Dopamine modulation of transient receptor potential vanilloid type 1 (TRPV1) receptor

in dorsal root ganglia neurons.

Saikat Chakraborty1, Mario Rebecchi1, Martin Kaczocha1 and Michelino Puopolo1*
1

Department of Anesthesiology, Stony Brook Medicine, Stony Brook, NY 11794

Running title: Dopamine modulation of TRPV1 channels

Key words: dorsal root ganglia neurons; dopamine; TRPV1 channels
*

Corresponding author:
Michelino Puopolo
Department of Anesthesiology
HSC L4 071
Stony Brook Medicine
Stony Brook, NY 11794
Competing Interest:
The authors declare no competing financial interests.
Author contributions:
Conception and design of the experiments: M.P, M.R., M.K.
Collection, analysis and interpretation of data: S.C., M.P, M.R., M.K.
Drafting the article: S.C., M.P, M.R., M.K.
All authors have approved the final version of the manuscript
Funding:
This work was supported by internal funds from the Department of Anesthesiology, Stony
Brook Medicine, Stony Brook, NY, to M.P.
Acknowledgements:
We thank Dr. Bruce Bean for helpful comments on the manuscript and William Galbavy for
technical assistance.

This is an Accepted Article that has been peer-reviewed and approved for publication in the The
Journal of Physiology, but has yet to undergo copy-editing and proof correction. Please cite this
article as an 'Accepted Article'; doi: 10.1113/JP271198.
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SUMMARY

Transient receptor potential vanilloid type 1 (TRPV1) receptors transduce noxious

thermal stimuli and are responsible for the thermal hyperalgesia associated with
inflammatory pain.

A large population of dorsal root ganglia (DRG) neurons, including the C low
threshold mechanoreceptors (C-LTMRs), express tyrosine hydroxylase, and likely
release dopamine.

We found that dopamine and SKF 81297 (an agonist at D1/D5 receptors), but not
quinpirole (an agonist at D2 receptors), down regulate the activity of TRPV1 channels
in DRG neurons.

The inhibitory effect of SKF 81297 on TRPV1 channels was strongly dependent on
external calcium and preferentially linked to calcium/calmodulin-dependent protein
kinase II (CaMKII).

We suggest that modulation of TRPV1 channels by dopamine in nociceptive neurons

may represent a way for dopamine to modulate incoming noxious stimuli.

ABSTRACT
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The transient receptor potential vanilloid type 1 (TRPV1) receptor plays a key role in the
modulation of nociceptor excitability. To address whether dopamine can modulate the
activity of TRPV1 channels in nociceptive neurons, the effects of dopamine and dopamine
receptor agonists were tested on the capsaicin-activated current recorded from acutely
dissociated small diameter (< 27 m) dorsal root ganglia (DRG) neurons. Dopamine or SKF
81297 (an agonist at D1/D5 receptors), caused inhibition of both inward and outward currents
by ~ 60% and ~ 48%, respectively. The effect of SKF 81297 was reversed by SCH 23390 (an
antagonist at D1/D5 receptors), confirming that it was mediated by activation of D1/D5
dopamine receptors. In contrast, quinpirole (an agonist at D2 receptors) had no significant
effect on the capsaicin-activated current. Inhibition of the capsaicin-activated current by SKF
81297 was mediated by G protein couples receptors (GPCRs), and highly dependent on
external calcium. The inhibitory effect of SKF 81297 on the capsaicin-activated current was
not affected when the protein kinase A (PKA) activity was blocked with H89, or when the
protein kinase C (PKC) activity was blocked with Bisindolylmaleimide II (BIM). In contrast,
when the calcium/calmodulin-dependent protein kinase II (CaMKII) was blocked with KN93, the inhibitory effect of SKF 81297 on the capsaicin-activated current was greatly reduced,
suggesting that activation of D1/D5 dopamine receptors may be preferentially linked to
CaMKII activity. We suggest that modulation of TRPV1 channels by dopamine in
nociceptive neurons may represent a way for dopamine to modulate incoming noxious
stimuli.

ABBREVIATIONS

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AADC,

aromatic

acid

decarboxylase;

DBH,

dopamine

-hydroxylase;

BIM,

Bisindolylmaleimide II; CaMKII, calcium/calmodulin-dependent protein kinase II; DOPAC,

dihydroxyphenylacetic acid; DRG, dorsal root ganglia; EPSC, excitatory post synaptic
current; GPCRs, G protein coupled receptors; HVA, homovanillic acid; IB4, isolectin B4;
PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C; TG, trigeminal
ganglia; TH, tyrosine hydroxylase; TRPV1, transient receptor potential vanilloid type 1;
TTX-R, tetrodotoxin-resistant.

INTRODUCTION
The transient receptor potential vanilloid type 1 (TRPV1) receptor is a polymodal
molecular integrator in the pain pathway preferentially expressed in A- and C-fiber
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nociceptors in dorsal root ganglia (DRG) and trigeminal ganglia (TG) (Szallasi et al., 1995;
Caterina et al., 1997; Cavanaugh et al., 2011). The TRPV1 channel is a non-selective cation
channel that can be activated by vanilloid compounds (i.e capsaicin and resineferatoxin),
endogenous lipids such as anandamide, protons (Ahern et al., 2005; Dhaka et al., 2009),
polyamines (Ahern et al., 2006), and noxious heat (Cesare & McNaughton, 1996; Caterina et
al., 1997; Caterina & Julius, 2001). Studies with TRPV1 knockout mice have provided strong
support for a major contribution of TRPV1 channels to thermal hyperalgesia associated with
inflammatory pain (Caterina et al., 2000; Davis et al., 2000).
A large population of DRG neurons, including the C low threshold
mechanoreceptors (C-LTMRs) specialized in detecting low-threshold mechanosensory
stimuli and those innervating pelvic organs (Price & Mudge, 1983; Philippe et al., 1993;
Brumovsky et al., 2006; Li et al., 2011; Brumovsky et al., 2012) express tyrosine
hydroxylase (TH), the rate limiting enzyme in the synthesis of catecholamines. The CLTMRs represent a molecularly distinct population of nonpeptidergic, small-diameter
sensory neurons which do not express TRPV1 channels (Li et al., 2011). They can be
activated by stretch or indentation of the skin or deflection of air follicles, and play a major
role in the affective component of touch and the mechanical hypersensitivity associated with
nerve injury (Seal et al., 2009; Olausson et al., 2010). Although it is not known which
catecholamine(s) are synthetized and/or released from TH(+) DRG neurons, dopamine and its
metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) have been
detected in DRG neurons and in dorsal spinal nerve roots, but not in satellite and Schwann
cells (Lackovic & Neff, 1980; Philippe et al., 1993; Weil-Fugazza et al., 1993). This supports
the possibility that TH(+) DRG neurons may release dopamine both in the dorsal root ganglia
and in the dorsal horn of the spinal cord. In addition to possible release of dopamine by
sensory neurons, dopamine is released in the dorsal horn of the spinal cord by supraspinal
descending dopaminergic projections (Skagerberg et al., 1982; Ridet et al., 1992; Holstege et
al., 1996; Qu et al., 2006; Benarroch, 2008; Koblinger et al., 2014).
A functional role for dopamine in the pain pathway is supported by several lines of
evidence: i) dopamine receptors are expressed in DRG neurons and spinal cord (Xie et al.,
1998; Zhu et al., 2007; Galbavy et al., 2013); ii) recordings from neurons in lamina II-III in
the dorsal horn spinal cord have suggested a post-synaptic effect of dopamine mediated by
D2 dopamine receptors (Tamae et al., 2005; Taniguchi et al., 2011); iii) behavioral studies in
intact animals support anti-nociceptive actions of dopamine during acute pain (Jensen &

