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of Physiology,
Biochemistry and
Pharmacology
169
Reviews of Physiology, Biochemistry
and Pharmacology
More information about this series at http://www.springer.com/series/112
Bernd Nilius Thomas Gudermann Reinhard Jahn
Roland Lill Ole H. Petersen Pieter P. de Tombe
Editors
Reviews of Physiology,
Biochemistry and
Pharmacology
169
Editor in Chief
Bernd Nilius
KU Leuven
Leuven
Belgium
Editors
Thomas Gudermann Reinhard Jahn
Ludwig-Maximilians-Universitat Munchen Max-Planck-Inst for Biophysical
Munich, Germany Chemistry
Gottingen
Roland Lill Germany
University of Marburg
Marburg Ole H. Petersen
Germany Cardiff School of Biosciences
Cardiff University
Pieter P. de Tombe Cardiff
Loyola University Chicago United Kingdom
Maywood, Illinois
USA
v
Rev Physiol Biochem Pharmacol (2015) 169: 124
DOI: 10.1007/112_2015_25
Springer International Publishing Switzerland 2015
Published online: 19 September 2015
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Chemical Stability of Hyperforin and Stable Analogues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
3 Hyperforin as Protonophore . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
4 Hyperforin as Inducer of Cytochrome P450 Enzymes, Hyperforin as Pregnane X
Receptor Ligand . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
5 Hyperforin as Activator of Ion Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
K. Friedland (*)
Department of Molecular and Clinical Pharmacy, Friedrich-Alexander University Erlangen/
Nuremberg, Cauerstr. 4, 91058 Erlangen, Germany
e-mail: kristina.leuner@fau.de
C. Harteneck (*)
Institute of Pharmacology and Toxicology and Interfaculty Centre for Pharmacogenomics
and Drug Research, Eberhard Karls Universitat, Wilhelmstr. 56, 72074 Tubingen, Germany
e-mail: christian.harteneck@uni-tuebingen.de
2 K. Friedland and C. Harteneck
1 Introduction
St. Johns wort, Hypericum perforatum, is used for centuries to treat mild and
moderate depression (Gastpar 2013). Depression is one of the most frequent disease
worldwide and the most common psychiatric diseases (Duman and Aghajanian
2012; Penninx et al. 2013). Approximately 1 of 5 women and 1 of 8 men suffer from
a depressive episode during their lifetime (Mu~noz et al. 2010; Murray et al. 2012).
Depression refers to a set of mental symptoms such as loss of interest and pleasure,
decreased cognition and memory, and disrupted sleeping, eating, ambulation, and
sexual activity (Duman and Aghajanian 2012). A large number of clinical trials
(Lecrubier et al. 2002; Gastpar et al. 2006; Kasper et al. 2010; Singer et al. 2011) as
well as a recent Cochrane meta-analysis (Linde et al. 2008) confirm the antidepres-
sant activity in patients of the plant and its extracts. St. Johns wort extracts contain
several constituents like hyperforin (Fig. 1a), a phloroglucinol derivative, the
naphtodianthrone hypericin (Fig. 1b), and some flavonoids such as isoquercitrin,
biapigenine, or rutin, which were discussed to represent the antidepressive principle
(Noldner and Sch otz 2002; Muller 2003; Butterweck and Schmidt 2007; Paulke
et al. 2008). The first molecule in focus of representing the antidepressive principle
was hypericin mediating its effect via the inhibition of the monoamine oxidase
(Suzuki et al. 1984). These results could not be validated by other groups arguing
for a minor role of hypericin in the antidepressant effects of St. Johns wort extract
(Bladt and Wagner 1994; Thiede and Walper 1994; Cott 1997). Other mechanisms
were investigated such as interaction of hypericin with monoamine receptors
(Butterweck et al. 2002; Caccia and Gobbi 2009). However, the concentration
gap of hypericin affinities in the micromolar range for binding these receptors
and nanomolar plasma concentrations measured in humans after intake of thera-
peutic doses made it unlikely to account for the antidepressive principle (Staffeldt
et al. 1994; Kerb et al. 1996; Brockmoller et al. 1997). Phototoxicity is a feature
attracting attention to hypericin (Boiy et al. 2008; Davids et al. 2008), being
currently under investigation regarding its antimetastatic and antiangiogenic prop-
erties in the treatment of glioblastomas or melanoma (Davids et al. 2008; Barliya
et al. 2011; Dror et al. 2013). The flavonoids, biapigenine, hyperoside, and
isoquercitrin, showed moderate antidepressive activity in the forced swim test, a
behavioral animal model for depression (Noldner and Schotz 2002; Paulke
et al. 2008) with yet unknown mechanism.
Hyperforin (Fig. 1a) was long neglected as the putative antidepressant active
constituent of H. perforatum due to its chemical instability in response to light and
air. Under appropriate storage conditions, 15% hyperforin can be found in the
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 3
A H3 C CH 3 B OH O OH
H 3C
CH 3
OH
O
CH 3 HO CH 3
O
HO CH 3
H3 C H3 C O CH 3
CH 3
H3C OH O OH
CH 3
hypericin
hyperforin
H3 C CH 3
C D
H 3C
CH 3
O O
O O HO O
HO CH 3
O
H3 C CH 3
H 3C H 3C O CH 3 OH
HO
CH 3
Hyp9
H 3C
CH 3
aristoforin
E
O
O CH 3
O
CH 3
OH
O
1-stearoyl-2-arachidonoyl-sn-glycerol
Fig. 1 Structure of hyperforin (A), hypericin (B), aristoforin (C), Hyp9 (D), and 1-stearoyl-2-
arachidonoyl-sn-glycerol (E)
St. Johns wort extracts (Chatterjee et al. 1998). The phloroglucinol derivative
hyperforin showed pronounced effects in behavioral models for depression includ-
ing the forced swim test (15 mg/kg BW), the learned helplessness test (15 mg kg/
BW), the elevated plus maze test, or the light/dark test (Chatterjee et al. 1998;
Zanoli et al. 2002). Importantly, one clinical trial showed loss of antidepressant
properties of a St. Johns wort extract containing 0.5% hyperforin instead of 5%
hyperforin (Laakmann et al. 1998). On the biochemical level, several groups
showed hyperforin-mediated inhibition of neurotransmitter uptake such as seroto-
nin (IC50 205 nM in whole brain rat synaptosomes), norepinephrine (IC50 102 nM in
rat synaptosomes from the occipital cortex), dopamine (IC50 80 nM in synapto-
somes isolated from rat striatum), GABA (IC50 184 nM in whole brain rat
4 K. Friedland and C. Harteneck
synaptosomes), and L-glutamate (IC50 143 nM in rat synaptosomes isolated from rat
forebrain) (Chatterjee et al. 1998; Singer et al. 1999; Wonnemann et al. 2000;
Marsh and Davies 2002). All transmitters are known for their potential role in the
pathogenesis of depressive disorders (Duman and Aghajanian 2012; Duric and
Duman 2013). Increases in extracellular neurotransmitter levels in response to
hyperforin were shown in rat synaptosomes (Chatterjee et al. 2001) and in different
brain areas using microdialysis (Philippu 2001; Buchholzer et al. 2002; Coskun
et al. 2004; Kiewert et al. 2004; Yoshitake et al. 2004) or pushpull analysis
(Kaehler et al. 1999). In rat hippocampus, hyperforin (10 mg/kg BW i.p. or
10 M via the dialysis probe) resulted in increased acetylcholine levels which
was completely reverted by local perfusion with calcium-free buffer or in the
presence of tetrodotoxin, an inhibitor of voltage-dependent sodium-channels
(Buchholzer et al. 2002; Kiewert et al. 2004). Hence, hyperforin-mediated neuro-
transmitter release is a calcium-dependent mechanism requiring intact neuronal
communication and cell firing (Buchholzer et al. 2002; Kiewert et al. 2004). The
impact of hyperforin in increased extracellular accumulation of neurotransmitters
like dopamine, norepinephrine, serotonin, and glutamate was measured in the rat
locus coeruleus upon application of hyperforin (10 mg/kg BW, i.p. application)
(Kaehler et al. 1999). In rat synaptosomes, hyperforin (5 M) also increased the
release of several neurotransmitters such as glutamate or GABA (Marsh and Davies
2002). These observations argued for the modulation of synaptosomal neurotrans-
mitter transport by hyperforin.
Pharmacological relevant concentrations regarding inhibition of neurotransmit-
ter uptake are achieved in human plasma. The intake of a single dose of 300 mg
Hypericum extract containing 14.8 mg hyperforin by healthy volunteers resulted in
human plasma levels of 150 ng/mL hyperforin (280 nM) (Biber et al. 1998). The
application of the clinical dosage (3 300 mg St. Johns wort extract per day) given
for one day resulted in hyperforin concentration of 100 ng/mL or 180 nM in plasma
of healthy volunteers (Biber et al. 1998). Comparable plasma level as well concen-
trations in brain tissues can be achieved by the application of hyperforin sodium
salt. In rats, the application of 300 mg/kg BW Hypericum extract (WS5572, 5%
hyperforin) results in plasma concentrations of 370 ng/mL hyperforin (690 nM).
