Shinshu Med J, 70⑵：81～90, 2022

The Combination of α1-adrenergic Receptor Antagonist andPhosphodiesterase 5 Inhibitor Mitigates Cold Stress-induced
Detrusor Overactivity through Resiniferatoxin-Sensitive Nerves
in Bladder Outlet Obstructed Rats
Ataru Suzuki, Tetsuya Imamura＊, Hiroaki Hara, Manabu Ueno
Tomonori Minagawa, Teruyuki Ogawa and Osamu Ishizuka
Department of Urology, Shinshu University School of Medicine

Background : We determined if the α1-adrenergic receptor (AR) antagonist naftopidil, the phosphodiesterase 5(PDE5) inhibitor tadalafil, or the combination inhibited cold stress-induced detrusor overactivity in bladder outlet
obstructed rats. We also investigated the role of resiniferatoxin (RTX)-sensitive nerves in detrusor overactivity.
Methods : The urethras of 10-week-old female Sprague-Dawley rats were loosely ligated to create a partial
bladder outlet obstruction. After 4 weeks, at room temperature (RT, 27 ℃), the rats were randomly assigned toreceive an intraperitoneal infusion of vehicle control (n＝11), 0.15 mg/kg-body weight naftopidil (n＝7), 0.5 mg/
kg-body weight tadalafil (n＝7), or the combination of naftopidil and tadalafil (n＝11). The treated rats were thenexposed to low temperature (LT, 4 ℃) for cystometry. Other rats were subcutaneously injected with 0.3 mg/kg
RTX (n＝8), and then two days later underwent cystometric investigations. The number of calcitonin generelated peptide (CGRP)-positive neurons was examined by immunohistochemistry.
Results : After transfer from RT to LT, the vehicle-, naftopidil-, and tadalafil-treated rats had decreased voiding
intervals and bladder capacity. These decreases were inhibited by the combined naftopidil-tadalafil treated rats.
RTX caused similar cystometric decreases as the combination-treated rats. The number of the CGRP-positiveafferent nerves in the RTX-treated rats was significantly reduced.
Conclusion : The combination of an α1-AR antagonist and a PDE5 inhibitor mitigated the cold stress-induced
detrusor overactivity in bladder outlet obstructed rats. RTX treatment also inhibited the cold stress responses
while reducing the presence of CGRP in afferent nerves. α1-AR antagonists and PDE5 inhibitors could act
efficiently, and may affect RTX-sensitive nerves, to reduce cold stress-induced detrusor overactivity. Shinshu
Med J 70 : 81―90, 2022
(Received for publication October 5, 2021 ; accepted in revised form November 26, 2021)
Key words : cold stress, bladder outlet obstruction, α1-adrenergic receptor antagonist, phosphodiesterase
5 inhibitors, resiniferatoxin
Ⅰ

Introduction(LUTS) that occur due to bladder outlet obstruc‑tion. Thus, patients with BPH are treated with an

Patients with benign prostatic hyperplasia (BPH)

α1-adrenergic receptor (AR) antagonist and/or aoften complain about lower urinary tract symptoms

phosphodiesterase 5 (PDE5) inhibitor to release theobstruction. In clinical practice, combination therapy＊

Corresponding author : Tetsuya Imamura
Department of Urology, Shinshu University School of
Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621,
Japan
E-mail : imatetu@shinshu-u.ac.jp

No. 2, 2022with both an α1-AR antagonist and a PDE5 inhibitor
is often effective for overactive bladder symptoms1）-4）.
One of the factors that exacerbates LUTS is cold
stress due to a sudden drop in temperature or re‑81

Suzuki・Imamura・Hara et al.

peated exposure to a low temperature environment.