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Yaksh, 1984; Barasi & Duggal, 1985), as well as in the setting of injury or inflammation
(Gao et al., 2001; Cobacho et al., 2010).(Wei et al., 2009); iv) a recent behavioral
pharmacologic study supports a role for dopamine in a model of hyperalgesic priming (Kim
et al., 2015).
In the dorsal root ganglia, dopamine has been shown to modulate the activity of
calcium channels (Marchetti et al., 1986; Formenti et al., 1993; Formenti et al., 1998) and the
intrinsic excitability of DRG neurons (Gallagher et al., 1980; Abramets & Samoilovich,
1991; Molokanova & Tamarova, 1995). Recently we have provided evidence that stimulation
of D1/D5 dopamine receptors results in reduction of tetrodotoxin-resistant (TTX-R) sodium
current in nociceptors (Galbavy et al., 2013). Here, using an in vitro preparation of acutely
dissociated DRG neurons, we show that dopamine down regulates TRPV1 channels
expressed in small diameter (< 27 m) DRG neurons. Pharmacological studies indicate that
the effect of dopamine is mediated by G protein coupled receptors (GPCRs), requires binding
of dopamine to D1/D5 dopamine receptors, and is mainly dependent on calcium influx and
activation of CaMKII.
METHODS
Animals
Sprague-Dawley rats (both male and female) postnatal days 14-28 were used in this study.
All procedures were performed in strict accordance with the recommendations in the Guide
for the Care and Use of Laboratory Animals of the National Institutes of Health and were
approved by Stony Brook University Institutional Animal Care and Use Committee. Rats
were anesthetized with isoflurane prior to decapitation.
Dissociated DRG neurons
Rats were deeply anesthetized with isoflurane and decapitated. Both thoracic and lumbar
segments of the spinal cord were removed and placed in a cold Ca2+, Mg2+-free (CMF)
Hanks solution containing (in mM): 137 NaCl, 5.3 KCl, 0.33 Na2HPO4, 0.44 KH2PO4, 5
HEPES, 5.5 Glucose, pH = 7.4 with NaOH. The bone surrounding the spinal cord was
removed and dorsal root ganglia were exposed and pulled out. After removing the roots,
ganglia were cut in half and incubated for 20 min at 34C in Ca2+, Mg2+-free Hanks solution
containing 20 U/ml Papain (Worthington Biochemical, Lakewood, NJ) and 5 mM DLcysteine. Ganglia were then treated for 20 min at 34C with 3 mg/ml collagenase (Type I,
Sigma-Aldrich, St. Louis, MO) and 4 mg/ml Dispase II (Boehringer Mannheim, Indianapolis,
IN) in in Ca2+, Mg2+-free Hanks solution. Ganglia were then washed with Leibovitzs L-15

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medium (Invitrogen, San Diego, CA) supplemented with 10% fetal calf serum and 5 mM
HEPES. Individual cells were dispersed by mechanical trituration using fire-polished Pasteur
pipettes with decreasing bore size and plated on glass coverslip treated with 30-50 g/ml
poly-D-lysine. Cells were incubated in the supplemented L-15 solution at 34 C (in 5% CO2)
for 2 hours, and then stored at room temperature in Neurobasal medium (Gibco) and used
over the next 4-6 hours. This protocol yields spherical cell bodies without neurites. The cells
can be lifted from the cover slip after establishing the whole-cell configuration in order to
facilitate rapid solution changes using flow pipes. Small DRG neurons (diameters < 27 m)
were chosen for recording. Small DRG neurons were initially selected by measuring the
diameter from images captured to a computer by a CCD camera Oly-150 (Olympus Imaging
America Inc., Center Valley, PA) using a video acquisition card (dP dPict Imaging, Inc.,
Indianapolis, IN). A more accurate measurement of cell diameter was obtained from
measurements of whole-cell capacitance assuming a membrane capacitance of 1 F/cm2 and
spherical shape. Cell capacitance was measured by integrating the average of 510 current
responses to a 5mV step from 80 mV filtered at 10 KHz and acquired at 50 KHz.
Cell classification
DRG neurons were rst tested for capsaicin sensitivity, and only those responding to
capsaicin (52% of those tested), corresponding to a subset of nociceptors (Cardenas et al.,
1995; Caterina et al., 1997; Petruska et al., 2000), were used for further experiments. Because
nociceptors are neurochemically and functionally distinct (Nagy & Hunt, 1982; Silverman &
Kruger, 1990; Stucky & Lewin, 1999; Dirajlal et al., 2003), a further classification was made
by testing the ability of TRPV1 positive DRG neurons to bind the isolectin B4 (IB4) FITC
conjugate and classified as peptidergic [IB4(-)] or non-peptidergic [IB4(+)]. Because there
were no obvious differences in the ability of dopamine or SKF 81297 (agonist at D1/D5
dopamine receptors) to inhibit the capsaicin-activated current between IB4(-) and IB4(+)
DRG neurons, data were pooled.
Electrophysiology
Whole-cell recordings were made with a Multiclamp 700B amplifier (Molecular Devices,
Sunnyvale, CA). Patch pipettes were pulled from borosilicate glass (A-M Systems, Sequim,
WA) using a Sutter P97 puller (Sutter Instrument, Novato, CA). The resistance of the patch
pipette was 1.8-2.5 M when filled with the standard internal CsCl-based solution. The
shank of the patch pipette was wrapped with Paralm to reduce pipette capacitance. In
whole-cell mode, the capacity current was reduced by using the amplifier circuitry. To reduce

