples were collected using the
passive drool technique, in which all participants allowed saliva to
accumulate within their mouths. Each participant then allowed the
accumulated saliva to flow into a Dixie cup until ~1 ml of saliva was
collected. The saliva was then immediately expelled into an Eppen-
dorf tube, closed, and stored at —80°C until electrolyte analyses
could be performed. Saliva [Na+] and [K+] were measured as in the
plasma samples.
Urine samples. All urine samples were collected in plastic contain-
ers. Two 1.5-ml samples were removed and placed into Eppendorf
tubes, which were immediately closed and stored at —80°C until
R369
previously estimated that 5– 8 participants would provide adequate
statistical power (þ = 0.2) to detect changes in sweat electrolyte
concentrations that would exceed the within-participant coefficient of
variation based on previous studies (25).
RESULTS
Ten participants successfully completed the study trial (8
males and 2 females). One female reported regular menses and
was tested only during the follicular phase of her cycle while
the other female ceased menstruation and was tested every
other week, like the male subjects. Since previous pilot testing
demonstrated no sex differences in any of the variables of
interest (13), male and female data were combined for analy-
ses. The mean age of the participants was 35.5 ± 13.1 yr with
8.8 ± 8.7 yr of running experience and a body mass index
(BMI) of 22.5 ± 3.3 kg/cm2. The average V˙ O2peak of
all participants was 58.7 ± 6.2 ml·kg—1·min—1, with a peak
treadmill running speed of 11.0 ± 1.4 mph and average time
to V˙ O2peak of 11.2 ± 2.4 s. The average speed that
represented 60% of V˙ O2peak (the designated steady-state
running speed) was 6.6 ± 0.8 mph.
Changes in [Na+] and [K+] from plasma, urine, saliva, and
sweat samples over both exercise (baseline, steady-state, and
performance) and condition (placebo, V2R agonist, and V2R
antagonist) are shown in Figs. 2 and 3. For plasma [Na+] (Fig. 2A),
there was a significant exercise effect [F(2,79) = 11.14] and
condition effect [F(2,79) = 3.81]. For plasma [K+] (Fig. 2B),
Fig. 2. Two-way repeated-measures ANOVA results for plasma and urine sodium ([Na +]) and potassium ([K+]) concentrations. Exercise is represented on the x-
axis (Baseline, Steady-State, and Performance) and represents the temporal order in which the samples were taken. Condition (Placebo, V2R Agonist, and V2R
Antagonist) is represented on the y-axis and represents the values obtained during each of the three separate pharmacological treatments. All data are represented
by means (symbols) and standard deviation (bars). The standard deviation for the placebo trial (open circle) is represented by the wide “T” and solid line; the
V2R Agonist trial (open square) is represented by the more narrow “T” and dashed line; while the V2R Antagonist (open triangle) is represented by the bold
“I” and dotted line to distinguish between the overlapping error bars. Significant differences from post hoc analyses are represented on the graphs as follows:
a, Significantly different from Placebo; b, significantly different from V2R agonist; and c, significantly different from V2R Antagonist.
 R370 AVP EFFECTS ON SWEATING AND FLUID REGULATION

