- Email: [email protected]
The influence of enteral obestatin administration to suckling rats on intestinal contractility
Accepted Manuscript The influence of enteral obestatin administration to suckling rats on intestinal contractility M. Słupecka, P. Grzesiak, J. Kwiatkowski, M. Gajewska, A.Kuwahara, I. Kato, J. Woliński PII: DOI: Reference:
S0016-6480(17)30125-9 http://dx.doi.org/10.1016/j.ygcen.2017.02.006 YGCEN 12587
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
14 August 2016 24 January 2017 13 February 2017
Please cite this article as: Słupecka, M., Grzesiak, P., Kwiatkowski, J., Gajewska, M., A.Kuwahara, Kato, I., Woliński, J., The influence of enteral obestatin administration to suckling rats on intestinal contractility, General and Comparative Endocrinology (2017), doi: http://dx.doi.org/10.1016/j.ygcen.2017.02.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
The influence of enteral obestatin administration to suckling rats on intestinal
2
contractility
3 4
M.Słupecka1*, P. Grzesiak1, J. Kwiatkowski1, M. Gajewska2, A.Kuwahara3, I. Kato 4,
5
J.Woliński1
6
1
7
Academy of Sciences, Jabłonna, Poland
8
2
9
Sciences Warsaw, Poland.
Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life
10
3
11
Nutritional and Enviromental Science, University of Shizuoka, Japan
12
4
Laboratory of Physiology, Institute for Environmental Sciences and Graduate School of
Department of Medical Biochemistry, Kobe Pharmaceutical University, Japan
13 14 15
Running head: Obestatin affects on intestinal contractility in rats
16 17
*Corresponding author: Monika Słupecka, PhD
18
Department of Endocrinology,
19
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences
20
Instytucka 3, 05-110 Jabłonna
21
E-mail: [email protected]
22
Phone: +48 22 765 33 18
23
Fax: +48 22 765 33 00
24 25 26 1
27
Abstract
28
This study investigated the effect of enteral administration of obestatin on the contractility of
29
whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology
30
of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated
31
with: C- saline solution; LO- obestatin (125 nmol/kg b.wt); HO-obestatin (250 nmol/kg b.wt).
32
Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life.
33
Sections were studied in an organ bath, for isometric recording in the presence of
34
acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal
35
muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin
36
sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was
37
performed in the intestinal segments.
38
In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction
39
compared to that observed in the C group. In the middle jejunum, the LO treatment also
40
decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous
41
contraction in the jejunum of LO and HO-treated animals. Compared to the C group,
42
duodenal sections from HO animals and middle jejunum sections from LO and HO groups
43
displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in
44
the thickness of the muscularis layer was observed in the duodenum of LO and HO groups
45
whereas the number ICC did not change significantly after treatment with obestatin.
46
Moreover, the enteral administration of obestatin did not effect significantly on the
47
cytoplasmic expression of M2 receptor in the jejunum.
48
Our study demonstrated that enteral administration of obestatin to suckling rats influences
49
small intestine contractility in the segment specific manner.
50 51 52 2
53
1.Introduction
54
Obestatin is a 23-amino acid peptide which was first identified in the rat stomach as a ghrelin-
55
accompanying peptide generated during the posttranslational processing of preproghrelin
56
(polypeptide precursor of ghrelin) (Zhang et al., 2005). At present, the functional receptor for
57
obestatin is unknown. The initially proposed G-protein-coupled receptor GPR39, has been
58
questioned due to a series of studies which failed to demonstrate the ability of obestatin to
59
bind to and activate this receptor (Lauwers et al., 2006; Chartrel et al., 2007; Host et al.,
60
2007). Studies by Zhang et al. (2005) reported that obestatin behaves as a physiological
61
opponent to ghrelin and inhibits food intake, body weight gain, gastric emptying and jejunal
62
contractility. With respect to gastrointestinal tract (GIT) motility, several studies were
63
undertaken to elucidate the effects of obestatin on GIT contractility and transit time in
64
rodents. The first study by Zhang et al. (2005) reported that peripheral injection of obestatin
65
decreases gastric emptying and contractile activity of jejunal muscular strips in vitro. Since
66
then, the inhibitory effects of obestatin on gastrointestinal motility have remained
67
controversial. For example, Gourcerol et al. (2006) reported that obestatin injected
68
peripherally, either alone or in combination with a peripheral injection of CCK, did not
69
influence gastric motor function in fasted rats and mice. Similarly, Bassil et al. (2007) and De
70
Smet et al. (2007) showed that in adult rats obestatin neither inhibited nor promoted GIT
71
motility, either in vitro or in vivo. However, Ataka et al. (2008) reported the inhibitory action
72
of obestatin given IV on the motor activity in the antrum and duodenum of conscious rats in
73
the fed state. These contrasting results may be partially explained by the different
74
experimental conditions, specifically the duration of monitoring, the route of administration
75
of obestatin and the state of feeding and consciousness of the animals.
76
These abovementioned results on GIT motility were obtained using adult mice and rats. A
77
recent study by our group (2014) has shown that the effect of obestatin on intestinal
3
78
contractility is both dependent on the age of the animals and the segment of intestine studied.
79
In suckling rats we observed a significant effect of obestatin administration on the
80
contractility of full thickness intestinal wall segments. Interestingly, in the duodenum of 14
81
and 21 day old rats, treatment with obestatin significantly decreased responsiveness to all
82
doses of Ach, while in the middle jejunum, the opposite effect was observed in rats from 7 to
83
21 days old. In both intestinal segments the obestatin effect was fully abolished by atropine,
84
indicating that the obestatin effect is coupled with a cholinergic pathway (Słupecka et al.,
85
2014). Intestinal sensitivity to obestatin in suckling rats seems to be physiologically
86
reasonable as substantial amounts of obestatin have been found in human and rat milk.
87
Moreover, the presence of obestatin immunoreactive (IR) cells was shown in the GIT of
88
newborn rats starting from the 1st day of life (20). These findings strongly support the
89
importance of obestatin (both endogenous and exogenous) in the regulation of gastrointestinal
90
function in neonates. Therefore, it would be intriguing to investigate the effect of enteral
91
administration of obestatin on intestinal motor function and the morphology of the enteric
92
nervous system (ENS) in suckling rat neonates.
93 94 95
2. Materials and Methods
96
The experiments and treatments were conducted in compliance with the European Union
97
regulations concerning the protection of experimental animals (EC Directive 86/609/EEC
98
with amendments). The study protocol was approved by the 3rd Local Ethics Committee in
99
Warsaw, according to the Polish Law for the Care and Use of Animals (Resolution no
100
50/2012).
101 102
2.1. Chemicals
4
103
Rat obestatin was synthesized at the Yanaihara Institute using a solid phase method with an
104
Fmoc-strategy and an automated peptide synthesizer (Applied Biosystem 9030 Pioneer,
105
Foster, CA, USA). Analytical HPLC and MALDI-TOF MS confirmed the homology of the
106
product.
107
The hormone was kept in powder form at -20 ˚C and then dissolved in saline solution (0.9%
108
NaCl) to the final concentration, just before use. Acetylcholine chloride and atropine were
109
purchased from Sigma–Aldrich (Germany). Tetrodotoxin (TTX) was purchased from Abcam
110
(Great Britain).
111 112
2.2. Animals
113
At the start of the experiment 12 male and 12 female Wistar Han rats (13 weeks old) were
114
obtained from the Center of Experimental Medicine at the Medical University of Bialystok
115
and the rats were mated. After mating, females were separated from males and they were
116
allowed commercial rat breeding chow (5% fat; 3.1 kcal/g, Wytwornia Pasz Morawski,
117
Poland) and water ad libitum in a humidity- and temperature-controlled room on a 12-h:12-h
118
light:dark cycle. Twenty-four hours after delivery litters were standardized to 10 pups. On the
119
14th day of life the rat pups were randomly assigned to one of the 3 treatment groups (݊ = 12
120
for each group): C- control animals- treated with saline solution; LO- pups treated with a
121
lower dose of obestatin at 125 nmol/kg b.wt.; HO-pups treated with a high dose of obestatin
122
at a 250 nmol/kg b.wt. Both pharmacological doses of obestatin were constructed based on
123
the previous in vitro studies on intestinal contractility in rats (De Smet et al., 2007; Słupecka
124
et al., 2014). Prior to euthanasia, all pups were housed with their mothers and breast-fed ad
125
libitum. Saline solution or obestatin were administered enterally via oral gavage twice a day
126
staring on the 14th day of life until the 21 st day of life. On the 21 st day of life (30 min after the
5
127
morning NaCl/obestatin treatment) three rat pups from each litter were euthanized by CO2.
128
The remaining rats were kept for use in another experiment.