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voltage errors, 70-80% of series resistance compensation was applied. After the whole cell
conguration was established, the cell was lifted up and placed in front of an array of quartz
ber ow pipes (320 m internal diameter) glued on an aluminum rod and containing the test
solutions. Solutions were changed in ~1 s by moving the cell from one pipe to another. All
experiments were done at room temperature (22 1 C), with the exception of those in
Figure 5 that were carried out at physiological temperature (35 1 C). Solutions were heated
with a temperature controller (Warner TC-344B, Warner Instruments, Hamden, CT).
Solutions
In voltage clamp experiments a Cs-based internal solution was used to block outward
currents through potassium channels. This solution contained (in mM): 125 CsCl, 10 NaCl, 2
MgCl2, 10 EGTA, 10 HEPES, 14 Tris-creatine PO4, 4 Mg-ATP, and 0.3 Na-GTP, pH= 7.2
with CsOH.
The standard external solution was a modied Tyrodes solution containing (in mM): 151
NaCl, 2 CaCl2, 10 HEPES, 13 glucose, pH 7.4 with NaOH.
Voltage clamp protocols
TRPV1 current was determined as the capsaicin-activated current by subtracting currents
before and after application of 1 M capsaicin. The current-voltage relationship for TRPV1
current was determined using 2 mV/ms voltage ramps. Ramps were delivered in the same
sweep from -100 to +100 mV and then from +100 to -100 mV (Fig. 1A, inset), with the
down ramp preceded by 50-200 ms at +100 mV. With this procedure, currents carried by
voltage-dependent sodium and calcium channels were largely inactivated before the down
ramp, which allowed more precise determination of TRPV1 currents by eliminating
overlapping sodium and calcium channel currents (Puopolo et al., 2013). Current-voltage
relationships for TRPV1 channels were therefore determined by using the down ramp.
TRPV1 channels showed some degree of desensitization upon sustained or repeated
application of the agonist capsaicin (Caterina et al., 1997). Usually, the desensitization of
TRPV1 channels reached its maximum during the first application of capsaicin, with small
additional desensitization during the second and third applications (Fig.1D). Thus, in order to
allow reliable comparison between capsaicin-activated currents before and after drug
treatment, the effect of drugs on the capsaicin-activated currents was measured during the
third application of capsaicin. Capsaicin was applied three times for 40 s with 1 min interval.
The typical protocol is illustrated in Figure 1D.
Data acquisition and analysis

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Currents and voltages were controlled and sampled using a Digidata 1440A interface and
pCLAMP 10.3 software (Molecular Devices, Sunnyvale, CA). Current or voltage signals
were ltered at 10 kHz (3 dB, 4-pole Bessel) and digitized at 50 kHz. Analysis was
performed using Clampfit 10.3 and IGOR Pro (version 6.2; WaveMetrics, Lake Oswego, OR)
using DataAccess (Bruxton, Seattle, WA) to import pCLAMP les into IGOR.
Statistics
Statistical differences between data sets were analyzed using Students t-test or one-way
ANOVA followed by Dunnetts post hoc comparison. Differences were considered
significant at *p < 0.05 or **p < 0.01. Data are reported as mean SD.

RESULTS:
Dopamine inhibition of TRPV1 current
Ionic current through TRPV1 channels were determined by challenging small DRG
neurons with 1 M capsaicin, and by subtracting currents before and after application of
capsaicin (Fig.1A). The capsaicin activated-current showed the typical outward rectification
and reversal potential close to 0 mV (1.7 2.4 mV, n=39), as expected for a non-selective
cation channel (Caterina et al., 1997). When dopamine was applied on top of capsaicin to a
small DRG neuron sensitive to capsaicin, there was a substantial inhibition of both outward
and inward currents, indicating a strong modulation of TRPV1 channels by dopamine
(Fig.1B). In collected results, 20 M dopamine inhibited the inward current (measured at
100 mV) by 56 8 % (n=10) and the outward current (measured at + 100 mV) by 44 9 %
(Fig.1C). As previously reported (Caterina et al., 1997), with 2 mM Ca2+ in the external
solution, there was some desensitization of TRPV1 channels upon sustained application of
capsaicin (usually about 40 sec in our experimental conditions). The desensitization of
TRPV1 channels induced by capsaicin was reflected in the incomplete recovery of the
capsaicin-activated current upon washing out dopamine (Fig.1B), and reduced size during the
second and third application of capsaicin (Fig.1D). The degree of desensitization usually
reached its maximum during the first application of capsaicin, with little additional change
during the second and third applications. On average, the capsaicin-activated current during
the second capsaicin application was 70 19 % of that during the first application, and the
current during a third application was 62 20 % of that during the first application (n=14).
Though smaller, capsaicin-activated current was typically maintained without decay during
the second and third applications of capsaicin, allowing a clearer assay of the effect of

