PAPER
www.rsc.org/analyst | Analyst
Understanding changes in uptake and release of serotonin from
gastrointestinal tissue using a novel electroanalytical approach†
Gianluca Marcellia and Bhavik Anil Patel*bc
Received 22nd April 2010, Accepted 10th June 2010
DOI: 10.1039/c0an00260g
Serotonin (5-HT) is well known to be a key neurotransmitter within the gastrointestinal (GI) tract,
where it is responsible for influencing motility. Obtaining dynamic information about the
neurotransmission process (specifically the release and reuptake of 5-HT) requires the development of
new approaches to measure the extracellular 5-HT concentration profile. In this work constantpotential amperometry has been utilised at +650 mV vs. Ag|AgCl to measure in vitro the overflow of 5HT. Steady-state levels of 5-HT have been observed, due to continuous mechanical stimulation of the
tissue from the experimental protocol. Measurements are conducted at varying tissue–electrode
distances in the range of 5 to 1100 mm. The difference in the current from the bulk media and that from
each tissue–electrode distance is obtained, and the natural log of this current is plotted versus the tissue–
electrode distance. The linear fit to the log of the current is derived, and its intercept, I0, with the vertical
axis and its slope are calculated. The reciprocal of the slope, indicated as slope1, is used as a marker of
reuptake. The ratio between intercept, I0, and the reciprocal of the slope, I0/slope1, is a measure of the
flux at the tissue surface and it can be used as a marker for the 5-HT release rate. Current measurements
for ileum and colon tissue indicated a significantly higher reuptake rate in the colon, showed by a lower
slope1. In addition, the ratio, I0/slope1, indicated that the colon has a higher 5-HT flux compared to
the ileum. Following the application of the serotonin selective reuptake inhibitor (SSRI), fluoxetine,
both tissues showed a higher value of slope1, as the reuptake process is blocked preventing clearance of
5-HT. No differences were observed in the ratio, I0/slope1, in the ileum, but a decrease was observed in
the colon. These results indicate that ileum and colon are characterised by different reuptake and
release processes. The new approach we propose provides pivotal information on the variations in the
signalling mechanism, where steady state levels are observed and can be a vital tool to study differences
between normal and diseased tissue and also the efficacy of pharmacological agents.
Introduction
Serotonin (5-HT) plays a key role in the gastrointestinal (GI)
tract and is found in two locations: the myenteric plexus and the
enterochromaffin (EC) cells located in mucosa. EC cells act as
sensory transducers and respond to mechanical and chemical
stimulation of the mucosal epithelial by releasing serotonin.1,2 5HT is essential for influencing local motility activity and acts on
mucosal endings of intrinsic primary afferent neurons and
extrinsic primary afferent neurons to initiate motor reflexes and
intestinal sensation.3–5
5-HT is released from vesicles in the EC cells in a calcium
dependent manner. Once released, it binds to its receptors to
cause changes in motility of the GI tract. It is then cleared
from extracellular space by the 5-HT transporter (SERT) but
5-HT molecules can also diffuse into extracellular space or be
a
Biomedical Engineering Group, Division of Engineering, King’s College
London, Strand, London, WC2R 2LS, UK
b
Centre for Biomedical and Health Sciences Research, University of
Brighton, Brighton, BN2 4GJ, UK
c
Department of Pharmacy and Biomolecular Sciences, University of
Brighton, Brighton, BN2 4GJ, UK. E-mail: [email protected];
Fax: +44 (0)1273 679333; Tel: +44 (0)1273 641912
† Electronic supplementary information (ESI) available: Results from
Brownian Dynamics Model. See DOI: 10.1039/c0an00260g
2340 | Analyst, 2010, 135, 2340–2347
metabolised. The SERT has been identified to be present in
many of the epithelial cells located in the mucosa.6,7 The
SERT is a major target for a class of drug known as serotonin-selective reuptake inhibitors (SSRIs), such as Prozac
(fluoxetine) and Citalopram. SSRIs are involved widely in the
treatment of all forms of depression.8 Alterations in the
release of 5-HT or clearance by SERT can have major
implications on the function of the GI tract and can lead to
the onset of various GI diseases. Alterations in the levels,
structure or function of the SERT, are implicated in the
symptoms of irritable bowel syndrome (IBS).9,10 Alterations in
5-HT and SERT have also been implicated in other irritable
bowel diseases (IBDs).11–13
Electroanalytical methods have been widely used to measure
neurotransmitter release from various biological environments14–16 as they offer excellent spatial and temporal resolution.