129 130
2.3 Study design for in vitro contractility
131
Upon euthanasia, duodenal and middle jejunum segments (15 mm long) were excised
132
promptly from the rats and immediately placed in cold Krebs–Henseleit buffer (in mM: NaCl
133
18, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, NaHCO3 25, glucose 11). The segments
134
were then placed vertically in 25 ml organ bath chambers (Letica Scientific Instruments,
135
Spain) that were filled with Krebs–Henseleit solution (37 ˚C, pH 7.4) and continuously
136
saturated with carbogen (95% O2, 5% CO2). The intestinal segments were attached to
137
isometric transducers (Letica Scientific Instruments, Spain) under a load of 0.5 g. The
138
transducers were coupled with a PowerLab recording system (ADInstruments, Sydney,
139
Australia). The tissues were allowed to equilibrate for 30 min (the solution in the chambers
140
was changed once after 15 min) to regain spontaneous activity. The segments were then
141
subjected to a procedure which started with the addition of ACh 10 -5 M to assess the viability
142
of the preparations. ACh was left in the solution for 1 min, after which the tissues were
143
washed and allowed to equilibrate. Next, spontaneous or ACh-stimulated contractility was
144
recorded. ACh-stimulated contractility was recorded as the response to ACh 10 -5 M. In some
145
experiments, before ACh was added, jejunal strips were pre-treated with atropine (ATR, 10 -5
146
M). The neural contractions were also examined by studying the tissue response to electrical
147
field stimulation (EFS). After equilibration, the electrical field stimulation (EXP-ST-01,
148
Experimetria, Budapest, Hungary) was performed (voltage 90 V, duration 10 s) at three
149
frequencies: 0.5, 5 and 50 Hz with 1 min intervals between each pulse. EFS parameters were
150
chosen based on previous studies performed on rat whole-thickness intestinal preparations
151
[9,11]. In some experiments jejunal segments were pretreated with tetradotoxin (TTX, 10 -5
6
152
M). Each experiment was completed by the administration of ACh 10-5 M in order to check
153
the viability of the tissue, followed by isoproterenol (10-5 M) in order to control its relaxation.
154 155
2.4 Immunostaining of Cajal cells
156
The duodenal and middle jejunum segments (15 mm long) were transected and samples of
157
each section were collected and immediately fixed in a 10% neutral formalin solution. After
158
the 24h fixation period, the intestinal samples were routinely embedded in paraffin. The
159
paraffin-embedded samples were cut into 4.5 µm sections and applied to silane-treated glass
160
slides. Next, the sections were dewaxed in xylene and rehydrated in decreasing grades of
161
ethanol and then washed in PBS buffer and then the antigen retrieval was performed by 20
162
min microwave heating (500 W) in citrate buffer. For the immunostaining of the Cajal cells
163
the antibodies for c-kit were applied. The sections were incubated with rabbit c-kit polyclonal
164
antibodies (Abcam, UK), 50 times diluted in 1% BSA-PBS. After 0.5 h incubation performed
165
in a humidified chamber at room temperature, the slides were washed in PBS buffer and then
166
incubated with goat anti-rabbit secondary FITC-conjugated antibodies, 2000 times diluted in
167
PBS buffer. After a 1 h incubation period performed in a dark humidified chamber at room
168
temperature, the slides were washed in PBS. For the nuclei staining, the sections were
169
immersed in a 1% solution of 7-AAD for 25 min at room temperature, under dark conditions.
170
Control immunostaining was carried out using the same procedures, except for the
171
replacement of the primary antibodies with PBS. C-kit positive cells were observed using
172
400x total magnification and their number was counted across either submucosal surface of
173
the circular muscle layer (ICC-SM), within circular muscle (ICC-IM) or between circular and
174
longitudinal muscle layer (ICC-MY) were counted in the field of view (0.4 mm). Ten
175
randomly selected fields of view were observed from each slide. C-kit positive cells were
176
analyzed according to their localization.
7
177 178
2.5 Histology of the intestinal muscularis layer
179
For histometrical studies, slides were prepared as described above and then stained with
180
hematoxylin and eosin. Three slides were randomly selected for each of the studied intestinal
181
sections and then 30 measurements of the muscularis layer were performed using a light
182
microscope (Axioskop 40, Zeiss, Germany), coupled with computer software for image
183
analysis (Axio Vision 4.2 Release, Zeiss, Germany) and a digital camera.
184
2.6 Immunodetection of the Muscarinic Acetocholine receptor 2 (M2 receptor)
185
For the immunostaining of M2 receptors the duodenal and middle jejunum samples were
186
prepared as described in section 2.4. The sections were incubated with rabbit polyclonal
187
antibody for cytoplasmic domain of Muscarinic Acetylocholine Receptor 2 (Abcam, Great
188
Britain) 250 times diluted in 1% BSA-PBS. After 24 h incubation performed in a humidified
189
chamber at 40C, the slides were incubated with goat anti-rabbit secondary FITC-conjugated
190
antibodies, 2000 times diluted in PBS buffer (Abcam, Great Britain). For the nuclei staining,
191
the sections were immersed in 7-AAD.
192
The same primary antibody was used for immunoblotting of M2 receptors in the
193
segments of jejunum (middle part). Dissected out jejunum segments were stored in -800C
194
until analysis. Then, they were thawed and about 0.5 g of intestinal samples from each
195
experimental group were homogenized in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1
196
mM EDTA, 1% NP-40, 0.25% Na-deoxycholate and 1 mM PMSF) supplemented with
197
protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Then the
198
samples were left on ice for 30 min, during which the process of cell lysis took place. Lysates
199
were cleared for 30 min at 14000 rpm, and supernatants containing extracted proteins were
200
collected. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent
201
according to the producer’s instructions (Bio-Rad Laboratories Inc., Hercules, CA,, USA).
8
202
Proteins (50 µg) were resolved by SDS-PAGE and transferred onto PVDF membrane (Sigma-
203
Aldrich). For immunostaining membranes were blocked with 5% nonfat dry milk in TBST
204
(20 mM Tris-HCL, 500 mM NaCl, 0.5% Tween 20). The membranes were incubated with
205
antibodies against M2 receptor (Abcam, cat. no: ab188891; 1:250 dilution), or β-actin (Santa
206
Cruz Biotechologies, Inc., cat. no: sc-47778, 1:1000 dilution) at 4oC overnight. Next, the
207
membranes were washed three times for 15 min and incubated with appropriate secondary
208
antibodies conjugated with IR fluorophores: IRDye® 680 or IRDye® 800 CW (at 1:5000
209
dilution). After incubation the membranes were washed three times in TBST. Subsequently
210
the protein expression was analyzed using Odyssey Infrared Imaging System (LI-COR
211
Biosciences). Scan resolution of the instrument was set at 169 µm, and the intensity at 4.
212
Quantification of the integrated optical density (IOD) was performed with the analysis
213
software provided with the Odyssey scanner (LI-COR Biosciences). Results were expressed
214
as a M2 receptor ratio to β-actin densitometry units. For the purpose of publication the color
215
immunoblot images were converted into black and white images in the Odyssey software.
216 217
2.7. Statistics
218
The data are expressed as means ± SEM (standard error of the mean). A one-way ANOVA
219
followed by a Tukey-Kramer post-hoc test, Kruskal-Wallis test followed by a Dunn’s
220
Multiple Comparison post-hoc test, Unpaired t-test or Mann-Whitney test were used to
221
determine statistically significant differences between the groups (Prism 6 for Mac OS X,
222
Version 6.0h, Graph Pad Software, San Diego, CA, USA). The level of significance p<0.05
223
was assumed in all statistical analyses.
224
3. Results
225
3.1.Effect of obestatin on spontaneous contractility
9
226
The amplitude and frequency of contraction was analyzed in order to investigate the effects of
227
obestatin on spontaneous intestinal contractility (Table 1). In both intestinal segments
228
treatment with obestatin significantly decreased the amplitude of contraction in the HO group
229
compared to the control animals (C) (Table 1A). In the middle jejunum, the lower dose of
230
obestatin also significantly decreased the amplitude of contraction (P ˂ 0.031). Only in the
231
middle jejunum of LO animals the frequency of contraction was significantly affected in
232
comparison to the control animals (P ≤ 0.05), (C). Injection of TTX had no effect on the
233
amplitude of spontaneous contraction in the duodenum and middle jejunum of animals treated
234
with obestatin (LO and HO). In the duodenum, pretreatment with TTX significantly increased
235
frequency of contraction in all treatment groups studied (Table 1B). Atropine significantly
236
decreased the amplitude of duodenal contractions only in the group of control animals (C),
237
(Table 1A).
238 239
3.2 Effect of obestatin on ACh-stimulated contractility
240
After the injection of ACh 10-5, the amplitude and frequency of contraction increased in all
241
intestinal segments studied. Compared to the control animals (C), the duodenal sections from
242
the HO animals and the middle jejunum sections from both obestatin treated groups (LO and
243
HO) had significantly lower amplitudes of Ach-evoked contraction. However, treatment with
244
higher doses of obestatin significantly increased the frequency of ACh stimulated contraction
245
in both the intestinal segments studied (Fig.1 and Fig.2).