The maximum concentration was detected after 3 h and an estimated half-life time
of 6 h could be calculated (Biber et al. 1998). The effective dosage used in
behavioral test like the forced swim test given as hyperforin sodium salt (15 mg/
kg BW) or as St. Johns wort extract (containing 5% hyperforin), resulted in organ
concentrations of 28.8 and 15.8 ng/g (hyperforin per brain tissue) respectively
(Keller et al. 2003). Based on its lipophilic structure, hyperforin might accumulate
in the brain reaching higher nanomolar concentrations in the brain than found in the
plasma.
The diversity of neurotransmitter being extracellularly increased in response to
hyperforin makes it unlikely that hyperforin directly modulates neurotransmitter
transport comparable to amitriptyline, a tricyclic antidepressant, or fluoxetine, a
selective serotonin reuptake inhibitor (Chatterjee et al. 1998; Singer et al. 1999).
This is underscored by the fact that the neurotransmitter being modulated by
hyperforin is mediated by neurotransmitter transporters of different classes. The
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 5
3 Hyperforin as Protonophore
H3C CH 3
H 3C
A
CH 3
H+
OH
O
CH 3
O
H3C H3C O CH 3
CH 3
H 3C
CH 3
outside H 3C CH 3
H+
H 3C
CH 3
H3C CH 3 H3C CH 3
+ H3C CH 3 OH + H3C CH 3
N H +
H H3C CH N H +
H
H N H 3 O H 3C C H3 H N H
CH 3 N
+
N
+ CH 3 CH 3 N
+
N
+
CH 3 CH 3
H 3C O
H H H H
O O CH H 3C O O
O O 3
CH O O
P O CH
3 P
O O O P O O H 3C OH H 3C 3
OH O O O P O O
O P O P O P O P
O O O O
O O CH 3 CH O O
O O O 3 H 3C C H3 C H3 O O
O
HH33CC CH 3
O H3C 3C O
O
O O H3C OH CH 3 C H3
C H3
O CH
O
O O
H 3C H 3C H 3C 3
O O O O O O O O O O O O
O O CH CH 3
OH O O
3
O O O O CH 3 O O O O
OH O
H 3C CH 3 CH 3
H 3C O O H 3C CH 3
CH 3 CH H 3C
O 3 CH 3
H 3C
CH C
CH 3 H 3C H 3C HO3 C H3 3 CH 3 CH 3
H3C OH H3C O CH 3 OH
CH 3 H 3C
O
CH 3 CH 3 CH 3 O
C H3
O H 3C OH
CH 3
O
H C O
H 3C H 3C O C H3 3 CH
CH
3
O H 3C H 3 CCH 3 O CH 3
H 3C 3
CH 3
H3C CH 3
H 3C H3C O CH
H+
3
CH 3 H 3C
OH C H3 CH 3
H 3C
CH 3
O H3C CH
C H3 H 3C
3
O H 3C CH 3 CH 3
H 3C
C H3
H 3C H 3C H 3C O CH 3
CH 3 OH
H3C CH 3 OH O H3C
H3C H 3C CH 3 CH3 CH H3C
H 3 CO 3
H 3C H 3C O H 3C H 3C
CH 3
CH CH H 3C CH CH
3
H3C 3 H 3C H 3HC CH 3 HO 3C H 3C CH 3 3
H3C 3
H3C
C 3 CH H3C H3C O CH
3 3
CH 3 CH 3 CH 3 CH 3
H 3C OH H 3C O CH
OH H 3C
H 3C H 3C H 3C 3
CH H 3C
CH 3 CH 3 CH 3 3 CH 3 CH 3
O O
C H3 CH H
OCH
CH 3 CH 3 3 3 CH 3 CH 3
O H 3C CH 3 O H 3C
O C H3
CH 3
O CH H 3C
H 3C H 3C H 3C
3 H 3C H 3 C CH 3 O O CH 3 H 3C CH 3
CH 3
CH 3 OH CH
H 33C H 3C H 3C O C H3
CH 3
O
H 3C CH 3 CHH 3C OH
C H3 3
O CH 3 O
H+
CH 3
H 3C
H 3C H 3C O CH 3 C H3 O
C HC3H H 3C H 3C O CH 3
H 3C 3
H 3C C H3
H 3C CH3
H 3C CH 3 H 3C C H3
CH 3 H 3C
CH 3 H 3C
OH H 3C CH
OH 3
CH 3
O
C H3 O OH
O C H3
C C H3
OH 3 O
CH 3
H 3C H 3C O CH 3 H 3C O CH 3
C
O O O O H 3C H 3 C
H 3CH 3
O C H3
H 3C C H3 O O O O
O O O CH 3 O O HH3C
3C H 3C O CH 3
O O O O
O O O O CH3 CH O O O O
3
O H 3C
O O O O C H3 OH CH O O O O
H 3 CH 3 C CH3 3
CH 3
C H3 O H 3C OH
CH 3
O
O O H 3C H 3C CH 3 O O O
O O O O
CH 3 H 3C H 3C O CH 3O CH 3 C H3
O P O P O O P O P
O
O O O
O O OH O
O O O
O O
P P H 3C H 3C O CH P P
O CH 3 3
O O H O O H CH 3 H 3C H 3C O CH
O O 3 H O O H
+ + O CH3 + +
H3C N H3C N H 3C H3C N H3C N
H H CH 3 H 3C C H3 3
CH H H
+ H + H + H + H
N N H 3C H 3C O CH 3 H 3C N N
H+
H3C CH H3C CH CH CH CH
3 3 H 3C 3 H 3HC3 C 3 H3C 3
CH 3
C H3 C H3
OH
H 3C O
C H3 CH 3
O
inside
H 3C H 3C O CH 3
CH 3
H 3C
CH 3
H+
B
physiological processes pharmacological interference
of serotonin re-uptake by protonophoric activity
& vesicle loading of hyperforin
+
ATP H ADP
H -ATPase P
5-HT H+ H+ [H +]
+ +
H H +
+ H
H + HYP H
+
HYP
VMAT H
+
H +
H
5-HT H
+
SERT
SLC6A4 + H +
+ [H +] Na
+
Na + H
H [Na+] NHE
5-HT
+
H
+ +
Na Na
Fig. 2 Model of hyperforin as protonophore. (a) Adapted to the models known for the action
mechanisms of other ionophores, the association of hyperforin molecules is shown forming a
proton-permeable tube of the plasma membrane. (b) The model of physiological processes of
serotonin reuptake and vesicular loading as well as pharmacological interference by the
protonophoric activity of hyperforin is adapted from the scheme developed by Sell et al. (2014).
The reuptake of neurotransmitter across the presynaptic plasma membrane is mediated by various
monoaminesodium symporter. Here, we show the reuptake of serotonin (5-HT) by the serontonin
transporter (SERT/SLC6A4). Cytosolic neurotransmitter is transported into vesicles against a
concentration gradient by the activity of the vesicular monoamine transporter (VMAT), a
monoamineproton antiporter. The proton gradient across the vesicular membrane is established
by the activity of the vacuolar proton ATPase (H+-ATPase) transporting protons across the
8 K. Friedland and C. Harteneck
suggest that hyperforin might mediate its effects on APP processing via a different
mechanism.
Recently, Sell et al. (2014) published that hyperforin acts as a protonophore.
Analyzing whole-cell currents in HEK293 cells stably expressing TRPC6, they
observed differences in currentvoltage relationship depending on the activating
ligand they use (Sell et al. 2014). While OAG and flufenamic acid resulted in the
typical double-rectifying currentvoltage relationship known for currents mediated
by TRPC6, hyperforin activated a current with a divergent currentvoltage rela-
tionship which could also be found in response to hyperforin in untransfected
HEK293 cells as well as in microglia. Although TRPC6 expression is absent in
microglia, they tested microglia isolated from TRPC6/TRPC3-deficient mice where
they found identical hyperforin-induced currents as they recorded in their initial
experiments. BCECF imaging used for monitoring intracellular pH showed that
extracellular solutions of different pH induced changes in intracellular pH in a
hyperforin-dependent manner. Based on these data, they conclude that hyperforin
acts as a protonophore in microglia and artificial lipid bilayers. In order to confirm
this hypothesis, carbonyl cyanide m-chlorophenylhydrazone (CCCP), a well-
documented protonophore, was used in side-by-side experiments. In microglia,
CCCP-induced current resulted in currentvoltage relationship which could be
nevertheless distinguished from hyperforin-induced currents. Surprisingly, CCCP
was inefficient to induce currents in the lipid bilayer experiments using an
undefined commercial lipid mix. Based on their data, the authors provide a concept
for the antidepressive effect of hyperforin by dually interfering with intracellular
proton and ion homeostasis (Fig. 2b) (Sell et al. 2014). At the plasma membrane,
hyperforin induces an increased proton transport being compensated by the
sodiumproton exchanger resulting in increased intracellular sodium concentra-
tions and thereby indirectly inhibiting the monoamine transporter, a monoamine
sodium symporter protein. At the membrane of intracellular monoamine storage
vesicles, hyperforin-mediated proton flux disturbs the proton gradient across the
vesicular membrane and thereby inhibits the vesicular monoamine transporter, a
monoamineproton antiporter. In summary, the authors presume that the antide-
pressant activity is mediated by hyperforin-dependent indirect inhibition of trans-
mitter reuptake and vesicular transmitter loading based on the protonophoric
activity of hyperforin. These results dont exclude other effects of hyperforin
such as TRPC6 activation (see below).
