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Received: 26 July 2019    Revised: 13 February 2020    Accepted: 2 March 2020

DOI: 10.1111/1440-1681.13297

ORIGINAL ARTICLE
Neurobiology

Stimulation of brain cannabinoid CB1 receptors can ameliorate

hypertension in spontaneously hypertensive rats

Takahiro Shimizu  | Masaki Yamamoto | Suo Zou | Shogo Shimizu |

Youichirou Higashi | Motoaki Saito

Department of Pharmacology, Kochi Medical

School, Kochi University, Nankoku, Japan
Correspondence
Takahiro Shimizu, Department of
Pharmacology, Kochi Medical School, Kochi
University, Nankoku 783-8505, Japan.
Email: shimizu@kochi-u.ac.jp
Funding information
The Smoking Research Foundation in Japan;
Japan Society for the Promotion of Science,
Grant/Award Number: 26460909; The
Japan Health Foundation
Abstract
Excessive activation of the sympatho-adrenomedullary system plays a pathogenic
role in triggering and sustaining essential hypertension. We previously reported
that, in normotensive rats, intracerebroventricularly (i.c.v.) administered neuropep-
tides, corticotropin-releasing factor and bombesin induced activation of the sympa-
tho-adrenomedullary system, and that brain cannabinoid CB1 receptors negatively
regulated this activation. In this study, we investigated the effects of brain CB1 re-
ceptor stimulation on blood pressure and the sympatho-adrenomedullary outflow in
spontaneously hypertensive rats (SHRs), commonly used animal models of essential
hypertension, and in Wistar-Kyoto (WKY) rats, normotensive controls of SHRs. In
18-week-old SHRs and WKY rats under urethane anaesthesia (1.0 g/kg, i.p.), SHRs
exhibited significantly higher systolic, mean and diastolic blood pressures and plasma
noradrenaline and adrenaline, and a lower heart rate than WKY rats. Single admin-
istration of arachidonyl 2′-chloroethylamide (ACEA, CB1 agonist, 1.4  µmol/animal,
i.c.v.) significantly but partially reduced mean and diastolic blood pressures and the
plasma level of noradrenaline in SHRs compared to vehicle (N,N-dimethylformamide)-
treated SHRs. These ACEA-induced reductions were abolished by central pretreat-
ment with rimonabant (CB1 antagonist, 300 nmol/animal, i.c.v.), which alone showed
no significant effect on blood pressures or plasma noradrenaline and adrenaline lev-
els of SHRs. On the other hand, ACEA had no significant effect on blood pressure or
plasma noradrenaline and adrenaline levels in WKY rats. These results suggest that
stimulation of brain CB1 receptors can ameliorate hypertension accompanied by en-
hanced sympathetic outflow without affecting blood pressure under normotensive
conditions.

KEYWORDS

brain, cannabinoid CB1 receptor, central sympatho-adrenomedullary outflow, hypertension,

noradrenaline, spontaneously hypertensive rat

Abbreviations: ACEA, arachidonyl 2′-chloroethylamide; CRF, corticotropin-releasing factor; DBP, diastolic blood pressure; DMF, N,N-dimethylformamide; dPAG, dorsal periaqueductal
gray; FAAH, fatty acid amide hydrolase; HR, heart rate; i.c.v., intracerebroventricularly; MBP, mean blood pressure; NTS, nucleus tractus solitarius; PVN, paraventricular nucleus; RVLM,
rostral ventrolateral medulla; SBP, systolic blood pressure; SHR, spontaneously hypertensive rat; WKY, Wistar-Kyoto.
The peer review history for this article is available at https://publo​ns.com/publo​n/10.1111/cep.13297