Shinshu University School of Medicine

We have established a rat model for cold stress LUTS

Ｂ

5）

Preparation of bladder outlet obstructed ratsthat elicits detrusor overactivity . The cold stress-

The SD rats were anesthetized with midazolam

exacerbated LUTS is mediated by cross talk among(2.0 mg/kg-body weight, Sandoz International GmbH,neurological pathways, including unmyelinated C

Tokyo, Japan), medetomidine hydrochloride (0.15 mg/

5）fibers within the urinary bladder , enhancement of
6）

kg-body weight, Kyoritsu Seiyaku Co., Tokyo, Japan),sympathetic nerve activity , and expression of tran‑

and butorphanol tartrate (2.5 mg/kg-body weight,

sient receptor potential cation channel, subfamily M,

Meiji Seika Pharma Co., Ltd., Tokyo, Japan). A midline

7）

incision was made to expose the urethra, and a metal

member 8 (TRPM8) in the skin .

We have developed a second rat model for testing

rod with an outer diameter of 1.1 mm was placed

the pharmacological effects of drugs on cold stress-

alongside it. To produce bladder outlet obstruction,8）

induced LUTS . The model, which is based on partial

the urethras were loosely ligated to the metal rod

obstruction of the bladder outlet, mimics the humanwith 5-0 silk. Afterwards, the metal rod was carefully

form of LUTS associated with BPH. In that model,

removed, leaving the ligature to create a partial

the cold stress-induced detrusor activity and changes

bladder outlet obstruction. The incision was thenin bladder storage characteristics are mitigated byclosed. The rats with bladder outlet obstruction weretreatment with an α1-AR antagonist . In addition, wehoused for 4 weeks (as above). Based upon cystomet‑

showed that PDE5 inhibition reduces unmyelinat‑

ric investigation (see below) to identify effectively

ed C fiber-related detrusor overactivity elicited byligated rats with bladder obstruction, only animals

8）9）

acetic acid in nicotine-treated rats . In normal healthy

with a bladder volume between 2 and 5 ml at room

rats, treatment with resiniferatoxin (RTX), a capsaicin

temperature (RT, 27±2 ℃) were selected for the fol‑

analogue that reduces the content of calcitonin gene-

lowing experiments.

related peptide (CGRP) in unmyelinated C fibers,

Ｃ

suppresses C fiber activation and inhibits cold stressinduced detrusor overactivity5）10）-13）.

Drugs
We used naftopidil, kindly provided by Asahi Kasei

Pharma Co. (Tokyo, Japan), as an α1-AR blocker. The

Based on these previous findings, in this study wenaftopidil powder was completely dissolved with 0.1

determined if the combination of an α1-AR antagonist

M phosphate buffer solution in half of the final vol‑and a PDE5 inhibitor, at lower doses than either

ume. Then several drops of 0.1 M sodium dihydrogenalone, could inhibit the cold stress-induced detrusor

phosphate solution were slowly added with vortex‑

overactivity in rats with partial bladder outlet ob‑

ing and ultrasonication to achieve the final volume

struction. We also investigated the effects of RTX

and pH 4.0. The dissolved naftopidil solution was di‑treatment on the CGRP content of the bladder un‑

luted to the desired concentration with 0.9 ％ saline.

myelinated C fibers and on cold stress-induced de‑Tadalafil powder (Toronto Research Chemical Inc.,

trusor overactivity.

Toronto, Canada), a PDE5 inhibitor, was completely

Ⅱ
Ａ

Material and Methods

Animals

dissolved with dimethyl sulfoxide (DMSO, Fujifilm
Wako Pure Chemical Co., Osaka, Japan). The dissolved
tadalafil solution was diluted to the desired concen‑

Ten-week-old female Sprague-Dawley (SD) rats

tration with DMSO. Resiniferatoxin powder (RTX,(Japan SLC Inc., Shizuoka, Japan) were housed for 4

Sigma-Aldrich, Steinheim, Germany), a capsaicin ana‑

weeks under a 12-hour alternating light-dark cycle

logue, was completely dissolved with DMSO. The

with freely available food and water. The animals

dissolved RTX solution was diluted with DMSO towere treated in accordance with National Institutes

the deliver 0.3 mg/kg by subcutaneous injection.