Ionophores forming ion-permeable pores in lipid bilayers (Fig. 2a) like
natamycin, nystatin, amphotericin B as potassium ionophores, or nigericin or
monensin as protophores are therapeutically used as antimicrobiotics. Based on
Fig. 2 (continued) vesicular membrane. The protonophoric activity of hyperforin (HYP) results in
acidification of the cytosol by increased proton concentration resulting in the activity of the
sodiumproton exchanger (NHE). With the activity of the sodiumproton exchanger, a sodium
proton antiporter, the sodium gradient across the plasma membrane is reduced diminishing the
activity of the serotoninsodium symporter
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 9
Cost-intensive safety pharmacology and clinical studies necessary for the approval
of modern drugs are substituted in the marketing of plant-derived remedies of the
traditional medicine by an evolutionary selection process over centuries. Under
these evolutionary conditions, not all aspects of modern safety pharmacology can
be identified and therefore the discovery of drug interference by St. Johns wort
extracts was a big surprise. Several case reports provided evidence for interference
reactions reporting reduced cyclosporine and theophylline levels during simulta-
neous intake of St. Johns wort extract (Ernst 1999; Breidenbach et al. 2000a, b;
Novelli et al. 2014). The initial suspicion was confirmed by subsequent clinical
studies showing that St. Johns wort extract induces a decline in the concurrent
therapy with immunosuppressants cyclosporine A, tacrolimus, and mycophenolic
acid (Bauer et al. 2003; Mai et al. 2003). The mechanism leading to increase
expression levels of cytochrome P450 enzymes (CYP) was soon deciphered by
experiments showing that constituents of St. Johns wort extracts in particular
hyperforin induce hepatic drug metabolism through activation of the pregnane X
receptor (PXR, Fig. 3) (Moore et al. 2000; Wentworth et al. 2000; Watkins
et al. 2003). Hyperforin binds to PXR with an EC50 value of 27 nM (Moore
et al. 2000). The binding of hyperforin to PXR results in transcriptional activation
and increased expression of proteins involved in drug metabolism. CYP enzymes,
mainly CYP3A4, as well as other proteins like the multidrug resistance-associated
protein (MDR1), are upregulated [for review see (Zhou et al. 2004)] and represent
the cause for the unwanted effects of St. Johns wort in therapy. While the
upregulation of multidrug resistance-associated protein (MDR1) is critical in can-
cer therapy by triggering the efflux of cytotoxic compounds, the upregulation of
CYP3A4 affects 50% of our therapeutic drug armamentarium.
Several hyperforin-derived compounds have been developed and analyzed.
Derivatives resulting from chemically oxidation processes of the instable
phloroglucinol, hyperforin, lost their antidepressive effects. In symmetric
phloroglucinol structures, the antidepressive effect of hyperforin was preserved
(Leuner et al. 2010). 2,4-Diacylphloroglucinol compounds (Hyp9; see Fig. 1d)
blocked serotonin uptake in murine synaptosomes and were able to mimic nearly
all features shown for hyperforin, e.g., induction of neurite growth and TRPC6
10 K. Friedland and C. Harteneck
HYP
MDR1
DRUG
ATP ATP
DRUG
HYP DRUG DRUG
DRUG
DRUG
HYP CYP DRUG
PXR
RUG
RUG
RUG
PXR HYP
PXR HYP
other CYP
mRNA mRNAs
Fig. 3 Illustration of hyperforin-triggered transcriptional regulation of MDR-1 and CYP450
enzymes. Hyperforin binds to the pregnane X receptor, thereby modulating the transcriptional
activity of CYP and MDR genes. The hyperforin-induced activation of pregnane X receptor results
in increases in mRNA species coding for CYP enzymes and transporters like MDR1. PXR
pregnane X receptor, POL DNA-dependent RNA polymerase, MDR1 multidrug resistance trans-
porter 1, CYP cytochrome P450 enzymes
activation [(Leuner et al. 2010) for further aspects see below]. However, the
absence of PXR binding is the most impressive feature of the
2,4-diacylphloroglucinol compounds (Kandel et al. 2014). Docking studies, model-
ing the compounds into the crystal structure of PXR provided evidence for the
inefficiency of the compounds to bind to PXR (Kandel et al. 2014). The modeling
results could be validated by biochemical experiments testing the induction of the
expression of drug metabolizing proteins. While hyperforin and rifampicin effi-
ciently elevated CYP3A4 mRNA transcription, this effect was absent in side-by-
side experiment using 2,4-diacylphloroglucinol compounds (Kandel et al. 2014).
The 2,4-diacylphloroglucinol structure provide an interesting lead in the develop-
ment of a new generation of antidepressants acting via a quite different mechanism
(see below) compared to the known synthetic antidepressants of today.
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 11
The impact of hyperforin on the modulation of ion channels became obvious by the
work of Chatterjee et al. (1999). In acutely dissociated hippocampal neurons, they
found a complex modulation of ion channels by the application of hyperforin
(Chatterjee et al. 1999). Hyperforin induced a current, which they pharmacologi-
cally characterized by the determination of an EC50 value of 9.1 M, the hyperforin
concentration needed for half-maximal activation (Chatterjee et al. 1999). On the
other hand, steady-state conductances of a variety of ion channels were blocked by
the application of hyperforin. Ligand-induced conductances like AMPA-, NMDA-,
and GABA-currents were blocked by hyperforin as well as voltage-dependent K+,
Na+, and Ca2+ channels (Chatterjee et al. 1999; Fisunov et al. 2000). While the
inhibition of K+ or Na+ channels was left without further characterization, the
authors showed inhibition of N-type and P-type voltage-dependent Ca2+ channels
at low micromolar concentrations (Chatterjee et al. 1999; Fisunov et al. 2000;
Krishtal et al. 2001).
The activation of a sodium-permeable ion channel leading to intracellular
sodium increase by hyperforin was shown by Singer et al. reporting an EC50 of
2 M for this process and an inhibition of serotonin uptake in human platelets as a
model system for serotonin transport in neurons (Singer et al. 1999). Based on the
sodium entry measured and the inhibition of the uptake of GABA and L-glutamate
in synaptosomes by monensin and ouabain, an inhibitor of the Na+/K+-ATPase, the
modulation of the Na+/K+-ATPase by hyperforin has been discussed (Wonnemann
et al. 2000). However the inability to substantiate the interaction of hyperforin with
the Na+/K+-ATPase resulted in the falsification of this hypothesis (Wonnemann
et al. 2000).
Different other features of hyperforin-induced currents were stepwise
complemented by imaging and pharmacological approaches. The imaging
approaches using SBFI-AM demonstrated hyperforin-mediated sodium influx
(EC50 0.72 M), while the use of fura-2/AM allowed the recording of increases
in intracellular Ca2+ concentrations (EC50 1.16 M) (Treiber et al. 2005). The
permeability of the hyperforin target in PC12 cells for sodium and calcium as
well as other divalent cations argued for a nonselective cation channel to be the
target of hyperforin. Several inhibitors of nonselective cation channels such as the
organic inhibitors SK&F96365 and LOE 908 as well as the anorganic inhibitors
lanthanum and gadolinium ions validated this hypothesis and pointed to the partic-
ipation of a TRP channel in mediating hyperforin-induced currents (Treiber
et al. 2005; Leuner et al. 2007).
TRP channels comprise a superfamily of 28 mammalian members (27 in
humans) subdivided in 6 subfamilies the TRPC, TRPV, TRPM, TRPP, TRPML,
and TRPA subfamilies (Harteneck et al. 2000; Clapham et al. 2001; Montell
et al. 2002; Flockerzi 2007; Wu et al. 2010; Nilius and Szallasi 2014). Several
TRP channel inhibitors such as 2-APB, ACA (Harteneck et al. 2007; Harteneck and
Gollasch 2011), and ruthenium red allowed to narrow down the spectrum of
12 K. Friedland and C. Harteneck
putative candidates to members of the TRPC family and finally to TRPC6 (Leuner
et al. 2007). The relevance for TRPC6 in hyperforin-induced cation entry was
validated in analyses of recombinantly expressed TRPC6 by imaging and electro-
physiological methods (whole-cell, inside-out, and outside-out configurations)
showing that TRPC6 is a target of hyperforin (Leuner et al. 2007, 2010; Muller
et al. 2008). Approaches using siRNA knockdown and the expression of a selective
dominant-negative TRPC6 mutant enabled to unravel the physiological role of
TRPC6 as hyperforin target in the neuronal-like PC12 cell line. The knockdown
of TRPC6 resulted in reduced growth of neurites and thereby argued for a role of
TRPC6 in neuronal plasticity and differentiation processes (Leuner et al. 2007).
The relevance of hyperforin-triggered TRPC6 activation in differentiation pro-
cesses was validated in the quite different cell model of keratinocytes, which
express TRPC6. During differentiation of keratinocytes, the expression pattern of
keratins changes. The change as measure of keratinocytes differentiation was
modulated by hyperforin in a TRPC6-dependent manner in these experiments
(Muller et al. 2008).