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1254     © 2020 John Wiley & Sons Australia, Ltd wileyonlinelibrary.com/journal/cep Clin Exp Pharmacol Physiol. 2020;47:1254–1262.
SHIMIZU et al.
1 |  I NTRO D U C TI O N
Activation of the central sympatho-adrenomedullary system is es-
sential for adaptation to stressful conditions.1-3 In contrast, in re-
sponse to excessive or sustained stress, activation of the central
sympatho-adrenomedullary system can play a pathogenic role
in triggering and sustaining essential hypertension.4-7 Therefore,
it is necessary to clarify the regulatory mechanisms that control
sympatho-adrenomedullary outflow, by focusing on the brain,
to elucidate fundamental mechanisms for the “establishment” of
hypertension.
We have investigated central regulatory mechanisms for sym-
patho-adrenomedullary outflow using several stress-related
neuropeptides, such as corticotropin-releasing factor (CRF), argi-
nine-vasopressin or bombesin. We reported that each peptide ad-
ministered centrally induced elevations of plasma noradrenaline
and adrenaline, and that these elevations were potentiated by cen-
tral pretreatment with antagonists of cannabinoid CB1 receptors in
rats.8-10 Other groups reported that administration of anandamide,
an endogenous ligand for CB receptors,11 into the nucleus tractus
solitarius (NTS) prolonged baroreflex inhibition of renal sympathetic
nerve activity through CB1 receptors in rats, 12
and that anandamide
or CP55940, a synthetic CB receptor agonist, administered intrace-
rebroventricularly (i.c.v.) or into the hypothalamic paraventricular nu-
cleus (PVN), a regulatory centre of the sympatho-adrenomedullary
13,14
outflow, decreased blood pressure through CB1 receptors in rats.
These lines of evidence suggest a possibility that brain CB1 recep-
tors might decrease blood pressure by inhibiting the central sym-
patho-adrenomedullary outflow. On the other hand, some groups
reported CB1 receptor-mediated increases in blood pressure. For
example, administration of anandamide or WIN 55212-2, a synthetic
CB receptor agonist, into the cisternal system, the rostral ventrolat-
eral medulla (RVLM) or the dorsal periaqueductal gray (dPAG) of rats
15-17
induced pressor response through CB1 receptors. Therefore,
central roles of CB1 receptors in the regulation of blood pressure are
still controversial.
The spontaneously hypertensive rat (SHR) is the most commonly
used animal model of essential hypertension with elevated sympa-
thetic nerve activity.18-20 In SHRs, alterations of central CB1 receptor
expression can contribute to hypertension and elevated sympathetic
tone,21,22 indicating a possibility that modulation of central CB1 re-
ceptor-mediated signalling can ameliorate hypertension in SHRs.
Therefore, based on our previous reports,8-10 we hypothesized that
TA B L E 1   Basal values of blood pressure and heart rate and body weight of SHR and WKY
  SBP (mm Hg) MBP (mm Hg)
SHR (n = 13) 168.2 ± 4.1* 137.2 ± 3.5*
WKY (n = 8) 138.6 ± 3.8 119.2 ± 4.1
Note: Values collected from SHR in Figure 1 and WKY in Figure 2 indicate means ± SEM
Abbreviations: DBP, diastolic blood pressure; HR, heart rate; MBP, mean blood pressure; SBP, systolic blood pressure; SHR, spontaneously
hypertensive rats; WKY, Wistar-Kyoto rats.
*P < .05, when compared to the WKY group using an unpaired Student's t-test.
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      1255
stimulation of central CB1 receptors might reduce blood pressure
by inhibiting central sympatho-adrenomedullary outflow in SHRs. In
this study, we compared the effects of centrally administered ara-
chidonyl 2′-chloroethylamide (ACEA), an agonist of CB1 receptors
on blood pressure and the central sympatho-adrenomedullary out-
flow between SHRs and their normotensive controls, Wistar-Kyoto
(WKY) rats.
2 | R E S U LT S
2.1 | General features of experimental rats
At the age of 18 weeks, systolic, mean and diastolic blood pres-
sure (SBP, MBP and DBP) were significantly higher in the SHR
group than in the WKY group. Heart rate and body weight in
the SHR group were significantly lower than in the WKY group
(Table 1).
2.2 | Effects of centrally administered ACEA or
rimonabant (a CB1 receptor antagonist) on blood
pressure and heart rate in SHRs and WKY rats
Central treatment with ACEA (1.4  µmol/animal, i.c.v.) in SHRs
significantly reduced MBP and DBP compared to SHRs centrally
treated with vehicle (3 µL N,N-dimethylformamide (DMF)/animal,
i.c.v.), and SBP showed a tendency to be reduced in the ACEA-
treated SHR group compared to the vehicle-treated SHR group,