there was a significant exercise effect [F(2,79) = 6.60]. For

urine [Na+] (Fig. 2C), there was a significant interaction
between exercise and condition [F(4,79) = 3.73] with a sig-
nificant exercise effect [F(2.79) = 7.76] and condition effect
[F(2,79) = 38.29]. For urine [K+] (Fig. 2D), there was a
significant exercise effect [F(2,79) = 11.74] and condition
effect [F(2,79) = 52.29]. For saliva [Na+] (Fig. 3A), there was
a significant exercise effect [F(2,75) = 11.61]. There were no
significant interaction, condition, or exercise effects for saliva
[K+] (Fig. 3B), sweat [Na+] (Fig. 3C), or sweat [K+] (Fig. 3D)
as measured by the sweat patch.
For plasma osmolality (Fig. 4A), there was a significant
exercise effect [F(2,77) = 26.00] and condition effect [F(2,77)
= 5.13]. For urine osmolality (Fig. 4B), there was a
significant condition effect [F(2,77) = 84.98]. For [AVP]P
(Fig. 4C), there was a significant exercise effect [F(2,27) =
22.33]. There were no interaction, exercise, or condition
effects for [OT]P (Fig. 4D). For peak core temperature, there
was a significant exer- cise effect [F(2,78) = 103.29; P <
0.0001] between the placebo versus the V2R agonist
conditions during the perfor- mance exercise point. Linear
correlation coefficients (Pear-
son’s) between [AVP]P versus related variables are shown in
Table 1.
For fluid balance and exercise performance parameters,
one-way ANOVA analyses revealed significant differences in
total urine produced and total water ingested, but not in overall
water balance, performance time, or plasma volume change
(Table 2). When a two-way repeated measures ANOVA was
performed for plasma volume change across both exercise and
condition, a significant exercise effect was noted [F(1,53) =
21.83; P < 0.0001]. For body weight change, there was also a
significant exercise effect [F(1,52) = 6.26; P = 0.02].
To further assess whether or not there was an effect of
V2R manipulation on sweat (patch) and saliva [Na+] con-
tent, mean differences from the placebo condition were
calculated individually for both the V2R agonist and V2R
antagonist conditions after the steady-state and performance
exercise time points (Fig. 5). For sweat [Na+], a nonsignif-
icant increase was seen with V2R antagonist treatment,
whereas a nonsignificant decrease was seen with V2R ago-
nist treatment (Fig. 5A). For saliva [Na+], greater increases
were seen during V2R antagonist treatment compared with

Fig. 3. Two-way repeated-measures ANOVA results for saliva and sweat sodium ([Na +]) and potassium ([K+]) concentrations. Exercise is represented on the x-
axis (Baseline, Steady-State, and Performance) and represents the temporal order in which the samples were taken. Condition (Placebo, V2R Agonist, and V2R
Antagonist) is represented on the y-axis and represents the values obtained during each of the three separate pharmacological treatments. All data are represented
by means (symbols) and standard deviation (bars). The standard deviation for the placebo trial (open circle) is represented by the wide “T” and solid line; the
V2R Agonist trial (open square) is represented by the more narrow “T” and dashed line; while the V2R Antagonist (open triangle) is represented by the bold
“I” and dotted line to distinguish between the overlapping error bars. A statistically significant Exercise Effect was seen in saliva [Na +] (A) without a significant
 difference between conditions in post hoc analyses.
AVP EFFECTS ON SWEATING AND FLUID REGULATION R371

Fig. 4. Two-way repeated-measures ANOVA results for plasma and urine osmolality, plasma arginie vasopressin (AVP) concentration ([AVP]P) and plasma
oxytocin concentration ([OT]P). Exercise is represented on the x-axis (Baseline, Steady-State, and Performance) and represents the temporal order in which the
samples were taken. Condition (Placebo, V2R Agonist, and V2R Antagonist) is represented on the y-axis and represents the values obtained during each of the
three separate pharmacological treatments. All data are represented by means (symbols) and standard deviation (bars). The standard deviation for the placebo
trial (open circle) is represented by the wide “T” and solid line; the V2R Agonist trial (open square) is represented by the more narrow “T” and dashed line;
while the V2R Antagonist (open triangle) is represented by the bold “I” and dotted line to distinguish between the overlapping error bars. Significant differences
from post hoc analyses are represented on the graphs as follows: a, Significantly different from Placebo; b, significantly different from V2R Agonist; and c,
significantly different from V2R Antagonist.