246 247
3.3 Effect of obestatin on electrical field stimulation
248
The amplitude of contraction after EFS impulses (0.5, 5, 50 Hz) was analyzed in order to
249
investigate the effects of obestatin on electrical field stimulation (Fig. 3). Among the groups
250
tested, the duodenum sections from the HO animals had significantly lower responsiveness
10
251
then duodenum sections from control animals, whereas in the middle jejunum a significantly
252
lower responsiveness for rising EFS frequencies was observed in sections from both obestatin
253
treated groups (LO, HO). Moreover, we observed that treatment with TTX and ATR
254
significantly reduced the amplitude of contraction. Following pre-incubation with TTX and
255
ATR the effects of obestatin were still observed in the duodenum sections from LO and HO
256
groups exposed to 50 Hz. Of the three studied frequencies of EFS, the 5 Hz applied after pre-
257
incubation with both blockers did not show any differences between the groups studied (Table
258
2).
259 260
3.4 Thickness of muscularis layer
261
In the duodenum, enteral treatment with both doses of obestatin (LO, HO) increased the
262
thickness of the muscularis layer in comparison to that of the control group (P ˂ 0.0001), (C).
263
In the middle jejunum, treatment with obestatin did not influence the thickness of the
264
muscularis layer (Table 3).
265 266
3.5 Interstitial Cajal cells
267
In order to show the interstitial Cajal cells (ICC) in the intestinal segments studied, we
268
performed immunostaining using a c-kit antibody. The immunostaining showed the presence
269
of ICC in the small intestinal mucosa of all experimental groups (Fig. 4). The c-kit positive
270
cells were observed mostly in the submucosal surface layer of the circular muscle layer (ICC-
271
SM), or between the circular and longitudinal muscle layers (ICC-MY). Only a few cells were
272
observed within the circular muscle layer (ICC-IM, data not shown). The number of ICC,
273
estimated separately in the duodenum and middle jejunum sections for ICC-SM and ICC-MY
274
did not change significantly after enteral treatment with obestatin (Table 4).
275
3.6 Muscarinic Acetylocholine Receptor 2 (M2 receptor)
11
276
The immunostaining showed the presence of M2 receptors in muscularis layers of both
277
studied intestinal segments of all groups. Immunostatinig of the receptors unable to observed
278
any differences in their expression and localization between studied groups (Fig. 5). To obtain
279
quantitative results on the expression of M2 receptors the immunoblotting was performed.
280
The enteral administration of obestatin did not effect significantly on the cytoplasmic
281
expression of M2 receptor in the middle jejunum (Fig. 6).
282 283
4. Discussion
284
Previous results from our lab have shown that the peripheral action of obestatin influences
285
intestinal contractility in rats, in both an age-dependent and intestinal segment specific
286
manner (Słupecka et al., 2014). In particular, we have shown that the small intestine of
287
suckling rats (until 21 day of life) is more sensitive to obestatin than that of adult animals. We
288
speculated that obestatin, of which substantial amounts have been found in rat milk, could be
289
an important factor regulating the function of the gastrointestinal tract, during the early
290
postnatal period. Thus, in the present study we aimed to investigate whether enteral
291
administration of pharmacological doses of obestatin (the same route of administration as if
292
obestatin was entering the intestine with the rat milk) to suckling rats would have an influence
293
on the contractile properties of the small intestine. We found that administration of obestatin
294
for a 7 day period decreased the amplitude of contraction in both the intestinal segments
295
studied. This is an interesting observation as we have previously shown that injection of a
296
single dose of obestatin directly into the organ bath chamber significantly increased the
297
amplitude of spontaneous contraction in the middle jejunum of 14 and 21 day old rats, while a
298
decrease in spontaneous contraction was observed in both the intestinal segments studied in
299
adult rats. This effect may indicate that obestatin, when administered into the stomach (the
300
main source of endogenous obestatin), triggers a different mechanism to that when obestatin 12
301
is injected directly into the intestine or that some of the obestatin is lost during its’ passage
302
through the stomach and duodenum, and the amount of hormone that reaches the middle
303
intestine is insufficient to induce the effects seen in the experiment with obestatin injected
304
directly into the organ bath. It is also possible that unlike with the single injection of
305
obestatin, continuous (7 day long) administration of this peptide may influence either the
306
synthesis of ACh or the sensitivity of the neurons to ACh, especially since it has been shown
307
that during the second and third weeks of life, major changes in cholinergic phenotype and
308
muscular transmission occur in the rat intestine (Matini et al., 1997). This is the more
309
important as nearly all obestatin immunoreactive cells in the myenteric plexus of the rat are
310
acetylotransferase (ChAT) positive. It should be mentioned that the expression of obestatin
311
receptors and/or their sensitivity may be altered after treatment with obestatin. Unfortunately,
312
the lack of discovery of the functional obestatin receptor makes further investigation in this
313
field impossible. Thus, in this study we focused on the possible influence of obestatin
314
administered into the stomach for consecutive days on the morphological changes of the
315
enteric nervous system (ENS). First of all, we decided to study full–thickness intestinal
316
segments instead of only muscle preparations, as previous in vitro studies have revealed that
317
different types of ion channels and receptors, as well as interneurons and non-muscle cells
318
occur along the intestinal wall and may be crucial for the contractile response (Matini et al.,
319
1997; Mondal et al., 2013). Gut motility occurs through the action of the interstitial Cajal cells
320
and smooth muscle layers. Thus, we analyzed both the expression and localization of ICC in
321
the gut wall, together with the thickness of the muscularis layer and the expression of
322
Muscarinic Acetylocholine Receptors 2 which play an essential role in the generation of
323
rhythmic motor activity (Tanahashi et. al., 2013).
324
In the gut wall, the Cajal cells play a double role. They generate the electrical slow
325
waves needed for motility and are required for effective neurotransmission. The morphology 13
326
and localization of the ICC determines their function. In our study we wanted to focus on the
327
ICC-MY as they form a network closely connected with MY neurons and have receptors for
328
neurotransmitters and circulating hormones (e.g. cholecystokinin). We did not observe any
329
changes in the number of these cells between the various treatment groups studied, which
330
brings about the hypothesis that the effects of obestatin on intestinal contraction occur
331
independently from the effect on the number of ICC. This is also confirmed by results with
332
spontaneous contractility, where after blocking neural activity by TTX the basal activity of
333
the jejunum segments did not differ between groups.
334
Interestingly, in duodenum and middle jejunum sections from 21 day old rats we also
335
observed ICC localized in the submucosal surface. So far, these type of ICC have been
336
observed in the canine colon and they are involved in the pace maker activity within the
337
gastrointestinal tract (Wurner et al., 2013). However, studies on ICC-SM structure and
338
functions within the small intestine of rats are very limited and there is no such data for
339
neonatal rats. Even though we did not observe any significant changes in the number of ICC
340
in our study, further studies on these cells could yield interesting observations on their
341
function in the small intestine of suckling animals. In the line of expectations we did not
342
observe Cajal cells in the circular muscle layer (ICC-CM) as they are c-kit negative (Streutker
343
et al., 2007; Chen et al., 2007). Although in the current study we did not observe any
344
significant changes in the number of c-kit positive cells, we did record a significant increase
345
in the thickness of the muscularis layer in the duodenum of pups treated with both doses of
346
obestatin. It is well documented that the hypertrophy of smooth muscle is a physiological
347
response to the increased functional demands placed on an organ. In the small intestine the
348
hypertrophy of the muscle wall has been observed in the intestine of rat dams during
349
pregnancy and lactation, but also in studies on its partial obstruction (Boass et al., 1992;
350
Bertoni et al., 2001).
14
351
To understand the nature of obestatin’s influence on intestinal contractility it is crucial
352
to investigate whether the hypertrophy observed within the duodenum is a primary or
353
secondary effect of administration of the peptide. Taking into account all the results obtained
354
from the current study and previous observations from our lab, where we observed changes in
355
intestinal contractility after a single obestatin injection, we can conclude that obestatin does
356
influence intestinal contractility and this effect is ICC independent. Weaker intestinal
357
contractility influences on nutrient transit resulted in increased availability of nutrients in the
358
intestinal lumen. Intestinal absorption increases and this is reflected in the histomorphometry
359
changes in the intestinal wall, the hypertrophy of the muscle layer and the increased thickness
360
of the mucosa (Slupecka et al, unpublished data). We observed that both the intestinal
361
segments studied from animals treated with obestatin were significantly less responsive to
362
ACh. However, duodenum was less “sensitive” to obestatin than middle jejunum. As we did
363
not observed the effect of obestatin administration neither on its expression (Słupecka et al,
364
unpublished data) nor the expression of Muscarinic Acetylocholine Receptor 2 in the small
365
intestinal wall this weaker responsiveness could be a result of the “dilution” of motor neurons
366
in the hypertrophic duodenal muscular layer. Since the relatively weak responsiveness to
367
electrical field stimulations was observed in both intestinal segments, we could speculate that
368
this is the effect of decreased number of enteric motor neurons or ACh release from these
369
neurons. Further studies are needed to elucidate this mechanism. In this study the frequency
370
of spontaneous contraction was unaffected and sometimes even increased following
371
pretreatment with atropine and TTX. These somewhat surprising observations are a result of
372
using whole thickness intestinal sections. It has been previously shown that both cholinergic
373
and adrenergic inhibitors as well as the neural blocker- TTX, evoke different responses in the
374
characteristics of spontaneous contractions, depending on the region of intestine, as well as
375
the specific part of the intestine wall being investigated (Grasa et al., 2004; Postorino et al.,
15
376
1990). For example in the studies using the rabbit, atropine and TTX decreased the amplitude
377
of spontaneous contraction only in the longitudinal muscles (Grasa et al., 2004), whereas in
378
the rat duodenum pretreatment with TTX, even in the presence of atropine and guanethidine,
379
caused an increase in amplitude and frequency of electrical and mechanical intestinal activity
380
(Postorino et al., 1990). This finding indicates the presence of tonically active inhibitory
381
intramural non adrenergic, non cholinergic (NANC) nerves in the rat duodenum. Moreover,
382
Postorino et al. showed diversity in contractile actions among the intestinal muscle layers. In
383
this study duodenal longitudinal strips showed a spontaneous mechanical activity resembling
384
that one recorded from isolated segment. Instead, circular strips were quiescent under resting
385
condition and a contractile activity was detected only after TTX pretreatment. In the current
386
study, in the duodenum of rats treated with the low dose of obestatin (LO, 125nmol/kg b.wt),
387
after pretreatment with TTX, a significantly higher frequency of contractions was observed
388
compared to that observed in the control group (C). This indicates that in duodenum the
389
obestatin may influences on the tonically active inhibitory intramural nonadrenergic
390
noncholinergic (NANC) nerves.