The current concept for TRPC6-dependent antidepressive activity of hyperforin
is visualized in Fig. 4. TRPC6 localized in the presynaptic membranes mediates
sodium and calcium entry upon activation by hyperforin. The sodium entry medi-
ated by TRPC6 impairs the sodium gradient across the plasma membrane and
thereby indirectly reduces the driving force for the serotoninsodium symporter,
while calcium entry via calcium-dependent kinase IV as well as the activation of the
MAPK and PI3K pathways is involved in differentiation processes modulating
neuronal plasticity (Heiser et al. 2013).
TRPC1TRPC7 channels (TRPC2 is a pseudogene in humans) are permeable for
mono- as well as divalent cations such as Ca2+, Ba2+, Sr2+, Zn2+, or Mn2+ and are
integrated in G protein-coupled receptor-mediated signaling cascades leading to
phospholipase C activation. Phospholipase C isoforms mediate the breakdown of
phosphatidyl-3,4-bisphosphate (PIP2) to inositol-1,4,5-trisphosphate and
diacylglycerol (DAG), intracellular signaling molecules activating downstream
processes. Inositol-1,4,5-trisphosphate acts as a second messenger on inositol-
1,4,5-trisphosphate receptors mediating calcium release from the intracellular
endoplasmic storage compartment. Diacylglycerols, 1,2-fatty acid esters of glyc-
erol, stimulate protein kinase C on one hand and on the other directly activate
TRPC3, TRPC6, and TRPC7 channels (Hofmann et al. 1999). Stearoyl-
arachidonoyl-sn-glycerol (SAG, Fig. 1e) represents a naturally occurring
diacylglycerol, whereas in most of the in vitro experiments, the oleoyl-acetyl-sn-
glycerol (OAG) is used as diacylglycerol analogue due to better solubility.
The expression of TRPC channels in the developing as well as in the adult brain
makes TRPC channels to interesting targets in the control of growth cone guidance,
neurotransmitter release, regulation of synaptic plasticity, and brain development
(Strubing et al. 2001; Fusco et al. 2004; von Bohlen Und Halbach et al. 2005;
Chung et al. 2006; Faber et al. 2006; McGurk et al. 2011). With respect to the
pharmacological use of hyperforin as antidepressant, the activation of TRPC3 and
TRPC6 channels by brain-derived neurotrophic factor via tyrosine kinase receptors
Hyperforin: To Be or Not to Be an Activator of TRPC(6) 13
P P
CREB
CREB
POL POL
CREB
CaMKII
2+
Ca 2+
5-HT 2+ Ca
Ca
2+ +
Na +Ca , Na
+
5-HT Na
+
Na
5-HT TRPC6
2+ +
SERT Ca , Na
SLC6A4
5-HT
+
Na Hyperforin
synthetic
antidepressants
Fig. 4 Illustration of hyperforin-triggered, TRPC6-dependent indirect inhibition of neurotrans-
mitter reuptake. TRPC6 localized in the presynaptic membranes mediates sodium and calcium
entry upon activation by hyperforin. The sodium entry mediated by TRPC6 impairs the sodium
gradient across the plasma membrane and thereby indirectly reduces the driving force for the
serotoninsodium symporter (SERT/SLC6A4: solute carrier family 6A4 transporter), while cal-
cium entry via calcium/calmodulin-dependent kinase II (CaMKII) is involved in differentiation
processes modulating neuronal plasticity via phosphorylation of cAMP response element-binding
protein (CREB). 5-HT serotonin, CREB P phosphorylated form of the cAMP response element-
binding protein, POL DNA-dependent RNA polymerase
(TrK), e.g., TrKB and phospholipase C, is of particular interest (Li et al. 2005,
2012; Amaral and Pozzo-Miller 2007; Sossin and Barker 2007). Reduced BDNF
levels have been observed in the hippocampus of patients with major depression,
and reduced levels of BDNF are considered to be an important trigger in the
pathophysiology of major depression. The antidepressant treatment results in
increased BDNF levels (Duman and Aghajanian 2012; Duric and Duman 2013;
Duman and Duman 2015).
Interested in neuronal zinc homeostasis, Gibon et al. studied TRPC6-mediated
currents in HEK293 cells and in cortical neurons (Gibon et al. 2011b; Bouron and
Oberwinkler 2014). Analyses of recombinantly expressed TRPC6 in HEK293 cells
elicited a pronounced uptake of Zn2+ in the presence of OAG or hyperforin. In
14 K. Friedland and C. Harteneck
latencies in the group also treated with tetrahydrohyperforin. The role of TRPC6
channels for Alzheimers disease pathology is also supported by Lessard
et al. (2005) demonstrating that Alzheimers disease-linked presenilin 2 variants
reduce agonist-induced TRPC6 activation when co-expressed in HEK293 cells.
7 Summary
The data analyzing the molecular mechanism of St. Johns wort antidepressive
action are divers and controversial. Despite older data, there is growing evidence
that hyperforin represents the pharmacological principle of St. Johns wort and its
extracts. The transcriptional activation via pregnane X receptor is mainly seen as
the major drawback in therapy reducing steady-state levels of simultaneously
applied remedies. The increase in CYP enzyme might contribute to the normaliza-
tion of cortisol levels, reported to be increased in depression; however the relevance
has not been studied so far. An obvious discrepancy in the studies analyzing the
activation mechanism of hyperforin results from electrophysiological data provid-
ing different currentvoltage relationships and current characteristics reported in
the absence of divalent cations (Leuner et al. 2007; Muller et al. 2008; Gibon
et al. 2010, 2011a, b; Tu et al. 2010; Sell et al. 2014). The discrepancy is linked to
the question whether or not the activity of hyperforin is dependent on TRPC6. A
recent report revived data on the protonophoric character of hyperforin, in contrast
to many showing the dependence on TRPC6 of hyperforin-stimulated currents.
Currently, the basis for the discrepancies in the shape of the currentvoltage
relationships is mysterious as well as the dependence of hyperforin-triggered
currents on divalent cation. It is obvious that the report claiming protonophoric
activity of hyperforin is able to show hyperforin-induced currents in the absence of
divalent cations in particular calcium, whereas the report providing data on cal-
cium- and zinc-based currents is unable to record currents in the absence of the
divalent cation. It will be interesting to understand the origin of these discrepancies
in the future.
With respect to drug developments, the view of hyperforin as protonophore-
mediating currents in a protein-independent manner is problematic. The side effects
reported for protonophoric and ionophoric compounds are serious. The problematic
is becoming much more evident in the light of the concentrations needed. TRPC6-
dependent modulation of synaptic plasticity by hyperforin is induced at concentra-
tions of 100300 nM, whereas the effect on the intracellular pH changes was
induced in the presence of much higher hyperforin levels. The view of TRPC6-
dependent, hyperforin-induced antidepressive activity provides a more optimistic
view as already lead structures have been described integrating the antidepressant
activity in animal models with simultaneously eliminating the therapy-limiting
pregnane X receptor-dependent side effects.
18 K. Friedland and C. Harteneck
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24 K. Friedland and C. Harteneck
Abstract Piezo1 and Piezo2 are critically required for nonselective cationic
mechanosensitive channels in mammalian cells. Within the last 5 years, tremen-
dous progress has been made in understanding the function of Piezo1/2 in
embryonic development, physiology, and associated disease states. A recent break-
through was the discovery of a chemical opener for Piezo1, indicating that
mechanosensitive ion channels can be opened independently of mechanical stress.
We will review these new exciting findings, which might pave the road for the
identification of novel therapeutic strategies.
Contents
1 Piezo1/2 Are Essential Components of Distinct Mechanically Activated Cationic
Channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2 Piezo1/2 Are Pore-Forming Subunits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3 Mapping the Ionic Pore in Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
4 The Small Synthetic Molecule Yoda1 Is an Opener of Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5 Upregulation of Piezo1 by Phosphoinositides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
6 Dehydrated Stomatocytosis (Xerocytosis) Is Caused by Gain-of-Function Mutations
in Piezo1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
7 A Possible Link Between Piezo1 and Sickle Cell Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Piezo1 was identified using a siRNA knockdown screening in the N2A cell line that
is characterized by a particularly high endogenous SAC activity (Coste et al. 2010).
A second protein Piezo2 was subsequently discovered by sequence homology
(Coste et al. 2010). Piezos are already present in paramecium, protozoan parasites,
chordates, plants, and invertebrates, although no Piezo homologue is found in
bacteria or yeast (Coste et al. 2010). The predicted Piezo proteins contain between
2,100 and 4,700 amino acids, depending on the species. In the adult mouse, the
strongest expression of Piezo1/2 was found in the lung and bladder, while expres-
sion is low in the heart and absent in the brain (Coste et al. 2010). Of note, Piezo2 is
also abundantly expressed in DRG neurons, unlike Piezo1 (Coste et al. 2010;
Ranade et al. 2014b). Interestingly, Piezo1 is also expressed in brain-derived
human neural stem/progenitor cells, and its activation was proposed to be involved
in neurogenesis and enhanced astrogenesis (Pathak et al. 2014).
