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\begin{document}

\title{Modulation of G-protein activation, calcium currents and opioid receptor
phosphorylation by the pH-dependent agonist NFEPP}



\author[1]{Melih Celik}%
\author[1]{Viola Seitz}%
\author[1]{Fatih Yergoz}%
\author[1]{Sandeep Dembla}%
\author[2]{Nina Blum}%
\author[2]{Stefan Schulz}%
\author[1]{Christoph Stein}%
\affil[1]{Charite Universitatsmedizin Berlin}%
\affil[2]{Universitätsklinikum Jena}%


\vspace{-1em}



  
  \date{May 18, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract Background and Purpose NFEPP is a newly-designed pain killer
selectively activating G-protein coupled mu opioid receptors in injured
tissues, and therefore devoid of central side effects. However, the
cellular mechanisms underlying NFEPP's antinociceptive effects were not
examined in sufficient detail so far. Here we investigated the effects
of NFEPP on G-protein activation, on voltage gated calcium channels and
on mu opioid receptor phosphorylation. Experimental Approach HEK293
cells stably transfected with mu opioid receptors were used to study
{[}35S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding and mu opioid receptor phosphorylation. Voltage
dependent calcium currents and intracellular calcium signals were
examined in rat sensory neurons. All experiments were performed at
acidic and physiological pH values using NFEPP compared to the
conventional mu opioid receptor agonist fentanyl. To investigate the
role of G protein subunits, we used pertussis toxin and gallein. Key
Results At low pH, NFEPP produced more efficient G-protein activation
and reduction of calcium currents in depolarized sensory neurons. The
latter was mediated by G protein \selectlanguage{greek}βγ \selectlanguage{english}subunits and NFEPP-mediated MOR
phosphorylation was pH-dependent. Fentanyl-induced signaling was not
affected by pH changes. Conclusion and Implications Our study shows
that, at low pH, MOR signaling induced by NFEPP is more effective and
neuronal calcium channels are directly modulated by G protein \selectlanguage{greek}βγ
\selectlanguage{english}subunits dissociated from G protein \selectlanguage{greek}α\selectlanguage{english}i/o subunits. Apparently, the
enhanced efficacy of NFEPP is dependent on extra- rather than
intracellular effects on opioid receptor function.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Opioids are the strongest painkillers to date. Clinically used opioid
drugs (e.g. morphine, fentanyl) mainly target the mu opioid receptor
(MOR), a G-protein coupled receptor (GPCR). The activation of MORs in
the peripheral (PNS) and central nervous system (CNS) produces strong
pain relief, but their activation in the CNS also results in serious
side effects including addiction and respiratory depression (Imam et
al., 2018). Recently, our group designed a new MOR agonist,
(\selectlanguage{ngerman}±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide
(NFEPP), with a lower acid dissociation constant (pK\textsubscript{a})
(Spahn et al., 2017; Rosas et al., 2019; Le\selectlanguage{polish}š\selectlanguage{english}nik et al., 2020; Ray et
al., 2020). NFEPP selectively activated peripheral MORs in inflamed
tissues at low pH values (common in most painful injuries) whereas a
conventional opioid (fentanyl) was equally active at both low and normal
pH values (Spahn et al., 2017; Rodriguez-Gaztelumendi et al., 2018;
Stein, 2018; Del Vecchio et al., 2019; Jim\selectlanguage{ngerman}énez-Vargas et al., 2022).
Targeting opioid receptors in the PNS at the site of injury avoids CNS
side effects, while still contributing to strong analgesia, as
previously demonstrated in preclinical and clinical studies: (i) locally
applied opioids relieve severe clinical pain and a large proportion of
the analgesic effects of systemically administered opioids is mediated
by opioid receptors in the PNS (Jagla et al., 2014; Machelska and Celik,
2018; Martínez and Abalo, 2020); (ii) input from peripheral sensory
neurons is essential in numerous pain syndromes (Berta et al., 2017;
Sexton et al., 2018); (iii) upon injury, MOR expression is upregulated
in peripheral sensory neurons and the perineurial barrier is disrupted,
eventually increasing accessibility of the drugs' target (reviewed in
Machelska and Celik, 2018; Jeske, 2019).

So far, the mechanisms underlying NFEPP-induced antinociception were not
examined in sufficient detail. At the level of sensory neurons, voltage
dependent Ca\textsuperscript{2+} channels (VDCC) play a major role in
the generation and inhibition of pain. These channels regulate the
excitation of neurons by allowing Ca\textsuperscript{2+} influx into the
neuron (Lu and Ikeda, 2016; Weiss and Zamponi, 2021). Following the
activation of MOR and dissociation of the heterotrimeric G-protein
complex, G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits block VDCC function, leading to
decreased excitability of the sensory neuron (Proft and Weiss, 2015; Lu
and Ikeda, 2016). Finally, MOR is phosphorylated and internalized
(Machelska and Celik, 2018; Jeske, 2019; Mann et al., 2015).

In this study, we investigated the effects of NFEPP at acidic and
physiological pH in comparison to the conventional agonist fentanyl on
G-protein activation, on the activity of endogenous VDCCs and on MOR
phosphorylation.

\textbf{Methods}

\emph{Animals}

Animal experiments were approved by the State animal care committee
(Landesamt f\selectlanguage{ngerman}ür Gesundheit und Soziales, Berlin, Germany) and followed
the ARRIVE guidelines (Kilkenny et al., 2010). Male Wistar rats (200-300
g; Janvier, Le Genest-Saint-Isle, France) were housed in groups of 1-2
per cage in a 12 h light/dark cycle, lined with ground corncob bedding
and with free access to standard laboratory rodent chow and tap water.
Room temperature was 22 ± 0.5\selectlanguage{english}*C and humidity was 60--65\%. We
investigated males only because the current evidence on gender- or
sex-differences in opioid analgesia is not strong enough to warrant
gender- or sex-specific therapeutic interventions (Bruehl et al., 2013;
Fullerton et al., 2018).