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dopamine without the complication of concurrent desensitization. When neurons were

challenged by dopamine during the third application of capsaicin, there was still a robust
inhibition of the capsaicin-activated current induced by dopamine, but also a complete
recovery of the effect upon washing out dopamine (Figs.1D, 1E). Thus, to allow the most
accurate measurement of the effect of dopamine and other treatments on TRPV1 current
without the confounding factor of concurrent desensitization, test compounds were applied
during the third application of capsaicin. In collected results (Fig.1F), 20 M dopamine
applied during the third application of capsaicin inhibited the capsaicin-activated inward
current by 60 18 % (n=9).
The next step was to identify which dopamine receptors mediate the effect of
dopamine. The D1/D5 dopamine receptor agonist SKF 81297 induced inhibition of the
capsaicin-activated current very similar to dopamine, in a dose-dependent manner (Fig.2).
Capsaicin-activated inward current was reduced by 57 21 % by 1 M SKF 81297 (n=5),
with no further effect at 3 M SKF 81297 (reduction by 61 13 %, n=8) or at 10 M SKF
81297 (reduction by 66 24 %, n=10). The potency and saturation of the effects of SKF
81297 suggest mediation by a receptor rather than a direct blocking effect on the channel.
Mediation by a receptor was further tested by applying SKF 81297 in combination with SCH
23390, a selective antagonist at D1/D5 dopamine receptors (Schiffmann et al., 1995; Cantrell
et al., 1997). In this series of experiments, the inhibitory effect of 1 M SKF 81297 on the
capsaicin-activated inward current was reduced from 63 18 % (n=5) when used alone to 14
7 % (n=6) when applied in combination with 100 nM SCH 23390 (Fig. 3). In contrast, the
D2 dopamine receptor agonist quinpirole had very little effect on the capsaicin-activated
inward and outward and currents (reduction of inward current by 11 7 % after 20 M
quinpirole, Fig.4, n=6). Taken together, the data suggest that the inhibitory effect of
dopamine on the capsaicin-activated outward and inward currents is mediated predominantly
by D1/D5 dopamine receptors, with little contribution of D2 dopamine receptors.
Change in temperature can affect the response of TRPV1 channel to agonists
(Neelands et al., 2008), its modulation by protons (Neelands et al., 2010), and the voltage
dependence of activation (Voets et al., 2004). We therefore determined whether the
inhibitory effect of SKF 81297 on the capsaicin-activated current was affected by
temperature. To this purpose, we tested the effects of SKF 81297 on the capsaicin-activated
current at a more physiological temperature of 35 1 C. As shown in Figure 5, 10 M SKF

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81297 inhibited the inward current by 56 9 % and the outward current by 47 6 %, similar
to that observed at room temperature.
Our standard intracellular solution contained 0.3 mM GTP (see methods). To test if
the effect of SKF 81297 was dependent on GTP, we tested intracellular solutions in which
GTP was either removed or replaced by the non-hydrolyzable analog GDP--S. When GTP
was removed from the intracellular solution, the inhibitory effect of 10 M SKF 81297 on the
capsaicin-activated inward current was reduced from 64 23 % (n=11) with GTP included in
the intracellular solution, to 24 7 % (n=6) with intracellular solution lacking GTP.
Similarly, when GTP was replaced by the non-hydrolyzable analog GDP--S, the inhibitory
effect of 10 M SKF 81297 on the capsaicin-activated inward current was reduced to 24 6
% (n=10) (Fig.6). Taken together, these results confirm that the coupling between dopamine
receptors and TRPV1 channels is dependent on GTP acting via G-proteins.
The TRPV1 channel is a non-selective cation channel with higher permeability to
Ca2+ than Na+ (PCa/PNa= ~10) (Caterina et al., 1997; Pedersen et al., 2005). To test whether
the inhibitory effect of SFK 81297 on the capsaicin-activated outward and inward currents
was dependent on external calcium, we replaced external Ca2+ (2 mM) with an equimolar
concentration of Mg2+, such that the external solution contained a final concentration of 0
mM Ca2+ and 3 mM Mg2+. Using 10 mM EGTA as the intracellular calcium chelator, the
inhibitory effect of 10 M SKF 81297 on the capsaicin-activated inward current was reduced
from 66 24 % (n=10) in 2 mM Ca2+/1 mM Mg2+ to 14 7 % (n=9) in 0 mM Ca2+/3 mM
Mg2+ (Fig.7). The residual inhibitory effect of SKF 81297 on the capsaicin-activated current
in zero external calcium/internal EGTA could reflect the contribution from free calcium that
was not chelated by the slow calcium buffer EGTA, or the contribution from calcium
released from internal stores, or a calcium-independent effect. To test these possibilities, first
we replaced 10 mM internal EGTA with 10 mM BAPTA, a faster calcium chelator. With
internal BAPTA and 0 mM Ca2+/3 mM Mg2+ in the external solution, the inhibitory effect of
10 M SKF 81297 on the capsaicin-activated inward current was reduced to 11 5% (n=11),
which was not different from the effect of SKF 81297 observed when the intracellular
solution contained the slow calcium buffer EGTA. Second, we used thapsigargin, an inhibitor
of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). When the intracellular calcium
stores were depleted by pre-incubating DRG neurons with 1M thapsigargin for 15 min, the
residual inhibitory effect of 10 M SKF 81297 on the capsaicin-activated currents observed
in zero external calcium/internal EGTA was completely abolished. Taken together, the data

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suggest that the inhibitory effect of SKF 81297 on the capsaicin-activated current is strongly
Ca2+-dependent.
Protein phosphorylation by protein kinase A (PKA), protein kinase C (PKC), and
calcium/calmodulin-dependent kinase II (CaMKII) has been shown to play a prominent role
in the modulation of TRPV1 channels (Vellani et al., 2001; Bhave et al., 2002; Jung et al.,
2004; Bangaru et al., 2015). Therefore, we tested whether PKA, or PKC, or CaMKII activity
were linked to D1/D5 dopamine receptor activation. When the PKA activity was blocked by
pre-incubating DRG neurons with 1 M H89 for 30 min, the inhibitory effect of 10 M SKF
81297 on the capsaicin-activated current was not affected: 10 M SKF 81297 inhibited the
capsaicin-activated inward current by 60 17 % (n=10) in control and by 63 14 % (n=9) in
H89 (Fig.8A). Similar results were obtained when the PKC activity was blocked by preincubating DRG neurons with 1 M Bisindolylmaleimide II (BIM) for 30 min: 10 M SKF
81297 inhibited the capsaicin-activated inward current by 57 12 % (n=9) in control and by
49.43 12.17% (n=9) in BMI (Fig.8B). In contrast, when the CaMKII activity was blocked
by including in the patch pipette 10 M KN-93, the inhibitory effect of 10 M SKF 81297 on
the capsaicin-activated current was significantly affected: 10 M SKF 81297 inhibited the
capsaicin-activated inward current by 66 24 % (n=10) in control and by 24 4% (n=8) with
intracellular KN-93 (Fig.8C). When KN-93 was replaced by the inactive analog KN-92 (10
M), 10 M SKF 81297 inhibited the capsaicin-activated inward current by 66 24 %
(n=10) in control and by 56 8% (n=8) with intracellular KN-92. Taken together, these
results suggest that activation of D1/D5 dopamine receptors is linked mainly to CaMKII
activity.