Measurements can be made from single cells through to intact
animals. Recordings obtained for biogenic amine or catecholamine transmitters are observed as dynamic responses, where the
signal can be analysed to obtain the information about changes
in uptake and release from a single event. Generally the amplitude is utilised as a marker for the amount of transmitter released
and the signal decay with time provides a marker of clearance of
the neurotransmitter by transporters.17–19 Although other
processes such as diffusion and metabolism can influence these
This journal is ª The Royal Society of Chemistry 2010
levels, reuptake is the major means of neurotransmitter clearance.
Boron-doped diamond (BDD) microelectrodes have been
previously used for the selective detection of serotonin overflow
from mucosal tissue.20 When electrodes were held at +650 mV,
no interference was observed from other released electroactive
signalling molecules such as melatonin.21–23 The steady-state
levels obtained of 5-HT cannot be interpreted in the same fashion
as dynamic events; therefore a different analytical approach is
required. Only one previous study has been developed to
measure reuptake from GI tissue, where the clearance of
endogenous 5-HT was investigated using a carbon fibre microelectrode.24 This method provides no information on release, and
by adding additional levels of 5-HT, the behaviour of the tissue is
compromised as well as the performance of the electrode which is
prone to fouling. Therefore there is a need for a new approach to
study release and uptake without altering the physiological
behaviour of the tissue.
In this manuscript we propose a new experimental approach to
obtain information on 5-HT release and reuptake. An electrochemical technique is used to obtain information on the release
and reuptake during steady-state release of 5-HT from EC cells
in the guinea pig ileum and colon. The fact that release and
reuptake in the GI tract are two different mechanisms allowed us
to isolate and characterise the two mechanisms separately from
the overall tissue responses. Differences in the reuptake and
release between ileum and colon tissues will be investigated and
discussed. Alterations in the release and reuptake will be assessed
using two different concentrations of the SERT inhibitor fluoxetine.
Experimental section
Chemicals
All experiments were carried out in oxygenated (95% O2 and 5%
CO2) Krebs buffer (composition: 117 mM NaCl, 4.7 mM KCl,
2.5 mM CaCl2, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 25 mM
NaHCO3 and 11 mM glucose). Chemicals for making this buffer
solution were purchased from Sigma-Aldrich. 5-Hydroxytryptamine (serotonin, 5-HT) and fluoxetine were purchased from
Sigma-Aldrich and analytical solutions were prepared freshly
prepared on the day of use.
Electrode fabrication
Boron-doped diamond (BDD) thin film was deposited on a 40
mm diameter Pt wire (99.99%, Aldrich Chemical) by microwaveassisted chemical vapour deposition (CVD) (1.5 kW, 2.54 GHz,
ASTeX, Woburn, MA, USA), as detailed previously.20,23–25 The
diamond-coated Pt wire was affixed to a longer copper wire using
conductive Ag epoxy and the entire assembly was insulated with
polypropylene from a pipette tip. The insulation was applied by
inserting the microelectrode into a pipette tip with about 500 mm
exposed from the end and carefully heating the tapered end using
the coil of micropipette puller. This softened the polypropylene
and caused it to conformally coat the rough, polycrystalline
diamond surface.25–27 The resulting microelectrode was cylindrical with a diameter of ca. 40 mm. The length of the exposed
electrode was 100–200 mm. The electrode can be reproducibly
This journal is ª The Royal Society of Chemistry 2010
insulated with a thin and continuous polymer film using this
method, but precise control of the exposed electrode length is
difficult to achieve.
