Mol Cell Biochem (2014) 388:135–147
DOI 10.1007/s11010-013-1905-2
The action of p-synephrine on hepatic carbohydrate metabolism
and respiration occurs via both Ca2+-mobilization and cAMP
production
Andrea Luiza de Oliveira • Jurandir Fernando Comar
Anacharis Babeto de Sá-Nakanishi •
Rosane Marina Peralta • Adelar Bracht
Received: 21 September 2013 / Accepted: 15 November 2013 / Published online: 28 November 2013
 Springer Science+Business Media New York 2013
Abstract Citrus aurantium extracts, which contain large
amounts of p-synephrine, are widely used for weight loss
purposes and as appetite suppressants. In the liver, C. aur-
antium (bitter orange) extracts affect hemodynamics, car-
bohydrate metabolism, and oxygen uptake. The purpose of
the present work was to quantify the action of p-synephrine
and also to obtain indications about its mechanism of action,
a task that would be difcult to accomplish with C. auran-
tium extracts due to their rather complex composition. The
experimental system was the isolated perfused rat liver. p-
Synephrine signicantly stimulated glycogenolysis, gly-
colysis, gluconeogenesis, and oxygen uptake. The com-
pound also increased the portal perfusion pressure and the
redox state of the cytosolic NAD?/NADH couple. A Ca2?-
dependency for both the hemodynamic and the metabolic
effects of p-synephrine was found. p-Synephrine stimulated
both cAMP overow and the initial Ca2? release from the
cellular stores previously labeled with 45Ca2?. The meta-
bolic and hemodynamic actions of p-synephrine were
strongly inhibited by a-adrenergic antagonists and moder-
ately affected by b-adrenergic antagonists. The results allow
to conclude that p-synephrine presents important metabolic
and hemodynamic effects in the liver. These effects can be
considered as both catabolic (glycogenolysis) and anabolic
(gluconeogenesis), they are mediated by both a- and b-
adrenergic signaling, require the simultaneous participation
of both Ca2? and cAMP, and could be contributing to the
overall stimulation of metabolism that usually occurs during
weight loss periods.
A. L. de Oliveira  J. F. Comar  A. B. de Sá-Nakanishi 
R. M. Peralta  A. Bracht (&)
Department of Biochemistry, University of Maringá, Avenida
Colombo 5790, Maringá 87020900, Brazil
e-mail: adebracht@uol.com.br
•
Keywords Adrenergic signaling  Liver
metabolism  Liver hemodynamics  Weight loss
Introduction
Citrus aurantium is a plant that belongs to the Rutaceae
family. The leaves, the peel, and the edible part of the fruits
of C. aurantium contain elevated amounts of alkaloids. The
fruit of the plant, also known as bitter orange, has been used
for preparing extracts sold worldwide in the form of phyto-
products to promote weight loss [1]. One of the main active
components in C. aurantium extracts [2], p-synephrine
(Fig. 1), also known simply as synephrine or oxedrine, has
been used worldwide in the treatment of hypotensive states
and as an ocular decongestant [3]. Formed by a pathway
involving tyramine and N-methyltyramine [4], p-synephrine
is structurally similar to ephedrine and epinephrine, and
widely distributed among plants, bacteria, invertebrates, and
vertebrates. In humans, p-synephrine is found only in the
adrenal gland and can be considered a trace bioamine [2].
In line with the proposed weight loss activity of the C.
aurantium extracts, it has been found that p-synephrine has
lipolytic activity in both human and rat adipocytes [1]. This
activity of p-synephrine is stronger in rat than in human
adipocytes. Considering that p-synephrine is the main
alkaloid in C. aurantium extracts, the possible lipid-
mobilizing effect of this extract in humans has been
attributed to this amine [1]. A lipolytic effect that is
probably elicited by p-synephrine has also been noted in a
study using Citrus unshu extracts, popularly known as
Satsuma mandarin [5].
Regarding the mechanism of action of p-synephrine,
studies have reported the activity of this compound on
specic receptors. It has been reported, for example, that p-
123
136
Fig. 1 Chemical structure of p-synephrine
synephrine binds to serotoninergic receptors [6]. Likewise,
an action of p-synephrine via adrenergic receptors has been
well-documented [7–9]. Acute oral administration of ele-
vated doses of a C. aurantium extract or p-synephrine
produced reversible toxic effects, probably due to unspe-
cic adrenergic stimulation [3]. In addition, it is also
known that extracts of C. aurantium containing p-syneph-
rine, can produce effects on the cardiovascular system
through adrenergic stimulation [10].
Besides the lipolytic effects of p-synephrine, there are
also reports about an inuence of this compound on car-
bohydrate metabolism. It has been shown, for example, that
p-synephrine increases glucose consumption and lactic
acid production in cultured skeletal muscle cells. These
effects have been attributed to stimulation of adrenergic
receptors [11]. A clinical trial has revealed that in addition
to the effects on blood pressure and exercise tolerance, the
ingestion of a dietetic supplement containing p-synephrine
increases blood glucose postexercise. This effect is sig-
nicant, but it is difcult to attribute it solely to p-sy-
nephrine, since the referred supplements also contained
caffeine in its composition [12]. In a recent work, it was
shown that C. aurantium extracts containing p-synephrine
in the range up to 190 lM increased glycogen catabolism,
oxygen uptake, and perfusion pressure in the isolated per-
fused rat liver, and inhibited gluconeogenesis [13]. The
effects on glycogen catabolism and perfusion pressure were
sensitive to several adrenergic antagonists. In the same
work, preliminary experiments with pure p-synephrine at a
concentration of 200 lM reproduced most but not all of the
effects of the C. aurantium extract. The latter contains
many substances which could be contributing to its overall
effect. Consequently, at this stage of the investigations, it
seems more appropriate to use puried compounds for
characterizing further the effects of the active principles of
C. aurantium. This characterization comprises the con-
centration dependences of the effects and also their sensi-
tivity to various hormone antagonists and their possible
dependence on co-factors such as calcium ions and cAMP.
This was exactly the purpose of the present work, in which
the effects of p-synephrine on catabolic and anabolic routes
of carbohydrate metabolism in the rat liver were quantied.
Experiments were also done with the purpose of obtaining
information about the participation of Ca2? and cAMP in
123
Mol Cell Biochem (2014) 388:135–147
the signaling cascades leading to the hemodynamic and
metabolic effects of p-synephrine.
Materials and methods
Materials
The liver perfusion apparatus was built in the workshops of
the University of Maringá. p-Synephrine, adrenergic
antagonists, enzymes, and coenzymes used in the enzy-
matic assays were purchased from Sigma Chemical Co.