DISCUSSION
The data presented here provide strong evidence that dopamine and SKF 81297 (an
agonist at D1/D5 dopamine receptors) down regulate the capsaicin-activated current and the
activity of TRPV1 channels expressed in nociceptors. Only small DRG neurons (diameter of
24.4 2.5 m and cell capacitance of 18.9 3.9 pF, n=247) sensitive to capsaicin (52% of
those tested) were included in the study, suggestive of nociceptors (Cardenas et al., 1995;
Petruska et al., 2000; Ho & O'Leary, 2011). Although nociceptors are neurochemically and
functionally distinct (Nagy & Hunt, 1982; Silverman & Kruger, 1990; Stucky & Lewin,
1999; Dirajlal et al., 2003), no obvious differences were observed in the inhibitory effect of
dopamine or SKF 81297 on the capsaicin-activated current between peptidergic [IB4(-)] and

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non-peptidergic [IB4(+)] DRG neurons. This is consistent with the expression of D1 and D5
dopamine receptors both in peptidergic and non-peptidergic DRG neurons (Galbavy et al.,
2013).
The effect of SKF 81297 on the capsaicin-activated current was strongly reduced by
removing GTP from the intracellular solution or by replacing GTP with GDP--S, a nonhydrolysable analog of GDP

(Fig.6), confirming that the effect of dopamine requires

activation of GPCRs as expected for dopamine receptors (Beaulieu & Gainetdinov, 2011).
Several observations support a functional role for dopamine in the dorsal root ganglia.
1) TH, the rate limiting enzyme for the synthesis of cathecolamines, is expressed in a large
population of DRG neurons (~ 8-10 %), including the C-LTMRs, specialized in detecting
low-threshold mechanosensory stimuli, and those innervating pelvic organs (Price & Mudge,
1983; Philippe et al., 1993; Brumovsky et al., 2006; Li et al., 2011; Brumovsky et al., 2012;
Lou et al., 2013). 2) Dopamine and its metabolites dihydroxyphenylacetic acid (DOPAC) and
homovanillic acid (HVA) have been detected in DRG neurons and in dorsal spinal nerve
roots (Lackovic & Neff, 1980; Philippe et al., 1993; Weil-Fugazza et al., 1993; Bertrand &
Weil-Fugazza, 1995). 3) Dopamine receptors are expressed in DRG neurons (Xie et al.,
1998; Galbavy et al., 2013). A question however remains why it has been difficult to detect
other enzymes necessary for the synthesis of catecholamines, i.e. aromatic acid decarboxylase
(AADC) and dopamine -hydroxylase (DBH) (Price & Mudge, 1983; Kummer et al., 1990;
Vega et al., 1991; Brumovsky et al., 2006), even though in same cases AADC and DBH have
been detected in TH-negative [TH(-)] DRG neurons (Brumovsky et al., 2006). A possible
explanation could be that the protein levels of AADC and DBH are too low to be detected by
immunocytochemistry in these neurons. Alternatively, TH(+) DRG neurons may synthesize
L-DOPA, which after release is converted to dopamine by AADC localized in other cell
types.
Using Western blotting and immunofluorescence, we recently showed that DRG
neurons express D1 and D5 (both in peptidergic and non-peptidergic DRG neurons), but not
D2 dopamine receptors (Galbavy et al., 2013). This is consistent with our pharmacological
studies showing that SKF 81297 (an agonist at D1/D5 dopamine receptors), but not
quinpirole (an agonist at D2 dopamine receptors) can mimic the inhibitory effect of dopamine
on the capsaicin-activated current, as well as on the TTX-R sodium current (Galbavy et al.,
2013). Using polymerase chain reaction (PCR) and DNA sequencing, transcripts for D1-D5
dopamine receptors, including D2 receptors, have been detected in dorsal root ganglia (Xie et

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al., 1998; Peiser et al., 2005). A possible explanation for this discrepancy could be that, even
though the transcript for D2 dopamine receptors is detectable with PCR, the protein level
could be too low to be detected by Western blotting and pharmacological analysis.
Alternatively, it is possible that once translated into protein, D2 dopamine receptors are
rapidly transported from the cell body to the terminals, and thus are missed with our analysis.
D1/D5 dopamine receptors may be linked to different protein kinases, according to
cell type or tissue specificity, including PKA (Cantrell et al., 1997; Maurice et al., 2001)
(Schiffmann et al., 1995), PKC (Young & Yang, 2004; Galbavy et al., 2013), and CaMKII
(Chen et al., 2007; Anderson et al., 2008; Xing et al., 2010). Protein phosphorylation plays a
major role in the modulation of TRPV1 channels. Several serine and threonine residues have
been reported to be phosphorylated by PKC (Tominaga et al., 2001; Numazaki et al., 2002;
Bhave et al., 2003; Premkumar et al., 2004), or by PKA (De Petrocellis et al., 2001; Bhave et
al., 2002; Rathee et al., 2002; Vlachova et al., 2003; Mohapatra & Nau, 2005; Jeske et al.,
2006), or by CaMKII (Jung et al., 2004; Price et al., 2005; Hucho et al., 2012).
Phosphorylation by PKC sensitizes TRPV1 channels by increasing the channel open
probability (Vellani et al., 2001), while phosphorylation by PKA may reverse the
desensitization of TRPV1 channels induced by repeated application of the agonist (Bhave et
al., 2002). Activation of PKC or PKA by dopamine would likely produce an increase of the
capsaicin-activated current. However, our data show that dopamine or SKF 81297
consistently down regulated the capsaicin-activated current, arguing against a direct
involvement of PKC or PKA. In contrast, the inhibitory effect of SKF 81297 on the
capsaicin-activated current was reduced by ~ 50% when the CaMKII activity was blocked,
suggesting

that

D1/D5

receptors

may

be

linked

to

CaMKII.