of Health Animal Care Guidelines and the protocolＤ

was approved by the Animal Ethics Committee of82

Cystometric investigations

Four weeks after creating the partial bladder outlet
Shinshu Med J

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Combination treatment for LUTS with bladder outlet obstruction

obstruction and 2 days prior to the cystometric in‑

kg-body weight naftopidil and 0.5 mg/kg-bodyvestigations, the animals were anesthetized (as above)

weight tadalafil (n＝11). Cystometric measurements

to insert a catheter for cystometric investigations.

were then conducted to obtain baseline measure‑The urinary bladder and ligated urethra were ex‑ments for approximately 20 minutes at RT. Throughposed, and the ligature thread was then removed. A

the intraperitoneal catheter, each rat received either

polyethylene catheter (PE50, Becton Dickinson andthe control vehicle, naftopidil, tadalafil, or the combi‑

Company, Sparks, MD, USA) was inserted at thenation of naftopidil and tadalafil. Twenty minutes

center of the bladder dome. The catheter was fixed

after the treatments, the rats were quickly andat that site with a 5-0 suture. For delivery of vehicle

smoothly transferred in the metabolic cages to aor drugs during the cystometry experiments, anotherrefrigerator (MPR-513, SANYO Tokyo Manufactur‑

catheter (PE90, Becton Dickinson and Company), wasing Co., Ltd., Tokyo, Japan) for exposure to low tem‑inserted into the intraperitoneal space. Both catheters

perature (LT, 4±2 ℃ ). The bladder pressure andwere brought out subcutaneously to the back andmicturition volume of the rats were again recorded

fixed with 3-0 silk sutures. After the operation, eachfor 40 min. After the cystometric investigations, therat was caged individually for two days.

rats were anesthetized as above, and then the urinary

For cystometry, the bladder catheter was connect‑
ed through a T-tube to a pressure transducer (P23

bladders were removed, and the rats were then eu‑
thanized by inhalation of diethyl ether.

DC ; Nihon Kohden, Tokyo, Japan) and a syringe

Two days prior to the cystometric investigations,pump (TE-351, Terumo, Tokyo, Japan). Saline (0.9 ％other rats with partial bladder outlet obstructions

NaCl) was infused continuously into the bladder atwere catheterized as above and then subcutaneously

a rate of 10 ml/hr. A urine collector connected to

injected with 0.3 mg/kg-body weight RTX. Two days

a force displacement transducer (type 45196 ; NEClater, the RTX-treated rats underwent the sameSan-ei Instruments, Tokyo, Japan) enabled measure‑LT-exposure cystometric investigations.

ment of micturition volume. The bladder pressureＥ

Immunohistochemistry investigations

and micturition volume were continuously recorded

The harvested urinary bladders were fixed in 4 ％with LabChart system (AD Instruments, BRC Biore‑

paraformaldehyde phosphate buffer solution (Nalkalai

search, Inc., Nagoya Japan) through a PowerLab

Tesque, Inc., Kyoto, Japan) for 12 hours at 4 ℃. The

system (AD Instruments).

tissues were embedded in paraffin, and cut into 5-μm

The following cystometric parameters were mea‑

thick serial sections. The sections were deparaffinized,

sured : basal pressure (cmH2O), micturition pressure

and then antigen retrieval was achieved by immer‑(cmH2O), voiding interval (min), and bladder capacity

sion of the sections in 0.01 M sodium citrate (pH 6.0,

(ml). The bladder capacity was calculated by adding

Mitsubishi Chemical Medience Co., Tokyo, Japan)

the micturition volume and the residual volume thatand microwaving at 100 ℃ for 5 minutes. The speci‑

was determined as the difference between the salinemens were coated with 1.5 ％ normal donkey serum

infusion volume and micturition volume. The rats

(Chemicon International Inc., Temecula, CA, USA)

were not given food or water during the cystometricand 1.5 ％ non-fat milk in 0.01 M phosphate bufferedinvestigations.