391
In conclusion, our study demonstrated that enteral stomach administration of obestatin
392
to suckling rats influences the small intestinal contractility. Enteral administration of obestatin
393
to suckling rats effects on cholinergic neurotransmission in the small intestine rather than the
394
network of peacemaker cells of Cajal. In the duodenum, also nonadrenergic noncholinergic
395
(NANC) nerve fibres could possibly be affected by obestatin, but further studies in this field
396
are required.
397 398 399
Grants
400
This research was supported by National Science Center Grant no. 2011/03/D/NZ9/03697.
16
401 402 403 404 405 406
Author contribution M.S. designed the research study, performed the research, analyzed the data and wrote the paper; P.G, J.K and M.G performed the research; I.K. and A.K. contributed essential reagents; J.W. performed the research, analyzed the data and revised the article
407
Acknowledges
408
The authors wish to thank Żaneta Dzięgelewska for her support in the immunoblotting of M2
409
receptors.
410 411
References
412 413 414
Ataka, K., Inui, A., Asakawa, A., Kato, I., Fujimiya, M., 2008. Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. Am J Physiol Gastrointest. Liver Physiol. 294 (5), G1210-1218.
415 416 417
Bassil, A.K., Haglund, Y., Brown, J., Rudholm, T., Hellstrom, P.M., Naslund, E., Lee, K., Sanger, G., 2007. Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract. Br. J. Pharmacol. 150 (1), 58-64.
418 419 420 421 422 423 424 425 426 427
Bertoni, S., Gabella, G., 2001. Hypertrophy of mucosa and serosa in the obstructed intestine of rats. J. Anat. 199 (6), 725-734. Boass, A., Lovdal, J.A., Toverud S.U., 1992. Pregnancy- and lactation-induced changes in active intestinal calcium transport in rats. Am. J. Physiol. 263 (1), G127-134.
428 429 430
Chen, H., Redelman, D., Ro, S., Ward, S.M., Ordog, T., Sanders, K.M., 2007. Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine. Am. J. Physiol. Cell. Physiol. 292 (1), C497-507.
431 432 433
De Smet, B., Thijs, T., Peeters, T.L., Depoortere, I., 2007. Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents. Neurogastroenterol Motil. 19 (3), 211217.
434 435 436
Gourcerol, G., Million, M., Adelson, D.W., Wang, Y., Wang, L., Rivier, J., St-Pierre, D.H., Tache, Y., 2006. Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents. Peptides 27 (11), 2811-2819.
437 438
Grasa, L., Rebollar, E., Arruebo, M.P., Plaza, M.A., Murillo, M.D., 2004. The role of Ca2+ in the contractility of rabbit small intestine in vitro. J. Physiol. Pharmacol. 55 (3), 639-650.
Chartrel, N., Alvear-Perez, R., Leprince, J., Alvear-Perez, R., Leprince, J., Iturrioz, X., Reaux-Le Goazigo, A., Audinot, V., Chomarat, P., Coge, F., Nosjean, O., Rodriguez, M., Galizzi, J.P., Boutin, J.A., Vaudry, H., Llorens-Cortes, C., 2007. Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake". Science 315 (5813), author reply 766.
17
439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458
Holst, B., Egerod, K.L., Schild, E., Vickers, S.P., Cheetham, S., Gerlach, L.O., Storjohann, L., Stidsen, C.E., Jones, R., Beck Sickinger, A.G., Schwartz, T.W., 2007. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 148 (1), 13-20. Korczynski, W., Ceregrzyn, M., Kato, I., Wolinski, J., Zabielski, R., 2006. The effect of orexins on intestinal motility in vitro in fed and fasted rats. J. Physiol. Pharmacol. 57 (Suppl 6), 43-54. Lauwers, E., Landuyt, B., Arckens, L., Schoofs, L., Luyten, W., 2006. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem. Biophys. Res. Commun. 351 (1), 21-25. Matini, P., Mayer, B., Faussone-Pellegrini, MS., 1997. Neurochemical differentiation of rat enteric neurons during pre- and postnatal life. Cell Tissue Res. 288 (1), 11-23. Mondal, A., Aizawa, S., Sakata, I., Goswami, C., Oda, S., Sakai, T., 2013. Mechanism of ghrelin-induced gastric contractions in Suncus murinus (house musk shrew): involvement of intrinsic primary afferent neurons. PLoS One 8 (4), e60365. Postorino, A., Mancinelli, R., Racanicchi, C., Adamo, E.B., Marini, R., 1990. Spontaneous electromechanical activity in the rat duodenum in vitro. Arch. Int. Physio.l Biochim. 98 (1), 35-40.
459 460 461 462 463 464 465
Slupecka, M., Pierzynowski, S.G., Kuwahara, A., Kato, I., Wolinski, J., 2014. Age-dependent effect of obestatin on intestinal contractility in Wistar rats. Gen. Comp. Endocrinol. 208, 109115.
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
Tanahashi, Y., Waki, N., Unno, T., Matsuyama, H., Iino, S., Kitazawa, T., Yamada, M., Komori, S., 2013. Roles of M2 and M3 muscarinic receptors in the generation of rhythmic motor activity in mouse small intestine.Neurogastroenterol Motil. 25 (10), e687-97.
Streutker, C.J., Huizinga, J.D., Driman, D.K., Riddell, R.H., 2007. Interstitial cells of Cajal in health and disease. Part I: normal ICC structure and function with associated motility disorders. Histopathology 50 (2), 176-189.
Wurner, L., Diener, M,. Receptors and mechanisms mediating the biphasic response evoked by bradykinin in rat colonic smooth muscle. Neurogastroenterol. Motil. 25 (9), e581-590. Zhang, J.V., Ren, P.G., Avsian-Kretchmer, O., Luo, C.W., Rauch, R., Klein, C., Hsueh, A.J., 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 310 (5750), 996-999. Zhao, C.M., Furnes, M.W., Stenstrom, B., Kulseng, B., Chen, D, 2008. Characterization of obestatin- and ghrelin- producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study. Cell. Tissue Res. 331 (3), 575-587. .
481 482 483
18
484 485 486
List of tables
487 488 489 490 491
Table 1. Effect of TTX-6M and ATR-6M on the amplitude (mm) (A), and frequency (number of contractions per 1s) (B) of spontaneous contraction according to the small intestinal segment examined and treatments A Duodenum -6 TTX
Basal
ATR
-6
Middle jejunum -6 TTX
Basal **
ATR
-6
C LO
0.64 ± 0.11 0.46 ± 0.20ab
a
0.40 ± 0.22 0.27 ± 0.09
*
0.33 ± 0.13 0.30 ± 0.06
0.51 ± 0.18 0.25 ± 0.04b
a
0.15 ± 0.09 0.15 ± 0.10
***
0.30 ± 0.18 0.19 ± 0.09
HO
0.37 ± 0.17
b
0.36 ± 0.19
0.31 ± 0.12
0.26 ± 0.16
b
0.12 ± 0.09
*
0.24 ± 0.08
P
0.021
0.494
0.919
0.031
0.780
Duodenum -6 TTX
-6
Basal
Middle jejunum -6 TTX
0.386
492 493
B Basal
494 495 496 497 498 499 500
ATR a**
C LO HO
0.86 ± 0.06 1.03 ± 0.07 1.17 ± 0.16
1.34 ± 0.04 * 2.29 ± 0.55b ab* 1.99 ± 0.18
P
0.173
0.011
***
2.34 ± 0.15 * 3.15 ± 0.55 ** 2.63 ± 0.17 0.304
a
ATR
-6
1.07 ± 0.08 ab 1.61 ± 0.17 b 1.76 ± 0.18
3.04 ± 0.54 * 3.74 ± 0.62 3.42 ± 0.85
3.29 ± 0.74 ** 3.94 ± 0.56 * 3.56 ± 0.72
0.034
0.889
0.801
C—saline solution (twice a day), LO- intra stomach obestatin (125 nmol/kg BW, twice a day); HO-intra stomach obestatin (250 nmol/kg BW twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups within type of contraction; *- indicates statistical differences between basal and TTX or ATRblocked contraction, (* P ≤ 0.05, ** P ≤ 0.001, ***P ≤ 0.0001).