Tissue preparation
Animal use procedures were carried out in compliance with the
relevant laws and institutional guidelines at Imperial College
London. All experiments were carried out using male guinea pigs
weighing 300–400 g, which were euthanased using CO2 gas. A
segment of ileum or colon tissue was removed and placed in
oxygenated (95% O2 and 5% CO2) Krebs’ buffer solution. A 1 cm
long segment of ileum or colon was then transferred to a flow
bath containing oxygenated Krebs buffer solution. The bottom
of the recording chamber was lined with a silicone elastomer
(Sylgard, Dow Corning, USA). The segment of ileum or colon
was then cut open along the mesenteric border, lightly stretched
and pinned flat in the flow bath using stainless steel pins (50 mm
diameter). The mucosal surface was face-up at the bottom of the
chamber. This tissue was constantly perfused with warm (37 C)
oxygenated Krebs’ buffer. Experiments were commenced after
the tissue was exposed to these conditions for 30 min.
Biological measurements
To gain information on 5-HT release and uptake, constant
potential amperometry was carried out for tissue measurements.
A Pt wire was used as the auxiliary electrode and a ‘‘no leak’’
Ag|AgCl reference electrode (3 M KCl, model EE009, Cypress
Systems Inc., USA) served as the reference electrode in a three
system configuration. All the electrodes were placed in a flow
bath, of which the fabrication has been described in detail
previously.20,21 The flow bath was mounted on the stage of an
inverted microscope (Model 3030, Accu-Scope, USA) and
superfused continuously with warm (37 C) oxygenated Krebs’
buffer solution at a flow rate of 2 ml min1. The solution
temperature was controlled with an immersion heating circulator
(Model 1130A, VWR Scientific, USA) and the solution flow was
controlled with a peristaltic pump (Masterflex, Cole Parmer,
USA).
For measurements, the BDD microelectrode was affixed to
a micromanipulator (Model 25033, Fine Scientific Tools, USA)
and placed >5 millimetres over the centre of tissue piece in the
bulk of the media. The BDD electrode was poised at a potential
of +650 mV vs. Ag|AgCl, which has been shown to be sufficient
to oxidise 5-HT and at this potential no interferences from other
known released transmitters or matrix components have been
observed.21 During measurements, the electrode was carefully
positioned over the tissue for a range of tissue–electrode
distances: from 5 to 1100 mm. A fine micromanipulator was used
for precise location of the BBD electrode. Initially the electrode
was used to gently touch the villi and was then retracted to a fixed
distance. The measurements were commenced as the electrode
was brought towards the tissue and positioned at each tissue–
electrode distance for 40 s. This multi-step measurement was
repeated over two or three regions of the same tissue section. As
the electrode is positioned at an angle, the corrected tissue–
electrode distance was utilised for analysis. Such measurements
were carried in repeated fashion in both ileum and colon tissue.
Analyst, 2010, 135, 2340–2347 | 2341
Following control measurements in each tissue piece, measurements were carried out in the same tissue sample to assess the
influence of the serotonin selective reuptake inhibitor fluoxetine
(500 nM and 1 mM) in Krebs’ buffer.
Data analysis
The log plot of the values of the current vs. electrode–tissue
distances was generated and a linear fit was derived using Igor
Pro (WaveMetrics Inc., USA). The slope of the linear fit and its
intercept, I0, with the vertical axis were calculated with the same
program. The results for ileum and colon tissue and the differences between control measurements and those in the presence of
the reuptake inhibitor fluoxetine were compared using a Students
t-test.
Results and discussion
Approach to obtain uptake and release from in vitro GI tissue
Fig. 1 shows measurements of 5-HT release at +650 mV vs.
Ag|AgCl from the mucosal surface from ileum and colon tissue.
From 0 to 50 s the electrode is positioned >5 mm away from the
tissue in the bulk of the media and the current observed is the
capacitive response from the background media. After 50
seconds the electrode is moved to an electrode–tissue distance of
500 mm and held within this position for 60 s to record the steadystate current response. After this stage the electrode is retracted
back into the bulk of the media. Thus only the current difference
from the background and when the sensor is placed on the tissue
surface (shown as the grey band in Fig. 1) can be utilised to gain
information on the activity of the tissue. For this reason, this
transmission is called steady-state because when the electrode is
placed over the tissue a constant current is observed. This steadystate level is observed as under the flow of the constantly perfused
buffering media, the villi are continuously brushing one another,
inducing constant 5-HT release through mechanical stimulation.