saline (PBS, pH 7.4, Mitsubishi Chemical Medience

Cystometric measurements of the unanesthetized,Co.) for 1 hour at 4 ℃. The sections were then incu‑

unrestricted rats were made under the following en‑

bated with primary antibodies, for CGRP (1 : 800,

vironmental temperature conditions. They were ran‑guinea pig polyclonal, Progen Biotechnik GmbH, Hei‑

domly separated into the control and three experi‑delberg, Germany) as a marker of afferent nerves,

mental groups as follows : (1) vehicle control (n＝11),

and smooth muscle actin (SMA, 1 : 100, mouse mono‑(2) 0.15 mg/kg-body weight naftopidil (n＝7), (3) 0.5

clonal, Progen Biotechnik GmbH, Heidelberg, Germa‑

mg/kg-body weight tadalafil (n＝7), and (4) 0.15 mg/ny) for 12 hours at 4 ℃ . The sections were rinsed

No. 2, 2022

83

Suzuki・Imamura・Hara et al.

with PBS at 4 ℃, and then incubated with donkey

naftopidil-, tadalafil-, or combination-treated rats

anti-guinea pig IgG secondary antibody conjugatedexhibited increased micturition frequency and lower

with Alexa Fluor 594 (1 : 250, Life Technology Co.,micturition volume compared to RT condition. Voiding

Molecular Probes, Eugene, OR, USA) and donkey

intervals at RT were significantly decreased at LT in

anti-mouse IgG secondary antibody conjugated withthe vehicle-treated (62.24±5.48 ％), naftopidil-treatedAlexa Fluor 488 (1 : 250, Life Technology Co.) for 1(66.25±0.56 ％), tadalafil-treated (67.99±2.05 ％), andhour at 4 ℃. Following rinsing, cell nuclei were coun‑combination-treated (34.54±12.31 ％) rats (Fig. 2A).

terstained with 5 μg/ml 4’, 6-diamidino-2-phenyl­

Similarly, bladder capacities at RT in the vehicle-,indole dihydrochloride (DAPI, Life Technology Co.).

naftopidil-, tadalafil-, and combination-treated rats

The slides were coated with Fluorescent Mounting

were also less at LT (64.84±4.13 ％, 64.08±10.46 ％,Medium (Dako Cytomation, Carpinteria, CA, USA)69.35±2.10 ％, 42.98±6.72 ％, respectively ; Fig. 2B).

and observed with a fluorescence microscope (Keyence,

However, the decreases of both voiding interval andOsaka, Japan). CGRP-positive cells were detected

bladder capacity in the combination-treated rats wereand counted among the SMA-positive smooth muscle

significantly less than in the vehicle-, naftopidil-, orlayers. With a x60 objective lens, the counting areas

tadalafil-treated rats.

were randomly viewed from the top of the bladder to

Ｂ Cold stress-induced bladder overactivity

the trigone, and the number of CGRP-positive cells

in resiniferatoxin-treated bladder outlet

were counted in 5-10 locations per tissue sample.

obstructed rats

Ｆ

Two days prior to cystometric investigations, an‑

Statistical analysis

The results were expressed as means±standard

other group of bladder obstructed rats (n＝8) waserror of the means. The significance of statistical dif‑treated with RTX. At RT, both bladder pressure andferences between cystometric variables were deter‑

micturition volume in the RTX-treated rats did notmined by Student’s paired t-tests before and after

differ from the control and experimental groups de‑the drug administration at RT, or between RT and

scribed above. After transfer from RT to LT, unlike

LT. Two-way non-repeated measures analysis of

the vehicle-, naftopidil-, tadalafil-, and combina‑

variance (ANOVA) followed by Student-Newman-

tion-treated rats, the RTX-treated rats did not have

Keuls (SNK) test for multiple comparisons were per‑increased micturition frequency and lower micturi‑

formed for comparison of variables among groups.

tion volume (Fig. 3A). During LT exposure, the basal

P-values less than 0.05 were considered significant.