501 502 503 504 505 506 507 508
19
509 510 511 512 513
Table 2. The amplitude of EFS-induced off-contraction (mm) (0.5 Hz, 5Hz, 50Hz) in the presence or absence of TTX (10-6M) (A), or ATR (10 6 M) (B) according to small intestinal segment examined and treatments A
C LO HO P
514 515
0.5Hz a 2.18 ± 0.98 a 2.18 ± 1.06 b 1.04 ± 0.76 0.0126
5Hz a 2.48 ± 1.19 ab 2.46 ± 1.06 b 1.62 ± 0.72 0.0233
50Hz a 2.80 ± 1.25 a 2.73 ± 0.78 b 1.20 ± 0.72 0.0036
Duodenum -6 TTX 0.5Hz 5Hz ** ** 0.71 ± 0.40 0.73 ± 0.42 * ** 0.44 ± 0.24 0.52 ± 0.24 * 0.47 ± 0.20 0.52 ± 0.22 0.1740 0.1980
50Hz
Middle jejunum -6 TTX 0.5Hz 5Hz
-6
50Hz a* 1.65 ± 0.64 b*** 0.93 ± 0.50 b 0.63 ± 0.25 0.0010
0.5Hz ** 0.59 ± 0.25 * 0.35 ± 0.10 0.43 ± 0.17 0.0973
ATR 5Hz ** 0.61 ± 0.27 ** 0.43 ± 0.15 * 0.49 ± 0.19 0.3245
0.5Hz
ATR 5Hz
50Hz a* 1.50 ± 0.59 b*** 0.81 ± 0.45 b* 0.57 ± 0.17 0.0026
B 0.5Hz C LO
5Hz
0.95 ± 0.38
a
b
0.25 ± 0.14
b
HO
0.36 ± 0.16
P
0.0005
1.22 ± 0.62
a
b
0.26 ± 0.19
ab
0.59 ± 0.29 0.0013
a
1.80 ± 0.90
b
0.60 ± 0.39
b
0.93 ± 0.33 0.0102
a***
0.29 ± 0.24
0.11 ± 0.06
ab
b***
0.10 ± 0.08 0.0365
0.23 ± 0.16
-6
50Hz ***
0.12 ± 0.07 0.12 ± 0.08 0.1006
***
a*
0.56 ± 0.32
b*
0.15 ± 0.08 0.23 ± 0.15 0.0010
ab**
0.40 ± 0.23
a** b
0.25 ± 0.14 0.33 ± 0.12 0.0490
ab
0.42 ± 0.26
50Hz **
0.16 ± 0.06 0.23 ± 0.10 0.0551
**
1.08 ± 0.50
a
b
0.21 ± 0.07
b***
0.26 ± 0.15 0.0105
516 517 518 519
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups * indicates differences between EFS- induced contraction without blockers and with either TTX or ATR (* P ≤ 0.05, ** P ≤ 0.001, ***P ≤ 0.0001)
20
520 521 522
Table 3. Thickness of muscularis layer (µm) of the small intestine of suckling rats according to small intestinal segment examined and treatments C
523 524 525 526 527 528
LO
HO
P
Duodenum
a
33 ± 7
b
b
38 ± 8
38 ± 9
<0.001
Middle jejunum
30 ± 8
28 ± 7
27 ± 7
0.09
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups
529 530 531 532 533 534 535 536
Table 4. Number of Cajal cells (ICC) distributed across submucosal surface of the circular muscle layer (ICC-SM), or between circular and longitudinal muscle layers (ICC-MY) of the small intestine of suckling rats according to small intestinal segment examined and treatments. C
OL
OH
P
Duodenum ICC-SM
0.50 ± 0.28
0.25 ± 0.25
1.20 ± 0.48
0.2325
ICC-MY
2.16 ± 0.54
2.37 ± 0.32
2.77 ± 0.52
0.6560
P
0.048*
0.002**
0.07
Middle jejunum
537 538 539 540 541 542
ICC-SM
0.0 ± 0.0
1.25 ± 0.75
0.87 ± 0.29
0.2014
ICC-MY
1.33 ± 0.37
1.80 ± 0.80
0.74 ± 0.35
0.3413
P
0.04*
0.639
0.733
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); * indicates statistical difference in ICC number between the locations (* P ≤ 0.05, ** P ≤ 0.001).
543 544 545 546 547
21
548
List of figures
549 550
Fig. 1. Effect of ACh -5M on the amplitude (mm) and frequency (number of contractions per
551
1s) of spontaneous contraction according to the small intestinal segment examined and
552
treatments
553
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
554
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given
555
as means ± SEM (n=12); different superscript letters indicate statistical differences between
556
the groups within type of contraction. *- indicates statistical differences between spontaneous
557
and ACh-evoked contraction.
558 559
Fig. 2. Representative tracing of Ach-evoked contraction in duodenal and middle jejunum
560
segments
561
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
562
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day).
563 564
Fig. 3. Representative tracing of EFS- induced contraction in duodenal and middle jejunum
565
segments
566
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
567
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day).
568 569
Fig. 4. C-kit expression on confocal images in representative cross-sections from the
570
duodenum of suckling rats
22
571
Legend: C—saline solution (twice a day), (A), LO—intra stomach obestatin (125 nmol/kg
572
BW, twice a day), (B); HO—intra stomach obestatin (250 nmol/kg BW, twice a day), (C);
573
Cajal cells (green fluorescence) are pointed by arrows. Nuclei were stained with 7-AAD (red
574
fluorescence)
575 576
Fig. 5. M2 receptors expression on confocal images in representative cross-sections from the
577
duodenum of suckling rats
578
Legend: C—saline solution (twice a day), (A), LO—intra stomach obestatin (125 nmol/kg
579
BW, twice a day), (B); HO—intra stomach obestatin (250 nmol/kg BW, twice a day), (C); M2
580
receptors - green fluorescence.
581 582
Fig. 6. (A) Western blot analysis of M2 receptor expression in the mucosa of middle jejunum
583
segments in suckling rats. (B) Optical density of the M2 receptor β-actin ratio in the study
584
groups.
585
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
586
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day). Results are
587
presented as means ± SEM from 6 replications.
588 589 590 591
23
592 593 594 595
Fig. 1
596 597
24
598 599 600 601
Fig. 2
602 603
25
604 605 606 607
Fig. 3
608 609
26
610 611 612 613
Fig. 4
614 615
27
616 617 618
Fig. 5
619 620
28
621 622 623 624
Fig. 6
625 626 627
29
628
Highlights:
629
Enteral obestatin administration effects on intestinal spontaneous contractility.
630
In suckling rats obestatin influences on response to ACh and EFS-evoked contractions.
631
Number of Cajal cells was unaffected by obestatin administration.
632
The effect of enteral obestatin on intestine contractility is segment specific.
633
30
S0016-6480(17)30125-9 http://dx.doi.org/10.1016/j.ygcen.2017.02.006 YGCEN 12587
To appear in:
General and Comparative Endocrinology
Received Date: Revised Date: Accepted Date:
14 August 2016 24 January 2017 13 February 2017
Please cite this article as: Słupecka, M., Grzesiak, P., Kwiatkowski, J., Gajewska, M., A.Kuwahara, Kato, I., Woliński, J., The influence of enteral obestatin administration to suckling rats on intestinal contractility, General and Comparative Endocrinology (2017), doi: http://dx.doi.org/10.1016/j.ygcen.2017.02.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
The influence of enteral obestatin administration to suckling rats on intestinal
2
contractility
3 4
M.Słupecka1*, P. Grzesiak1, J. Kwiatkowski1, M. Gajewska2, A.Kuwahara3, I. Kato 4,
5
J.Woliński1
6
1
7
Academy of Sciences, Jabłonna, Poland
8
2
9
Sciences Warsaw, Poland.
Department of Endocrinology, The Kielanowski Institute of Animal Physiology and Nutrition, Polish
Department of Physiological Sciences, Faculty of Veterinary Medicine, Warsaw University of Life
10
3
11
Nutritional and Enviromental Science, University of Shizuoka, Japan
12
4
Laboratory of Physiology, Institute for Environmental Sciences and Graduate School of
Department of Medical Biochemistry, Kobe Pharmaceutical University, Japan
13 14 15
Running head: Obestatin affects on intestinal contractility in rats
16 17
*Corresponding author: Monika Słupecka, PhD
18
Department of Endocrinology,
19
The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences
20
Instytucka 3, 05-110 Jabłonna
21
E-mail: [email protected]
22
Phone: +48 22 765 33 18
23
Fax: +48 22 765 33 00
24 25 26 1
27
Abstract
28
This study investigated the effect of enteral administration of obestatin on the contractility of
29
whole-thickness preparations of duodenum and middle jejunum, as well as on the morphology
30
of the enteric nervous system (ENS). Suckling rats were assigned to 3 groups (n=12) treated
31
with: C- saline solution; LO- obestatin (125 nmol/kg b.wt); HO-obestatin (250 nmol/kg b.wt).