while there were no significant changes in heart rate between
the ACEA and vehicle groups (Figure  1A-D). These reductions
in blood pressure induced by ACEA in the SHR group were not
observed in the rimonabant (300  nmol/animal, i.c.v.)-pretreated
SHR group (Figure 1B-C). In SHRs, at 180 minutes after the cen-
tral administration of ACEA or vehicle, increments to SBP, MBP,
DBP (mm Hg) and heart rate (bpm) from these values at 0 minutes
were as follows: 1.0 ± 1.0, 1.0 ± 1.5, 1.0 ± 2.0 and 0.3 ± 11.1 in the
vehicle-treated group (n = 4), −4.0 ± 1.4, −8.2 ± 0.2, −10.0 ± 0.6
and −3.7 ± 1.8 in the ACEA-treated group (n = 4), and 4.0 ± 2.3,
3.6 ± 2.3, 3.0 ± 2.4 and 11.0 ± 8.1 in the rimonabant- and ACEA-
treated group (n  =  5), respectively (Figure  1A-D). In addition, at
240  minutes after the administration of ACEA or vehicle, incre-
ments of SBP, MBP, DBP (mm Hg) and heart rate (bpm) from these
DBP (mm Hg) HR (bpm) Body weight (g)
121.6 ± 3.2* 345.4 ± 3.7* 363.1 ± 5.2*
109.7 ± 4.4 399.9 ± 6.0 391.3 ± 7.7
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1256       SHIMIZU et al.
values at 0 minute were as follows: 0.3 ± 1.2, −0.3 ± 3.0, 3.0 ± 2.0
and −2.5  ±  14.5 in the vehicle-treated group (n  =  4), −3.3  ±  1.9,
−6.5  ±  1.5, −7.3  ±  1.9 and −8.3  ±  7.7 in the ACEA-treated group
(n  =  4), and 3.3  ±  1.9, 4.0  ±  1.6, 4.3  ±  1.5 and 19.7  ±  2.3 in the
rimonabant- and ACEA-treated group (n  =  5), respectively
(Figure 1A-D). In the ACEA-treated group, values of MBP at 180
and 240  minutes and values of DBP at 240  minutes were signif-
icantly (P  <  .05) lower than values in the vehicle-treated group
(Figure 1B-C). Actual MBP (mm Hg) at 0, 180 and 240 minutes was
as follows: 141.7 ± 8.5, 150.4 ± 3.9, and 149.1 ± 2.3 in the vehicle-
treated group (n = 4), 141.3 ± 1.7, 133.9 ± 2.2, and 134.6 ± 3.7 in
the ACEA-treated group (n = 4), and 130.2 ± 5.3, 128.9 ± 1.9, and
128.8  ±  3.0 in the rimonabant- and ACEA-treated group (n  =  5),
respectively. Actual DBP (mm Hg) at 0, 180 and 240 minutes was
as follows: 125.8 ± 7.7, 133.7 ± 3.4, and 131.5 ± 2.5 in the vehicle-
treated group (n = 4), 126.3 ± 1.8, 116.7 ± 2.4, and 118.7 ± 4.1 in
the ACEA-treated group (n = 4), and 114.6 ± 4.6, 113.3 ± 1.3, and
114.7  ±  1.8 in the rimonabant- and ACEA-treated group (n  =  5),
respectively.
On the other hand, central treatment with ACEA (1.4  µmol/
animal, i.c.v.) in WKY rats showed no significant changes in SBP,
MBP, DBP or heart rate compared to WKY rats centrally treated
with vehicle (3 µL DMF/animal, i.c.v.) (Figure 1E-H). In WKY rats, at
240 minutes after the administration of ACEA or vehicle, increments
of SBP, MBP, DBP (mm Hg) and heart rate (bpm) from these values at
F I G U R E 1   Effects of centrally
administered arachidonyl
2′-chloroethylamide (ACEA) on blood
pressure and heart rate in spontaneously
hypertensive rats (SHRs) (A–D) and
Wistar-Kyoto (WKY) rats (E–H). ACEA
(an agonist of CB1 receptors) (1.4 µmol/
animal) or vehicle (3 µL DMF/animal)
was i.c.v. administered. In some SHRs,
rimonabant (Rimo, an antagonist of CB1
receptors) was pretreated (300 nmol/
animal, i.c.v.) 30 minutes before the
administration of ACEA (1.4 µmol/animal,
i.c.v.). ΔSBP (systolic blood pressure),
ΔMBP (mean blood pressure), ΔDBP
(diastolic blood pressure) and ΔHR (heart
rate): increments of SBP (A, E), MBP (B, F),
DBP (C, G) and HR (D, H) in comparison
with each value measured just before
the administration of ACEA or vehicle.
Values present means ± SEM. *P < .05,
when compared with the Bonferroni
method to the vehicle-treated group. The
number of animals per group is indicated
in parentheses. A–D:  Vehicle (n = 4),
ACEA (n = 4) and Rimo + ACEA (n = 5).
E–H: Vehicle (n = 4) and ACEA (n = 4)
0 minute were as follows: 2.0 ± 1.5, 1.6 ± 2.3, 1.3 ± 2.7 and 1.5 ± 1.5
in the vehicle-treated group (n = 4), and 4.7 ± 2.3, 4.9 ± 1.1, 5.0 ± 0.6
and −2.0  ±  8.0 in the ACEA-treated group (n  =  4), respectively
(Figure 1E-H). There was no significant difference in each increment
between these two groups.
Central treatment with rimonabant (300  nmol/animal, i.c.v.)
alone in SHRs showed no significant changes in SBP, MBP, DBP or
heart rate compared to SHRs centrally treated with vehicle (3  µL
DMF/animal, i.c.v.; Figure 2A-D). In SHRs, at 240 minutes after the
administration of rimonabant or vehicle, increments of SBP, MBP,
DBP (mm Hg) and heart rate (bpm) from these values at 0 minutes
were as follows: 0.3 ± 1.2, −0.3 ± 3.0, 3.0 ± 2.0 and −2.5 ± 14.5 in
the vehicle-treated group (n = 4), and 4.0 ± 1.6, −1.1 ± 2.3, −2.5 ± 1.9
and 13.4 ± 12.1 in the rimonabant-treated group (n = 5), respectively