\emph{Chemicals and Drugs}

NFEPP was synthesized and provided by ASCA GmbH (Berlin, Germany) as
described in (Spahn, Del Vecchio et al. 2017). Fentanyl citrate (F3886)
and naloxone hydrochloride (NLX; N7758), were purchased from
Sigma-Aldrich (Taufkirchen, Germany). Pertussis toxin (PTX) and gallein
were purchased from Tocris (Wiesbaden-Nordenstadt, Germany).
Radioactively labeled GTP\selectlanguage{greek}γ\selectlanguage{english}S kits were purchased from Perkin Elmer
(Freiburg, Germany).

Chemicals used for membrane preparation and
{[}\textsuperscript{35}S{]}-guanosine-5'-O-(3-thio)-triphosphate
({[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S) binding: Trizma\selectlanguage{ngerman}® Preset Crystals
(Sigma-Aldrich, Taufkirchen, Germany), bovine serum albumin (BSA),
dithiotreitol (DTT), GDP, {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S, GTP\selectlanguage{greek}γ\selectlanguage{english}S,
optiphase HISAFE 3 (Perkin Elmer, Waltham, USA); HEM G-protein buffer: 8
mM HEPES, 8 mM EPPS, 8 mM MES, 100 mM NaCl, 0.2 mM EGTA, 5 mM
MgCl\textsubscript{2} ; all from Sigma-Aldrich (Taufkirchen, Germany).

Chemicals used for dissociation and culture of sensory neurons:
Dulbecco's Modified Eagles Medium (DMEM)/HAM's F-12 medium (Biochrom
F4815, Berlin, Germany), Penicillin (10,000 U), Streptomycin (10 mg/ml),
1.25\% Collagenase (Sigma-Aldrich, Taufkirchen Germany), 2.5\% Trypsin
(Sigma-Aldrich, Taufkirchen, Germany), acridine orange/propidium iodide
(Logos, Villeneuve, France).

Chemicals used for patch clamp experiments: Extracellular buffer (ECS)
(10 mM CaCl\textsubscript{2}~\selectlanguage{ngerman}· 6H\textsubscript{2}O, 130 mM
TEA-Cl\textsubscript{2}, 5 mM HEPES, 25 mM d-glucose; adjusted to pH 7.4
or 6.5; all from Sigma-Aldrich, Taufkirchen, Germany). Intracellular
buffer (ICS) (105 mM CsCl, 2.5 mM MgCl\textsubscript{2}, 40 mM HEPES, 10
mM EGTA, 2 mM Mg-ATP, 0.5 mM GTP, 5 mM d-glucose; adjusted to pH 7.4 or
6.5; all from Sigma-Aldrich, Taufkirchen, Germany).

Chemicals used for calcium imaging: Fura 2\selectlanguage{english}-AM (Thermo-Fisher,
Hennigsdorf, Germany). Extracellular solution: 130 mM~NaCl, 3 KCl mM, 2
mM~CaCl\textsubscript{2}, 0.6 mM~MgCl\textsubscript{2}, 1 mM
NaHCO\textsubscript{3} 10 mM~HEPES, 5 mM~D\selectlanguage{english}-glucose (adjusted to pH 7.4
or 6.5). High K\textsuperscript{+} solution contained: 61~NaCl mM,
75~KCl mM, 2~CaCl\textsubscript{2} mM, 0.6~MgCl\textsubscript{2} mM, 1
NaHCO\textsubscript{3} mM\textsubscript{,} 10~Hepes mM, 5~D-glucose mM
(adjusted to pH 7.4 or 6.5); all from Sigma-Aldrich (Taufkirchen,
Germany).

Chemicals used for MOR phosphorylation experiments: The
phosphosite-specific MOR antibodies against pT370-MOR (7TM0319B),
pS375-MOR (7TM0319C), pT376-MOR (7TM0319D), and pT379-MOR (7TM0319E) as
well as the phosphorylation-independent antibodies against np-MOR
(7TM0319N) and rabbit polyclonal anti-HA antibodies (7TM000HA) were
provided by 7TM Antibodies (Jena, Germany). {[}D-Ala2, N-MePhe4,
Gly-ol{]}-enkephalin (DAMGO) acetate salt and dimethyl sulfoxide (DMSO)
were purchased from Sigma-Aldrich (Darmstadt, Germany). Fentanyl citrate
was obtained from Rotexmedica (Trittau, Germany).

\emph{Culture and Transfection of Human embryonic kidney (HEK) cells}

Human embryonic kidney (HEK293) cells (German Collection of
microorganisms and Cell Cultures, Braunschweig, Germany;
RRID:CVCL\_0045) were maintained in full medium containing DMEM media
supplemented with fetal bovine serum (Biochrom, Berlin, Germany),
penicillin (100 U/ml, Biochrom) and streptomycin (100 \selectlanguage{greek}µ\selectlanguage{english}g/ml, Biochrom)
with or without geneticin (G418, 100 \selectlanguage{greek}µ\selectlanguage{english}g/ml, Biochrom), in 5\% CO2 at 37
\selectlanguage{ngerman}°C as described before\{Spahn, 2017, nontoxic\}. Cells were passaged 1:3
- 1:20 every second to third day from p8 and p28 depending on
confluence.