Plasma membrane expression of tagged Piezo1 was reported in various cell
types (Coste et al. 2010; Peyronnet et al. 2013). Moreover, nonselective cationic
SAC activity (with a reversal near 0 mV) was observed upon heterologous expres-
sion of Piezo1 or Piezo2, with a single-channel conductance of about 2535 pS and
activation in response to mechanical stress, including cell poking, membrane
stretch, substrate deflexion, or fluid flow (Coste et al. 2010; Poole et al. 2014;
Ranade et al. 2014a, b). Sodium, potassium, calcium, and magnesium all permeate
Piezo1 with a slight preference for calcium (Coste et al. 2010; Gnanasambandam
et al. 2015). Piezo1 is inhibited by ruthenium red and Gd3+ (despite the low
specificity of these agents for SACs), as well as the spider peptide GsTMx4, a
more specific inhibitor of mechanosensitive cationic channels (Suchyna et al. 2000;
Bae et al. 2011). GsMTx4 produces a 30 mmHg rightward shift in the pressure-
gating curve of Piezo1, thus acting as a gating modifier (Bae et al. 2011). Both Piezo
isoforms show pronounced inactivation, with a relatively faster kinetics of inacti-
vation for Piezo2. Interestingly, protonation of human Piezo1 stabilizes inacti-
vation, thus inhibiting channel activity (Bae et al. 2015). Of note, Drosophila Piezo
(dPiezo) isoform shows a much smaller single-channel conductance of about 3 pS
and is in addition resistant to inhibition by ruthenium red (Coste et al. 2012).
A mPiezo1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
LNLFLFQGFRLVPFLVELRAVMDWVWTDTTLSLSNWMCVEDIYANIFIIKC
2133
C
M 2225R R2456H
Hydrophobic T2127M
5 hPiezo1
R1358P A2020T E2496ELE
Hydropathicity scale
Xerocytosis mutations
2.5
2.5
5
Hydrophilic
0 250 500 750 1000 1250 1500 1750 2000 2250 2500
Fig. 1 Hypothetical transmembrane topology of mPiezo1. (A) Cartoon showing the proposed
topological structure of mPiezo1, as predicted by bioinformatical analysis. The conserved PFEW
motif is located within a hydrophobic domain at the C-terminal region. (B) Putative structural
models of the C-terminal domain of mPiezo1 comprising the PFEW motif, with green segments
representing hydrophobic stretches. The E2133 residue involved in permeation is indicated by a
red sphere. (C) Hydropathicity plot of hPiezo1 with gain-of-function mutations associated with
xerocytosis (indicated in red). The mutant R2456H (squared in red) produces the most dramatic
impairment of inactivation. Peaks with scores greater than 1.8 on the hydrophobicity scale indicate
possible transmembrane segments. Adapted with permission from Coste et al. (2015)
activity, further indicating that this region plays a key role in pore properties (Coste
et al. 2015). Altogether these findings indicate that E2133 is either within or at least
in close proximity to the Piezo1 pore domain. However, since this conserved
residue is also present in dPiezo (E2091), the major difference in pore properties
between mPiezo1 and dPiezo has to be explained by other differences present in the
C-terminal domain that remain to be identified.
Yoda1
Xerocytosis
Piezo1 knock-out
Force Sickle cell
disease
Piezo1
H2O H2O
Ca2+
K+
K+
H2O
KCa3.1 KCa3.1
open closed
Fig. 2 The opening of Piezo1 shrinks red blood cells. The opening of Piezo1 by the agonist Yoda1
or by mechanical stress promotes calcium influx that stimulates the opening of the Gardos channel
KCa3.1 in RBCs (left panel). The resulting K+ efflux leads to water loss and RBC shrinkage. Gain-
of-function mutations of Piezo1 slowing down inactivation similarly result in dehydration of
RBCs, causing xerocytosis. Inhibition of Piezo1 by antagonists might become valuable for the
treatment of the sickle cell disease, when Piezo1 activity in RBCs appears to be enhanced. By
contrast, red blood cells from Piezo1 knockout mice (right panel) are overhydrated since water is
retained because of low intracellular calcium and closing of the Gardos channels
The Piezo Mechanosensitive Ion Channels: May the Force Be with You! 31
Piezo1 expression is particularly strong in the bladder and in the urothelium, both in
mice and humans (Coste et al. 2010; Miyamoto et al. 2014). The urothelium is
centrally involved in bladder mechanosensation. A Piezo1-dependent and
GsMTx4-sensitive increase in cytosolic Ca2+ concentrations in response to stretch
was demonstrated in urothelial cells (Miyamoto et al. 2014). Furthermore, this
effect was associated with the release of ATP. These findings suggest that urothelial
Piezo1 is involved in bladder mechanotransduction and might represent a putative
pharmacological target for the treatment of bladder dysfunction (Miyamoto
et al. 2014).
they are etiologically related (Coste et al. 2013; McMillin et al. 2014). Thus, both
Piezo1 and Piezo2 GOF mutations are associated with inherited diseases
(Zarychanski et al. 2012; Bae et al. 2013a, b; Coste et al. 2013; IAndolfo
et al. 2013; McMillin et al. 2014).
Touch dome
Moving stimulus
Keratinocytes
Static displacement
SAI response
Merkel cells
Piezo2 IA firing
(Transduction in afferents)
Afferents
SA firing
(Transduction in Merkel cells)
Fig. 3 The Merkel-cell neurite complexes in touch domes of the skin. Tactile stimuli evoke
responses from sensory afferents innervating touch domes. A two-receptor-site model has been
proposed for type I slowly adapting firing responses (Ranade et al. 2014b; Woo et al. 2015). The
opening of Piezo2 in Merkel cells mediates sustained firing during static displacement, while the
opening of Piezo2 in neurite afferents mediate fast-adapting firing in response to moving mecha-
nical stimuli. Adapted with permission from Woo et al. (2015)
The Piezo Mechanosensitive Ion Channels: May the Force Be with You! 35
Merkel cells are essential to induce sustained neuronal activity in tactile afferents
(Fig. 3) (Ikeda et al. 2014; Maksimovic et al. 2014; Woo et al. 2014).
Thus, the opening of Piezo2 in neuronal afferents underlies the transient (fast-
adapting) response to moving mechanical stimuli, while the opening of Piezo2 in
Merkel cells mediates the sustained discharge in response to static displacement
(Fig. 3) (Maksimovic et al. 2014; Ranade et al. 2014b; Woo et al. 2014). Although
Piezo2 is a fast-inactivating SAC, the small plateau current of Piezo2 might be
sufficient to mediate the sustained activation of Merkel cells by static mechanical
stimuli, because of a high resistance of the membrane (Coste et al. 2010; Woo
et al. 2014). In the same line, Piezo2 was shown to be required for
mechanotransduction in human stem cell-derived touch receptors (Schrenk-
Siemens et al. 2015).
Kidney epithelial cells respond to both changes in intraluminal pressure and fluid
flow (Patel and Honore 2010; Weinbaum et al. 2010). Peristaltic pressure generated
by rhythmic papillary contractions varies between 15 and 45 mmHg, resulting in
the stretching of both apical and basolateral membranes (Jensen et al. 2007).
Intraluminal pressure can also be abnormally elevated in various kidney diseases
(Patel and Honore 2010). For instance, obstructive uropathy leads to an increase in
intratubular pressure, in excess of 60 mmHg (Wyker et al. 1981; Cachat et al. 2003;
Power et al. 2004; Quinlan et al. 2008; Rohatgi and Flores 2010). Stretching, as well
as compression, of renal epithelial cells is also common in PKD patients (Patel and
Honore 2010). Abnormal fluid accumulation in renal cysts causes the cyst wall to
stretch (Derezic and Cecuk 1982; Tanner et al. 1995; Praetorius et al. 2009).
Moreover, compression of healthy tubules by neighboring cysts results in the
upstream accumulation of urine and consequent tubular distension. Thus, the
stretch of tubular epithelial cells is relevant to both physiological and pathological
renal conditions (Patel and Honore 2010). Moreover, the apical side of tubular
epithelial cells is also continuously subjected to urine flow stimulation (i.e., shear
stress).
Nonselective SACs were recorded at the basolateral side of renal tubular epi-
thelial cells and were characterized by a lack of inactivation and a very slow
deactivation when recorded in the cell-attached patch clamp configuration
(Peyronnet et al. 2013). Piezo1 is critically required for SAC activity in mouse
The Piezo Mechanosensitive Ion Channels: May the Force Be with You! 37
renal tubular cells (Peyronnet et al. 2013). The lack of Piezo1 inactivation in renal
epithelial cells remains unexplained.
Unexpectedly, overexpression of TRPP2 (polycystin-2; PC2), or to a greater
extent its pathogenic mutant PC2-740X (which lacks interaction with polycystin-1;
PC1), impaired native SACs in renal tubular epithelial cells, as well as in arterial
myocytes (Sharif Naeini et al. 2009; Peyronnet et al. 2013). The inhibitory effect of
PC2 on native SACs could be reversed by overexpressing PC1, while it was
mimicked by Pkd1 deletion (Sharif Naeini et al. 2009; Peyronnet et al. 2013).