V2R agonist treatment, but these differences were not sta- agonist, and V2R antagonist conditions, respectively (P =
tistically significant (Fig. 5B). 0.75). A weak correlation (r = 0.39; P < 0.05) was demon-
For thirst rating, there was a significant exercise effect
strated in sweat [Na+] between the sweat patch versus WBW
[F(2,81) = 78.21; P < 0.0001] and condition effect [F(2,81) technique.
= 4.49; P = 0.01] between the V2R antagonist versus the
placebo condition during the steady-state point. For the whole
body washdown sweat collection, the mean sweat [Na+] was DISCUSSION
14.1 ± 6.6, 12.7 ± 4.8, and 12.4 ± 3.8 meq/l for the The primary hypothesis that sweat water content, as re-
placebo, V2R flected by sweat [Na+], is regulated by the vasopressin V2R at
the level of the sweat gland was not supported by the results of
Table 1. Linear correlations (r) with endogenous [AVP]P this study. Confirmation of V2R stimulation by the selective
under the three randomized trial conditions
V2R agonist desmopressin was evidenced by urine osmolality
Variable Placebo V2R Agonist V2R Antagonist trial conditions: Placebo, pretreatment with the V2R Agonist desmopressin;
and pretreatment with the V2R Antagonist tolvaptan. *P < 0.05.
Plasma osmolality, mosmol/kg H2O 0.20 0.69* 0.69*
Plasma [Na+], meq/l 0.20 0.61* 0.55*
Sweat [Na+], meq/l —0.07 —0.23 —0.06
Saliva [Na+], meq/l 0.23 0.20 0.42*
Urine [Na+], meq/l —0.17 —0.45* 0.60*
Plasma Volume A, % —0.33 —0.62* —0.69*
Body Weight A, % —0.18 —0.41 —0.42
Plasma [Oxytocin], pg/ml 0.37 0.81* 0.29
Thirst Rating, 1–10 0.27 0.50* 0.45*
([AVP]P, plasma arginine vasopressin concentrations. Three randomized
 values that were higher than the values obtained in either the
placebo and V2R antagonist condition at all exercise points
tested (Fig. 4B). Similarly, confirmation of V2R antagonism
by the selective V2R antagonist tolvaptan was evidenced by
urine osmolality values that were significantly lower than
those obtained in either the placebo and V2 agonist condition
at all exercise points tested (Fig. 4B). Blocking the V2R
augmented urinary free-water excretion, as verified by a urine
volume production that was threefold greater than in the
placebo and V2R agonist trials (Table 2). The lack of a
significant decrease in urine volume with desmopressin
treatment likely reflects near-maximal exercise-induced
antidiuresis in the placebo
 R372 AVP EFFECTS ON SWEATING AND FLUID REGULATION

Table 2. One-way ANOVA results for fluid balance and exercise performance under the three randomized trial conditions
Variable Placebo V2R Agonist V2R Antagonist

Total water ingested (during trial), ml 314.3 ± 323.5 (0–870) 436.3 ± 324.3 (0–855) 746.0 ± 322.0a (295–1,175)
Total urine produced (during trial), ml 37.5 ± 29.3 (0–93) 39.4 ± 20.7b (12–71) 140.4 ± 106.8a (27–337)
Overall water balanced (end of trial), ml —903.2 ± 579.8 (—1,593–57) —523.1 ± 732.0 (—1,914–329) —526.0 ± 645.1 (—1,852–
668)
Body weight change (after steady state), % —1.3 ± 0.6 (—2–0) —0.8 ± 0.8 (—2–0) —0.9 ± 0.8 (—2–0)
Body weight change (after performance), % —1.7 ± 0.4 (—2– —1) —1.3 ± 1.0 (—3–0) —1.3 ± 0.8 (—3–0)
Plasma volume change (after steady state), % —5.4 ± 5.2 (—16–4) —4.9 ± 2.9 (—10–0) —2.9 ± 3.3 (—8–2)
Plasma volume change (after performance), % —10.3 ± 3.9 (—17– —2) —9.3 ± 5.1 (—15–2) —9.5 ± 5.0 (—15–2)
Performance time, s 585.5 ± 122.3 (450–813) 612 ± 105.4 (485–780) 617 ± 110.4 (482–793)
Values are means ± SD with minimum through maximum values in parentheses. The change values represent the percent change from baseline after
both the steady-state and performance exercise time points. aSignificantly different from placebo; bsignificantly different from V2R antagonist.

state. Thus the intended activation and blockade of the V2R by points.
the treatment protocols were successfully confirmed by
urinary output measures, and particularly highlighted by the
changes in urine osmolality (Fig. 4B). These expected changes
in urinary free water excretion, however, were not
accompanied by par-