For MOR transfection, wild-type HEK293 cells were plated on 30 mm
diameter plastic culture dishes coated with poly-L-lysine 24 h before
transfection. Confluent HEK293 cells (70--90\%) were transfected with 1
\selectlanguage{greek}µ\selectlanguage{english}g per 200 \selectlanguage{greek}µ\selectlanguage{english}l transfection mix of each plasmid containing the different
cDNAs using ``X-tremeGENE HP DNA Transfection Reagent'' (Roche;
Mannheim, Germany) following the supplier's recommendations. The plasmid
containing the cDNA encoding the FLAG-epitope-tagged rat MOR (oprm1, NM
013071.2) in pcDNA3.1 vector with geneticin resistance gene was
provided by Prof. Christian Z\selectlanguage{ngerman}öllner (University Hamburg, Germany). The
cells were later grown in T175 flasks to have enough membranes for
further experiment.

\emph{Membrane Preparation}

Membrane fractions were prepared from transfected HEK293 cells as
described previously (Zöllner, 2003). The cells were grown in 175 cm2
tissue culture flasks to approximately 90 \% confluence. Cells were then
washed with Tris buffer (50 mM, Trizma preset crystals, pH 7.4; Sigma
Aldrich, Darmstadt, Germany), harvested with a scraper, homogenized
using a mechanical disperser (Dispergierstation T8.10, IKA-Werke,
Staufen, Germany) at maximum speed for 10 s and centrifuged at 42K x g
for 20 min at 4oC (Avanti JXN-26 ultracentrifuge, Beckmann Coulter,
Krefeld, Germany). Cellular pellets including membranes with embedded
and anchored proteins were then resuspendend in Tris buffer for washing
to separate them from cytosolic components by homogenization and
centrifugation at the same settings. Supernatants were discarded and the
pellets were stored at -80 oC. On the day of usage, the pellets were
thawed on ice in Tris buffer and homogenized. Total protein
concentrations were determined according to the Bradford method (Bio-Rad
Laboratories GmbH, München, Germany). (Bradford, 1976) and homogenates
were split according to the number of conditions tested in respective
assay buffers.

\emph{Isolation and culture of sensory neurons}

Dorsal root ganglia (DRGs) were harvested from naïve male Wistar rats
following their sacrifice by an overdose of isoflurane (5\%) (AbbVie,
Wiesbaden, Germany). Immediately thereafter, the thoracic and lumbar
spinal regions were exposed and all DRGs were collected in a working
medium (DMEM/HAM's F-12 without penicillin/streptomycin). 1.25\%
collagenase was added just before incubation. Following their incubation
at 37°C for 60 min, DRGs were washed three times with PBS and incubated
in the working medium digestive solution with trypsin for 10 min at
37°C. Thereafter, the tissue was further triturated using plastic
pipette tips and filtered through a 40~\selectlanguage{greek}µ\selectlanguage{english}l filter. The filtrate was
centrifuged and the pellet was resuspended in 1 ml culture medium
(DMEM/HAM's F12 supplemented with 1\% penicillin/streptomycin and 10\%
horse serum). Neurons were then seeded onto poly-L-lysine coated plastic
culture dishes (35 mm) and placed in an incubator (5\%
CO\textsubscript{2}~at 37\selectlanguage{ngerman}°C). One hour later, 2 ml of culture medium
were added to the cell culture and neurons were incubated for 24--48 h
until patch clamp recordings or calcium imaging, as previously described
(Nockemann et al., 2013; Dembla et al., 2017).

\selectlanguage{greek}\emph{{[}\textsuperscript{35}S{]}-GTPγS binding experiments}\selectlanguage{english}

{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S experiments were performed on crude
membrane fractions of MOR-expressing HEK293 cells to determine
mu-agonist-induced G-protein activation (as reflected by the exchange
rate of GDP for GTP) at different pH values (6.5-7.4). GTP was replaced
by a high concentration of {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S in the
assay solvent, and the accumulation of
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S-bound G proteins in the membrane was
measured. To this end, membranes were homogenized and dissolved in HEM
G-protein buffer at pH 7.4 or 6.5, including freshly added 0.1 \% (w/v)
BSA and 1 mM dithiotreitol (DTT). In analogy to (Ludwig et al., 2003),
50 \selectlanguage{greek}µ\selectlanguage{english}g of membrane fractions in duplicates were incubated with GDP (30
\selectlanguage{greek}µ\selectlanguage{english}M) and {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S (0.05 nM) for 90 min at
30\textsuperscript{o}C to determine dose-response curves and
EC\textsubscript{50} values. Unspecific
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding in the presence of
non-radioactive GTP\selectlanguage{greek}γ\selectlanguage{english}S (10 \selectlanguage{greek}µ\selectlanguage{english}M) was subtracted to yield specific binding.
Bound and free ligands were separated by rapid filtration under vacuum
through Whatman GF/B glass fibre filters soaked in water followed by 6
washes with Tris Buffer. Bound radioactivity was determined by liquid
scintillation spectrophotometry for \textsuperscript{35}S after
overnight extraction of the filters in scintillation
fluid\emph{optiphase HISAFE3} . Concentrations of radioactive compound
were calculated based on the half-life of \textsuperscript{35}S (87.4
days). Experiments were randomized to compensate for position effects in
the filter apparatus or unequal sample processing times. Data processing
and analysis were blinded for pH values (6.5 or 7.4) with the help of a
colleague.