Levels of 5-HT are unlikely to be depleted because 80–90% of the
bodies’ 5-HT content is known to be present within these EC
cells.1
Fig. 1 Steady-state responses from ileum and colon tissue. The grey box
indicates the time period during which the sensor is placed 0.5 mm over
the tissue. The rise and decay in the current are influenced by the
movement of the electrode close to and far away from the tissue surface.
2342 | Analyst, 2010, 135, 2340–2347
As Fig. 1 shows, the measured current is different for the ileum
and colon tissues at the fixed distance chosen. The ileum current
is twice than that obtained from the colon, which indicates
differences in the transmission mechanism. The lower coloncurrent could be due to one or both of the following factors: (i)
higher reuptake rate or number of uptake transporters or (ii)
lower release of 5-HT. Either of these two points could explain
the differences in current responses between the tissues, thus
a new experimental approach is required to gain more information on the specific changes in the signalling mechanism.
Fig. 2A shows an experimental trace from ileum tissue where
measurements are made at a wide range of tissue–electrode
distances, in a similar fashion to typical approach curve utilised
in scanning electrochemical microscopy (SECM). Fig. 2A shows
steady-state current profile, which gradually increases at
distances between 1100 and 700 mm, but from 710 mm to 140 mm
the current drastically increases, and nearly doubles during each
step. The concentration profile is influenced by mechanisms that
regulate clearance, such as reuptake, diffusion and convection,
but also by the amount of 5-HT released. Thus analysing this
concentration profile provides a means of understanding differences in these various processes.
Once the experimental trace is obtained the current difference between each electrode–tissue distance and the response
from the bulk media is obtained and plotted against the electrode–tissue distances. This is reported in Fig. 2B which shows
an exponential response of current versus tissue–electrode
distance. To gain a better understanding of the response, the
natural log of the current in Fig. 2B is calculated and reported
in Fig. 2C. From this response a linear fit can be conducted
and a slope and intercept can be derived. From the slope and
intercept, information on the uptake and release of 5-HT can
be obtained.
In this work we consider the reciprocal of the slope, indicated
as slope1, as the parameter that is affected by the reuptake rate
and any other mechanism that removes 5-HT. The higher the
removal rate the smaller the slope1. In our experimental
protocol the only factor affecting slope1 that may change is the
uptake rate of the different tissues. Thus, we infer that changes in
slope1 would indicate changes in uptake rate. To provide
a justification for using the slope as a marker of the uptake rate,
in the ESI† we present the results of a Brownian Dynamics model
which has been applied to study other signalling processes.28–30
The intercept, I0, and slope1 can be used together to obtain
information about the release rate. This is possible if it is
assumed that the 5-HT is produced at a zero-order reaction rate
and if the exponential decay (Fig. 2B) measured from the
distance range we used is representative of the 5-HT concentration at the tissue. The assumption of a zero-order reaction
allowed us to express the 5-HT flux, J, from the tissue as Fick’s
law:
vC J ¼ D (1)
vz z¼0
where D is the diffusivity of 5-HT, C the 5-HT concentration and
the z the distance from the tissue. Whereas the assumption that
the concentration close to the tissue follows the same exponential
decay found with the current measurements allowed us to estimate the first derivative of C in z, right-hand side of eqn (1), and
This journal is ª The Royal Society of Chemistry 2010
then express the flux, J, as proportional to I0/slope1. Given the
two above assumptions, the exponential behaviour of the data
can be utilised to characterise alterations in neurotransmitter
release. A similar approach is used in the self-referencing, ionselective probe technique, measuring local extracellular ion
gradients;31,32 in this technique, however, only the net ion flux can
be derived, while in our approach, most importantly, information regarding the uptake mechanism can be obtained as well.