32
Saline solution or obestatin were administered twice daily, from the 14th to the 21st day of life.
33
Sections were studied in an organ bath, for isometric recording in the presence of
34
acetylocholine (ACh), atropine (ATR) and tetradotoxin (TTX). Thickness of intestinal
35
muscularis layer, the number of interstitial cells of Cajal (ICC) were measured in the paraffin
36
sections. The immunodetection of Muscarinic Acetylocholine Receptor 2 (M2 receptor) was
37
performed in the intestinal segments.
38
In both intestinal segments HO treatment decreased the amplitude of spontaneous contraction
39
compared to that observed in the C group. In the middle jejunum, the LO treatment also
40
decreased the amplitude. TTX and ATR had no effect on amplitude of spontaneous
41
contraction in the jejunum of LO and HO-treated animals. Compared to the C group,
42
duodenal sections from HO animals and middle jejunum sections from LO and HO groups
43
displayed a lower amplitude in response to ACh and EFS evoked contraction. An increase in
44
the thickness of the muscularis layer was observed in the duodenum of LO and HO groups
45
whereas the number ICC did not change significantly after treatment with obestatin.
46
Moreover, the enteral administration of obestatin did not effect significantly on the
47
cytoplasmic expression of M2 receptor in the jejunum.
48
Our study demonstrated that enteral administration of obestatin to suckling rats influences
49
small intestine contractility in the segment specific manner.
50 51 52 2
53
1.Introduction
54
Obestatin is a 23-amino acid peptide which was first identified in the rat stomach as a ghrelin-
55
accompanying peptide generated during the posttranslational processing of preproghrelin
56
(polypeptide precursor of ghrelin) (Zhang et al., 2005). At present, the functional receptor for
57
obestatin is unknown. The initially proposed G-protein-coupled receptor GPR39, has been
58
questioned due to a series of studies which failed to demonstrate the ability of obestatin to
59
bind to and activate this receptor (Lauwers et al., 2006; Chartrel et al., 2007; Host et al.,
60
2007). Studies by Zhang et al. (2005) reported that obestatin behaves as a physiological
61
opponent to ghrelin and inhibits food intake, body weight gain, gastric emptying and jejunal
62
contractility. With respect to gastrointestinal tract (GIT) motility, several studies were
63
undertaken to elucidate the effects of obestatin on GIT contractility and transit time in
64
rodents. The first study by Zhang et al. (2005) reported that peripheral injection of obestatin
65
decreases gastric emptying and contractile activity of jejunal muscular strips in vitro. Since
66
then, the inhibitory effects of obestatin on gastrointestinal motility have remained
67
controversial. For example, Gourcerol et al. (2006) reported that obestatin injected
68
peripherally, either alone or in combination with a peripheral injection of CCK, did not
69
influence gastric motor function in fasted rats and mice. Similarly, Bassil et al. (2007) and De
70
Smet et al. (2007) showed that in adult rats obestatin neither inhibited nor promoted GIT
71
motility, either in vitro or in vivo. However, Ataka et al. (2008) reported the inhibitory action
72
of obestatin given IV on the motor activity in the antrum and duodenum of conscious rats in
73
the fed state. These contrasting results may be partially explained by the different
74
experimental conditions, specifically the duration of monitoring, the route of administration
75
of obestatin and the state of feeding and consciousness of the animals.
76
These abovementioned results on GIT motility were obtained using adult mice and rats. A
77
recent study by our group (2014) has shown that the effect of obestatin on intestinal
3
78
contractility is both dependent on the age of the animals and the segment of intestine studied.
79
In suckling rats we observed a significant effect of obestatin administration on the
80
contractility of full thickness intestinal wall segments. Interestingly, in the duodenum of 14
81
and 21 day old rats, treatment with obestatin significantly decreased responsiveness to all
82
doses of Ach, while in the middle jejunum, the opposite effect was observed in rats from 7 to
83
21 days old. In both intestinal segments the obestatin effect was fully abolished by atropine,
84
indicating that the obestatin effect is coupled with a cholinergic pathway (Słupecka et al.,
85
2014). Intestinal sensitivity to obestatin in suckling rats seems to be physiologically
86
reasonable as substantial amounts of obestatin have been found in human and rat milk.
87
Moreover, the presence of obestatin immunoreactive (IR) cells was shown in the GIT of
88
newborn rats starting from the 1st day of life (20). These findings strongly support the
89
importance of obestatin (both endogenous and exogenous) in the regulation of gastrointestinal
90
function in neonates. Therefore, it would be intriguing to investigate the effect of enteral
91
administration of obestatin on intestinal motor function and the morphology of the enteric
92
nervous system (ENS) in suckling rat neonates.
93 94 95
2. Materials and Methods
96
The experiments and treatments were conducted in compliance with the European Union
97
regulations concerning the protection of experimental animals (EC Directive 86/609/EEC
98
with amendments). The study protocol was approved by the 3rd Local Ethics Committee in
99
Warsaw, according to the Polish Law for the Care and Use of Animals (Resolution no
100
50/2012).
101 102
2.1. Chemicals
4
103
Rat obestatin was synthesized at the Yanaihara Institute using a solid phase method with an
104
Fmoc-strategy and an automated peptide synthesizer (Applied Biosystem 9030 Pioneer,
105
Foster, CA, USA). Analytical HPLC and MALDI-TOF MS confirmed the homology of the
106
product.
107
The hormone was kept in powder form at -20 ˚C and then dissolved in saline solution (0.9%
108
NaCl) to the final concentration, just before use. Acetylcholine chloride and atropine were
109
purchased from Sigma–Aldrich (Germany). Tetrodotoxin (TTX) was purchased from Abcam
110
(Great Britain).
111 112
2.2. Animals
113
At the start of the experiment 12 male and 12 female Wistar Han rats (13 weeks old) were
114
obtained from the Center of Experimental Medicine at the Medical University of Bialystok
115
and the rats were mated. After mating, females were separated from males and they were
116
allowed commercial rat breeding chow (5% fat; 3.1 kcal/g, Wytwornia Pasz Morawski,
117
Poland) and water ad libitum in a humidity- and temperature-controlled room on a 12-h:12-h
118
light:dark cycle. Twenty-four hours after delivery litters were standardized to 10 pups. On the
119
14th day of life the rat pups were randomly assigned to one of the 3 treatment groups (݊ = 12
120
for each group): C- control animals- treated with saline solution; LO- pups treated with a
121
lower dose of obestatin at 125 nmol/kg b.wt.; HO-pups treated with a high dose of obestatin
122
at a 250 nmol/kg b.wt. Both pharmacological doses of obestatin were constructed based on
123
the previous in vitro studies on intestinal contractility in rats (De Smet et al., 2007; Słupecka
124
et al., 2014). Prior to euthanasia, all pups were housed with their mothers and breast-fed ad
125
libitum. Saline solution or obestatin were administered enterally via oral gavage twice a day
126
staring on the 14th day of life until the 21 st day of life. On the 21 st day of life (30 min after the
5
127
morning NaCl/obestatin treatment) three rat pups from each litter were euthanized by CO2.
128
The remaining rats were kept for use in another experiment.
129 130
2.3 Study design for in vitro contractility
131
Upon euthanasia, duodenal and middle jejunum segments (15 mm long) were excised
132
promptly from the rats and immediately placed in cold Krebs–Henseleit buffer (in mM: NaCl
133
18, KCl 4.7, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, NaHCO3 25, glucose 11). The segments
134
were then placed vertically in 25 ml organ bath chambers (Letica Scientific Instruments,
135
Spain) that were filled with Krebs–Henseleit solution (37 ˚C, pH 7.4) and continuously
136
saturated with carbogen (95% O2, 5% CO2). The intestinal segments were attached to
137
isometric transducers (Letica Scientific Instruments, Spain) under a load of 0.5 g. The
138
transducers were coupled with a PowerLab recording system (ADInstruments, Sydney,
139
Australia). The tissues were allowed to equilibrate for 30 min (the solution in the chambers
140
was changed once after 15 min) to regain spontaneous activity. The segments were then
141
subjected to a procedure which started with the addition of ACh 10 -5 M to assess the viability
142
of the preparations. ACh was left in the solution for 1 min, after which the tissues were
143
washed and allowed to equilibrate. Next, spontaneous or ACh-stimulated contractility was
144
recorded. ACh-stimulated contractility was recorded as the response to ACh 10 -5 M. In some
145
experiments, before ACh was added, jejunal strips were pre-treated with atropine (ATR, 10 -5
146
M). The neural contractions were also examined by studying the tissue response to electrical
147
field stimulation (EFS). After equilibration, the electrical field stimulation (EXP-ST-01,
148
Experimetria, Budapest, Hungary) was performed (voltage 90 V, duration 10 s) at three
149
frequencies: 0.5, 5 and 50 Hz with 1 min intervals between each pulse. EFS parameters were
150
chosen based on previous studies performed on rat whole-thickness intestinal preparations
151
[9,11]. In some experiments jejunal segments were pretreated with tetradotoxin (TTX, 10 -5
6
152
M). Each experiment was completed by the administration of ACh 10-5 M in order to check
153
the viability of the tissue, followed by isoproterenol (10-5 M) in order to control its relaxation.