(St. Louis, MO, USA). 45Ca2? (1.7 mCi mmol-1) and the
biodegradable counting scintillant solution (BCSÒ) were
purchased from Amersham Life Science (Buckingham-
shire, UK). All other chemicals were from the best avail-
able grade.
Animals
Male Wistar rats, weighing 200–280 g, were used in all
experiments. Animals were fed ad libitum with a standard
laboratory diet (NuvilabÒ, Colombo, Brazil) and main-
tained on a regulated light–dark cycle. In accordance with
the requirements of the experimental protocols, fed rats as
well as 18 h fasted rats were used. For the surgical pro-
cedure, the rats were anesthetized by intraperitoneal
injection of sodium pentobarbital (50 mg kg-1). The cri-
terion of anesthesia was the lack of body or limb movement
in response to a standardized tail clamping stimulus. All
experiments were done in accordance with the world-wide
accepted ethical guidelines for animal experimentation.
Liver perfusion
Hemoglobin-free, nonrecirculating perfusion was per-
formed [14, 15]. After cannulation of the portal and cava
veins, the liver was positioned in a plexiglass chamber. The
constant perfusate ow was provided by a peristaltic pump
(Minipuls 3, Gilson, France) and adjusted between 30 and
33 mL min-1, depending on the liver weight. The perfu-
sion uid was Krebs/Henseleit-bicarbonate buffer (pH 7.4)
containing 25 mg % bovine-serum albumin, saturated with
a mixture of oxygen and carbon dioxide (95:5) by means of
a membrane oxygenator with simultaneous temperature
adjustment (37 °C). The composition of the Krebs/Hense-
leit-bicarbonate buffer is the following: 115 mM NaCl,
25 mM NaHCO3, 5.8 mM KCl, 1.2 mM Na2SO4,
1.18 mM MgCl2, 1.2 mM NaH2PO4, and 2.5 mM CaCl2.
The perfusion uid enters the liver via the portal vein
cannula and leaves the organ through the cava vein can-
nula. Samples of the efuent perfusion uid were collected
and analyzed for their metabolite contents. p-Synephrine
Mol Cell Biochem (2014) 388:135–147
was added to the perfusion uid at the desired concentra-
tions (up to 500 lM).
Analytical
In the efuent perfusion uid, the following compounds
were assayed by means of standard enzymatic procedures:
glucose, lactate and pyruvate [16]. The oxygen concentra-
tion in the outowing perfusate was monitored continuously
continuously, employing a Teon-shielded platinum elec-
trode adequately positioned in a plexiglass chamber at the
exit of the perfusate [14]. Metabolic rates were calculated
from input–output differences and the total ow rates, and
were referred to the wet weight of the liver.
The portal perfusion pressure was monitored by means
of a pressure transducer (Hugo Sachs Elektronic-Harvard
Apparatus GmbH, March-Hugstetten, Germany). The sen-
sor was positioned near the entry of the portal vein, and the
transducer was connected to a recorder [17]. The pressure
changes were computed from the recorder tracings and
expressed as millimeters of mercury (mm Hg).
Quantication of 45Ca present in perfusate samples was
done by liquid scintillation counting. A commercial scin-
tillation liquid formed by an aqueous biodegradable
emulsion (BCSÒ, Biodegradable Counting Scintillant,
Amersham) was used. Measurement of adenosine 2,5
cyclic monophosphate (cAMP) in perfusate samples was
done by an ELISA commercial kit (cAMP EIA kit from
EnzoÒ Life Sciences).
Statistical analysis and calculations
The error parameters presented in the graphs are standard
errors of the means. Statistical analysis was performed with
the GraphPad Prism SoftwareÒ (version 5.0; Graph Pad
Software, San Diego, CA, USA). The Student’s t test was
applied, and the 5 % level (p \ 0.05) was adopted
as a criterion of signicance. Numerical interpolation
(Stineman’s interpolation formula) was done using formula
in the Scientist Program from MicroMathÒ (Saint Louis,
MO, USA).
Results
Effects of p-synephrine on glycogen catabolism,
glycolysis and oxygen uptake
The rst experiments were planned to evaluate the effects
of p-synephrine on carbohydrate catabolism and oxygen
uptake in the liver of fed rats. When perfused with
137
substrate-free medium, livers from fed rats survive at the
expense of the oxidation of endogenous fatty acids (major
route) and glycolysis from endogenous glycogen (minor
route). Under these conditions, the livers release glucose,
lactate, and pyruvate as a result of glycogen catabolism
[14]. Figure 2a illustrates the responses of perfused rat
livers to the infusion of p-synephrine at the concentration
of 200 lM. It also illustrates a typical experimental pro-
tocol, which was used for all other drug concentrations.
After a preperfusion period of 10 min, 200 lM p-syneph-
rine was infused during 20 min. This was followed by
additional 10 min of drug-free perfusion. Four parameters
were measured: glucose release, lactate, and pyruvate
production and oxygen consumption. As can be noticed in
Fig. 2a, all parameters were stable before the initiation of
p-synephrine infusion. After the onset of the infusion,
however, oxygen uptake increased and remained so during
the entire infusion period. It was signicantly stimulated
with an increment of 0.54 ± 0.10 lmol min-1 g-1 above
the basal rates (p = 0.04, n = 3). Glucose output was
rapidly stimulated with an increase of 120 % above the
basal level and remained stable during the whole time of
drug infusion. Lactate production was also stimulated, but
the effect was not stable. After a peak increment of 72 %,
the stimulation decreased progressively, but it was still
signicant at the end of the p-synephrine infusion period.
Pyruvate production was diminished with a maximal
decrease of 32 % just after initiation of the p-synephrine
infusion. Removal of p-synephrine from the perfusion uid
at 30 min perfusion time caused rapid responses that are
indicative of reversibility.
Experiments as those illustrated by Fig. 2a were repe-
ated with p-synephrine 10, 25, 50, 100, and 500 lM in
order to investigate the concentration dependence of the
effects. The mean results are shown in Fig. 2b and repre-
sent the nal values of each parameter at the end of the
drug infusion period (25–30 min perfusion time in Fig. 2a).
All variables were represented against the portal p-sy-
nephrine concentration in Fig. 2b. The effects of p-sy-
nephrine on glucose output and oxygen uptake increased in
the range up to 100 lM, and remained approximately the
same until 500 lM. Half-maximal stimulation of glucose
output and oxygen uptake can be expected at p-synephrine
concentrations of 26.1 and 8.95 lM, respectively, as
revealed by numerical interpolation. Stimulation of lactate
production was also saturable with the half-maximal effect
at the concentration of 63.3 lM. Pyruvate production was
stimulated at low concentrations, but stimulation turned
into inhibition at high concentrations. This fact and the
lactate production stimulation leaded to increased lactate to
pyruvate ratios, from 4 (basal) to 10.5 at the p-synephrine
concentrations of 200 and 500 lM.
123
138 Mol Cell Biochem (2014) 388:135–147