This is due to the fact that release and uptake in the gut consist of
two different mechanisms, as highlighted in the Introduction.
The assumption that release is a zero-order reaction, while the
uptake is a first-order reaction (see ESI†), allows us to characterise separately the two mechanisms from the measurements.
Thus the reciprocal of the slope, slope1, and the ratio, I0/
slope1, will be utilised to study differences between ileum and
colon tissue and the influence of the SERT-inhibitor fluoxetine.
However, these parameters will not be used to quantify specific
uptake or release rates, since this would require precise knowledge of other quantities, such as the full geometry of the experimental apparatus and convection.
Characterisation of factors influencing the slope and intercept
Responses from ileum and colon tissue obtained from a wide
range of tissue–electrode (1100 to 70 mm) distances are shown in
Fig. 3. The exponential responses are shown in Fig. 3A, where
the colon tissue is shown to have a steeper decay in the current
over the distance range than the ileum tissue. However, when the
log of the current is obtained as in Fig. 3B, this variation in the
slope seems to be less evident. In order to obtain accurate
comparative responses between tissues, we need to sample at
tissue–electrode distances which provide a uniform response of
the tissue, rather than sampling from distances that are influenced by experimental conditions. For these reasons the wide
tissue–electrode range was subjected to error analysis to investigate aspects of the experimental protocol which may influence
the overall response.
Fig. 2 Analytical methodology to determine release and uptake
responses from experimental data: (A) a typical current trace at various
tissue–electrode distances on the ileum tissue where the numbers are in
millimetres, whereas in (B), the difference in current from the bulk media
and that from a particular tissue–electrode distance is plotted as a function of that tissue–electrode distance, and finally this response in (B) is
plotted again in (C) where the natural log (ln) of the current is plotted
versus the electrode–tissue distance. From the linear fit, the slope is
utilized to provide information on the uptake and the ratio of the
intercept to slope is utilized to understand the amount of 5-HT released.
This journal is ª The Royal Society of Chemistry 2010
Fig. 3 Responses obtained from ileum and colon tissue over a wide
electrode–tissue distance range. In (A) the current versus electrode–tissue
distance plots for ileum and colon tissue are shown and in (B) the ln of the
current is shown for both ileum and colon tissue. The grey boxes shown in
(B) indicate, from experimental analysis, where experimental error is
observed. At low tissue–electrode distances, variation in tissue topology
introduces error and at larger tissue–electrode distances, the analytical
signal is at the limit of detection, relative to the noise signal, inducing high
error on the measurement. Results expressed as mean SD, n ¼ 5.
Analyst, 2010, 135, 2340–2347 | 2343
At the poles of the measurement (distances less than 100 mm
and greater than 750 mm from the tissue surface), the variation in
the current observed for both tissue types is high, as shown in
Fig. 3B. The grey boxes indicate regions where the error has
a major influence on the slope and intercept as explained in the
following paragraphs.
At tissue–electrode distances greater than 750 mm (percentage
error of mean values was greater than 36% in both tissues), the
current levels observed are low and prone to high error and
variability. In particular, in the colon at distances greater than
750 mm, the analytical signal is at the limit of detection, relative
to the background signal. Therefore the upper limit for accurate
measurements of the current is chosen at a tissue–electrode
distance less 750 mm, where a uniform behaviour of the tissue is
observed.
At tissue–electrode distances less than 120 mm (where the
percentage error of mean values was greater than 14% in both
tissues), the error in the response is influenced by the topology of
the tissue due to the random locations of EC cells and 5-HT
transporters. This variation is high as villi are randomly
distributed on a scale of 100 mm in length and the EC cell
distribution along the villi is varied as EC cells are constantly
being formed and migrate to the tip of the villi.33 We are interested in characterising uptake and release as quantities of the
whole tissue; therefore, we have chosen 150 mm as a lower limit
distance for our measurements, as this provides a uniform
response from the tissue.
For these reasons, we will estimate the slope and intercept
using current measurements of tissue responses taken from
distances between 120 and 750 mm, where the maximum
percentage error for both tissue types was 10%. These measurements would reflect the uniform properties of the tissue that we
are interested in characterising, and allow a means to study
alterations during the application of therapeutic agents.