(Figure 2A-D). There was no significant difference in each increment
between these two groups.
2.3 | Effects of centrally administered ACEA or
rimonabant on plasma noradrenaline and adrenaline
in SHRs and WKY rats
Basal values for plasma noradrenaline and adrenaline at 0 minutes
were 544 ± 40 and 321 ± 49 pg/mL in the SHR group (n = 18), and
394 ± 44 and 139 ± 33 pg/mL in the WKY group (n = 9), respectively.
SHIMIZU et al.
F I G U R E 2   Effects of centrally administered rimonabant on
blood pressure and heart rate in spontaneously hypertensive
rats (SHRs). Rimonabant (Rimo, 300 nmol/animal) or vehicle
(3 µL DMF/animal) was i.c.v. administered. The vehicle-treated
group is the same as in Figure 1. ΔSBP (systolic blood pressure),
ΔMBP (mean blood pressure), ΔDBP (diastolic blood pressure)
and ΔHR (heart rate): increments of SBP (A), MBP (B), DBP (C)
and HR (D) in comparison with each value measured just before
the administration of rimonabant or vehicle. Values present
mean ± SEM. The number of animals per group is indicated in
parentheses: Vehicle (n = 4) and Rimo (n = 5)
These values were collected from SHRs in Figures 3 and 4 and from
WKY rats in Figure 3. Both values in the SHR group were significantly
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      1257
higher than those in the WKY group (P < .05, unpaired Student's t-
test or Welch's test).
Central treatment with ACEA (1.4  µmol/animal, i.c.v.) in SHRs
significantly reduced plasma noradrenaline, but not adrenaline,
compared to SHRs centrally treated with vehicle (3  µL DMF/ani-
mal, i.c.v.) (Figure 3A-B). The reduction in noradrenaline induced by
ACEA in the SHR group was not observed in the rimonabant-pre-
treated SHR group (300 nmol/animal, i.c.v.) (Figure 3A-B). In SHRs,
at 180  minutes after the central administration of ACEA or vehi-
cle, rates of plasma noradrenaline and adrenaline (%) relative to
those at 0  minute were as follows: 101.8  ±  2.4 and 104.7  ±  18.0
in the vehicle-treated group (n  =  4), 71.4  ±  9.0 and 92.7  ±  9.3 in
the ACEA-treated group (n = 6), and 116.1 ± 7.9 and 114.4 ± 11.2
in the rimonabant- and ACEA-treated group (n  =  4), respectively
(Figure 3A-B). In addition, at 240 minutes after the administration
of ACEA or vehicle, rates of plasma noradrenaline and adrenaline
(%) relative to those at 0 minute were as follows: 104.7 ± 3.6 and
115.4  ±  28.2 in the vehicle-treated group (n  =  4), 75.2  ±  7.0 and
98.2 ± 12.6 in the ACEA-treated group (n = 6), and 103.6 ± 5.3 and
121.8  ±  33.2 in the rimonabant- and ACEA-treated group (n  =  4),
respectively (Figure  3A-B). In the ACEA-treated group, rates of
plasma noradrenaline at 180 and 240  minutes were significantly
(P < .05) lower than those in the vehicle-treated group (Figure 3A-
B). Actual plasma noradrenaline (pg/mL) at 0, 180 and 240 minutes
was as follows: 566.3 ± 113.9, 577.7 ± 119.6 and 586.3 ± 103.3 in
the vehicle-treated group (n = 4), 536.3 ± 56.1, 345.0 ± 62.3, and
412.0 ± 67.2 in the ACEA-treated group (n = 6), and 454.9 ± 88.2,
517.5 ± 94.5, and 500.0 ± 20.6 in the rimonabant- and ACEA-treated
group (n = 4), respectively.
On the other hand, central treatment with ACEA (1.4  µmol/
animal, i.c.v.) in WKY rats showed no significant changes in plasma
noradrenaline or adrenaline compared to WKY rats centrally treated
with vehicle (3  µL DMF/animal, i.c.v.) (Figure  3C-D). In WKY rats,
at 240 minutes after the central administration of ACEA or vehicle,
rates of plasma noradrenaline and adrenaline (%) relative to those
at 0  minutes were as follows: 96.0  ±  1.0 and 101.2  ±  34.9 in the
vehicle-treated group (n  =  4), and 106.7  ±  6.3 and 116.5  ±  5.6 in
the ACEA-treated group (n  =  5), respectively (Figure  3C-D). There
was no significant difference in each increment between these two
groups.
Central treatment with rimonabant (300  nmol/animal, i.c.v.)
alone in SHRs tended to increase plasma noradrenaline or adren-
aline compared to SHRs centrally treated with vehicle (3 µL DMF/
animal, i.c.v.), but there was no significant change between these
two groups (Figure 4A-B). In SHRs, at 240 minutes after the cen-
tral administration of rimonabant or vehicle, rates of plasma nor-
adrenaline and adrenaline (%) relative to those at 0 minutes were
as follows: 104.7  ±  3.6 and 115.4  ±  28.2 in the vehicle-treated
group (n  =  4), and 112.8  ±  6.4 and 110.9  ±  35.3 in the rimon-
abant-treated group (n = 4), respectively (Figure 4A-B). There was
no significant difference in each increment between these two
groups.
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1258       SHIMIZU et al.