Moreover, PC2 inhibited exogenous Piezo1/SAC activity expressed in a variety of
cell types (Peyronnet et al. 2013). PC2 co-immunoprecipitated with Piezo1 and
deletion of its N-terminal domain prevented both this interaction and inhibition of
SAC activity. Altogether, these findings indicate that renal SACs depend on Piezo1
but are critically conditioned by the PC1/PC2 ratio. Interestingly, Piezo1 opening is
thought to control cell extrusion by tissue overcrowding in developing epithelia of
the zebrafish (Eisenhoffer et al. 2012). Thus, inhibition of Piezo1, as observed upon
Pkd1 deletion or expression of the PC2-740X mutant, might contribute to
cystogenesis by impairing cell extrusion from developing cysts (Peyronnet
et al. 2013).
These findings, together with the previously reported downstream activation of
Painless by dPiezo, indicate a possible functional interaction between TRP chan-
nels and Piezos (Kim et al. 2012; Peyronnet et al. 2013).
The discovery of Piezos is definitely an amazing finding in the ion channel field
and more surprises are certainly to be expected in the near future.
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The Piezo Mechanosensitive Ion Channels: May the Force Be with You! 41
AII and aldosterone (ALD), while increasing renin activity for about 24 h. From the
results of recent investigations in human, it is hypothesized that reduction of AII
and ALD is one of the drivers of increased survival and improved quality of life in
dogs receiving ACE inhibitors. To support and consolidate this hypothesis, addi-
tional efforts should be directed toward the collection of circulating RAAS peptides
in spontaneous cases of canine CHF. If such a link could be established, profiling of
these biomarkers could support determination of the severity of heart failure,
complement clinical and echocardiographic findings, and be used for therapeutic
drug monitoring purposes.
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2 An Overview of the ReninAngiotensinAldosterone System: Past and Present . . . . . . . . . 46
2.1 A Complex and Highly Regulated Machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.2 ReninAngiotensinAldosterone Activation in Vascular Inflammation,
Remodeling, and Congestive Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3 Modeling and Simulation: A Basis for Optimizing the Dosing Schedule of RAAS
Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4 Components of the Renin Cascade, Blood Pressure, and Urinary Electrolytes Fluctuate
with Clear Circadian Changes in Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
5 The Chronobiology of Renin Activity, Blood Pressure, and Urinary Electrolytes Is
Synchronized to the Dogs Feeding Schedule . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
6 Benazepril Markedly Influences the Dynamics of the Circulating RAAS . . . . . . . . . . . . . . . . . 58
7 Conclusions and Research Perspectives in Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Abbreviations
1 Introduction
Congestive heart failure (CHF) is a major cause of morbidity and mortality with an
increasing prevalence in human and canine populations (Guglielmini 2003; George
et al. 2014). In dogs, CHF most often develops consequent to chronic valvular heart
defect, also known as myxomatous mitral valve disease (MMVD) or chronic left
valvular heart disease (CVHD) (Borgarelli and Buchanan 2012). MMVD is char-
acterized by thickening and shortening of the atrioventricular valves and affects
about 75% of dogs over the age of 16 (Guglielmini 2003). While MMVD has been
recognized in dogs for over a century, histopathological and clinical studies have
not been able to reveal its cause or why it occurs ten times more frequently in dogs
than in humans (Borgarelli and Buchanan 2012). In humans, the majority of heart
diseases are caused by atherosclerosis, a condition which is not spontaneously
observed in dogs even under a high-cholesterol regime (Mahley et al. 1974). The
first large animals used to study heart failure were dogs, in which models of
myocardial infarction and serial microembolization of the coronary artery were
developed (Zaragoza et al. 2011). Similar to humans, the -myosin heavy chain
isoforms predominate in the dog myocardium (Hasenfuss 1998), such that the
excitation/contraction coupling in the myocardium of dogs appears to be similar
to that in the human myocardium. More importantly, the pathophysiological
scheme of renin activation, as observed in the course of CHF, is similar between
dogs and humans, which motivated the choice of this animal species in the
experimental work on the reninangiotensinaldosterone system (RAAS) and
blood pressure (BP) pioneered by Guyton, Hall, and co-workers (Cowley and
Guyton 1972; Guyton et al. 1972; McCaa et al. 1975; Young and Guyton 1977;
DeClue et al. 1978; Lohmeier et al. 1978; Hall et al. 1980, 1984; Wilczynski and
Osmond 1983). Renin release from the juxtaglomerular apparatus is a common
compensatory mechanism to the reduced cardiac output observed in symptomatic
stages of canine and human heart failure (Watkins et al. 1976; Hall 1991). Recog-
nition of the dysregulation of the RAAS in the pathophysiology of CHF has led to
significant medical advances (McMurray et al. 2012). Reduction of angiotensin II
46 J.P. Mochel and M. Danhof
Various authors have amply reviewed the role of the RAAS in the regulation of BP
and volume homeostasis (Ferrario and Strawn 2006; Moon 2013; Sayer and Bhat
2014). The expression of certain RAAS components even in simple organisms like
crustaceans, insects, and leeches underscores the importance of the renin cascade in
the control of cell volume and water homeostasis throughout evolution (De Mello
2014). The history of the RAAS and its discovery has recently been retraced with
great accuracy in a review paper by Tsukamoto and Kitakaze (2013).
A common description of the functioning of the systemic RAAS cascade begins
with the release of renin from granular cells of the juxtaglomerular apparatus, in
response to changes in sodium chloride concentrations, decreased renal blood flow,
and sympathetic stimulation. Many studies have established that renin secretion is
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 47
Angiotensinogen Renin
Angiotensin I
Bradykinin
Compensatory
Chymase ACE
feedback loop
Inactive
fragments
Angiotensin II
AT1 Receptor
End-Organ Damage
(heart, vasculature, kidneys)
Aldosterone
Fig. 2 Classic view of reninangiotensin system cascade (blue) and recent view of renin
angiotensin system cascade (green). AP aminopeptidase, APA aminopeptidase A, APN
aminopeptidase N, CP carboxypeptidase, EP endopeptidase, ACE angiotensin-converting
enzyme, ACE2 angiotensin-converting enzyme 2, CPP carboxypeptidase P, PRCP prolyl car-
boxypeptidase, NEP neprilysin, PO prolyl oligopeptidase, Mas Ang-(1-7) Mas receptor, Mrg
Mas-related G-protein-coupled receptor, AT4 angiotensin type 4 receptor. Letters in green repre-
sent amino acids using the one-letter code. Source: Ferr~ao et al. (2014). Reprinted with permission
from Baishideng Publishing Group
homeostasis via sodium, potassium, and hydrogen ion exchanges in the distal renal
tubules and collecting ducts of Bellini (Quinn and Williams 1988). Note that the effect
of ALD on the regulation of natriuresis and BP would be quantitatively less important
than the action of AII on proximal tubular sodium reabsorption. This direct intrarenal
effect of AII further results in reduced urinary flow in the tubular segments of the
medulla, thereby increasing medullary osmolality and fluid reabsorption in the
descending loop of Henle and the collecting ducts of Bellini (Hall, 1991).
Next to the systemic (circulatory) renin cascade, several RAAS components are
also produced at the tissue level, in the heart, the vascular endothelium, or the
kidneys (Danser 1996; Danser et al. 1997). This local RAAS functions as an
autocrine or paracrine system and regulates tissue growth and repair processes.
It is now recognized that the conventional renin/ACE/AII/AT1 cascade is no
longer the sole signaling pathway of the RAAS. At least three new axes have
recently been identified in the kidneys and other tissues (Zhuo et al. 2013)
(Fig. 2). These include (i) the ACE2/ANG(1-7)/Mas receptor pathway that may
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 49
play an opposing role to the renin/ACE/AII/AT1 axis (Esteban et al. 2009), (ii) the
prorenin/PRR/MAP kinases ERK1/2 axis, which appears to be pivotal in the
development of diabetic nephropathy in rodents (Ichihara et al. 2004, 2006), and
(iii) the ANGIV/AT4/IRAP cascade, whose implication in the regulation of BP and
renal modulation remains controversial. With the discovery of these additional
pathways, the action of the RAAS has been extended beyond the regulation of
BP, sodium, and fluid homeostasis by the AT1 receptor.
for modeling of the placebo data would result in overestimating the effect of ACE
inhibition on AII and ALD, while underestimating its effect on RA (Mochel
et al. 2015). Circadian changes in the functioning of the circulating RAAS provide
a strong scientific rationale for tailored administration of cardioactive drugs at a
time that would optimize efficacy. However, additional influences to the underlying
biological rhythm may further contribute to temporal changes in the effectiveness
of drug therapies. Several physiological factors such as gastrointestinal pH and
motility, cardiac blood flow, or liver enzyme activity are prone to diurnal variations
(Reinberg and Smolensky 1982; Bruguerolle 1998; Ohdo 2007) and could impact
the disposition kinetics (i.e., absorption, distribution, metabolism, and elimination)
and the related efficacy of RAAS inhibitors. These fluctuations could be taken into
account using ad hoc PB PKPD modeling techniques.
Fig. 3 The core oscillator of the mammalian circadian clock. (a) The transcriptionaltranslational
feedback loop that makes up the core oscillator of the mammalian circadian clock. CLOCK and
BMAL1 bind to E-box DNA sequences to activate clock-controlled genes (cogs), including those
that code for the oscillator proteins PER and CRY. PER and CRY are translated in the cytoplasm
and then cycle back into the nucleus to directly repress CLOCK:BMAL1. As PER and CRY level
drop, CLOCK:BMAL1 reactivates another round of transcription. (b) The CLOCK (green) and
BMAL1 (blue) proteins heterodimerize to bind DNA through their N-terminal bHLH domains.