\emph{Patch clamp experiments}

Patch clamp experiments were performed on rat sensory neurons (DRGs)
24--48 h after their dissection, following a modified protocol from
(Walwyn et al., 2007). First, cell viability was evaluated by an
automated cell counter (Luna, Villeneuve, France) using acridine
orange/propidium iodide dyes. During patch clamp recordings, cells were
superfused with ECS buffer and visualized using a Zeiss Axiovert 200
inverse microscope (Zeiss, Jena, Germany). Patch clamp pipettes
(resistance 3.5--8 M\selectlanguage{greek}Ω\selectlanguage{english}) were produced using the Sutter P-97 puller
(Sutter Instruments, Novato, CA, USA) from Borosilicate glass
capillaries (Harvard Bioscience, MA, USA). Before the experiment, the
glass pipettes were filled with an ICS buffer. Currents were amplified
by an EPC-10 patch amplifier and recorded using Pulse software (HEKA,
Lambrecht, Germany). Using a pressurized application system (Perfusion
Pressure Kit VPP-6; Warner Instruments, Hamden, CT, USA), ECS buffer was
added in a steady flow of 800--1,000~\selectlanguage{greek}µ\selectlanguage{english}l/min. After reaching the
``giga-seal'' at -60 mV, the membrane patch was breached to achieve the
whole-cell configuration. The currents were initially recorded at a
holding potential of -80 mV in ECS buffer in the absence of test
compounds. Subsequently, the cells were depolarized to +10 mV (for 100
ms) for eight times following 20 s intervals. During the first five
cycles, only ECS buffer continued to flow. On the sixth cycle, an opioid
agonist (fentanyl, NFEPP) was added to the solution. During the last two
cycles, the opioid antagonist naloxone was used to block/confirm opioid
receptor-mediated effects. Neurons were selected for analysis after the
8\textsuperscript{th} cycle. For experiments using PTX or gallein, the
blockers were added to culture media overnight before the day of the
experiment (Lu and Ikeda 2016). Opioid ligands were administered via a
perfusion valve system (VC-6; Warner Instruments). All recordings were
performed at room temperature.

\emph{Calcium Imaging}

Intracellular calcium imaging experiments were performed as previously
described (Dembla, Behrendt et al. 2017). Briefly, cultured rat sensory
neurons (DRGs) were incubated at room temperature with 5~\selectlanguage{greek}μ\selectlanguage{english}M Fura 2-AM
(1 mM stock in DMSO + 0.02\% pluronic F-127) for 30~min in growth
medium. Following the application of Fura 2-AM, cover-slips were
transferred to a closed recording chamber (Warner Instruments) and
continuously perfused with the extracellular solution for 5 min.
Fluorescence was monitored every 2~seconds and images were taken with
the DS-Qi1 (Nikon, Japan) at 510~nm wavelength. Alternate excitation at
340 and 380~nm wavelengths were filmed using a Polychrome V
monochromator, mounted on a Nikon TE2000 inverted microscope (with 20x
Sfluor objective; N.A. 0.5). After a baseline period of ca. 2 minutes,
ligands (as shown in figures) were superfused onto the cells, using a
gravity-driven perfusion system, which was controlled by ALA VC3-8 valve
control system. Fluorescence intensities of ratio images (340/380~nm) on
regions of interest (ROI) (including the whole cell) were quantified by
NIKON NIS-Elements software after subtraction of background. To
distinguish between neuronal and non-neuronal cells, a high potassium
(high K\textsuperscript{+}) solution was used to depolarize the cells at
the end of the superfusion protocol.

\emph{MOR phosphorylation experiments:}

HEK293 cells stably expressing MOR were cultivated at 37\selectlanguage{ngerman}°C, 5\%
CO\textsubscript{2} in Dulbecco's Modified Eagle's Medium (DMEM),
supplemented with 10\% (v/v) fetal bovine serum (FBS), 1\% (v/v)
penicillin/streptomycin and 1\% (v/v) glutamine (Capricorn Scientific,
Ebsdorfergrund, Germany). Cells were seeded into poly-L-lysine-coated 60
mm culture dishes and grown until a confluency of 95\% was reached. MOR
agonists were diluted in PBS buffer of different pH values ranging from
6.0 to 8.0 and were carefully added onto the cell layer for 10 min at
room temperature. After the agonist solution was removed, cells were
washed with Dulbecco's PBS, with Ca\textsuperscript{2+} and
Mg\textsuperscript{2+} (Capricorn Scientific, Ebsdorfergrund, Germany)
and lysed with a detergent buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.4;
5 mM EDTA; 1\% Igepal CA-360; 0.5\% deoxycholic acid; and 0.1\% SDS)
containing phosphatase and protease inhibitors (PhosSTOP and cOmplete
mini tablets; Roche, Mannheim, Germany). Cell lysates were then
centrifuged at 21,000 x g and 4°C for 30 min to obtain the supernatant
which was mixed with HA epitope tag antibody agarose conjugates (Thermo
Fisher Scientific, Waltham, USA). This mixture was incubated for 2 h at
4°C. After washing the agarose beads with detergent buffer for three
times, SDS sample buffer (100 mM DTT, 62.5 mM Tris-HCl, 20\% glycerol,
2\% SDS, and 0.005\% bromophenol blue) was added and incubated at 43°C
for 25 min to release the precipitated receptor from the conjugates.