Lactate or pyruvateoutput (µmol min g )

Pyruvate output (µmol min -1 g -1)
0.5 2.0

-1
-1
0.4
1.6

0.3
1.2
0.2
2.5
Lactate output (µmol min g )
-1 -1

0.8
0.1
2.0
0 0.4
1.5
2.8 2.5
0

Oxygen uptake(µmol min -1 g-1 )

Oxygen uptake (µmol min-1 g-1)

1.0
2.3
2.4
0.5
2.1
0 2.0
Lactate
4.5 Pyruvate 1.9
3.2
Glucose output (µmol min g )

Oxygen
-1

1.6

Glucose output (µmol min-1 g -1)

Glucose
-1

2.8 1.7
3.5
1.2
2.4 1.5
2.5 Lactate
2.0
Pyruvate
1.5 1.6
Oxygen

p-Synephrine(200 µM)
1.2 Glucose
0.5
0.8
0 5 10 15 20 25 30 35 40
0 100 200 300 400 500
Perfusion time (minutes)
p-Synephrine concentration (µM)

A B
Fig. 2 Changes in glycogen catabolism and oxygen uptake caused by b Concentration dependence of the effects of p-synephrine. For each
p-synephrine. Livers of fed rats were perfused as described in concentration the metabolic rates found at the end of the infusion
‘‘Materials and methods’’ section. Samples of the efuent perfusate period (30 min perfusion time) were computed and represented
were taken for measuring glucose, lactate, and pyruvate. Oxygen in against the concentration; for zero p-synephrine concentration the
the efuent perfusion uid was monitored polarographically. a Time basal rates (10 min perfusion time) were represented. Data are
courses of the effects caused by 200 lM p-synephrine. means ± mean standard errors of 3–4 liver perfusion experiments

Effects of p-synephrine on gluconeogenesis p = 0.0005). Glucose production was rapidly stimulated and
and associated variables remained stable during the whole time of drug infusion (from
0.69 ± 0.01 to 0.96 ± 0.08 lmol min-1 g-1, n = 3,
Experiments were additionally done with livers from 18 h p = 0.03). The pyruvate production was clearly reduced
fasted rats with the intention of evaluating the effects of (-26 % at the end of the infusion). The effects were revers-
p-synephrine on gluconeogenesis (Fig. 3a). For this pur- ible, i. e., there was a clear tendency of returning to the basal
pose, 2 mM lactate was used as a gluconeogenic substrate. values after cessation of the p-synephrine infusion.
After the stabilization of oxygen consumption, lactate was The effects of p-synephrine under glucogenic conditions
infused during 30 min followed by an additional period of were further investigated for their concentration depen-
20 min in which 2 mM lactate plus 50 lM p-synephrine were dence. The mean results are shown in Fig. 3b. Oxygen
infused, as illustrated by Fig. 3a. The following parameters uptake was stimulated more strongly at low concentrations
were measured: oxygen uptake and glucose and pyruvate and maximal stimulation occurred at the concentration of
productions. The infusion of 2 mM lactate caused immediate 50 lM. Half-maximal stimulation, on the other hand, can
increases in all parameters. At 30 min of perfusion time, they be expected at the concentration of 8.8 lM. Stimulation
had already reached new steady-states. The infusion of clearly declined at the concentrations between 100 and
50 lM p-synephrine immediately elevated oxygen uptake, 500 lM. Gluconeogenesis stimulation also presented a
which remained so during the whole drug infusion time (from maximum at 50 lM and declined thereafter. The concen-
2.59 ± 0.05 to 3.29 ± 0.04 lmol min-1 g-1, n = 3; tration for half-maximal stimulation was 4.1 lM. Pyruvate