Phosphorylation/dephosphorylation of TRPV1 channels by CaMKII/calcineurin have been

suggested to modulate vanilloids binding to TRPV1 channels (Jung et al., 2004). Thus, an
intriguing possibility is that activation of D1/D5 dopamine receptors may modulate the
function of TRPV1 channels by modulating the vanilloids binding to the channel. Because
Ca2+ signaling regulates both CaMKII and calcineurin, different mechanisms have been
proposed to explain the fine balance between phosphorylation and dephosphorylation,
including different activation kinetics, different sensitivities to Ca2+ and calmodulin, or crosstalk between these two enzymes-signaling pathways (Hashimoto et al., 1988; Klee, 1991;
Tian & Karin, 1999). Future experiments are required to fully elucidate the interaction
between D1/D5 dopamine receptors and the CaMKII/calcineurin pathway.

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Our results in Figure 7 support the conclusion that the inhibitory effect of SKF 81297
on the capsaicin-activated current is highly dependent on calcium. Removal of Ca2+ from the
external solution strongly reduced the inhibitory effect of SKF 81297 on the capsaicinactivated current, arguing in favor of a major contribution of Ca2+ influx. While our
experiments were designed to minimize the contribution of calcium channels, activation of
voltage-dependent calcium channels during action potential firing in nociceptors likely
provides the major source of Ca2+ influx under physiological conditions (Carbone & Lux,
1984; Scroggs & Fox, 1991, 1992; Blair & Bean, 2002; Bell et al., 2004; Castiglioni et al.,
2006; Gemes et al., 2010), contributing to a subsequent rise in cytoplasmic Ca2+ sufficient to
engage downstream targets such as CaMKII. However, because of their high permeability to
Ca2+ (PCa2+/PNa+ = ~10), membrane-delimited TRPV1 channels (Caterina et al., 1997),
together with those expressed on the endoplasmic reticulum (Olah et al., 2001; Wong et al.,
2014), may contribute, at least in part, to the rise in cytoplasmic Ca2+. Indeed, experiments
using thapsigargin indicate that Ca2+ released from internal stores contributes to the effects of
SKF 81297.
D5 receptors have been shown to couple to phospholipase C (PLC) through Gq and
induce mobilization of Ca2+ from internal stores (So et al., 2009). In contrast, D1 receptors
appear to require the presence of D2 receptors to activate PLC (Lee et al., 2004; Rashid et al.,
2007). Because we find that D2 receptor levels are low in DRG neurons (Galbavy et al.,
2013), and quinpirole (D2 agonist) has very little effect on the capsaicin-activated current
(Fig.4), we infer that D5 receptors, coupled to PLC, may constitute the principal pathway for
dopamine modulation of TRPV1 channels. This pathway should stimulate hydrolysis of
plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4phosphate (PI4P), which are well known to modulate the activity of TRP channels (Rohacs &
Nilius, 2007), as well as release Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive stores
(Berridge, 2009).
The high permeability of TRPV1 channels to Ca2+ (PCa2+/PNa+ = ~10) (Caterina et al.,
1997) may be of particular significance for the modulation and integration of incoming
noxious stimuli. Calcium influx is suggested to play a prominent role in the desensitization of
TRPV1 channels upon repeated stimulation with the agonist (Cholewinski et al., 1993;
Docherty et al., 1996; Liu & Simon, 1996; Koplas et al., 1997; Jung et al., 2004; Rosenbaum
et al., 2004), which predicts a reduced response of nociceptors to repeated noxious stimuli.
Our data show that dopamine, not only can down regulate fully activated TRPV1 channels

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(e.g. during the first application of capsaicin, Fig.1B), but can also down regulate TRPV1
channels that are partially desensitized during repeated applications of the agonist (Fig.1D).
This suggests that the down regulation of TRPV1 channels by dopamine may occur through a
mechanism different from Ca2+-induced desensitization. The down regulation of TRPV1
channels by dopamine could provide an additional level of modulation to further reduce the
activity of TRPV1 channels and the response of nociceptors to repeated incoming noxious
stimuli.
In the retina and the olfactory bulb, spontaneously active dopaminergic neurons
provide tonic release of dopamine (Feigenspan et al., 1998; Puopolo et al., 2005), which
plays an important function in sensory adaptation. In the retina, dopamine sets the gain of the
retinal networks for vision in bright light (Witkovsky, 2004); in the olfactory system,
dopamine sets the gain for odor discrimination (Nowycky et al., 1983; Duchamp-Viret et al.,
1997; Hsia et al., 1999; Berkowicz & Trombley, 2000; Davison et al., 2004). The C-LTMRs
are usually silent, but in response to light mechanical force, such as stretch of the skin or
deflection of hair follicles, fire action potentials (Li et al., 2011) that may trigger the release
of dopamine in the dorsal root ganglia as well as in the dorsal horn of the spinal cord. In the
dorsal root ganglia, the cell bodies of DRG neurons are usually encapsulated by a single
satellite glial cell, rarely in direct contact, and synaptic inputs are virtually absent (Pannese et
al., 1991; Shinder et al., 1999). DRG neurons release various peptides and ATP from their
soma (Huang & Neher, 1996; Zhang et al., 2007), and when loaded with dopamine,
dopamine release can be evoked by high potassium stimulation and detected by amperometric
means (Zhang & Zhou, 2002), suggesting that DRG neurons may carry the necessary release
machinery for dopamine. Thus, once released from C-LTMRs, dopamine could modulate the
activity of neighboring cells by inter-neuron signaling (Amir & Devor, 1996; Rozanski et al.,
2012). Increasing or decreasing levels of dopamine may serve to adjust the sensitivity of
nociceptors to incoming noxious stimuli, with increasing levels of dopamine raising the
threshold for pain and reduced levels increasing the sensitivity to noxious stimuli.
TRPV1 channels are expressed not only on the cell body and peripheral terminals of
nociceptors, but also on central terminals that make synaptic contacts in the dorsal horn of the
spinal cord (Holzer, 1991; Winter et al., 1993; Szallasi et al., 1995; Tominaga et al., 1998;
Guo et al., 1999; Hwang et al., 2004). Activation of presynaptic TRPV1 channels on central
terminals with endovanilloids or capsaicin has been shown to trigger the release of peptides
and modulate glutamatergic transmission (Yang et al., 1998; Tognetto et al., 2001; Nakatsuka

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et al., 2002; Baccei et al., 2003; Labrakakis & MacDermott, 2003; Tong & MacDermott,
2006; Medvedeva et al., 2008). In the dorsal horn of the spinal cord, dopamine induces
antinociceptive actions by decreasing the frequency and amplitude of spontaneous and
miniature EPSC in substantia gelatinosa neurons (Taniguchi et al., 2011), depresses dorsal
root potentials evoked by stimulation of cutaneous and muscle afferents (Garcia-Ramirez et
al., 2014), and induces inhibition of the responses to noxious stimuli in superficial neurons
(Fleetwood-Walker et al., 1988). Thus, in the dorsal horn of the spinal cord, dopamine may
interact with presynaptic TRPV1 channels to modulate synaptic transmission between
nociceptors and dorsal horn neurons.