Fig. 5. Two-way repeated-measures ANOVA results for the change in sweat

(A) and saliva [Na+] (B) from the placebo trial (V2R Agonist minus the
placebo sodium concentration and V2R Antagonist minus the placebo sodium
concentration) after both the steady-state and performance trial exercise time
 allel changes in either sweat or salivary secretions. It
therefore appears unlikely that V2R activation stimulates
AQP5 water channel activity in sweat or salivary glands, in
contrast to AVP-mediated V2R activation with subsequent
insertion of AQP2 into the lumen of the kidney collecting
duct principal cells.
V2R effects on AVP secretion were physiologically com-
pensated by osmotically appropriate changes in endogenous
AVP secretion (Table 1). Furthermore, changes in [AVP]P
coincided with osmotically appropriate changes in behavioral
thirst ratings (Table 1). Hyperosmolality associated with max-
imal exercise intensity was likely attributed to the 9 –10%
decline in plasma volume, as documented previously (50). Of
note, hypernatremia (plasma [Na+] > 145 meq/l) was docu-
mented in seven athletes after the steady state and eight
athletes after the performance run during the V2R antagonist
trial (Fig. 2A). Drinking according to thirst, particularly in
the V2R antagonist trial, may have been attenuated by
stomach fullness (39) or distraction from completing the
assigned treadmill task. The augmented hypernatremia
demonstrated in the V2R antagonist condition, however, did
not adversely affect overall water balance, body weight
change, or per- formance compared with both the placebo
AVP EFFECTS ON SWEATING AND FLUID REGULATION
(37). Previous evidence has also shown that [OT]P can activate
the V2 receptor, causing APQ2 translocation and concomitant
antidiuresis (19). However, unlike in animal studies, a regula-
tory role for OT in human osmoregulation remains equivocal
(12, 37).
V2R Effects on Sweat Composition and Volume
Sweat [Na+] did not mirror the same pattern as urine [Na+]
in response to the V2R agonist and antagonist (Fig. 3C). Thus
it is clear that V2R does not influence sweat sodium concen-
tration, as originally hypothesized. The average sweat [Na+] of
~80 meq/l as measured in our participants is similar to those
reported by other investigators using the same region and
technique (13, 15). Sweat [K+] values (Fig. 3D) were around
the physiological range of ~4 meq/l, which suggested the
“leaching” effect of epidermal electrolytes was minimal (49).
The WBW technique is considered the gold standard mea-
surement of sweat electrolyte content during exercise (1, 2,
42). However, our sweat [Na+] values were sevenfold lower
than the patch measurements (between 12 and 14 meq/l) and
~50% lower than previously reported (2, 30). The large
discrepancy between our patch and WBW results may have
resulted from sweat droplets escaping from the measurement
area; electrolytes trapped in clothing, towel, and/or the collec-
tion pool; contamination from cleaning solutions or prior use;
or other unknown factors.
V2R Effects on Saliva
Salivary [Na+] mirrored the pattern of plasma [Na+] under
the three pharmacological conditions (i.e., highest with the
V2R antagonist and lowest with the V2R agonist; Fig. 3A).
This suggests water absorption occurred in salivary glands
after steady-state and performance running from a mechanism
other than V2R activation. Weakly significant correlations
were found between salivary [Na+] versus plasma [Na+] (r =
0.45), which offer limited support for the use of salivary
measurements as a potential surrogate for the determination of
changes in plasma [Na+] in real time (48) during exercise (26),
and the V2R agonist conditions (Table 2).
V2R Effects on Plasma Variables
Plasma [Na+] increased with increasing exercise intensity,
which accounted for ~20% of the overall variance seen in
plasma [Na+]. This corresponding hypernatremia appeared to
further stimulate endogenous AVP secretion, with plasma
AVP levels statistically higher at the performance time point
in the V2R antagonist condition (3.6 ± 2.9 pg/ml) compared
with both the placebo (2.0 ± 1.6 pg/ml) and V2R agonist (2.5
± 1.6 pg/ml) conditions (Fig. 4C). The significant increase in
both plasma [Na+] and [AVP]P would be expected secondary
to the significantly increased urinary free water losses seen
under the V2R antagonist condition. Parallel changes were not
seen between plasma [Na+] and [K+] (Fig. 2, A and B), which
confirm independent regulation of the body’s main extracellu-
lar and intracellular cations during exercise (40).
Plasma oxytocin concentrations did not mirror any discern-
able osmotic pattern related to plasma, urine, sweat, or saliva
[Na+] or [K+] (Fig. 4D). Previous investigations have demon-
strated an electrophysiological response in human immortal-
ized sweat glands (NCL-SG3 cells) when exposed to oxytocin
R373
possibly due to removal of parasympathetic impulses that
modify salivary secretion rates (26). However, the absolute
measurements of plasma and saliva [Na+] differed by a mag-
nitude of approximately sevenfold, which would invalidate any
clinical utility of using salivary measures to diagnose dysna-
tremia. Changes in salivary [K+] did not mirror changes in
plasma [K+] (Fig. 3B) and should not be used as a surrogate
marker of plasma [K+] change.
V2R Effects on Urine Composition and Volume
Urine [Na+] and osmolality values were highest during the
V2R agonist condition and lowest during the V2R antagonist
condition, as pharmacologically expected. Urine [Na+] and
osmolality increased after the performance trial under the V2R
antagonist condition but not following the placebo or V2R
agonist conditions (Figs. 2C and 4B). This paradoxical decline
in urine concentrating ability has been previously noted in
conjunction with relative decreases in urine excretion (16). Our
data demonstrate a reversal of this paradoxical renal finding
only during the V2R antagonist condition, suggesting that
either the sensitivity of the V2R receptor was altered during
high-intensity exercise (8, 23, 24), or the V2R was downregu-
 lated in response to dehydration (22). Urine output was also
sustained in the V2R antagonist trial, suggesting V2R antago-
nism can override the competing effects of limited renal
blood flow and reduced glomerular filtration rate in the
production of urine during exercise (16, 27, 36).