123
Mol Cell Biochem (2014) 388:135–147 139

0.6
0.6
Pyruvate production (µmol min -1 g-1)

Pyruvate production (µmol min-1 g-1 )

0.5

0.4
0.4

Pyruvate 4.0
Glucose 0.3

Glucose production (µmol min -1 g-1)

0.2 1.2
3.8
Oxygen

Oxygen uptake (µmol min g )

-1 -1
0.2 3.6
0.9

0 3.4
0.1
0.6
Pyruvate 3.2

1.3
3.4 Oxygen 3.0
0.3

Glucose production (µmol min-1 g-1)

Oxygen uptakte (µmol min-1 g-1)

1.2
Glucose 2.8
3.0 0 1.1
2.6

1.0
2.6
0.9

2.2 Synephrine 0.8

(50 µM)
0.7
1.8 Lactate (2 mM)
0.6
0 10 20 30 40 50 60 70
0 100 200 300 400 500
Perfusion time (minutes) p-Synephrine concentration (µM)

A B
Fig. 3 Changes in lactate gluconeogenesis and associated parameters p-synephrine the metabolic rates found at the end of its infusion
caused by p-synephrine. Livers from 18-h fasted rats were perfused as period (60 min perfusion time) were computed and represented
described in ‘‘Materials and methods’’ section. Samples of the efuent against the concentration; for zero p-synephrine concentration the
perfusate were taken for measuring glucose and pyruvate. Oxygen in rates in the presence of 2 mM lactate (40 min perfusion time) were
the efuent perfusion uid was monitored polarographically. a Time represented. Data represent the means ± mean standard errors of 3–4
courses of the effects caused by 50 lM p-synephrine. b Concentration liver perfusion experiments
dependence of the effects of p-synephrine. For each concentration of

production, nally, was inhibited with a half-maximal well as the basal rate of oxygen consumption. The effect of
effect at the concentration of 25.1 lM. 10 lM p-synephrine on the perfusion pressure was small
(0.49 ± 0.03 mmHg), but the effect of 25 lM was similar to
Effects of p-synephrine on hemodynamics and oxygen that found with 200 lM (4.80 ± 0.03 mmHg). There is,
consumption thus, a very sharp transition in the perfusion pressure
dependence on the p-synephrine concentration.
Experiments were done to verify if p-synephrine is active on
the liver hemodynamics, because this kind of action is typical Inuence of calcium on the actions of p-synephrine
for compounds that act as adrenergic agents [18]. In these and induction of 45Ca2? efux
experiments, p-synephrine was infused at a concentration of
200 lM during 20 min. Figure 4 shows that both perfusion Considering that the hemodynamic effects are essentially
pressure and oxygen consumption were stable before the p- due to muscle contraction or distention, both calcium-
synephrine infusion. Upon p-synephrine infusion, the portal dependent phenomena, a set of experiments was done to
perfusion pressure raised rapidly in parallel with the oxygen test a possible calcium dependence. The results of these
consumption rate and attained a maximum after 2 min experiments are shown in Fig. 5. Initially, the liver was
infusion. After this time, the perfusion pressure declined and perfused with a calcium-free perfusion uid. The infusion
tended to stabilize at a level that was 5.33 ± 0.55 mmHg of 200 lM p-synephrine in the absence of Ca2? produced
(n = 3) or 221 % above the basal level (before p-synephrine an increase in oxygen consumption that was approximately
infusion). The effect was reversible, i.e., cessation of the p- half that found in the presence of calcium, and a very small
synephrine infusion restored the basal perfusion pressure as increment in perfusion pressure. Glucose release was