154 155
2.4 Immunostaining of Cajal cells
156
The duodenal and middle jejunum segments (15 mm long) were transected and samples of
157
each section were collected and immediately fixed in a 10% neutral formalin solution. After
158
the 24h fixation period, the intestinal samples were routinely embedded in paraffin. The
159
paraffin-embedded samples were cut into 4.5 µm sections and applied to silane-treated glass
160
slides. Next, the sections were dewaxed in xylene and rehydrated in decreasing grades of
161
ethanol and then washed in PBS buffer and then the antigen retrieval was performed by 20
162
min microwave heating (500 W) in citrate buffer. For the immunostaining of the Cajal cells
163
the antibodies for c-kit were applied. The sections were incubated with rabbit c-kit polyclonal
164
antibodies (Abcam, UK), 50 times diluted in 1% BSA-PBS. After 0.5 h incubation performed
165
in a humidified chamber at room temperature, the slides were washed in PBS buffer and then
166
incubated with goat anti-rabbit secondary FITC-conjugated antibodies, 2000 times diluted in
167
PBS buffer. After a 1 h incubation period performed in a dark humidified chamber at room
168
temperature, the slides were washed in PBS. For the nuclei staining, the sections were
169
immersed in a 1% solution of 7-AAD for 25 min at room temperature, under dark conditions.
170
Control immunostaining was carried out using the same procedures, except for the
171
replacement of the primary antibodies with PBS. C-kit positive cells were observed using
172
400x total magnification and their number was counted across either submucosal surface of
173
the circular muscle layer (ICC-SM), within circular muscle (ICC-IM) or between circular and
174
longitudinal muscle layer (ICC-MY) were counted in the field of view (0.4 mm). Ten
175
randomly selected fields of view were observed from each slide. C-kit positive cells were
176
analyzed according to their localization.
7
177 178
2.5 Histology of the intestinal muscularis layer
179
For histometrical studies, slides were prepared as described above and then stained with
180
hematoxylin and eosin. Three slides were randomly selected for each of the studied intestinal
181
sections and then 30 measurements of the muscularis layer were performed using a light
182
microscope (Axioskop 40, Zeiss, Germany), coupled with computer software for image
183
analysis (Axio Vision 4.2 Release, Zeiss, Germany) and a digital camera.
184
2.6 Immunodetection of the Muscarinic Acetocholine receptor 2 (M2 receptor)
185
For the immunostaining of M2 receptors the duodenal and middle jejunum samples were
186
prepared as described in section 2.4. The sections were incubated with rabbit polyclonal
187
antibody for cytoplasmic domain of Muscarinic Acetylocholine Receptor 2 (Abcam, Great
188
Britain) 250 times diluted in 1% BSA-PBS. After 24 h incubation performed in a humidified
189
chamber at 40C, the slides were incubated with goat anti-rabbit secondary FITC-conjugated
190
antibodies, 2000 times diluted in PBS buffer (Abcam, Great Britain). For the nuclei staining,
191
the sections were immersed in 7-AAD.
192
The same primary antibody was used for immunoblotting of M2 receptors in the
193
segments of jejunum (middle part). Dissected out jejunum segments were stored in -800C
194
until analysis. Then, they were thawed and about 0.5 g of intestinal samples from each
195
experimental group were homogenized in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1
196
mM EDTA, 1% NP-40, 0.25% Na-deoxycholate and 1 mM PMSF) supplemented with
197
protease inhibitor cocktail and phosphatase inhibitor cocktail (Sigma-Aldrich). Then the
198
samples were left on ice for 30 min, during which the process of cell lysis took place. Lysates
199
were cleared for 30 min at 14000 rpm, and supernatants containing extracted proteins were
200
collected. Protein concentration was determined using Bio-Rad Protein Assay Dye Reagent
201
according to the producer’s instructions (Bio-Rad Laboratories Inc., Hercules, CA,, USA).
8
202
Proteins (50 µg) were resolved by SDS-PAGE and transferred onto PVDF membrane (Sigma-
203
Aldrich). For immunostaining membranes were blocked with 5% nonfat dry milk in TBST
204
(20 mM Tris-HCL, 500 mM NaCl, 0.5% Tween 20). The membranes were incubated with
205
antibodies against M2 receptor (Abcam, cat. no: ab188891; 1:250 dilution), or β-actin (Santa
206
Cruz Biotechologies, Inc., cat. no: sc-47778, 1:1000 dilution) at 4oC overnight. Next, the
207
membranes were washed three times for 15 min and incubated with appropriate secondary
208
antibodies conjugated with IR fluorophores: IRDye® 680 or IRDye® 800 CW (at 1:5000
209
dilution). After incubation the membranes were washed three times in TBST. Subsequently
210
the protein expression was analyzed using Odyssey Infrared Imaging System (LI-COR
211
Biosciences). Scan resolution of the instrument was set at 169 µm, and the intensity at 4.
212
Quantification of the integrated optical density (IOD) was performed with the analysis
213
software provided with the Odyssey scanner (LI-COR Biosciences). Results were expressed
214
as a M2 receptor ratio to β-actin densitometry units. For the purpose of publication the color
215
immunoblot images were converted into black and white images in the Odyssey software.
216 217
2.7. Statistics
218
The data are expressed as means ± SEM (standard error of the mean). A one-way ANOVA
219
followed by a Tukey-Kramer post-hoc test, Kruskal-Wallis test followed by a Dunn’s
220
Multiple Comparison post-hoc test, Unpaired t-test or Mann-Whitney test were used to
221
determine statistically significant differences between the groups (Prism 6 for Mac OS X,
222
Version 6.0h, Graph Pad Software, San Diego, CA, USA). The level of significance p<0.05
223
was assumed in all statistical analyses.
224
3. Results
225
3.1.Effect of obestatin on spontaneous contractility
9
226
The amplitude and frequency of contraction was analyzed in order to investigate the effects of
227
obestatin on spontaneous intestinal contractility (Table 1). In both intestinal segments
228
treatment with obestatin significantly decreased the amplitude of contraction in the HO group
229
compared to the control animals (C) (Table 1A). In the middle jejunum, the lower dose of
230
obestatin also significantly decreased the amplitude of contraction (P ˂ 0.031). Only in the
231
middle jejunum of LO animals the frequency of contraction was significantly affected in
232
comparison to the control animals (P ≤ 0.05), (C). Injection of TTX had no effect on the
233
amplitude of spontaneous contraction in the duodenum and middle jejunum of animals treated
234
with obestatin (LO and HO). In the duodenum, pretreatment with TTX significantly increased
235
frequency of contraction in all treatment groups studied (Table 1B). Atropine significantly
236
decreased the amplitude of duodenal contractions only in the group of control animals (C),
237
(Table 1A).
238 239
3.2 Effect of obestatin on ACh-stimulated contractility
240
After the injection of ACh 10-5, the amplitude and frequency of contraction increased in all
241
intestinal segments studied. Compared to the control animals (C), the duodenal sections from
242
the HO animals and the middle jejunum sections from both obestatin treated groups (LO and
243
HO) had significantly lower amplitudes of Ach-evoked contraction. However, treatment with
244
higher doses of obestatin significantly increased the frequency of ACh stimulated contraction
245
in both the intestinal segments studied (Fig.1 and Fig.2).
246 247
3.3 Effect of obestatin on electrical field stimulation
248
The amplitude of contraction after EFS impulses (0.5, 5, 50 Hz) was analyzed in order to
249
investigate the effects of obestatin on electrical field stimulation (Fig. 3). Among the groups
250
tested, the duodenum sections from the HO animals had significantly lower responsiveness
10
251
then duodenum sections from control animals, whereas in the middle jejunum a significantly
252
lower responsiveness for rising EFS frequencies was observed in sections from both obestatin
253
treated groups (LO, HO). Moreover, we observed that treatment with TTX and ATR
254
significantly reduced the amplitude of contraction. Following pre-incubation with TTX and
255
ATR the effects of obestatin were still observed in the duodenum sections from LO and HO
256
groups exposed to 50 Hz. Of the three studied frequencies of EFS, the 5 Hz applied after pre-
257
incubation with both blockers did not show any differences between the groups studied (Table
258
2).
259 260
3.4 Thickness of muscularis layer
261
In the duodenum, enteral treatment with both doses of obestatin (LO, HO) increased the
262
thickness of the muscularis layer in comparison to that of the control group (P ˂ 0.0001), (C).
263
In the middle jejunum, treatment with obestatin did not influence the thickness of the
264
muscularis layer (Table 3).