F I G U R E 3   Effects of centrally administered arachidonyl 2′-chloroethylamide (ACEA) on plasma levels of noradrenaline and adrenaline
in spontaneously hypertensive rats (SHRs) (A, B) and Wistar–Kyoto (WKY) rats (C, D). ACEA (1.4 µmol/animal) or vehicle (3 µL DMF/animal)
was i.c.v. administered. In some SHRs, rimonabant (Rimo) was pretreated (300 nmol/animal, i.c.v.) 30 minutes before the administration
of ACEA (1.4 µmol/animal, i.c.v.). Data were calculated as the ratio of plasma noradrenaline (A, C) and adrenaline (B, D) values measured
just before the administration of ACEA or vehicle. Values present mean ± SEM. *P < .05, when compared with the Bonferroni method
to the vehicle-treated group. The number of animals per group is indicated in parentheses. A, B: Vehicle (n = 4), ACEA (n = 6) and
Rimo + ACEA (n = 4). C, D: Vehicle (n = 4) and ACEA (n = 5)

which is in agreement with previous reports. 23-27 In addition, SHRs

3 |  D I S CU S S I O N
In this study, we confirmed that SHRs exhibited higher blood pres-
sures (SBP, MBP and DBP) and basal levels of plasma noradrenaline
and adrenaline, and lower heart rate and body weight than WKY
rats. Central treatment with ACEA partially reduced MBP and DBP
and the level of plasma noradrenaline in SHRs, while central treat-
ment with vehicle or rimonabant showed no effect on these values
in SHRs. These ACEA-induced reductions were abolished by central
pretreatment with rimonabant. On the other hand, centrally admin-
istered ACEA had no effect on blood pressure or plasma level of no-
radrenaline and adrenaline in normotensive WKY rats.
Our present data indicate that SHRs exhibited hypertension and
elevated sympathetic nerve activity as evidenced by higher levels of
basal plasma noradrenaline and adrenaline compared with WKY rats,
exhibited a lower heart rate than WKY rats in this study. In fact, our
group previously reported that SHRs exhibited a lower heart rate
than WKY rats at the age of 18 weeks (SHRs vs WKY rats; 299 ± 3 vs
328 ± 9 bpm26; 302 ± 4 vs 338 ± 10 bpm27). In line with our present
and previous data, Batkai et al also reported that SHRs exhibited a
lower heart rate than WKY rats at the age of 8–10 months. 25 On the
other hand, it was reported that SHRs exhibited a higher heart rate
than WKY rats at the age of 4 weeks (SHRs vs WKY rats; 441 ± 4
vs 407 ± 5 bpm28) and at the age of 12–16 weeks. 29 Tachycardia in
SHRs is explained at least in part by attenuation of baroreflex func-
tion. 28,30 However, bradycardia in SHRs is observed at compara-
tively older ages. 25-27 Therefore, long-term exposure to high blood
pressure might recruit other compensatory mechanisms which can
counteract attenuated baroreflex function in SHRs, thereby induc-
ing bradycardia.
SHIMIZU et al.
F I G U R E 4   Effects of centrally administered rimonabant on
plasma levels of noradrenaline and adrenaline in spontaneously
hypertensive rats (SHRs). Rimonabant (Rimo, 300 nmol/animal)
or vehicle (3 µL DMF/animal) was i.c.v. administered. The vehicle-
treated group is the same as in Figure 3. Data were calculated as
the ratio of plasma noradrenaline (A) and adrenaline (B) values
measured just before the administration of rimonabant or vehicle.
Values present mean ± SEM. The number of animals per group is
indicated in parentheses: Vehicle (n = 4) and Rimo (n = 4)
Cannabinoid CB receptors are members of the superfamily of G
protein-coupled receptors and are divided into two subtypes, CB1
and CB2. CB1 receptors are mainly present in the central nervous
system while CB2 receptors are predominant in immune cells. 31,32
In this study, central administration of ACEA in SHRs partially de-
creased MBP and DBP and reduced sympathetic tone, as indicated
by a reduction in plasma noradrenaline with rimonabant-sensitive
brain mechanisms. Centrally administered ACEA reduced plasma
noradrenaline, but not adrenaline, in SHRs, so ACEA may mainly re-
duce peripheral vascular resistance, thereby reducing DBP. On the
other hand, in WKY rats, ACEA had no effect on blood pressure or
on sympathetic tone. These data are supported by our previous re-
ports showing that centrally administered AM251, a CB1 receptor
antagonist, potentiated elevation of plasma noradrenaline induced
by centrally administered CRF- or bombesin, while AM251 alone
had no effect on the basal level of plasma noradrenaline in nor-
motensive Wistar rats.9,10 In addition, systemically administered
inhibitor of fatty acid amide hydrolase (FAAH), which catalyzes the
degradation of anandamide, in SHRs normalized the elevated blood
pressure and sympathetic tone without affecting these parameters
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      1259
in WKY rats. 25,29 These FAAH inhibitor-induced effects were re-