123
140
Oxygen
12.0 Portal pressure
Perfusion portal pressure (mm Hg)
10.0
8.0
6.0
4.0
2.0
Synephrine (200 µM)
0.0
0 5 10 15 20 25
Perfusion time (minutes)
Fig. 4 Actions of p-synephrine on oxygen uptake and portal
perfusion pressure. Livers from fed rats were perfused as described
in ‘‘Materials and methods’’ section. Oxygen in the efuent perfusion
uid was monitored polarographically. The perfusion pressure was
monitored by means of a pressure transducer. Data represent the
means ± mean standard errors of three liver perfusion experiments
stimulated to a lesser extent when compared to the stimu-
lation in the presence of Ca2?, the same applying to lactate
production. Pyruvate, production, suffered an initial stim-
ulation rather than inhibition. The subsequent introduc-
tion of Ca2? did not produce signicant alterations in
lactate or pyruvate production. However, it caused an
additional peak increment in glucose release and an addi-
tional stable increment in oxygen consumption (from
0.20 ± 0.06 lmol-1 min-1 in the absence of Ca2? to
0.47 ± 0.06 lmol-1 min-1 in the presence of Ca2?,
p = 0.04, n = 3). Furthermore, it produced a strong tran-
sient increase in perfusion pressure, which subsequently
stabilized at values that were approximately 70 %
(3.77 ± 0.44 mmHg, n = 5) of the response found in those
experiments in which Ca2? was present from the beginning
(Fig. 4).
Agents acting via Ca2?-dependent mechanisms also pro-
mote transient net Ca2? efux from the liver cells [19–21]. To
determine if the same occurs with p-synephrine, experiments
were performed in which the intracellular calcium pools of the
liver cells were labeled with 45Ca2? using recirculating per-
fusion. After labeling, the perfusion was continued with a
Ca2?-free medium in the nonrecirculating mode, and the
45
Ca2? efux was monitored. The mean results of all exper-
iments are shown in Fig. 6. Figure 6a reveals that the intro-
duction of p-synephrine during a short period of time (2 min)
produced a substantial, but transient, increment in 45Ca2?
123
Mol Cell Biochem (2014) 388:135–147
2.5 release. The latter was observed for several Ca2?-dependent
Oxygen consumption (µmol min-1 g-1)
agents and it is said to reect the initial mobilization of Ca2?
2.3 from the cellular stores [19, 20]. Figure 6b–d show the results
that were obtained when p-synephrine was infused in the
2.1
presence of three antagonists, namely prazosin (mainly
a1-antagonist), yohimbine (mainly a2-antagonist but also
1.9
a1-antagonist), and propranolol (non specic b-antagonist)
[22–24]. None of these antagonists had any signicant effect
1.7
on the rate of 45Ca2? release prior to the infusion of
p-synephrine. When p-synephrine was introduced in the pre-
1.5
sence of these antagonists at the same time and concentration
as in the experiment shown in Fig. 6a (no antagonists), it
failed to produce any increment in the release of 45Ca2?.
Effects of p-synephrine on cAMP overow
The overow of cAMP in the perfused rat liver is a well
30 35 40
known phenomenon when glucagon is infused [25]. The
experiments shown in Fig. 7 were done in order to verify if
a similar phenomenon occurs when p-synephrine is intro-
duced into the perfusion uid. Under basal conditions (zero
time in Fig. 7), the release of cAMP by the liver was very
low. It increased immediately, however, after the intro-
duction of p-synephrine and remained elevated during the
whole infusion period. When p-synephrine was infused in
the presence of the adrenergic antagonists prazosin,
yohimbine, and propranolol, the overow of cAMP was
strongly diminished. The least effective was prazosin, but
even with this antagonist stimulation was kept to minimal
levels. Propranolol completely eliminated cAMP release.
Effects of adrenergic antagonists on the actions of
p-synephrine
Considering the actions of the adrenergic antagonists on both
45
Ca2? and cAMP overow induced by p-synephrine, it is of
interest to investigate if the metabolic and hemodynamic
effects of this agent are equally affected by the same
antagonists. The results obtained in experiments using the
a-antagonists yohimbine and prazosin are shown in Fig. 8.
The effects of 100 lM yohimbine alone (Fig. 8a), which was
infused before p-synephrine, were not important except
perhaps for a small stimulation of lactate production. When
p-synephrine was infused in the presence of 100 lM
yohimbine no changes in glucose release, oxygen uptake and
perfusion pressure were found (compare Figs. 8a and 2a).
Yohimbine, thus, completely abolished the actions of p-sy-
nephrine on these variables. Pyruvate production, on the
other hand, was still inhibited by p-synephrine and lactate
production suffered some increment. The latter, however,
was much smaller than that one found in the absence of
yohimbine (compare Figs. 2a and 8a). The action of 10 lM
prazosin, which is illustrated by Fig. 8b, was similar to that
Mol Cell Biochem (2014) 388:135–147 141

Fig. 5 The inuence of calcium 0.3 Pyruvate

on the effects of p-synephrine. Lactate

Pyruvate production (µmol min-1 g-1)

Livers from fed rats were Glucose
perfused as described in
Oxygen
‘‘Materials and methods’’
section. The perfusion was 0.2 Portal pressure
2.5
initiated with Ca2?-free

Lactate production (µmol min-1 g-1)

medium. The introduction of
p-synephrine and Ca2? occurred
2.0
at 10 and 30 min perfusion
time, respectively. Samples of 0.1
the efuent perfusate were
collected for measuring glucose, 1.5
lactate, and pyruvate. The
perfusion pressure was
0.0
monitored by means of a 1.0
pressure transducer. Oxygen
5.5
consumption was monitored
polarographically. Data
Glucose production (µmol min-1 g-1)

0.5
points ± mean standard errors
4.5
are from 5 liver perfusion
experiments
3.5

2.5
2.5

Oxygen consumption (µmol min-1 g-1)

1.5
2.3

0.5
2.1

12.0
1.9
Perfusion portalpressure (mm Hg)

10.0

1.7
8.0

6.0 1.5

4.0

CaCl 2 (2.5 mM)

2.0

Synephrine (200 µM)

0.0

0 10 20 30 40 50 60
Perfusion time (minutes)

of yohimbine. Prazosin alone was without effect on the
variables measured except for small increments in pyruvate
and lactate production. With respect to the effects of p-sy-
nephrine on glucose release and perfusion pressure, they
were practically abolished by prazosin. A very small stim-
ulation of oxygen uptake was still found. The inhibition of
pyruvate production by p-synephrine, on the other hand, was
not substantially modied by prazosin.
Figure 9 shows the results obtained when p-synephrine
was infused in the presence of b-adrenergic antagonists.
The inuence of 50 lM propranolol, a non specic
b-adrenergic antagonist, is shown in Fig. 9a. Propranolol
alone caused an increase in oxygen uptake, but the other
parameters were not affected. The introduction of 200 lM
p-synephrine produced a further stable increase in oxygen
uptake, which was smaller, however, than that found in the

123
142 Mol Cell Biochem (2014) 388:135–147

A 12 B
12
10
10
8
8
6 S

Ca2+ in the venous perfusate (cpm/mL · 10 -2 )

Ca2+ in the venous perfusate (cpm/mL · 10 -2 )

6
4
4
2
2
S
0 Prazosin
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30

Time after restoring once-through perfusion (minutes) Time after restoring once-through perfusion (minutes)
12
12 C D
10 10