For electrophoresis, the supernatant was loaded onto an 8\%
polyacrylamide gel. Subsequently, the proteins were transferred to a
PVDF membrane using a semi-dry blotting system (Trans-Blot® Turbo
Transfer System, Bio-Rad Laboratories GmbH, Feldkirchen, Germany).
Unspecific binding sites were blocked by incubating the membranes in 5\%
BSA/TBS-T buffer solution for 2 h at room temperature, before the
primary antibody diluted in 5\% BSA/TBS-T was added overnight at 4°C. On
the next day, the membrane was washed three times with TBS-T and
incubated with the secondary antibody diluted in 5\% BSA/TBS-T for 2 h
at room temperature. After washing the membrane for another three times,
it was placed into SuperSignal West Dura Extended Duration Substrate
(Thermo Fisher Scientific, Waltham, USA) to induce a chemiluminescent
reaction. The signals were detected using Fusion Fx7 (Peqlab, Erlangen,
Germany). In order to reprobe the membrane, bound antibodies were
removed by Tris (2-carboxyethyl) phosphin containing stripping buffer.

Western Blot signals were quantified using the software ImageJ. The
intensity of each band was calculated by subtracting the background
signal from the mean intensity of the selected band area. To determine
the magnitude of protein phosphorylation, the ratio of the signal of the
phosphorylated protein to the one exhibited by the loading control
(total protein concentration) was calculated.

\emph{Data analysis and statistics}

Sample sizes were calculated using the G\selectlanguage{english}\textsuperscript{*}Power 3.1.2
program with~\selectlanguage{greek}α\selectlanguage{english}~\textless{} 0.05 and a power of 80\% based on a defined
effect size derived from pilot experiments. Experiments were randomized
to compensate for the position effects on plates or filter apparatus and
unequal sample processing time. All experiments were performed by an
investigator blinded to the sample assignments. The codes were broken
after the completion of the experiments. Statistical analyses were
performed using GraphPad Prism software (version 5 and 9 for Windows;
GraphPad Software Inc., San Diego, USA). Grubbs' test was performed to
identify potential outliers. No samples were excluded from the analysis.
All data were assessed for normal distribution and equal variances by
D'Agostino and Pearson or Kolmogorov--Smirnov tests. Analysis of
concentration-response relationships was performed with simple linear
regression using Prism 9 (where y = {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S
bound and x = {[}NFEPP{]} or {[}fentanyl{]}).
Ca\textsuperscript{2+}imaging time series of single cells were extracted
from stacks of ratio images as averages over the entire cell area in
excel-sheets, which were then plotted as time vs. ratio 340/380 graphs
using Prism 5. For quantitative analyses, the first 10 data points
(corresponding to the first 20 s of the experiment) were averaged to
form the baseline which was then subtracted from all high potassium
(high K\textsuperscript{+}) responding cells to obtain \selectlanguage{greek}Δ \selectlanguage{english}ratio 340/380
values. During each recording, we typically were able to record at least
from 10 cells, meaning that each trace in Ca\textsuperscript{2+} imaging
recordings represents the mean of more than 20 cells. The precise number
of cells is stated in the figures or in the figure legends. Data are
represented as means \selectlanguage{ngerman}± standard error of the mean (SEM). Detailed
statistical analyses are presented in figure legends.

\textbf{Results}

Both NFEPP and fentanyl produced dose-dependent increases in
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding (Figure 1). The maximum
effects of NFEPP were significantly higher and the
EC\textsubscript{50}values were significantly lower at pH 6.5 when
compared to the physiological pH 7.4 (see Table 1; *P \textless{} 0.05;
pH 6.5 versus pH 7.4; t test, N=6 and 7 membranes). In contrast,
fentanyl showed similar potencies (EC\textsubscript{50}) and similar
maximum effects at both pH values (Figure 1, Table 1).

Table 1. EC\textsubscript{50} values in
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding experiments\selectlanguage{english}
\begin{longtable}[]{@{}lllll@{}}
\toprule
& NFEPP & NFEPP & Fentanyl & Fentanyl\tabularnewline
\midrule
\endhead
pH & 6.5 & 7.4 & 6.5 & 7.4\tabularnewline
EC\textsubscript{50} (mean) & 1,340e-007* & 7,943e-007 & 2,905e-008 &
4,709e-008\tabularnewline
standard error & 3,594e-008 & 2,644e-007 & 4,103e-009 &
1,081e-008\tabularnewline
\bottomrule
\end{longtable}

In the patch clamp experiments, the DRG cells initially rested in the
whole-cell voltage clamp mode at a constant -80 mV holding potential.
When depolarized to +10 mV (100 ms), VDCCs opened to allow
Ca\textsuperscript{2+} ions to flow into the cell inducing an inward
current. NFEPP treatment dose-dependently decreased VDCC currents at
both pH values (6.5 and 7.4) but to different degrees. At pH 6.5, the
most effective NFEPP dose (100 \selectlanguage{greek}µ\selectlanguage{english}M) attenuated
Ca\textsuperscript{2+}currents by 41.8 \%. This effect was significantly
stronger than the effect of NFEPP at pH 7.4 (28.8\%) (Figure 2a). In
contrast to NFEPP, the dose-response curves upon fentanyl treatment were
not different and the most effective fentanyl dose (100 \selectlanguage{greek}µ\selectlanguage{english}M) attenuated
+10 mV-induced Ca\textsuperscript{2+} currents to the same degree
(\textasciitilde{}39\%) at both pH 7.4 and pH 6.5 (Figure 2b). These
findings show that NFEPP is more effective at low pH, whereas the
conventional opioid agonist fentanyl is equally effective at both acidic
and physiological pH values. Naloxone dose-dependently recovered the +10
mV-induced VDCC currents during NFEPP treatment. The effects of NFEPP
were completely reversed by the highest naloxone dose (100 \selectlanguage{greek}µ\selectlanguage{english}M) at both
pH values (Figure 3a). This dose of naloxone also abolished fentanyl
effects at both pH values (Figure 3b).