pressure of the RTX-treated rats also increased sig‑

Ⅲ

Results

Ａ Effects of naftopidil and tadalafil alone andnificantly, while micturition pressure did not change(Table 1). Both the voiding interval (Fig. 3B) andbladder capacity (Fig. 3C) tended to decrease, but

in combination on cold stress-induced bladderthe changes were not statistically significant. Howev‑

overactivity in bladder outlet obstructed rats

er, the percent decreases of voiding interval and

At RT, there were no differences in either bladderbladder capacity in the LT RTX-treated rats, 30.15

pressure or micturition volume among the rats in the±9.99 ％ and 24.33±11.24 ％ respectively (Fig. 3A, B),

control or experimental groups (Fig. 1). These were

were not significantly different from the decreases in

not altered after intraperitoneal administration of thethe combined naftopidil- and tadalafil-treated rats

vehicle control, naftopidil, tadalafil, or the combina‑(Fig. 2A, B).

tion of naftopidil and tadalafil.

Ｃ Expression of urinary bladder CGRP-positive

After transfer from RT to LT, basal pressure in allafferent nervesgroups significantly increased, but micturition pres‑We examined the expression and distribution ofsures did not change (Table 1). As seen for individu‑

CGRP-positive afferent nerves among the SMA-

al rats (Fig. 1), during LT exposure, the vehicle-,positive smooth muscle layers of each group. There84

Shinshu Med J

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Combination treatment for LUTS with bladder outlet obstruction

Fig. 1 Representative cystograms of changes in bladder pressure, micturition frequency, and micturition volume upontransfer from RT to LT. (A) After transfer from RT to LT, vehicle-treated rats exhibited cold stress-induced bladderoveractivity. Micturition frequency increased (upper tracing) and micturition volume decreased (lower tracing) comparedto RT condition. (B and C) At RT, naftopidil (B) or tadalafil (C) treatments did not alter micturition. During exposure toLT, (B) naftopidil- and (C) tadalafil-treated rats also exhibited cold stress-induced bladder overactivity that were similar
to the vehicle-treated ones. (D) At RT, combined treatment with naftopidil and tadalafil did not alter micturition.
After transfer to LT, the combination-treated rats also exhibited increased micturition frequency and lower micturi‑
tion volume compared to RT conditions ; however, these changes were partially inhibited compared to the vehicle-,
naftopidil-, or tadalafil-treated rats. Top : bladder pressure ; Bottom : micturition volume. Triangles : micturition during
LT exposure. ＊, Twenty minutes of waiting time after intraperitoneal injection of vehicle, 0.15 mg/kg-body weight naf‑
topidil, 0.5 mg/kg-body weight tadalafil, or the combination of naftopidil and tadalafil ; ＊＊, approximately 3 minutes oftransfer time from RT (room temperature) to LT (low temperature). Arrowheads, micturition events during LT exposure.
Table 1

Effects of temperature on basal and micturition pressures in obstructed bladder outlet rats

Parameter
Basal pressure (cmH2O)

Micturition pressure (cmH2O)

Condition

VehicleNaftopidil

Tadalafil

Naftopidil＋
Tadalafil

RTX

RT
LT
(RT-LT)

4.47±0.74
5.79±1.06
4.45±0.52
3.68±0.39
8.54±0.79
11.79±1.24
9.20±1.25
7.92±0.68-4.13±1.03＊＊ -5.18±1.10＊＊ -4.75±1.16＊＊ -4.24±0.63＊＊-3.11±1.26＊

RT
LT(RT-LT)

22.92±3.58
22.33±2.16
0.38±2.42

35.75±3.2836.55±3.11-0.78±4.56

26.03±3.51
26.38±2.59-0.38±3.05

28.95±4.02
23.00±3.72
5.95±2.64

24.64±2.2825.89±2.87-1.25±1.20

10.14±1.99
13.25±2.03

RTX, resiniferatoxin ; RT, room temperature, 27±2 ℃ ; LT, low temperature, 4±2 ℃ ; ＊P＜0.05, ＊＊P＜0.01 compared to
RT in each group.
No. 2, 2022

85

Suzuki・Imamura・Hara et al.