8 8
S
6 S 6

45
45

4 4

2 2

0 Yohimbine Propranolol
0
0 5 10 15 20 25 30 0 5 10 15 20 25 30
Time after restoring once-through perfusion (minutes) Time after restoring once-through perfusion (minutes)

Fig. 6 45Ca2? net efux caused by p-synephrine and the inuence of bicarbonate buffer, and perfusion was continued in the open mode.
adrenergic antagonists. Livers from fed rats were perfused initially in Samples (500 lL) for the measurement of radioactivity were
an open system with Krebs/Henseleit-bicarbonate buffer (pH 7.4) collected in 15–60-sec intervals. p-Synephrine (50 lM) was infused
containing 2.5 mM CaCl2, as described in ‘‘Materials and methods’’ during 2 min as indicated by the bar labeled as ‘‘S’’. In the
section. At approximately 20 min after the surgical procedure, experiments shown in b–d, prazosin (10 lM), yohimbine (100 lM),
[45Ca]CaCl2 was added to 100 mL of the perfusion uid and propranolol (50 lM), respectively, were infused prior to the
(0.5 lCi mL-1) and the system was switched to the recirculating introduction of p-synephrine, as indicated. The radioactivity in the
mode. Recirculation was continued for 40 min. After this time, the samples was plotted against the time after restoration of the
perfusion uid was switched to Ca2?-free Krebs/Henseleit- nonrecirculating perfusion

absence of the antagonist (Fig. 2a). The perfusion pressure production, and glucose release as well as the inhibition of
was also increased by p-synephrine, but to a lesser extent pyruvate production were still almost fully evident. The
when compared to the increase observed in the absence of increment of the portal perfusion pressure, however, was
propranolol (see Fig. 4). Glucose release and lactate pro- signicantly reduced (-60 %) (compare Figs. 9b, 4).
duction, however, were only transiently stimulated and to Additional experiments with livers from fasted rats (not
extents that were much smaller than those found in the shown) revealed that prazosin and yohimbine abolished the
absence of propranolol. Pyruvate production, nally, was stimulatory effects of 200 lM p-synephrine on glucose
no longer inhibited by p-synephrine. It should be recalled production (gluconeogenesis), oxygen uptake, and portal
that inhibition of pyruvate production by p-synephrine was perfusion pressure, as well as the inhibition caused by
not affected by either prazosin or yohimbine. Figure 9b p-synephrine on pyruvate production. Propranolol was less
shows the results obtained with SR59230A a b3 specic effective on the stimulatory effects of p-synephrine on
antagonist [26]. This experimental approach is justied by gluconeogenesis.
the fact that b3-adrenergic receptors are said to be involved
in lipolysis activation [8], and because oxygen uptake in
the substrate-free perfused liver is likely to represent Discussion
mainly oxidation of fatty acids derived from lipolysis [27].
Figure 9b reveals that 10 lM SR59230A alone had no The results of this work conrm that p-synephrine, the
actions on the parameters measured in the present work. main adrenergic amine in the C. aurantium extracts, is
Furthermore, the b3-antagonist showed little or no inu- highly active on liver metabolism and hemodynamics. The
ence on the modications normally caused by 200 lM compound signicantly stimulated glycogenolysis, glycol-
p-synephrine: the stimulations of oxygen uptake, lactate ysis (lactate plus pyruvate production), gluconeogenesis,