Figure legends
Figure 1. Inhibition of capsaicin-activated current by dopamine.
A) Ionic currents through TRPV1 channels were determined by subtracting currents before
and after application of 1 M capsaicin. Inset: voltage clamp protocol. Ramps (2 mV/ms)
were delivered in the same sweep from -100 to +100 mV and from +100 to -100 mV with the
down ramp preceded by 50-200 ms at +100 mV to inactivate voltage-dependent sodium
and calcium channels. The trace shown is the capsaicin-activated current during the down
ramp. B) Effects of dopamine on the capsaicin-activated current during a single application
of capsaicin. Inward and outward currents were measured at -100 and +100 mV, respectively.
Note that, because of desensitization of TRPV1 channels induced by capsaicin, the washout
after dopamine was not complete. C) In collected results, 20 M dopamine, applied on top of
capsaicin (first application), inhibited the inward current by 56 8 % (n=10, **p<0.01,
paired t-test), and the outward current by 44 9 % (n=10, **p<0.01, paired t-test), with
respect to control. D) Effects of dopamine on the capsaicin-activated current during repeated
application of capsaicin. Note a full recovery upon washing out dopamine. E) Capsaicinactivated currents from the cell in D: 1 M capsaicin (black trace), 20 M dopamine on top
of capsaicin (red trace), 1 M capsaicin (upon washing out dopamine, blue trace). F) In
collected results, 20 M dopamine, applied on top of capsaicin (third application), inhibited
the inward current by 60 18 % (n=9, **p<0.01, paired t-test) and the outward current by 48
16 % (n=9, **p<0.01, paired t-test), with respect to control.

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This article is protected by copyright. All rights reserved.

Figure 2. Inhibition of capsaicin-activated current by the D1/D5 dopamine receptor

agonist SKF 81297.
Dose-dependent inhibition of capsaicin-activated currents by the D1/D5 agonist SKF 81297,
showing similar effects on inward and outward currents. Each bar represents an independent
experiment. For each concentration, statistical significance was assessed with paired t-test by
comparing the effect of the drug to its own control. The IC50 was determined using a Log
inhibitor versus normalized response equation: Y=100/(1 + 10^((X-LogIC50))). A) The
inward current was reduced by 19 6 % (n=7, **p<0.01) by 10 nM SKF 81297; by 20 9 %
(n=8, *p<0.05) by 30 nM SKF 81297; by 45 15 % (n=6, *p<0.05) by 100 nM SKF 81297,
by 40 11 % (n=8, **p<0.01) by 300 nM SKF 81297; by 63 18 % (n=5, *p<0.05) by 1 M
SKF 81297; by 61 13 % (n=8, **p<0.01) by 3 M SKF 81297; by 66 24 % (n=10,
**p<0.01) by 10 M SKF 81297. The IC50 for the inward current was determined at 5.5 nM.
B) The outward current was reduced by 14 4 % (n=7, **p<0.01) by 10 nM SKF 81297; by
18 10 % (n=8, **p<0.01) by 30 nM SKF 81297; by 25 13 % (n=6, *p<0.05) by 100 nM
SKF 81297; by 40 10 % (n=8, **p<0.01) by 300 nM SKF 81297; by 54 27 % (n=5,
*p<0.05) by 1 M SKF 81297; by 47 6 % (n=8, **p<0.01) by 3 M SKF 81297; by 57
21 % (n=10, *p<0.05) by 10 M SKF 81297. The IC50 for the outward current was
determined at 3.3 nM. C) Representative capsaicin-activated currents in 1 M capsaicin
(black trace), 10 M SKF 81297 on top of capsaicin (red trace), 1 M capsaicin (washout,
blue trace).

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Figure 3. The effects of SKF 81297 (D1/D5 agonist) were reversed by SCH 23390 (D1/D5
antagonist).
A) Representative capsaicin-activated currents recorded in the presence of 100 nM SCH
23390 (black trace), after application of 1 M SKF 81297 on top of SCH 23390 (red trace),
and upon removal of SKF 81297 (blue trace). B) In collected results, the inward current was
reduced by 63 18 % by 1 M SKF 81297 (n=5) and by 14 7 % by 1 M SKF 81297 +
100 nM SCH 23390 (n=6), **p <0.01, unpaired t- test. C) The outward current was reduced
by 54 26 % by 1 M SKF 81297 (n=5) and by 7 4 % by 1 M SKF 81297 + 100 nM SCH
23390 (n=6), **p<0.01, unpaired t-test.

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Figure 4. Effects of quinpirole (D2 agonist) on the capsaicin-activated current.

A) Representative capsaicin-activated currents recorded in control (black trace), in the
presence of 20 M quinpirole (agonist at D2 dopamine receptors, red trace), and upon
washing out quinpirole (blue trace). B, C) Collected results show comparison of 10 M SKF
81297 and 20 M quinpirole effects on the capsaicin-activated currents. The inward current
was reduced by 66 24 % (n=10) by 10 M SKF 81297 and by 11 7 % (n=5) by 20 M
quinpirole, **p<0.01, unpaired t-test. The outward current was reduced by 57 21 % (n=10)
by 10 M SKF 81297 and by 17 8 % (n=5) by 20 M quinpirole, **p<0.01, unpaired t-test.

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Figure 5. Effects of SKF 81297 on the capsaicin-activated current at physiological

temperature.
A) Representative capsaicin-activated currents recorded at physiological temperature (35 1
C) in control (black trace), in the presence of 10 M SKF 81297 (agonist at D1/D5
dopamine receptors, red trace), and upon washing out SKF 81297 (blue trace). B) In collected
results, 10 M SKF 81297, applied on top of capsaicin (third application), inhibited the
inward current by 56 9 % (n=9), **p<0.01, paired t-test) and the outward current by 47 6
% (n=9), **p<0.01, paired t-test).