Responses from low tissue–electrode distances
By recording from a specific tissue–electrode distance range
(120–750 mm), we can obtain a uniform response for uptake and
release, but we need to confirm if these measurements are
reflective of the tissue behaviour. For this purpose, we took
multiple measurements in one tissue section near the tissue
surface (tissue–electrode distances of 5–50 mm) and compared
responses against measurements taken within the proposed range
(120–750 mm). Fig. 4 shows responses at closer distances taken
from 12 locations over ileum and colon tissue. The locations
where these recordings were carried out from are shown in
Fig. 4A and B, for ileum and colon tissue respectively.
Measurements were taken at locations corresponding to random
topology of the tissue and the average response from all these
regions was recorded. In Fig. 4C, the slope1 data from close and
far tissue–electrode distances are reported, showing no significant differences for both the ileum and colon. However, there is
a significant increase in the variance (p < 0.01) of measurements
carried out between 5 and 50 mm in both tissue types. This is
a reflection on the topological variation observed in the tissue. In
Fig. 4D the ratio, I0/slope1, is shown for measurements near and
far. Once again there is no significant difference in the responses
observed at tissue–electrode distances near and far from both
2344 | Analyst, 2010, 135, 2340–2347
Fig. 4 Differences between near (5–50 mm) and chosen (120–750 mm)
electrode–tissue distances range. (A and B) Images of the ileum and colon
tissue respectively. The black dots show the location where measurements
are carried out at tissue–electrode distances between 5 and 50 mm. Scale
bar in (A) is the same for (B). In (C) a bar graph of the slope1 for ileum
and colon tissue is shown where the average response of electrode–tissue
distances between 5 and 50 mm is compared to that from 120 to 750 mm. A
similar comparison of near and far tissue–electrode distances bar graph is
shown in (D), where the I0/slope1 response is shown. Electrode–tissue
distances between 5 and 50 mm carried out from 12 different locations,
whilst electrode–tissue distances between 120 and 750 mm carried out
from 5 different animals.
tissue types. However, there is significant increase in the variation between near and far electrode–tissue distances in the ileum
(p < 0.05).
Overall, in both the slope1 and ratio, I0/slope1, there is no
difference between measurements taken very close to the tissue
over multiple points and those from the tissue–electrode distance
range chosen to reflect on the uniform behaviour of the tissue.
This indicates that measurements in the tissue–electrode distance
range of 120 to 750 mm do provide a good estimation of
a uniform behaviour of 5-HT release and uptake from a tissue
section and that these measurements can be utilised to compare
tissue types.
Variations between ileum and colon tissue
Based upon the analysis of error, only the current values
measured from the tissue–electrode distances in the range of 120
to 750 mm were analysed for ileum and colon tissue. Experimental responses are shown for both tissue types in Fig. 5A.
Analysed responses for the ileum and colon tissue are shown in
Fig. 5B. Within the range of tissue–electrode distances chosen,
there is a significant difference in the slopes between the ileum
and colon than observed over the larger distance range shown in
Fig. 3B. Moreover, the intercept for both the ileum and colon is
similar for the shorter 141 to 707 mm distance range.
Fig. 6 reports bar graphs showing the response from multiple
experiments on ileum and colon tissue. When looking at the
values of slope1, there is a significant difference between the
ileum and colon tissue (p < 0.001, n ¼ 4). In ileum, slope1 is
302.1 23.8 mm, however, in the colon this decreases to 171.7 This journal is ª The Royal Society of Chemistry 2010
Fig. 5 Responses in the presence and absence of fluoxetine from ileum
and colon tissue. A representative experimental trace from ileum and
colon tissue is shown in (A), where the numerical values represent
distances in millimetres. The response from ileum and colon tissues is
shown in (B). ln of the current versus tissue–electrode distance plots for
ileum and colon tissue in the presence of 500 nM and 1 mM fluoxetine are
shown in (C) and (D) respectively. Results expressed as mean SD,
n ¼ 4.