Fig.2 Decreased voiding interval and bladder capacity during LT exposure. (A and B) After transfer to LT, voiding
interval (A) and bladder capacity (B) in all groups were significantly decreased. However, the decreases in voiding
interval and bladder capacity of the combination-treated rats were significantly inhibited compared to the vehicle-,
naftopidil-, and tadalafil-treated rats. White bar : RT (room temperature). Gray bar : LT (low temperature) ; naftopidil,
0.15 mg/kg-body weight ; tadalafil, 0.5 mg/kg-body weight ; ＊P＜0.05, ＊＊P＜0.01 ; compared to RT baseline values.

were numerous GCRP-positive afferent nerves in thein a large number of mammals, including rats andvehicle- (Fig. 4A), naftopidil- (Fig. 4B), tadalafil-

humans. We verified this effect again in our LUTS

(Fig. 4C), and combination- (Fig. 4D) treated rats.

rat model that had cold stress-induced detrusor

However, there were many fewer CGRP-positive af‑

overactivity following partial obstruction of the blad‑

ferent nerves in the RTX-treated rats (Fig. 4E). The

der outlet. The decreased voiding interval and blad‑number of afferent nerves detected by CGRP anti‑

der capacity elicited by exposure to LT in this model

body among the SMA-positive smooth muscle layers

were not inhibited by 0.15 mg/kg-body weight of thein the RTX-treated rats was significantly lower than

α1-AR antagonist naftopidil or by 0.5 mg/kg-bodyin any of the other treatment groups (Fig. 4F).

weight of the PDE5 inhibitor tadalafil. However, theⅣ

Discussion

It is well established that cold stress induces LUTS
86

combined treatment with the same naftopidil andtadalafil dosages partially inhibited the cold stressinduced detrusor overactivity. A previous study
Shinshu Med J

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Combination treatment for LUTS with bladder outlet obstruction

Fig. 3 Change of micturition and voiding interval and bladder capacity in RTX-treated rats with transfer from RT to
LT. (A) After transfer to LT, RTX-treated rats partially inhibited the cold stress-induced bladder overactivity
that increased micturition frequency (upper tracing) and decreased of micturition volume (lower tracing). ＊, ap‑
proximately 3 minutes of transfer time from RT to LT. (B) During LT exposure, the voiding interval of the RTXtreated rats tended to decrease, but the change was not statistically significant. (C) Bladder capacity of the RTXtreated rats was decreased compared to the RT. The decreases of voiding interval and bladder capacity were thesame as for the rats given the combination of 0.15 mg/kg-body weight naftopidil and 0.5 mg/kg-body weighttadalafil (see Figure 2). RTX, resiniferatoxin, 0.3 mg/kg ; RT, room temperature ; LT, low temperature ; ＊P＜0.05,
compared to RT. Arrowheads, micturition events during LT exposure.

showed that 0.3 mg/kg-body weight naftopidilthat mediates detrusor overactivity5）. The current

inhibits the cold stress-induced detrusor overactivity

study also showed that RTX inhibited the cold stress8）of the same rat model . In addition, 1.0 mg/kg-body

response in rats with bladder outlet obstruction. In

weight tadalafil improved bladder storage functions

addition, the urinary bladders of the RTX-treatedin rats with nicotine-induced bladder hypoxia9）. The

rats had fewer CGRP-positive afferent nerves com‑combined therapy, in which half the dose of eachpared to the controls. Thus, our evidence suggests

drug was used in our study, partially inhibited thethat the cold stress-induced detrusor overactivity ofcold stress responses. Thus, our data suggest thatthe bladder in outlet obstructed rats is also mediatedsome effects of these two drugs efficiently inhibit theby bladder unmyelinated C fibers that include RTX-

cold stress-induced detrusor overactivity.

sensitive nerves.

To investigate the mechanisms by which cold

In general, either naftopidil or tadalafil is used tostress detrusor activity can be treated, we focused

improve clinical voiding symptoms. Also, these drugs

on RTX-sensitive nerves. A previous study of RTX-

are reported to pharmacologically suppress afferent

treated normal rats showed that activation of unmy‑

nerve (myelinated Aδ and/or unmyelinated C fibers)

elinated C fibers is one of the neuronal pathways

activity14）-16）. These findings suggest a neurologicalNo. 2, 2022

87Suzuki・Imamura・Hara et al.