The structure reported by Huang et al. (2011) shows that the PAS A domains dimerize symmet-
rically; in contrast, the PAS B domains form a head-to-tail interaction, mediated by a conserved
Trp residue on BMAL1 that binds into CLOCK PAS B. The analogous Trp on CLOCK projects
into solvent for putative interactions with CRY. To inhibit transcription, the PER tandem PAS
domains may interact with those of CLOCK:BMAL1. Unstructured regions of CLOCK and
BMAL1 (dotted lines) are important for transcriptional activation and histone acetyltransferase
(HAT) activity. Gln glutamine, ccgs clock-controlled gene sequences. Source: Crane (2012).
Reprinted with permission from the American Association for the Advancement of Science
(AAAS)
levels measured in the morning and lowest values around midnight (Staessen
et al. 1992; Smolensky and Haus 2001). Several factors have been shown to
influence daynight variations of the systemic RAAS, including alterations in
posture (Muller et al. 1958), sleep cycles (Brandenberger et al. 1985, 1994), and
age (Cugini et al. 1985). However, investigations on the effect of feeding time on
the periodicity of the RAAS have led to conflicting results (Kunita et al. 1976;
Ikonomov et al. 1981).
Although few observations of time variations of RAAS peptides have been
reported in dogs (Corea et al. 1996; Gordon and Lavie 1985; Reinhardt
et al. 1996), no systematic characterization of the chronobiology of these variables
is presently available in the literature. In addition, the question of whether BP
oscillates over the 24-h span in dogs is still a matter of debate (Miyazaki et al. 2002;
Piccione et al. 2005; Soloviev et al. 2006; Mochel et al. 2013a, 2014a). Results from
our research (Mochel et al. 2013a) showed that plasma renin (Fig. 4), urinary
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 53
400
160
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Renin activity (% change from baseline)
140
150
300
120
200
100
100
80
100
50
60
Night Night
Night
40
0
0
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
400
160
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Renin activity (% change from baseline)
140
150
300
120
100
200
100
80
100
50
60
Night Night Night
40
0
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
Fig. 4 Circadian changes in renin activity, urinary sodium, and potassium fractional excretion
under various feeding schedules. Top. Average plasma renin activity (left pane), urinary sodium
(middle pane), and potassium (right pane) fractional excretion (% change from baseline) in dogs
fed a normal-sodium diet (0.5% sodium) at 07:00 h (continuous line) and 13:00 h (dashed line).
Bottom. Average plasma renin activity (left pane), urinary sodium (middle pane), and potassium
(right pane) fractional excretion (% change from baseline) in dogs fed a normal-sodium diet (0.5%
sodium) at 07:00 h (continuous line) and 19:00 h (dashed line). Source: Mochel et al. (2014a).
Reprinted by permission of Taylor & Francis LLC
aldosterone (UA:C), BP (Fig. 5), and urinary electrolytes (UK,fe and UNa,fe for
potassium and sodium fractional excretion, respectively) oscillate with a circadian
periodicity in trained and relaxed healthy dogs fed a regular diet at 07:00 h. An
approximately twofold (1.63.2-fold) difference between day and night measure-
ments was found for RA ( p < 0.01), UA:C ( p: 0.01), UK,fe ( p: 0.01), and UNa,fe
( p: 0.007).
Our results are consistent with previous investigations in dogs (Corea
et al. 1996), horses (Clarke et al. 1978, 1988), and humans (Cugini et al. 1981,
1985), which underlines the similarity of body fluid homeostasis in mammals. A
cosine model with a fixed 24-h period was found to fit the periodic variations of RA,
BP, urinary aldosterone, and potassium excretion well, as suggested by the quality
of the model diagnostics. In contrast, circadian changes in urinary sodium were best
characterized by means of a surge model, reflecting an afternoon peak sodium
excretion followed by a monotonous decay (Fig. 6). Here, NLME modeling
allowed borrowing information from the densely sampled plasma variables (i.e.,
RA) to improve parameter estimation of the (more sparse) urinary endpoints.
Similar to fluctuations in RA and UA:C, renal excretion of potassium was low in
the morning, increased in the afternoon, and peaked in the early evening. Systolic
54 J.P. Mochel and M. Danhof
140
140
Dogs fed at 07:00 h
Dogs fed at 07:00 h
Dogs fed at 19:00 h Dogs fed at 19:00 h
120
120
100
100
Night 80 Night
80
7:00 13:00 19:00 01:00 7:00 7:00 13:00 19:00 01:00 7:00
Fig. 5 Circadian changes in systolic and diastolic blood pressure under various feeding schedules.
Average systolic (left pane) and diastolic (right pane) blood pressure vs. time profile (% change
from baseline) in dogs fed a normal-sodium diet (0.5% sodium) at 07:00 h (continuous line) and
19:00 h (dashed line). Source: Mochel et al. (2014a). Reprinted by permission of Taylor &
Francis LLC
and diastolic BP rose during the first half of the night, before returning to baseline
values in the morning.
From a physiological standpoint, the morning decrease in RA would be related
to body fluid expansion consecutive to sodium and water intake, while the simili-
tude between RA, urinary aldosterone, and potassium fluctuations would reflect
aldosterone-stimulated secretion by the reninangiotensin pathway and
aldosterone-mediated excretion of potassium in the kidney distal tubules. Likewise,
the main contribution of the reninangiotensin cascade to the chronobiology of BP
would be related to the sodium-retaining effects of AII and ALD, associated with
the strong vasomotor effect of AII. Compared with urinary potassium, the peak
renal elimination of sodium occurred much earlier during the day (around 15:00 h).
This phenomenon is known as the impulse-response pattern of sodium excretion
and is characterized by a peak natriuresis 48 h after feeding (Boemke et al. 1995).
Finally, the increase in sodium excretion during daytime was not associated to an
elevated BP, which is consistent with earlier publications in dogs (Andersen
et al. 2000; Bie and Sandgaard 2000; Sandgaard et al. 2000), and suggests that
pressure natriuresis is not a prime determinant of sodium homeostasis in
intact dogs.
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 55
140
Observed variable (unit)
: period
120
A: amplitude
M : mesor
100
: acrophase
80
7 am 1 pm 7 pm 1 am 7 am 1 pm 7 pm 1 am 7 am
Time (hours)
140
Observed variable (unit)
: period
120
A: amplitude B : baseline
w : width
100
: acrophase
80
7 am 1 pm 7 pm 1 am 7 am 1 pm 7 pm 1 am 7 am
Time (hours)
Fig. 6 Model parameters of a cosine and a surge function. Top: The shape of a cosine model is
determined by a set of parameters: (M, A, , and ), where M is the mesor (daily average of
rhythm), A is the amplitude of the cosine, is the acrophase (or time of peak), and is the period
(herein fixed to a value of 24 h). Bottom: The structure of a surge function is similar to that of a
cosine, with the substitution of the mesor by the baseline (initial value of rhythm, B), and the
addition of another parameter: the width of the surge (w). Source: Mochel et al. (2013a). Reprinted
by permission of Taylor & Francis LLC
Stomach Melatonin
LH
Ghrelin Raphe
SCN
Leptin
ARC
GCs
Adiponectin Glucose
White adipose Adrenal
tissue
Pancreas
Fig. 7 Endocrine feedback to the circadian clock. Various hormones can directly or indirectly
feedback on central and peripheral clock function. In the brain endocrine targets with connections
to the SCN include the orexinergic neurons of the lateral hypothalamus (LH), the arcuate nucleus
(ARC), and the raphe nuclei of the brainstem. Other endocrine effects may be mediated via
peripheral tissues and clocks such as the liver and muscle. Source: Tsang et al. (2013). Reprinted
with permission from Bioscientifica Ltd
oscillators include autonomic outputs from the central nervous system, dietary
sodium, and feeding-dependent hormones (Mistlberger and Antle 2011; Tsang
et al. 2013) (Fig. 7). Although they are important to metabolic homeostasis,
glucocorticoids would not play a role. In contrast, ghrelin levels do exhibit a
clear circadian rhythm which appears to be aligned with the timing of food intake.
It has been hypothesized that ghrelin-secreting cells are themselves entrained by
feeding and that their endocrine signal serves as a messenger to other cells, both in
the brain and in peripheral tissues (LeSauter et al. 2009). Importantly, ghrelin can
also modify the phase of the SCN and its response to light (Brown and Azzi 2013).