versed by systemic pretreatment with the brain-penetrant CB1 re-
ceptor antagonist (AM251), but not by the peripherally restricted
antagonist. 29 These lines of evidence from our and other groups
suggest that stimulation of brain CB1 receptors can ameliorate hy-
pertension accompanied with enhanced sympathetic outflow in
SHRs without affecting blood pressure under normotensive condi-
tions. Godlewski et al reported that enhanced G protein activation
was observed in brain CB1 receptors of SHRs compared to WKY
rats, 29 suggesting that sensitivity of ACEA to brain CB1 receptors
might be higher in SHRs than in WKY rats. Therefore, at least in
our conditions, centrally administered ACEA may induce no ef-
fect on blood pressure or sympatho-adrenomedullary outflow in
normotensive WKY rats. However, based on previous reports, the
roles of brain CB1 receptors in regulating blood pressure seem to
be different depending on the brain regions, with a hypotensive
role in the hypothalamic PVN13,14 and NTS,12 and a hypertensive
role in the RVLM and dPAG.15-17 In addition, down-regulation of
CB1 receptors in the NTS and up-regulation of CB1 receptors in the
RVLM were reported in SHRs. 21,22 Therefore, these changes to CB1
receptors might be a cause of hypertension in SHRs. Since we used
i.c.v. administration of ACEA, a limitation of this study is that it is
unclear which central CB1 receptors were involved in the ACEA-
induced reduction of blood pressure in SHRs. Further studies are
therefore needed to examine the specific location in the brain that
contributes to CB1 receptor-mediated depressor responses under
hypertensive conditions.
Centrally administered AM251 alone showed no effect on
plasma levels of noradrenaline in normotensive Wistar rats in
our previous reports,9,10 so we speculate that blocking brain CB1
receptors shows no effect on normal sympathetic tone or basal
blood pressure of normotensive rats, but can further enhance
higher sympathetic tone and hypertension of SHRs. In this study,
central administration of rimonabant alone in SHRs tended to in-
crease plasma levels of noradrenaline and adrenaline but showed
no effect on blood pressure. Although the dose of rimonabant
in this study might be insufficient to block endogenous CB1 re-
ceptors-mediated modulation of hypertension in SHRs, the dose
seemed to be sufficient to cancel the exogenous ACEA-induced
attenuation of enhanced sympathetic tone and hypertension in
SHRs. Therefore, the dose of rimonabant was suitable for evaluat-
ing the involvement of brain CB1 receptors on the ACEA-induced
responses. Of course, since our data were insufficient to clarify
the roles of “endogenous” CB1 receptors in hypertension of SHRs,
further studies are necessary to investigate the “dose-depen-
dency” of rimonabant on enhanced sympathetic tone and hyper-
tension in SHRs.
Our present data indicate that in SHRs, stimulation of central
CB1 receptors can reduce blood pressure by inhibiting the cen-
tral sympatho-adrenomedullary outflow, especially reducing ele-
vated plasma noradrenaline. However, there are two limitations of
this study: (a) it is unclear whether the ACEA-induced reduction of
blood pressure in SHRs was mediated by changes of alpha and beta
 |
1260       SHIMIZU et al.
adrenoceptors-mediated responses; (b) the effects of centrally admin-
istered rimonabant alone on blood pressure in normotensive WKY rats
were not investigated. The former point (a) should be investigated in
the future by confirming whether each adrenoceptor agonist-induced
reversal of ACEA-induced reduction of noradrenaline-mediated sig-
nalling can attenuate the ACEA-induced reduction of blood pressure
in SHRs. The latter point (b) should also be investigated in the future
to clarify the roles of CB1 receptors in regulation of basal blood pres-
sure under a normotensive condition, although our previous data in-
dicated that centrally administered AM251 alone showed no effect
on plasma levels of noradrenaline in normotensive Wistar rats.9,10 In
addition, because Niederhoffer et al reported that in rabbit isolated
adrenal glands, electrically-evoked adrenaline release was inhibited by
33
WIN 55212-2, we cannot exclude a possibility that in SHRs, centrally
administered ACEA can leak into the systemic circulation and directly
stimulate CB1 receptors in the adrenal medulla, thereby inhibiting nor-
adrenaline secretion from the medulla. Previously, we preliminarily
confirmed that intravenously administered ACEA showed no signifi-
cant change of plasma noradrenaline in normotensive Wistar rats.9,10
However, the effects of intravenously administered ACEA on plasma
noradrenaline in SHRs have not been investigated yet. In the future,