V2R Effects on Overall Fluid Balance

Urinary water retention (V2R agonist condition) and water
losses (V2R antagonist condition) were adequately compen-
sated by drinking according to thirst during each study trial.
There were no statistically significant differences between
fluid balance and percent body weight change between the
three study conditions (Table 2). As expected, fluid intake
was greatest during the V2R antagonist condition with total
water ingestion ~50% higher than either the total amount of
water ingested during the placebo and V2R agonist condi-
tion to compensate for the threefold increase in urinary
losses (Table 2).

V2R Effects on Core Temperature and Performance

The only statistically significant difference in peak body
temperature was seen between the placebo versus V2R
agonist conditions after the performance time point (38.4°C
vs. 38.9°C, respectively). This finding appears to contradict
data obtained from rats suggesting that central AVP
administration acts as an antipyretic agent, at least in
response to fever (31). There was no difference in exercise
performance between the three pharmacological conditions
(Table 2). However, tread- mill running time to exhaustion
was extended by 32 s (5%) during the V2R antagonist over
the placebo trial despite the increased tonicity
(hypernatremia) and associated cellular de- hydration, which
persisted after the steady-state run into the performance test.

Limitations
We utilized both 1) the customary measurement (sweat
patch) and 2) gold standard measurement (WBW) to measure
changes in sweat [Na+] as a primary outcome measure. None-
theless, our inability to detect subtle, real-time, regulatory
changes in sweat gland output remains a study limitation. The
difference in urine osmolality between the V2R antagonist
versus the placebo and V2R agonist conditions were suffi-
ciently robust, however, to negate even modest measurement
errors in the interpretation of our sweat data. Other potential
study limitations include lack of control of room temperature,
daily exercise, or food intake, all of which could potentially
affect the results.