123
Mol Cell Biochem (2014) 388:135–147
16
cAMP release (pmol min −1 g−1)
12
⎯ ⎯ Synephrine alone
8
⎯ ⎯ Synephrine + prazosin
⎯ ⎯ Synephrine + yohimbine
⎯ ⎯ Synephrine + propranolol
4 lation in livers from fed rats was a saturable function of both
0 extract concentration [13]. Actually, even the concentrations
0 2 4 6 8 10
Time after starting synephrine infusion
(mM)
Fig. 7 cAMP overow caused by p-synephrine and the inuence of
adrenergic antagonists. Livers from fed rats were perfused in an open
system with Krebs/Henseleit-bicarbonate buffer (pH 7.4), as
described in ‘‘Materials and methods’’ section. After stabilization of
oxygen uptake, 100 lM p-synephrine was infused during 18 min in
the absence (control) or in the presence of the antagonists prazosin
(10 lM), yohimbine (100 lM), or propranolol (50 lM). The infusion
of the antagonists was initiated 5 min prior to the infusion of p-
synephrine. Samples were collected for quantifying cAMP in the
perfusion uid by means of an ELISA procedure. Results are the
means ± mean standard errors of 3–5 liver perfusion experiments for
each condition
and oxygen uptake. The compound also increased the
portal perfusion pressure and the redox state of the cyto-
solic NAD?/NADH couple, the latter indicated by the
increased lactate to pyruvate ratios [14]. Pyruvate pro-
duction from exogenous lactate under gluconeogenic con-
ditions was inhibited. It is noteworthy to mention that the
effects found in the present study were also reported for
other adrenergic agents such as epinephrine and norepi-
nephrine [19, 28–30].
Stimulation of glycogenolysis, glycolysis, and oxygen
uptake were also observed in the perfused liver when an
aqueous extract of C. aurantium was infused [13], and it is of
interest to compare concentration dependences obtained
with p-synephrine alone (this work) with those found using
the C. aurantium extract in terms of its p-synephrine content.
In the present work, p-synephrine alone increased glucose
output from glycogenolysis in a saturable manner, the half-
maximal effect occurring at a concentration around
26.1 lM. When the C. aurantium extract, was used a linear
relationship, was found in the range up to 400 mg L-1 which
corresponds to a p-synephrine concentration of 190 lM [13].
Stimulation of glycogenolysis at this extract concentration
was approximately 35 % above the maximal stimulation
found in the present work with p-synephrine alone and the
absence of saturation for concentrations up to 400 mg L-1
143
suggests that further increments in the extract concentration
are likely to produce stronger stimulations. It can be thus
concluded from this comparison that the stimulatory effect of
the C. aurantium extract on glycogenolysis can be only
partly explained by its p-synephrine content and that the
extract possibly contains other active agents that can equally
produce this kind of stimulation. In this respect it is worthy to
mention octopamine, an amine whose concentration in the C.
aurantium extract is signicant [31]. Oxygen uptake stimu-
the p-synephrine concentration as found in the present work
but it was also a saturable function of the C. aurantium
12 14 16 18 for half-maximal stimulation are similar in terms of the nal
p-synephrine concentrations, namely 8.95 lM in the present
work and *8 lM in the experiments with the C. aurantium
extract [13]. A more notable difference between the C.
aurantium extract and pure p-synephrine concerns the
actions of both preparations on gluconeogenesis. Pure p-
synephrine was stimulatory in the range up to 500 lM even
though the stimulation of gluconeogenesis decreased at
concentrations above 100 lM. The C. aurantium extract was
clearly inhibitory for gluconeogenesis at high concentrations
and at low concentrations stimulation was not signicant
[13]. Here again the conclusion is allowed that the extract
contains additional compounds that exert an inhibitory
action on gluconeogenesis thus effectively counteracting the
stimulatory action of p-synephrine.
The action of synephrine on pyruvate production
deserves a few additional comments. In the fed state, in
which pyruvate results from glycolysis, stimulation
occurred at low concentrations (up to 100 lM), but inhi-
bition was found at the higher concentrations. Pyruvate is
the minor component of the lactate dehydrogenase redox
equilibrium, and since the sum of lactate ? pyruvate pro-
duction (glycolysis) was still presenting stimulation, the
diminished pyruvate production was most likely reecting
the increased NADH/NAD? ratio [14] at high p-synephrine
concentrations. This inhibitory effect was not affected by
a-adrenergic antagonists (prazosin and yohimbine), but it
was sensitive to the b-adrenergic antagonist propranolol. It
is thus possible that the effect results from b-adrenergic
signaling. Under gluconeogenic conditions, on the other
hand, pyruvate production was inhibited by p-synephrine
even at low concentrations. Two factors could have con-
tributed: the increased NADH/NAD? redox potential and,
more important, the increased rates of glucose production.
The latter can only occur if the pyruvate carboxylation
reaction is increased, a phenomenon that should also result
in a decreased release of pyruvate due to its lower cellular
concentrations.
Metabolic and hemodynamic effects of p-synephrine
probably occur through the stimulation of adrenergic
123
144 Mol Cell Biochem (2014) 388:135–147

0.4

Pyruvate output (µmol min -1 g -1 )

0.4
Pyruvate output (µmol min-1 g -1)

0.3
0.3
2.5

Lactate output (µmol min-1 g -1)

0.2
0.2 2.5
2.0

Lactate output (µmol min-1 g -1)

0.1 0.1
1.5 2.0

0.0 0
1.0
Pyruvate 1.5
Lactate 4.0

Glucose output (µmol min-1 g -1 )

Glucose output (µmol min-1 g -1)

4.0
Glucose 0.5 3.5
3.5 Pyruvate
Oxygen 1.0
Lactate
3.0 Pressure 3.0
Glucose

Oxygen uptake (µmol min-1 g -1)

2.5 2.5 2.5 Oxygen
Pressure 0.5
2.0 2.0
2.3
1.5

Oxygen uptake (µmol min -1 g -1)

1.5 2.5
1.0 2.1
1.0
0.5 2.3
1.9 0.5
2.1
12 1.7
12
Perfusion pressure (mm Hg)
Perfusion pressure (mm Hg)

10 1.9
1.5 10
8 1.7
8
6 1.5
6
4
4
2 2 p-Synephrine(200 µM)
p-Synephrine (200 µM)
0 0 Prazosin(10 µM)
Yohimbine (100 µM)

0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35 40 45 50
Perfusion time (minutes) Perfusion time (minutes)
A B
Fig. 8 Inuence of the a-adrenergic antagonists yohimbine (a) and Samples were taken for the measurement of glucose, lactate, and
prazosin (b) on the metabolic and hemodynamic effects of p-syneph- pyruvate. Oxygen was monitored polarographically. The portal
rine in livers from fed rats. Livers were perfused as described in perfusion pressure was monitored simultaneously by means of a
‘‘Materials and methods’’ section. The infusions of yohimbine and pressure transducer. Data represent the means ± mean standard errors
prazosin were started 10 min prior to the p-synephrine infusion. of three liver perfusion experiments

receptors present in the liver and are at least partly Ca2?- This could be the reason why the perfusion pressure was
and cAMP-dependent. The sensitivity of the effects of the only effect of p-synephrine that was signicantly
p-synephrine to 10 lM prazosin is highly indicative of the affected by the antagonist SR59230A, but this is a matter
participation of a1-adrenergic receptors [32]. The same can deserving further investigations. The Ca2?-dependence, on
be said about the sensitivity to 100 lM yohimbine because the other hand, can also be regarded as evidence in favor of
at this concentration the latter compound, usually regarded a participation of a1-adrenergic receptors, since it is well
as a a2-antagonist, also binds to a1-adrenergic receptors known that these receptors act basically by pathways that
[32]. The participation of b-adrenergic receptors could be presuppose a series of Ca2? movements that culminate in
less important, as can be judged, in principle at least, from Ca2?-dependent activations. An analogous conclusion
the fact that propranolol was less effective in inhibiting the concerning the participation of b-adrenergic signaling can
action of p-synephrine than the a-adrenergic antagonists. be drawn from the observation that p-synephrine stimulates
The specic participation of the b3-adrenergic receptor on cAMP overow. However, it seems also apparent that
the metabolic effects is unlikely as indicated by the cAMP and Ca2? signaling in the case of p-synephrine
observation that they were practically insensitive to the present a pronounced degree of interdependence. This is
antagonist SR59230A [33]. Actually, the presence of the suggested by the fact that blocking of the a-adrenergic
b3-receptors in the liver is controversial. Possibly, they signaling by prazosin and yohimbine almost completely
exist mainly or even only in the vascular system [34, 35]. inhibited the effects of p-synephrine on cAMP overow.