To investigate the role of G-protein subunits in detail, we initially
used PTX which prevents the replacement of GDP by GTP at the
G\selectlanguage{greek}\textsubscript{αi}\selectlanguage{english} subunits and precludes subsequent
G\selectlanguage{greek}\textsubscript{αβγ} \selectlanguage{english}dissociation (Lu and Ikeda, 2016). PTX blocked
NFEPP's effect in a dose-dependent manner (Figure 4a), fully reversing
the +10 mV-induced VDCC currents at the highest PTX dose (100 ng/ml).
The effect of fentanyl was also blocked in a dose-dependent manner after
PTX incubation (Figure 4b). Because PTX does not allow to distinguish
between the involvement of G\selectlanguage{greek}\textsubscript{α} \selectlanguage{english}versus
G\selectlanguage{greek}\textsubscript{βγ}\selectlanguage{english}subunits (Lu and Ikeda, 2016), we sought to
selectively block G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits by gallein (Lu and Ikeda,
2016). Gallein dose-dependently decreased NFEPP-induced effects and
fully recovered +10 mV-induced Ca\textsuperscript{2+} currents at the
highest dose at both pH values (Figure 5a). Fentanyl's effects were also
decreased and fully blocked by the most effective dose of gallein at
both pH values (Figure 5b). Together, these results indicate that the
effects of both opioid agonists on VDCCs are mediated via activation and
subsequent dissociation of heterotrimeric G-protein complexes at both pH
values.

We then performed Ca\textsuperscript{2+} imaging experiments to
corroborate our electrophysiological results. Under our recording
conditions, both NFEPP and fentanyl decreased the Fura [?] ratio 340/380,
i.e. the Ca\textsuperscript{2+} signals were reduced. Naloxone (100 \selectlanguage{greek}µ\selectlanguage{english}M)
completely reversed the effect of fentanyl (Figure 6b, c). However, in
NFEPP-treated neurons, the Ca\textsuperscript{2+} signal was
significantly lower at pH 6.5 when compared to pH 7.4 (Figure 6a, c). We
also examined whether the extracellular pH by itself had a role in the
decrease of Ca\textsuperscript{2+} signals. During short (60 s) (Figure
7a) or long (20 min) (Figure 7b) term pH changes, there was no
difference in the Fura [?] ratio 340/380 at either pH 6, 6.5, 7 or 7.4.
This indicates that the pH difference by itself does not influence
Ca\textsuperscript{2+} signals in the cells and that NFEPP and fentanyl
were solely responsible for the decrease. The control experiments using
high potassium indicated that the signals indeed derived from the influx
of extracellular Ca\textsuperscript{2+} into neurons (Figure 7).

Finally, we examined the effects of NFEPP on MOR phosphorylation. MOR
expressing HEK293 cells were treated with either NFEPP or fentanyl at pH
6.0 - 8 (Figure 8a). The strongest NFEPP-mediated phosphorylation signal
was detected at pH 6.0. This signal diminished with increasing pH values
(Figure 8b). In contrast, fentanyl-induced phosphorylation was similar
at all pH values (Figure 8c).

\textbf{Discussion}

Developing new analgesics with less adverse effects is challenging
(reviewed in Yekkirala et al., 2017; Machelska and Celik, 2018).
Activation of peripheral opioid receptors by site-restricted opioid
administration was shown to produce strong analgesia in both acute and
chronic pain models as well as in patients with arthritis and
postoperative pain (reviewed in Machelska and Celik, 2018; Mart\selectlanguage{ngerman}ínez and
Abalo, 2020). Additionally, animal studies using conditional opioid
receptor knockout in primary afferent Nav1.8 nociceptors clearly
demonstrated the essential role of peripheral opioid receptors
(Gaveriaux-Ruff et al., 2011; Weibel et al., 2013). Accordingly, the
selective activation of peripheral MOR on DRG neurons innervating
injured (inflamed) tissues appears to be a promising strategy to inhibit
pain while avoiding central side effects. NFEPP is an opioid with a
lower dissociation constant (pK\textsubscript{a}) that was shown to bind
and activate MORs more effectively at low pH values. Therefore, even
though NFEPP is presumably distributed throughout the body (including
the CNS) after systemic (intravenous) application, it only activates
MORs in peripheral inflamed tissues where the pH is more acidic
(reviewed in Stein, 2018).