this possibility should be also investigated by using isolated adrenal
grands from SHRs.
Cannabinoid CB1 receptors are widely distributed in the
brain,31,32 therefore global stimulation of these receptors in the brain
by agonists is associated with unwanted psychoactive side effects
such as cognitive impairment, dysphoria/depression and tiredness/
fatigue.34,35 In addition, synthetic agonists of these receptors can
modulate behaviours related to drug addiction induced by morphine
36,37
or heroin. Interestingly, systemically administered hemopressins,
which are peptidic ligands for CB1 receptors,38,39 do not activate the
rat brain reward centres including the ventral tegmental area, unlike
other conventional synthetic ligands for these receptors.40 We re-
ported that centrally administered RVD-hemopressin, one type of
hemopressin, reduced centrally administered bombesin-induced el-
evation of plasma noradrenaline and adrenaline via rimonabant-sen-
sitive brain mechanisms, while this peptide alone had no effect on
41
the basal level of these catecholamines. RVD-hemopressin, but
not hemopressin, has agonist activity at CB1 receptors,38,39 so this
peptide may be a useful candidate to stimulate brain CB1 receptors
without addictive side effects. Furthermore, instead of exogenously
stimulating the brain CB1 receptor, enhancing the effects of brain
endocannabinoid by blocking its degradation may also be a useful
way to reduce aversive side effects induced by global stimulation of
these receptors.
In summary, we demonstrated that stimulation of brain canna-
binoid CB1 receptors can ameliorate hypertension and enhanced
sympathetic outflow in SHRs without affecting these parameters in
normotensive WKY rats. These findings indicate that agonists tar-
geting brain CB1 receptors, like RVD-hemopressin, would be useful
for hypertension treatment without the risk of addictive side effects
such as addiction and the risk of hypotension if these agonists were
to be administered to “normotensive” patients by mistake.
4 | M ATE R I A L S A N D M E TH O DS
4.1 | Animals
All animal experiments were conducted in accordance with NIH
guidelines and approved by the Institutional Animal Care and Use
Committee of Kochi University (#G-6, #H-38, #I-66). All efforts
were made to minimize the suffering of the animals and the number
of animals needed to obtain reliable results. A total of 53 animals,
36 male SHRs and 17 male WKY rats (the normotensive control of
SHRs), were used in this study. Eighteen SHRs and eight WKY rats
were used to monitor blood pressure and heart rate, and 18 SHRs
and nine WKY rats were used to monitor plasma noradrenaline and
adrenaline, as described below. Twelve-week-old male SHRs (SHR/
Izm) and WKY rats (WKY/Izm) were provided by the Disease Model
Cooperative Research Association (Kyoto, Japan). These rats were
housed at two per cage and were maintained in an air-conditioned
room at 22–24°C under a constant day–night rhythm and given food
and water ad libitum. After reaching 18  weeks of age, these rats
were used for experiments.
4.2 | Experimental procedures for i.c.v.
administration
In the morning (09:00–10:00), under urethane anaesthesia (1.0 g/kg,
i.p.), two catheters were inserted into the femoral artery and the vein
to collect blood samples and infuse saline (1.2  mL/h), respectively.
Subsequently, each rat was placed in a stereotaxic apparatus for the
brain (SR-6R; Narishige) until the end of each experiment, as described
previously in a published study from our laboratory.42 The skull was
drilled for intracerebroventricular administration of drugs using a
stainless-steel cannula (outer diameter of 0.3 mm). The stereotaxic co-
ordinates of the tip of the cannula were as follows (in mm): AP –0.8, L
1.5, V 4.0 (AP, anterior from the bregma; L, lateral from the midline; V,
below the surface of the brain), according to the rat brain atlas.43 Three
hours after surgery, the steel cannula was injected into the right lateral
ventricle and each drug was administered as described below.
4.3 | Drug administration
In a published study from our laboratory, ACEA (0.28, 0.7 or
1.4  µmol/animal, i.c.v.) dose-dependently reduced each stress-re-
lated neuropeptide (CRF or bombesin)-induced elevation of plasma
noradrenaline and adrenaline in normotensive Wistar rats.9,10 In ad-
dition, we preliminarily checked the dose-dependency of ACEA (0.7
or 1.4 µmol/animal, i.c.v.) in SHRs, then selected the effective dose
of 1.4 µmol in this study. Our preliminary experiments indicated that
the ideal dose of rimonabant was 300 nmol/rat.
Either ACEA or rimonabant dissolved in 3 µL of 100% DMF was
slowly administered into the right lateral ventricle using a cannula