18.1 mm. This decrease in the slope1 in the colon indicates that
there is a higher uptake rate, which may be due to either an
increase in the density or the affinity of SERT. 5-HT availability
is well known to have an influence on motility and as 5-HT
uptake varies between ileum and colon, the implications on
motility patterns and activity may be different.34
When looking at the ratio, I0/slope1, there is a significant
increase in the amount of 5-HT released from the colon (p < 0.05,
n ¼ 4) in comparison to the ileum. The ratio, I0/slope1, in the
ileum is 3.97 0.63 pA mm1, whilst in the colon this value is 8.30
2.78 pA mm1. There is also a greater variation in the colon,
which may be due to altered 5-HT levels (either due to EC cell
number or release) in patches of the colon. This allowed the
variations in rates of transit over the tissue, which is important to
the colon to allow for maximum water adsorption from the
bowel.35,36
Results indicate that 5-HT release is greater in the colon than
the ileum; however, as the reuptake rate is far greater in the
colon, current responses at particular tissue–electrode distances
are lower in the colon. This indicates that lower extracellular
levels of 5-HT are observed in the colon. This is an important
enhancement in our knowledge of the two tissue regions as
alterations in the amount of 5-HT released and activity of
reuptake transporters have been implicated during the onset of
GI diseases such as irritable bowel syndrome and ulcerative
colitis.11–13 Applying this method based upon our understanding
of the uptake and release of 5-HT from normal tissue to diseased
tissue will help unveiling which mechanism in the transmission
process is altered.
Influence of reuptake inhibitor
Fig. 6 Changes in uptake and release from ileum and colon tissue. In (A)
responses from the slope1 and (B) responses of the I0/slope1 ratio are
shown for the ileum and colon tissues in the presence of 500 nM and 1 mM
fluoxetine. Data are shown as mean SD, n ¼ 4, *p < 0.05, **p < 0.01
and ***p < 0.001.
This journal is ª The Royal Society of Chemistry 2010
In the presence of the SSRI, fluoxetine, there should be
a decrease or loss in the clearance of 5-HT from extracellular
space following release. In Fig. 5C and D, the response of the
ileum and colon tissue in the presence of 500 nM and 1 mM
fluoxetine is shown, respectively. For both the ileum and the
colon tissue there is a change in the slope and each response in the
presence of fluoxetine is shifted to a higher current value. An
increase in the value of the intercept is observed for the ileum
tissue in the presence of both concentration of fluoxetine
(Fig. 5C), however, only slight increase in the intercept in the
presence of fluoxetine is observed in the colon (Fig. 5D).
The overall data from responses in the presence of 500 nM and
1 mM fluoxetine are shown in Fig. 6. In Fig. 6A, the slope1 shows
a similar trend in the ileum and colon. The value of slope1 in the
ileum in buffer is 302.1 23.8 mm, which significantly increases
to 364.0 13.7 mm in the presence of 500 nM fluoxetine (p < 0.01,
n ¼ 4) and significantly further increases to 542.5 38.0 mm in
the presence of 1 mM fluoxetine (p < 0.01, n ¼ 4). There is also
significant increase in slope1 obtained from tissue treated with
500 nM to 1 mM fluoxetine (p < 0.01, n ¼ 4). A similar trend is
observed in the colon, where slope1 is 171.7 18.1 mm during
control measurements and significantly increases to 337.66 25.7 mm in the presence of 500 nM fluoxetine (p < 0.01, n ¼ 4) and
significantly further increases to 489.3 30.1 mm in the presence
of 1 mM fluoxetine (p < 0.001, n ¼ 4). A significant increase in the
value of slope1 between 500 nM and 1 mM fluoxetine (p < 0.001,
n ¼ 4) is also observed during measurements on colon tissue. As
Analyst, 2010, 135, 2340–2347 | 2345
expected, in general the values of slope1 increase in the presence
of fluoxetine as the clearance of 5-HT is decreased. Moreover,
there are no significant differences in the value of the slope1 for
a given concentration of fluoxetine in either tissue types, indicating that the influence of the drug is similar in both the ileum
and colon. This may suggest that the densities of SERT are
similar as the influence of the two drug concentrations is similar
in both tissues. This would implicate that differences in uptake
observed between control ileum and colon tissue are most likely
due to a variation in the rate of SERT for 5-HT in the two tissue
types.