In this study, we expanded our understanding of NFEPP's antinociceptive
effects by investigating MOR signaling in transfected HEK cells and
sensory neurons. Opioid receptors exert their intracellular actions
through G-proteins, which interact with various cellular effector
systems. First, we found that NFEPP produced more efficient G-protein
activation at low pH, while fentanyl induced comparable G-protein
activation at both physiological and low pH values, as shown by the
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding experiments. Opioid receptor
activation can result in blockade of membrane VDCCs, which are essential
for the excitation of sensory neurons (reviewed in Machelska and Celik,
2018; Weiss and Zamponi, 2021). To verify that NFEPP acts through the
same mechanisms, we examined NFEPP-induced inhibition of inward calcium
currents at both physiological (7.4) and acidic (6.5) pH values. Our
experiments revealed that the electrical excitation of depolarized
sensory neurons was more efficiently reduced following NFEPP treatment
at acidic pH (in contrast to pH 7.4). However, fentanyl, a conventional
opioid, attenuated Ca\textsuperscript{2+} currents to similar degrees at
both pH values. By using naloxone, we confirmed that the effects of
NFEPP and fentanyl were mediated by opioid receptors. Together, these
findings support our previous studies where fentanyl, possibly due to
its higher pK\textsubscript{a} value (8.44) and comparable protonation,
was equally effective at both pH values (Spahn et al., 2017, 2018). In
contrast, NFEPP (pK\textsubscript{a} = 6.82) showed increased G-protein
activation and VDCC inhibition at acidic conditions, where an increase
in protonation due to lower pH improves NFEPP-MOR interaction (Spahn et
al. 2017). To examine whether the dissociation of G-protein subunits
differs between pH values following NFEPP or fentanyl treatment, we
blocked the GDP-GTP exchange at G\selectlanguage{greek}\textsubscript{αi/o}\selectlanguage{english} subunits using
PTX, and we inhibited G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits using gallein in our
patch clamp analysis. G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits were previously shown
to directly deactivate VDCCs following MOR activation (reviewed in Proft
and Weiss, 2015). Here, we have shown that NFEPP-induced inhibition of
inward calcium currents were dependent on G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits.
Consistent with its indistinguishable effect on G\selectlanguage{greek}\textsubscript{α}\selectlanguage{english}versus
G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits (Lu and Ikeda, 2016), PTX also blocked
Ca\textsuperscript{2+} currents, indicating that, by blocking GDP-GTP
exchange, consequent G\selectlanguage{greek}\textsubscript{αiβγ} \selectlanguage{english}dissociation is also
impaired. Apparently, the extracellular pH changes did not significantly
affect intracellular activation of G-proteins since the dose ranges of
inhibitors (PTX, gallein) were similar for both opioid agonists and both
pH values.

Further validation of our electrophysiological data was performed using
Ca\textsuperscript{2+} imaging experiments.
Ca\textsuperscript{2+}signals were only reduced in NFEPP-treated DRG
neurons at pH 6.5. Fentanyl, however, significantly reduced
Ca\textsuperscript{2+} signals at both pH values, indicating no
difference of its efficacy between inflamed and non-inflamed
environments. Control experiments using naloxone and high potassium
confirmed that the signals were modulated by opioid receptors and
derived from influx of Ca\textsuperscript{2+} into neurons, similar to
previous studies (Hochstrate et al., 1995). To examine direct effects of
low pH (without drug treatment), we investigated VDCC activity at pH
6.5. Both calcium imaging and patch clamp experiments (data not shown)
demonstrated that pH alone did not affect Ca\textsuperscript{2+}
signals/ currents at low pH values.

Additionally, the ability of NFEPP to phosphorylate MOR under different
pH conditions was examined. Our western blot analysis showed that NFEPP
induced the strongest MOR phosphorylation at pH 6.0. The signal was most
pronounced at MOR residues T370 and S375 and decreased with increasing
pH levels. In comparison, fentanyl caused similar phosphorylation
signals of MOR regardless of increasing pH values. Notably, the
antibodies used in our experiments only detect intracellular
phosphorylation sites at the C-terminal of MOR (Mann et al., 2015).
Assuming that nonspecific pH effects would produce similar
phosphorylation signals for all pH values and both agonists, our
findings strongly suggest that the pH-dependent signals produced by
NFEPP were due to extracellular pH changes. Importantly, fentanyl
apparently stimulates MOR independently from extracellular pH, while
NFEPP activates MOR preferentially at low pH values.

In summary, our results show that during both NFEPP and fentanyl
treatment, VDCCs are directly modulated by G\selectlanguage{greek}\textsubscript{βγ} \selectlanguage{english}subunits
dissociated from G\selectlanguage{greek}\textsubscript{αi/o}\selectlanguage{english}. However, MOR signaling induced
by NFEPP is more effective at low pH. Given the known decrease of
extracellular pH in injured tissues (Stein, 2018) and the co-expression
of MOR and VDCC in sensory neurons (Proft and Weiss, 2015), our study
uncovers the neural mechanisms underlying the antinociceptive effects of
NFEPP in injured tissues, and strongly suggests that the enhanced
efficacy of NFEPP in acidic milieu is dependent on extra- rather than
intracellular effects on MOR function.

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\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 1}

Dose-dependent {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding induced by
NFEPP (a) and fentanyl (b) at pH 6.5 and 7.4.
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding is expressed as percent
increase in {[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding relative to
binding in unstimulated samples (n=6 membrane preparations). Data points
represent means \selectlanguage{ngerman}± standard error of the mean (SEM) of specific
{[}\textsuperscript{35}S{]}-GTP\selectlanguage{greek}γ\selectlanguage{english}S binding in \% of baseline. P
\textless{} 0.05 for Log EC\textsubscript{50} of NFEPP at pH 6.5 vs 7.4,
2-tailed \emph{t} test.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

\textbf{Figure 2}

Dose-dependent inhibition of +10mV-induced calcium currents in isolated
rat sensory neurons (DRG cells) by NFEPP (a) or fentanyl (b) at pH 6.5
and pH 7.4 measured by patch clamp (N=14-22 cells) (left). Maximum
inhibition by the highest agonist dose (Log \selectlanguage{greek}µ\selectlanguage{english}M) (right). *P \textless{}
0.05 NFEPP at pH 6.5 vs 7.4; 2-tailed \emph{t} test. Data are means \selectlanguage{ngerman}±
SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

\textbf{Figure 3}

Dose-dependent effects of naloxone on the maximum inhibition of
+10mV-induced calcium currents by NFEPP (a) or fentanyl (b) at pH 6.5
and pH 7.4 measured by patch clamp in isolated rat sensory neurons
(N=16-20 cells) (left). Effects of the highest naloxone doses (Log \selectlanguage{greek}µ\selectlanguage{english}M)
(right). ***P \textless{} 0.001 naloxone vs. control at pH 6.5 vs 7.4;
2-tailed \emph{t} test. Data are means \selectlanguage{ngerman}± SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4}