Ａ

Ｂ

Ｃ

Ｄ

Ｅ

Ｆ

Fig. 4 CGRP-positive afferent nerves within the detrusor of bladder outlet obstructed rats. (A-D),
There were numerous CGRP-positive afferent nerves (red, arrowheads) in the (A) vehicle-, (B)naftopidil-, (C) tadalafil-, and (D) combined naftopidil- and tadalafil-treated rats. (E) Within the
detrusor of the RTX-treated rats, there were fewer CGRP-positive nerve cells (red, arrowheads).
(F) Expression numbers of the CGRP-positive afferent nerves in the RTX-treated rats were thelowest among the groups. CGRP, calcitonin gene-related peptide ; Green : SMA-positive detrusor.
Blue : nuclei. Bar : 30 μm.

88

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Combination treatment for LUTS with bladder outlet obstruction

mechanism by which cold stress storage symptoms

suggest that the combination of naftopidil and tadala‑

are improved, and our data are consistent with these

fil has the potential to effectively treat cold stress-

observations. We found that treatment with the com‑exacerbated LUTS due to BPH.

bination of naftopidil and tadalafil, each at half theⅤeffective dose alone8）9）, partially reduced the cold

Conclusions

stress-induced LUTS symptoms of the bladder outlet

Neither the α1-AR antagonist naftopidil (0.15 mg/

obstructed rats. A previous study showed expressions

kg-body weight) nor the PDE5 inhibitor tadalafil (0.5of α1A- and α1D-AR within the CGRP-positive affer‑

mg/kg-body weight) alone inhibited detrusor over‑8）

ent nerves . Also, expressions of PDE5 activity were17）

activity in cold stressed, bladder outlet obstructed

detected within the CGRP-positive cells . This sug‑rats. However, the combination of naftopidil andgests that the combination of naftopidil and tadalafil

tadalafil inhibited the decrease in voiding interval by

suppressed activity of the afferent nerves, possibly

35 ％ and the decrease in bladder capacity by 43 ％.

including the RTX-sensitive nerves. The pharmaco‑

The cold stress response in bladder outlet obstructed

logical effects of the combination treatments mightrats was also inhibited by subcutaneous injection ofefficiently inhibit the cold stress-induced detrusor

RTX two days before cystometry. RTX treatment

overactivity in bladder outlet obstructed rats.

reduced the presence of CGRP in detrusor afferent

We recognize some limitations within this study.

nerves, and inhibited the decrease in cold stressFirst, naftopidil and tadalafil have anti-inflammatory,

voiding intervals and bladder capacity in the bladderanti-oxidative, anti-fibrotic, and vascular endothelial

outlet obstructed rats. This indicates that the cold

protective properties that could be caused by chronic

stress-induced detrusor overactivity in the bladder

18）
19）

. We did not investigate

outlet obstructed rats was mediated, at least in part,these potential modes of action in reducing LUTS

by RTX-sensitive nerves. We hypothesize that thesymptoms. Secondly, while both drugs are known to

combination of naftopidil and tadalafil acts efficiently

improve pelvic blood flow20）-22）, we did not directly

to inhibit cold stress-induced detrusor overactivity

investigate any improvement of blood flow withinin bladder outlet obstructed rats by suppressing

the urinary bladders as a result of the pharmacologi‑

afferent nerve activity, some of which may include

cal treatments. Finally, we did not estimate the ex‑

RTX-sensitive nerves. Thus, we conclude that thepression levels of endothelial and/or nerve-associatedcombination of an α1-AR antagonist and a PDE5

nitric oxide synthase or cyclic guanosine monophos‑

inhibitor has the potential to effectively treat clinicalphate, which were metabolites related with the PDE5

cases of cold stress-induced LUTS in patients with

inhibitor. Even with these limitations, our results

BPH.

ischemia related with BPH

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（2021. 10. 5 received ; 2021. 11. 26 accepted）

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