When looking at the values of the ratio, I0/slope1, which is
a marker of the 5-HT release, in the presence of fluoxetine
(Fig. 6B), there is a varying trend between the ileum and colon. In
measurements on ileum tissue, no significant differences were
observed between control measurements and those in the presence of fluoxetine. The value for I0/slope1 is 3.97 0.63 pA
mm1, which increased to 4.30 0.3 pA mm1 (p ¼ 0.52, n ¼ 4) in
the presence of 500 nM fluoxetine and decreases to 3.14 0.35
pA mm1 in the presence of 1 mM fluoxetine (p ¼ 0.13, n ¼ 4).
These results are as expected, as fluoxetine is known to block the
reuptake of 5-HT, but should have no influence on the release of
5-HT. Nevertheless, there was a significant difference in the I0/
slope1 between the two concentrations of fluoxetine in the ileum
(p < 0.05, n ¼ 4).
For the colon tissue a different trend is observed, as shown in
Fig. 6B. A significant decrease was observed in the response of
the ratio, I0/slope1, between the control value (8.3 2.78 pA
mm1) and that in the presence of 500 nM fluoxetine (4.26 0.29
pA mm1; p < 0.05, n ¼ 4). There was a greater significant
decrease in the I0/slope1 ratio between control response and that
of 1 mM fluoxetine (2.79 0.2 pA mm1; p < 0.01, n ¼ 4). There is
also a significant difference in the ratio, I0/slope1, between the
two concentrations of fluoxetine (p < 0.001, n ¼ 4).
These results indicate that there is a difference in the effect of
fluoxetine on the amount of 5-HT released from the ileum and
colon. The I0/slope1 decreases in the colon in the presence of 1
mM fluoxetine together with the decrease in reuptake rate,
therefore a feedback mechanism must be present to regulate
extracellular 5-HT levels. One possible explanation for this is
that following the accumulation of 5-HT, autoreceptors on the
EC cells are activated, which decrease the release rate of 5-HT in
order to keep concentration close to the tissue constant, thus
acting as an effective feedback. Autoreceptors for 5-HT have
been identified to be present in the GI tract,37 but limited
investigations have been conducted to understand their role in
the ileum and colon.
Overall new findings in the transmission process of 5-HT
between the ileum and colon have been identified using these new
experimental approaches. This method shows the potential to
study the efficacy of SERT inhibitors or other pharmacological
agents known to influence the mechanisms involved in 5-HT.
Conclusions
The application of this new multiple step electroanalytical technique has provided a means to understand the changes in uptake
and release from steady-state release. Measurements are conducted at various fixed distances over the tissue and the log of the
2346 | Analyst, 2010, 135, 2340–2347
current is plotted as a function of electrode–tissue distance. From
the linear fit, the reciprocal of the slope, slope1, is a marker of
reuptake, and the ratio between the intercept and the reciprocal
of the slope, I0/slope1, is a marker of the amount of 5-HT
released. From biological measurements, a higher reuptake rate
was observed in the colon with respect to the ileum, as well as
a higher release of 5-HT. The effect of the SERT inhibitor
fluoxetine varied in the two tissue types. Fluoxetine inhibited
reuptake of 5-HT in a similar fashion in the ileum and colon,
however, a feedback mechanism was observed in the colon, as the
amount of 5-HT released decreased in the presence of fluoxetine.
The application of this method can provide a means to understand changes in uptake and release and can thus provide indications in the changes observed during GI diseases or in the
presence of various pharmacological agents.
Acknowledgements
BAP would like to thank Prof. Greg Swain (Michigan State
University) for the kind donation of the boron-doped diamond
electrodes used within this study and scientific input. BAP
acknowledges support provided by an EPSRC LSI postdoctoral
research fellowship grant (EP/C532058/1).
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