123
Mol Cell Biochem (2014) 388:135–147 145

0.4
Glucose output (µmol min-1 g-1) Pyruvate output (µmol min-1 g-1)

Perfusion pressure (mm Hg) Glucose output (µmol min-1 g-1 ) Pyruvate output (µmol min-1 g -1)
0.4
2.5 Pyruvate
0.3
Lactate

Lactate output (µmol min-1 g-1)

0.3 Glucose
0.2 Oxygen
2.0 Pressure 2.5
0.2

Lactate output (µmol min -1g -1)

0.1 2.0
1.5 0.1
0 1.5
1.0 0
4.0
4.0
1.0
3.5 Pyruvate
Lactate 3.5
0.5
3.0 Glucose 0.5
Oxygen 3.0
2.5 Pressure
2.5 0.0
2.0
2.5 2.0

Oxygen uptake (µmol min -1 g -1)

1.5 2.5

Oxygen uptake (µmol min-1 g-1 )

1.5
1.0 2.3
1.0 2.3
0.5
2.1 0.5
12 2.1
12
Perfusion pressure (mm Hg)

10 1.9 1.9
10
8 1.7
1.7 8
6
6 1.5
1.5
4 4

2 p-Synephrine(200 µM) 2 p-Synephrine (200 µM)

0 Propranolol (50 µM) SR 59230A (10 µM)

0

0 5 10 15 20 25 30 35 40 45 50 0 5 10 15 20 25 30 35
Perfusion time (minutes) Perfusion time (minutes)

A B
Fig. 9 Inuence of the b-adrenergic antagonists propranolol (a) and continued until the end of the experiments. Samples were taken for
SR59230A (b) on the metabolic and hemodynamic effects of the measurement of glucose, lactate, and pyruvate. Oxygen was
p-synephrine in livers from fed rats. Livers were perfused as monitored polarographically. The portal perfusion pressure was
described in ‘‘Materials and methods’’ section. The infusion of monitored by means of a pressure transducer. Data represent the
propranolol was started 10 min and that of SR59230A 5 min prior to means ± mean standard errors of four liver perfusion experiments
the p-synephrine infusion. The infusion of both antagonists was

Furthermore, if cAMP and Ca2? signaling were occurring strongly to a-adrenoceptor subtypes as well as to
independently, the summation of the metabolic responses b-adrenoceptors when compared to other adrenergic
in the presence of prazosin (or yohimbine) and propranolol amines [36]. Consistently, p-synephrine was less active on
should correspond to the metabolic responses in the glycogenolysis than epinephrine and norepinephrine in the
absence of antagonists. Also, in the case of independent a- liver. When these agonists were used in the liver, strong
and b-adrenergic effects, propranolol should not affect the stimulation of glycogenolysis was observed at concentra-
action of p-synephrine on 45Ca2? release. All these tions up to 10 lM [19, 28–30]. These stimulations were
observations are difcult to interpret in terms of the cross- comparable to the maximal ones found with p-synephrine
talk events between the cAMP and Ca2? signaling path- whose half-maximal action occurred at the concentration of
ways in which glucagon, for example, affects Ca2? mobi- 26.1 lM. It seems, thus, that p-synephrine is less active
lization in the liver [21]. To our knowledge, there is no than epinephrine and norepinephrine in the liver.
parallel in the literature for the observation that the In conclusion, the results of this work support the
a-adrenergic antagonists (prazosin and yohimbine) were proposition that p-synephrine presents important metabolic
able to prevent the p-synephrine-induced increase in cAMP and hemodynamic effects in the liver. These effects can be
release by the liver. This is a point to be elucidated by considered as both catabolic (glycogenolysis) and anabolic
additional experimental work. (gluconeogenesis), are mediated by both a- and b-adren-
A recent review about the receptor-binding properties of ergic signaling, require the simultaneous participation of
p-synephrine emphasizes that this compound binds less both Ca2? and cAMP, and could be contributing to the

123
146
overall stimulation of metabolism that usually occurs dur-
ing weight loss periods. It should be remarked also that the
effects of p-synephrine are similar to those recently
reported for octopamine, another constituent of C. auran-
tium [38]. Thus, both compounds, p-synephrine and octo-
pamine, are likely to contribute to the reported weight loss
effects usually attributed to C. aurantium extracts [1].
Furthermore, effects as those described in the present work
for p-synephrine, and elsewhere for octopamine [37] are
also likely to have stimulating and performance-enhancing
properties. For octopamine, indeed this property has lead to
its prohibition in sports [38]. The contribution of the liver
in this respect resides mainly in enhancing glucose avail-
ability to other tissues, muscle for example [11], via both
glycogenolysis and gluconeogenesis.
Acknowledgments This work was supported by Grants from the
Conselho Nacional de Desenvolvimento Cientı́co e Tecnológico
(CNPq). Andrea Luiza de Oliveira is a fellowship holder of the Co-
ordenação de Aperfeiçoamento de Pessoal do Ensino Superior
(CAPES).
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