Perspectives and Significance

These data indicate that the V2R does not modulate water
losses from sweat or saliva during moderate to high-intensity
exercise stress. Despite the teleological advantage of reducing
sweat water and sodium losses during exercise, this appears
not to be a major homeostatic mechanism during acute
exercise. The present study used pharmacological
manipulation with a receptor-specific agonist and antagonist
to activate and inhibit the AVP V2R, thereby overriding the
effects of endogenous AVP secretion. As such, these results
provide indirect evidence that the V2R does not affect
translocation of AQP5 channels
 R374 AVP EFFECTS ON SWEATING AND FLUID REGULATION
that are present in sweat and salivary glands. In contrast,
this study verified that V2R activation or blockade produced
physiologically appropriate, osmotically induced, secondary
changes in endogenous [AVP]P and thirst stimulation
through activation or blockade of AQP2 channel insertion in
the kidney collecting duct. Although the V2R antagonist
produced mild hypernatremia unaccompanied by greater
plasma volume losses at the end of the exercise trial, overall
water balance, body weight change, and exercise perfor-
mance were not significantly different between conditions.
Future basic science and translational studies should assess
the contribution of muscarinic activation on the relationship
between AQP5 translocation and sweat water output (18, 28,
41). Alternatively, the translocation of APQ5 water channels
in sweat gland epithelium may be triggered by mechanical
stimuli related to cell volume changes that are secondary to
plasma tonicity changes (43). For example, stimulation of the
musca- rinic type 3 receptor by the acetylcholine receptor
agonist carbachol has been shown to cause an unexpected
decrease in cell volume with increased AQP5 trafficking
(18). Similarly, rats exposed to hypertonic stress
demonstrate an increase in AQP5 protein expression in lung,
submandibular, and lacrimal glands through an ERK-
dependent pathway (14). Hypotonicity has also been shown to
reduce APQ5 expression through mechanical activation of
transient receptor potential vanilloid 4 channels (21, 43). Thus
perhaps our original hypothesis should now be extended in the
direction of investigating a more direct relationship between
plasma tonicity and sweat water output (Fig. 5). While
central activation of muscarinic receptors may initiate eccrine
sweat secretion, it appears that hypertonicity increases, and
hypotonicity decreases, AQP-mediated plasma water
absorption independently of V2R stimulation. Future sweat
water output studies should target exercising humans and
horses as unique thermoregulatory models to best translate
molecular AQP5 water channel insights into a more coordi-
nated understanding of whole body fluid homeostasis, physical
performance, and secretory gland disease (3, 18).
ACKNOWLEDGMENTS
The authors thank Dr. Kristin Landis-Piwowar, Rachel Griffin, and Emma
Hurst for assistance with venipuncture; Jeff Cross, Lizzy Jones, and Brigid
Nash for enthusiastic assistance with exercise testing; Nik Sharma for speci-
men analysis and Qin Xu for AVP and OT assays; and last but not least, our
ten resilient participants to whom we are most indebted. Current address for
B. C. Rider: Dept. of Kinesiology, University of Tennessee, Knoxville, TN.
GRANTS
This study was funded by the University Research Committee (URC)
Summer Fellowship Award from Oakland University, an Investigator award
from Otsuka America Pharmaceutical, and grant UL1TR000101 from the
National Center for Advancing Translational Sciences (NCATS), National
Institutes of Health, through the Clinical and Translational Science Awards
Program (CTSA), a trademark of DHHS, part of the Roadmap Initiative, “Re-
Engineering the Clinical Research Enterprise.” Reference List.
DISCLOSURES
J. G. Verbalis has received grant support from Otsuka America Pharma-
ceuticals, as well as non-CME-related fees as a consultant from Otsuka
Pharmaceuticals, Cardiokine, and Cornerstone Therapeutics.
AUTHOR CONTRIBUTIONS
Author contributions: T.D.H.-B. and J.G.V. conception and design of
research; T.D.H.-B., J.H., and B.C.R. performed experiments; T.D.H.-B., J.H.,
and J.G.V. analyzed data; T.D.H.-B., J.H., B.C.R., and J.G.V. interpreted
results of experiments; T.D.H.-B. prepared figures; T.D.H.-B. drafted manu-
script; T.D.H.-B., J.H., B.C.R., and J.G.V. edited and revised manuscript;
T.D.H.-B., J.H., B.C.R., and J.G.V. approved final version of manuscript.
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