Dose-dependent effects of pertussis toxin (PTX) on the maximum
inhibition of +10mV-induced calcium currents by NFEPP (a) or fentanyl
(b) at pH 6.5 and pH 7.4 measured by patch clamp in isolated rat sensory
neurons (N=17-22 cells) (left). Effects of the highest PTX doses (Log
\selectlanguage{greek}µ\selectlanguage{english}M) (right). ***P \textless{} 0.001 PTX vs. control at pH 6.5 vs. 7.4;
2-tailed \emph{t} test. Data are means \selectlanguage{ngerman}± SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\textbf{Figure 5}

Dose-dependent effects of gallein on the maximum inhibition of
+10mV-induced calcium currents by NFEPP (a) or fentanyl (b) at pH 6.5
and pH 7.4 measured by patch clamp in isolated rat sensory neurons
(N=15-25 cells) (left). Effects of the highest gallein doses (Log \selectlanguage{greek}µ\selectlanguage{english}M)
(right). ***P \textless{} 0.001 gallein vs control at pH 6.5 vs 7.4;
2-tailed \emph{t} test. Data are means \selectlanguage{ngerman}± SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image6/image6}

\end{center}
\end{figure}

\textbf{Figure 6}

Inhibition of calcium signals by NFEPP at pH 6.5 in rat sensory neurons.
(a) Fura-2 imaging experiments with rat sensory neurons treated with 100
\selectlanguage{greek}µ\selectlanguage{english}M NFEPP (green trace, N=259 neurons, 9 recordings from 3 rats) and
non-treated (0 \selectlanguage{greek}µ\selectlanguage{english}M NFEPP) (black trace, N=256 neurons, 9 recordings from
3 rats) at pH 6.5 and pH 7. (b) Calcium responses in rat sensory neurons
treated with 100 \selectlanguage{greek}µ\selectlanguage{english}M fentanyl (red trace, N=445 neurons, 18 recordings
from 6 rats), treated with 100 \selectlanguage{greek}µ\selectlanguage{english}M fentanyl + 100 \selectlanguage{greek}µ\selectlanguage{english}M naloxone (blue
trace, N=454 neurons, 18 recordings from 6 rats) and non-treated
(vehicle control: DMSO) (black trace, N=463 neurons, 18 recordings from
6 rats) at pH 6.5 and pH 7. (c) Summary, delta ratio 340/360 from (a) \&
(b) at pH 6.5 and pH 7. *P \textless{} 0.05 NFEPP at pH 6.5 vs 7.4;
2-tailed \emph{t} test. Traces represent means \selectlanguage{ngerman}± SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image7/image7}

\end{center}
\end{figure}

\textbf{Figure 7}

Effects of inflammatory pH conditions on calcium signals in rat sensory
neurons. Fura-2 imaging experiments with rat sensory neurons treated
with (a) short (60s) and (b) long (20 min) application of extracellular
pH 7.4 (black trace, N=356 neurons), pH 7 (red trace, N=302 neurons), pH
6.5 (green trace, N=383 neurons) and pH 6 (blue trace, N=386 neurons).
Twelve recordings for each pH from 4 rats (1 rat per pH) are shown. The
right bar graphs represent delta ratio 340/380 for different pH values.
P \textgreater{} 0.05; One-way ANOVA on ranks. Traces represent means \selectlanguage{ngerman}±
SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image8/image8}

\end{center}
\end{figure}

\textbf{Figure 8}

Effects of different pH values on NFEPP- or fentanyl-induced MOR
phosphorylation in HEK293 cells. (a) MOR phosphorylation was analyzed
with antibodies specifically reacting with phosphorylated residues T370,
S375, T376 and T379. Controls were treated with 1 \selectlanguage{greek}μ\selectlanguage{english}M DMSO (negative
control) or 10 \selectlanguage{greek}μ\selectlanguage{english}M DAMGO (positive control). Total phosphorylation signal
recorded from residues (T370, S375, T376, T379) when stimulated with 1
\selectlanguage{greek}μ\selectlanguage{english}M NFEPP (b) or fentanyl (c) in a pH-dependent manner (N=16). **P
\textless{} 0.01; One-way ANOVA on ranks (specific sample pairs are not
analyzed). Data are means \selectlanguage{ngerman}± SEM.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image9/image9}

\end{center}
\end{figure}

\textbf{Graphical Abstract}

Abbreviations

CNS: central nervous system

DAMGO: {[}D-Ala2,N-Me-Phe4,Gly5-ol{]}-enkephalin

GPCR: G-protein coupled receptor

HEK MOR-WT: HEK293 cells stably transfected with mu opioid receptor wild
type

MOR: mu opioid receptor

NFEPP: (\selectlanguage{ngerman}±)-N-(3-fluoro-1-phenethylpiperidine-4-yl)-N-phenyl propionamide

NLX: naloxone

pK\textsubscript{a}: acid dissociation constant

PNS: peripheral nervous system

PTX: pertussis toxin

VDCC: voltage dependent calcium channel

M. Özgür Celik ORCID ID: 0000-0002-7706-4964

Viola Seitz ORCID ID: 0000-0002-8086-8090

Fatih Yergöz ORCID ID: 0000-0002-9709-8563

Sandeep Dembla ORCID ID: 0000-0003-2708-4390

Stefan Schulz ORCID ID: 0000-0002-5997-8885

Christoph Stein ORCID ID: 0000-0001-5240-6836

\selectlanguage{english}
\FloatBarrier
\end{document}