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This article is protected by copyright. All rights reserved.

Figure 6. The effects of SKF 81297 on the capsaicin-activated current are mediated by
G protein coupled receptors (GPCRs).
Collected results show reduction of the effect of 10 M SFK 81297 on the capsaicinactivated inward and outward currents when the usual 0.3 mM GTP was omitted from the
internal solution or replaced by the non-hydrolyzable analog GDP--S. A) The inward
current was reduced by 66 24 % (n=10) with GTP included in the intracellular solution, by
24 7 % (n=6) with intracellular solution lacking GTP, and by 24 6 % (n=10) when GTP
was replaced by equimolar concentration of the non-hydrolyzable analog GDP--S. No GTP
versus GTP: **p<0.01, one way ANOVA followed by Dunnetts post hoc comparison test.
GDP--S for GTP versus GTP: **p<0.01, one way ANOVA followed by Dunnetts post hoc
comparison test. B) The outward current was reduced by 57 21 % (n=10) with GTP
included in the intracellular solution, by 18 7 % (n=6) with intracellular solution lacking
GTP, and by 20 8 % (n=10) when GTP was replaced by equimolar concentration of the
non-hydrolyzable analog GDP--S. No GTP versus GTP: **p<0.01, one way ANOVA
followed by Dunnetts post hoc comparison test. GDP--S for GTP versus GTP: **p<0.01,
one way ANOVA followed by Dunnetts post hoc comparison test.

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Figure 7. The effects of SKF 81297 on the capsaicin-activated current are dependent on
calcium.
A) Representative capsaicin-activated currents recorded in 0 Ca2+/EGTA in control (black
trace), in the presence of 10 M SKF 81297 (red trace), and upon washing out SKF 81297
(blue trace). The reversal potential of the capsaicin-activated current in 0 Ca2+/EGTA was
determined at -3.8 2.4 mV (n=19). Collected results show the effect of 10 M SKF 81297
on the capsaicin-activated inward and outward currents with manipulation of external and/or
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internal calcium. The effect of 10 M SKF 81297 was determined with 2 mM Ca2+ in the
external solution and 10 mM EGTA in the internal solution (2 Ca2+/EGTA), or when 2 mM
Ca2+ in the external solution was replaced by 2 mM Mg2+ (0 Ca2+/EGTA), or when the 0 Ca2+
external solution was tested with an internal solution in which 10 mM BAPTA replaced 10
mM EGTA (0 Ca2+/BAPTA), or when the 0 Ca2+/EGTA solution was tested after depleting
the internal calcium stores by pre-incubating the cells with 1 M thapsigargin for 15 min (0
Ca2+/thapsigargin/EGTA). B) The inward current was reduced by 66 24 % (n=10) in 2
Ca2+/EGTA, by 14 6 % (n=9) in 0 Ca2+/EGTA, by 11 5 % (n=11) in 0 Ca2+/BAPTA, and
by -2 5 % (n=11) in 0 Ca2+/thapsigargin/EGTA. 0 Ca2+/EGTA versus 2 Ca2+/EGTA:
**p<0.01, one way ANOVA followed by Dunnetts post hoc comparison test. 0 Ca2+/BAPTA
versus 2 Ca2+/EGTA: **p<0.01, one way ANOVA followed by Dunnetts post hoc
comparison test. 0 Ca2+/thapsigargin/EGTA versus 2 Ca2+/EGTA: **p<0.01, one way
ANOVA followed by Dunnetts post hoc comparison test. C) The outward current was
reduced by 57 21 % (n=10) in 2 Ca2+/EGTA, by 16 10 % (n=9) in 0 Ca2+/EGTA, by 14
5 % (n=11) in 0 Ca2+/BAPTA, and by 2 6 % (n=11) in 0 Ca2+/thapsigargin/EGTA. 0
Ca2+/EGTA versus 2 Ca2+/EGTA: **p<0.01, one way ANOVA followed by Dunnetts post
hoc comparison test. 0 Ca2+/BAPTA versus 2 Ca2+/EGTA: **p<0.01, one way ANOVA
followed by Dunnetts post hoc comparison test. 0 Ca2+/thapsigargin/EGTA versus 2
Ca2+/EGTA: **p<0.01, one way ANOVA followed by Dunnetts post hoc comparison test.

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This article is protected by copyright. All rights reserved.

Figure 8. Intracellular pathways linked to D1/D5 dopamine receptors activation.

Collected results show the effect of 10 M SKF 81297 on the capsaicin-activated inward and
outward currents with manipulation of intracellular protein kinases. A) Blockade of PKA by
pre-incubation with 1 M H89 for 30 min. The inward current was reduced by 60 17 %
(n=10) in SKF 81297 alone, and by 63 15 % (n=9) following incubation with H89,
p=0.704, unpaired t-test. Similarly, the outward current was reduced by 51 16 % (n=10) in
SKF 81297 alone, and by 49 12 % (n=9) following incubation with H89, p=0.711, unpaired
t-test. B) Blockade of PKC by pre-incubation with 1 M Bisindolylmaleimide II (BIM) for
30 min. The inward current was reduced by 57 12 % (n=9) in SKF 81297 alone, and by 52
12 % (n=9) following incubation with BIM, p=0.362, unpaired t-test. Similarly, the
outward current was reduced by 48 13 % (n=9) in SKF 81297 alone, and by 41 10 %
(n=9) following incubation with BIM, p=0.233, unpaired t-test. C) Blockade of CaMKII with
10 M KN-93 or 10 M KN-92 (inactive control) included in the patch pipette. The inward
current was reduced by 66 24 % (n=10) in SKF alone, by 24 4 % (n=8) with KN-93, and
by 56 8 % (n=8) with KN-92. KN-93 versus control: **p<0.01, one way ANOVA followed
by Dunnetts post hoc comparison test. KN-92 versus control: p=0.306, one way ANOVA
followed by Dunnetts post hoc comparison test. The outward current was reduced by 57 21
% (n=10) in SKF 81297 alone, by 22 6 % (n=8) with KN-93, and by 43 5 % (n=8) with
KN-92. KN-93 versus control: **p<0.01, one way ANOVA followed by Dunnetts post hoc
comparison test. KN-92 versus control: p=0.058, one way ANOVA followed by Dunnetts
post hoc comparison test.

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