Timed feeding has the ability to synchronize the activity of central oscillators, as
shown by Kurumiya and Kawamura (1991) in a rodent experiment where the peak
activity of neurons located in the hypothalamus was driven by the time of food
intake. Entrainment signals may also be provided by the intracellular ratio of
reduced to oxidized nicotinamide adenine dinucleotide cofactors (Rutter
et al. 2001). Next to the effect of feeding time on the chronobiology of the
RAAS, food composition per se (i.e., dietary sodium) also influences the periodicity
of renin. This was shown (i) from the results of the covariate analysis performed
under regular diet conditions (0.5% sodium) (Mochel et al. 2013a, 2014a) and
(ii) from the comparison of the mesor and amplitude estimates of RA in dogs fed a
58 J.P. Mochel and M. Danhof
Inhibition of the RAAS, as part of a global therapeutic scheme to decrease AII and
ALD exposure, and lower BP for preventing or delaying end-organ damage has
proven to be effective in human and canine CHF (Chobanian et al. 2003; Lefebvre
et al. 2007). Among RAAS inhibitors, two classes of drug directly target AII
through complementary mode of actions: (i) ACE inhibitors prevent the formation
of AII and the degradation of bradykinin, which increases the stimulation of nitric
oxide and has positive effects on endothelial function, while (ii) angiotensin recep-
tor blockers (ARBs) selectively antagonize AII at AT1 receptors. A theoretical
advantage of ARBs lies in their ability to increase activation of the AT2 receptor
and modulate the effects of AII breakdown products (Liu 1997), while reducing the
risk of ALD escape. Nevertheless, the escape phenomenon has also been reported
during long-term use of ARBs (Naruse et al. 2002), and the use of non-peptide
ARBs in small animal patients was shown to be ineffective (Adams 2009).
More recently, ALD receptor antagonists (ARAs) have also been registered for
use in canine patients suffering from CHF. In a study from Bernay et al. (2010),
spironolactone reduced by a factor of 2 the risk of cardiac-related death, euthanasia,
or severe worsening when used in addition to conventional therapy (ACE inhibi-
tion, plus furosemide and digoxin if required) in dogs with CVHD. These results
were however disputed by Kittleson and Bonagura (2010) on the grounds of several
methodological flaws (e.g., patient categorization, definition of CHF). In addition,
Schuller et al. (2011) could not find any significant effect of low-dose
spironolactone on survival when used as adjunct treatment to conventional conges-
tive heart failure treatment in dogs. Interestingly enough, ARAs have shown a
significant reduction in mortality in human CHF patients when combined with ACE
inhibitors, whereas ARBs have not (Werner et al. 2010). Lately, results of the
PARADIGM-HF clinical trial comparing the angiotensin receptorneprilysin
inhibitor LCZ696 with enalapril in patients with reduced ejection fraction CHF
were disclosed in the New England Journal of Medicine (McMurray et al. 2014).
LCZ696 was found to be superior by ca. 20% to enalapril in reducing the risks of
death and of hospitalization for heart failure ( p < 0.001). In a preliminary dog
study, valsartan, LCZ696 at 15 and 45 mg/kg decreased ALD levels to a significant
extent ( 23%, 45%, and 43%, respectively, p < 0.05). The greatest reductions
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 59
were observed in the LCZ696 groups, where LCZ696 15 mg/kg at 2 h reduced ALD
twofold lower than valsartan ( p < 0.05) (Mochel et al. 2014b).
By decreasing systemic vascular resistance, ACE inhibitors are known to
improve cardiac hemodynamics and exercise capacity in human and dog patients
(Levine et al. 1984; Uretsky et al. 1988; Lefebvre et al. 2007). Benazepril, enalapril,
imidapril, and ramipril are currently approved for use in dogs with CHF. Benazepril
hydrochloride (Fortekor; Novartis Animal Health, Basel, Switzerland) is a
nonsulfhydryl prodrug which is converted in vivo by esterases into its active
metabolite, benazeprilat, a highly potent and selective inhibitor of ACE (Webb
1990) with well-documented effectiveness in symptomatic canine CHF (King
et al. 1995; Lefebvre et al. 2007). In the BENCH (BENazepril in Canine Heart
Disease) Study Group (1999), the mean survival time of benazepril-treated dogs
with mild to moderate CHF was improved by a factor of 2.7, as compared with the
placebo group (428 vs. 158 days, p < 0.05). Although most of the preclinical
investigations for dose selection of benazepril have used ACE activity as a surro-
gate marker of efficacy in dogs, recent literature suggests that this may not be a
sensitive approach to properly assess the modulatory effect of ACE inhibitors on
the RAAS (Van de Wal et al. 2006). Using a low-sodium diet (LSD) model of
RAAS activation, our research shows that benazeprilat markedly influences the
dynamics of the systemic RAAS following single and repeated oral administrations
of benazepril at its recommended dose (0.251.0 mg/kg q24 h) in dogs (Mochel
et al. 2013b). In this study, treatment with benazepril triggered an apparent decrease
in AII and ALD, together with a sustained elevation of RA, as a consequence of
benazeprilat-induced interruption of the AIIrenin negative feedback loop (Bussien
et al. 1986; Steele et al. 2002). As expected, changes in ALD were followed by a
significant reduction of potassium renal excretion. The modulatory action of
benazeprilat on the functioning of the circulating RAAS was further characterized
using an integrated mechanism-based PKPD modeling approach (Fig. 8) (Mochel
et al. 2015). The final model, which also described the natural time course of the
biomarkers in the absence of the ACE inhibitor (i.e., under placebo treatment),
predicted no time delay between the dynamics of benazeprilat, RA, AII, and ALD,
which is an indication of a rapid turnover of RAAS biomarkers in plasma. As
previously mentioned, the use of a straight line approximation of the mean (in lieu
of a time-varying function) for modeling of the placebo data would have resulted in
overestimating the effect of ACE inhibition on AII and ALD, while
underestimating its effect on RA. Simulations from the PKPD model allowed
quantifying the extent and duration of effect of benazepril (0.34 mg/kg PO,
q24 h) on the RAAS. Results showed a two- to threefold change in systemic
RAAS levels at steady-state benazeprilat peak concentrations, and a more
prolonged effect on RA (at least 16 h) compared with AII and ALD (between
5 and 10 h). Such discrepancies could be related to the production of AII by
upregulation of ACE-independent pathways in response to renin and AI accumu-
lation during acute and long-term use of ACE inhibitors (Geary et al. 1992;
Fyhrquist and Saijonmaa 2008). Alternatively, the relatively short-lasting effect
of benazeprilat on AII and ALD might have been the result of the opposite
60 J.P. Mochel and M. Danhof
Depot Tinf
Benazepril
compartment Renin
M(RA), A(RA), (RA)
Angiotensinogen
ka
Angiotensin I
Imax (AII), (AII)
k1
k10 Free ACE-bound IC50 (AII)
Benazeprilat Benazeprilat
Emax (RA), (RA)
k2
Angiotensin II EC50 (RA)
Total benazeprilat M(AII), A(AII), (AII)
[Angiotensin II]
Aldosterone
M(ALD), A(ALD), (ALD)
stimulatory effect of sodium depletion on the RAAS. The reduction of AII and ALD
systemic levels certainly explains part of the clinical efficacy observed in
benazepril-treated CHF dogs, but additional effects on bradykinin degradation
and BP are likely to also come into play.
Chronobiology and Pharmacologic Modulation of the ReninAngiotensin-. . . 61
Similar to humans, peptides of the systemic RAAS and BP oscillate with a clear
circadian periodicity in dogs. In both species, the timing of food intake appears to
be pivotal to the circadian organization of these biomarkers. Our data further show
that benazepril influences the dynamics of the reninangiotensinaldosterone cas-
cade, resulting in a profound but temporary decrease in AII and ALD, while
increasing RA for about 24 h.
Results from our model-based approach provide new insights into the relation of
dietary sodium to the chronobiology of the renin cascade, which would have been
impossible using standard statistics. Specifically:
1. The amount of sodium intake was shown to influence the tonic (i.e., mesor) and
the phasic (i.e., amplitude) secretion of renin; the greater the intake of sodium,
the smaller the mesor and amplitude of RA.
2. The time of food (i.e., sodium) intake appeared to exert a synchronizing effect on
the acrophase of RA and BP oscillations, which consolidates preliminary data
from the literature (Itoh et al. 1996).
Based on our findings on the dynamics of the circulating RAAS under physio-
logical (Mochel et al. 2013a, 2014a) and RAAS-activated conditions (Mochel
et al. 2013b, 2015), various strategies could in theory improve the therapeutic
management of cardiovascular diseases in dogs. Essentially, one could think of:
1. Adjusting the time of dosing. In dogs, cardioactive medications are commonly
given with morning food for the sake of convenience. However, results from our
chronobiological investigations with morning feeding indicate that the peak RA
and BP occurs in the evening and at night. Assuming that drug efficacy is
maximum when the peak effect time of the drug is synchronized with the peak
of the underlying biological rhythm, one would expect optimized efficacy with
bedtime dosing.
2. Adjusting dietary sodium intake. Because high dietary sodium is thought to play
a role in the development of HT and cardiovascular and renal diseases in
humans, a common practice in veterinary cardiology was to restrict sodium
intake in the diet of CHF dogs. There is however no substantial evidence that
elevated sodium intake increases the risk of HT in dogs (see results from
Anderson et al. 1986 and Greco et al. 1994 showing that fluctuations in sodium
intake has no apparent effect on BP and heart rate), and the current recommen-
dation is to avoid highly elevated dietary salt intake, without making a specific
effort to restrict it (Chandler 2008). Furthermore, because the mesor and ampli-
tude value of RA oscillations was found to be much greater in dogs fed a
low-sodium regime (Mochel et al. 2015), we could assume that CHF dogs
would rather benefit from a normal-sodium diet.
The research summarized herein is not a static and completed piece of work but
is, instead, a starting point for further data integration and hypothesis testing. As the
62 J.P. Mochel and M. Danhof
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