connected to a 10-µL Hamilton syringe at a rate of 10 µL/min, and
SHIMIZU et al.
the cannula was retained until the end of the experiment. In some
SHRs, rimonabant dissolved in 3  µL of DMF was i.c.v. pretreated
30  minutes before the administration of ACEA. In this combined
administration, the cannula was retained in the ventricle for 15 min-
utes to avoid leakage of the antagonist and then removed from the
ventricle. Subsequently, ACEA was i.c.v. administered. The exact lo-
cation of the cannula was confirmed at the end of each experiment
by verifying that a Cresyl Violet solution, injected through the can-
nula, had spread throughout the entire ventricular system.
4.4 | Experimental groups for i.c.v. administration
In monitoring blood pressure and heart rate, 26 rats placed in a stere-
otaxic apparatus were divided into six groups: vehicle (3  µL DMF/ani-
mal, i.c.v.)-administered SHR group (n  =  4); vehicle (3  µL DMF/animal,
i.c.v.)-administered WKY group (n = 4); ACEA (1.4 µmol [500 µg]/animal,
i.c.v.)-administered SHR group (n = 4); ACEA (1.4 µmol/animal, i.c.v.)-ad-
ministered WKY group (n = 4); rimonabant (300 nmol [139 µg]/animal,
i.c.v.)- and ACEA (1.4 µmol/animal, i.c.v.)-administered SHR group (n = 5);
and rimonabant (300 nmol/animal, i.c.v.)-administered SHR group (n = 5).
In the measurement of plasma noradrenaline and adrenaline, 27
rats placed in a stereotaxic apparatus were divided into six groups:
vehicle (3  µL DMF/animal, i.c.v.)-administered SHR group (n  =  4);
vehicle (3  µL DMF/animal, i.c.v.)-administered WKY group (n  =  4);
ACEA (1.4 µmol/animal, i.c.v.)-administered SHR group (n = 6); ACEA
(1.4 µmol/animal, i.c.v.)-administered WKY group (n = 5); rimonabant
(300 nmol/animal, i.c.v.)- and ACEA (1.4 µmol/animal, i.c.v.)-adminis-
tered SHR group (n = 4); and rimonabant (300 nmol/animal, i.c.v.)-ad-
ministered SHR group (n = 4).
4.5 | Monitoring of blood pressure and heart rate
The monitoring was performed as described previously.44 Briefly, after
the surgery described in Section 4.2, the arterial catheter was con-
nected to a pressure transducer (DX-100; Nihon Koden) that was con-
nected to a carrier amplifier (AP-601G; Nihon Koden) and to a heart
rate counter (AT-601G; Nihon Koden). Signals provided by the trans-
ducer were monitored using a flat pen recorder (FBR-253A; Hioki EE
Corporation). Monitoring was started after placement in a stereotaxic
apparatus, and was performed until 240 minutes after ACEA adminis-
tration described in Section 4.3. MBP was calculated by the following
formula: MBP = DBP + (SBP − DBP)/3. Pressure transducers were cali-
brated daily using a mercury manometer.
4.6 | Measurement of
plasma noradrenaline and adrenaline
Blood samples (250 µL) were collected through the arterial catheter
at 0, 60, 120, 180 and 240 minutes after the administration of ACEA,
rimonabant or vehicle (3 µL DMF). These samples were preserved
|
      1261
on ice during experiments. Plasma was prepared immediately after
the final sampling. Noradrenaline and adrenaline in the plasma were
extracted by the method of Anton and Sayre 45 with a slight modifi-
cation and were assayed electrochemically with high performance
liquid chromatography.42
4.7 | Treatment of data and statistics
All values are expressed as mean ± SEM. Statistical differences were
determined using repeated-measure (treatment × time) analysis of vari-
ance, followed by post hoc analysis with the Bonferroni method. When
only two means were compared, an unpaired Student's t-test or Welch's
test was used. P values <.05 were considered statistically significant.
4.8 | Drugs and chemicals
ACEA (arachidonyl 2′-chloroethylamide) and rimonabant
[5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-N-1-piperi-
dinyl-1H-pyrazole-3-carboxamide] were purchased from Cayman
Chemical. All other reagents of the highest grade were purchased from
Nacalai Tesque.
AC K N OW L E D G E M E N T S
The authors would like to thank SciRevision for linguistical reviewing of
the manuscript. This work was supported in part by a Grant-in-Aid for
Scientific Research (C) (No. 26460909 to TS) from the Japan Society for
the Promotion of Science, a grant from The Japan Health Foundation,
and a grant from The Smoking Research Foundation in Japan.
C O N FL I C T O F I N T E R E S T
The authors have no conflicts of interest to declare.
ORCID
Takahiro Shimizu  https://orcid.org/0000-0002-1555-2284
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How to cite this article: Shimizu T, Yamamoto M, Zou S,
Shimizu S, Higashi Y, Saito M. Stimulation of brain cannabinoid
CB1 receptors can ameliorate hypertension in spontaneously
hypertensive rats. Clin Exp Pharmacol Physiol. 2020;47:1254–
1262. https://doi.org/10.1111/1440-1681.13297