265 266
3.5 Interstitial Cajal cells
267
In order to show the interstitial Cajal cells (ICC) in the intestinal segments studied, we
268
performed immunostaining using a c-kit antibody. The immunostaining showed the presence
269
of ICC in the small intestinal mucosa of all experimental groups (Fig. 4). The c-kit positive
270
cells were observed mostly in the submucosal surface layer of the circular muscle layer (ICC-
271
SM), or between the circular and longitudinal muscle layers (ICC-MY). Only a few cells were
272
observed within the circular muscle layer (ICC-IM, data not shown). The number of ICC,
273
estimated separately in the duodenum and middle jejunum sections for ICC-SM and ICC-MY
274
did not change significantly after enteral treatment with obestatin (Table 4).
275
3.6 Muscarinic Acetylocholine Receptor 2 (M2 receptor)
11
276
The immunostaining showed the presence of M2 receptors in muscularis layers of both
277
studied intestinal segments of all groups. Immunostatinig of the receptors unable to observed
278
any differences in their expression and localization between studied groups (Fig. 5). To obtain
279
quantitative results on the expression of M2 receptors the immunoblotting was performed.
280
The enteral administration of obestatin did not effect significantly on the cytoplasmic
281
expression of M2 receptor in the middle jejunum (Fig. 6).
282 283
4. Discussion
284
Previous results from our lab have shown that the peripheral action of obestatin influences
285
intestinal contractility in rats, in both an age-dependent and intestinal segment specific
286
manner (Słupecka et al., 2014). In particular, we have shown that the small intestine of
287
suckling rats (until 21 day of life) is more sensitive to obestatin than that of adult animals. We
288
speculated that obestatin, of which substantial amounts have been found in rat milk, could be
289
an important factor regulating the function of the gastrointestinal tract, during the early
290
postnatal period. Thus, in the present study we aimed to investigate whether enteral
291
administration of pharmacological doses of obestatin (the same route of administration as if
292
obestatin was entering the intestine with the rat milk) to suckling rats would have an influence
293
on the contractile properties of the small intestine. We found that administration of obestatin
294
for a 7 day period decreased the amplitude of contraction in both the intestinal segments
295
studied. This is an interesting observation as we have previously shown that injection of a
296
single dose of obestatin directly into the organ bath chamber significantly increased the
297
amplitude of spontaneous contraction in the middle jejunum of 14 and 21 day old rats, while a
298
decrease in spontaneous contraction was observed in both the intestinal segments studied in
299
adult rats. This effect may indicate that obestatin, when administered into the stomach (the
300
main source of endogenous obestatin), triggers a different mechanism to that when obestatin 12
301
is injected directly into the intestine or that some of the obestatin is lost during its’ passage
302
through the stomach and duodenum, and the amount of hormone that reaches the middle
303
intestine is insufficient to induce the effects seen in the experiment with obestatin injected
304
directly into the organ bath. It is also possible that unlike with the single injection of
305
obestatin, continuous (7 day long) administration of this peptide may influence either the
306
synthesis of ACh or the sensitivity of the neurons to ACh, especially since it has been shown
307
that during the second and third weeks of life, major changes in cholinergic phenotype and
308
muscular transmission occur in the rat intestine (Matini et al., 1997). This is the more
309
important as nearly all obestatin immunoreactive cells in the myenteric plexus of the rat are
310
acetylotransferase (ChAT) positive. It should be mentioned that the expression of obestatin
311
receptors and/or their sensitivity may be altered after treatment with obestatin. Unfortunately,
312
the lack of discovery of the functional obestatin receptor makes further investigation in this
313
field impossible. Thus, in this study we focused on the possible influence of obestatin
314
administered into the stomach for consecutive days on the morphological changes of the
315
enteric nervous system (ENS). First of all, we decided to study full–thickness intestinal
316
segments instead of only muscle preparations, as previous in vitro studies have revealed that
317
different types of ion channels and receptors, as well as interneurons and non-muscle cells
318
occur along the intestinal wall and may be crucial for the contractile response (Matini et al.,
319
1997; Mondal et al., 2013). Gut motility occurs through the action of the interstitial Cajal cells
320
and smooth muscle layers. Thus, we analyzed both the expression and localization of ICC in
321
the gut wall, together with the thickness of the muscularis layer and the expression of
322
Muscarinic Acetylocholine Receptors 2 which play an essential role in the generation of
323
rhythmic motor activity (Tanahashi et. al., 2013).
324
In the gut wall, the Cajal cells play a double role. They generate the electrical slow
325
waves needed for motility and are required for effective neurotransmission. The morphology 13
326
and localization of the ICC determines their function. In our study we wanted to focus on the
327
ICC-MY as they form a network closely connected with MY neurons and have receptors for
328
neurotransmitters and circulating hormones (e.g. cholecystokinin). We did not observe any
329
changes in the number of these cells between the various treatment groups studied, which
330
brings about the hypothesis that the effects of obestatin on intestinal contraction occur
331
independently from the effect on the number of ICC. This is also confirmed by results with
332
spontaneous contractility, where after blocking neural activity by TTX the basal activity of
333
the jejunum segments did not differ between groups.
334
Interestingly, in duodenum and middle jejunum sections from 21 day old rats we also
335
observed ICC localized in the submucosal surface. So far, these type of ICC have been
336
observed in the canine colon and they are involved in the pace maker activity within the
337
gastrointestinal tract (Wurner et al., 2013). However, studies on ICC-SM structure and
338
functions within the small intestine of rats are very limited and there is no such data for
339
neonatal rats. Even though we did not observe any significant changes in the number of ICC
340
in our study, further studies on these cells could yield interesting observations on their
341
function in the small intestine of suckling animals. In the line of expectations we did not
342
observe Cajal cells in the circular muscle layer (ICC-CM) as they are c-kit negative (Streutker
343
et al., 2007; Chen et al., 2007). Although in the current study we did not observe any
344
significant changes in the number of c-kit positive cells, we did record a significant increase
345
in the thickness of the muscularis layer in the duodenum of pups treated with both doses of
346
obestatin. It is well documented that the hypertrophy of smooth muscle is a physiological
347
response to the increased functional demands placed on an organ. In the small intestine the
348
hypertrophy of the muscle wall has been observed in the intestine of rat dams during
349
pregnancy and lactation, but also in studies on its partial obstruction (Boass et al., 1992;
350
Bertoni et al., 2001).
14
351
To understand the nature of obestatin’s influence on intestinal contractility it is crucial
352
to investigate whether the hypertrophy observed within the duodenum is a primary or
353
secondary effect of administration of the peptide. Taking into account all the results obtained
354
from the current study and previous observations from our lab, where we observed changes in
355
intestinal contractility after a single obestatin injection, we can conclude that obestatin does
356
influence intestinal contractility and this effect is ICC independent. Weaker intestinal
357
contractility influences on nutrient transit resulted in increased availability of nutrients in the
358
intestinal lumen. Intestinal absorption increases and this is reflected in the histomorphometry
359
changes in the intestinal wall, the hypertrophy of the muscle layer and the increased thickness
360
of the mucosa (Slupecka et al, unpublished data). We observed that both the intestinal
361
segments studied from animals treated with obestatin were significantly less responsive to
362
ACh. However, duodenum was less “sensitive” to obestatin than middle jejunum. As we did
363
not observed the effect of obestatin administration neither on its expression (Słupecka et al,
364
unpublished data) nor the expression of Muscarinic Acetylocholine Receptor 2 in the small
365
intestinal wall this weaker responsiveness could be a result of the “dilution” of motor neurons
366
in the hypertrophic duodenal muscular layer. Since the relatively weak responsiveness to
367
electrical field stimulations was observed in both intestinal segments, we could speculate that
368
this is the effect of decreased number of enteric motor neurons or ACh release from these
369
neurons. Further studies are needed to elucidate this mechanism. In this study the frequency
370
of spontaneous contraction was unaffected and sometimes even increased following
371
pretreatment with atropine and TTX. These somewhat surprising observations are a result of
372
using whole thickness intestinal sections. It has been previously shown that both cholinergic
373
and adrenergic inhibitors as well as the neural blocker- TTX, evoke different responses in the
374
characteristics of spontaneous contractions, depending on the region of intestine, as well as
375
the specific part of the intestine wall being investigated (Grasa et al., 2004; Postorino et al.,
15
376
1990). For example in the studies using the rabbit, atropine and TTX decreased the amplitude
377
of spontaneous contraction only in the longitudinal muscles (Grasa et al., 2004), whereas in
378
the rat duodenum pretreatment with TTX, even in the presence of atropine and guanethidine,
379
caused an increase in amplitude and frequency of electrical and mechanical intestinal activity
380
(Postorino et al., 1990). This finding indicates the presence of tonically active inhibitory
381
intramural non adrenergic, non cholinergic (NANC) nerves in the rat duodenum. Moreover,
382
Postorino et al. showed diversity in contractile actions among the intestinal muscle layers. In
383
this study duodenal longitudinal strips showed a spontaneous mechanical activity resembling
384
that one recorded from isolated segment. Instead, circular strips were quiescent under resting
385
condition and a contractile activity was detected only after TTX pretreatment. In the current
386
study, in the duodenum of rats treated with the low dose of obestatin (LO, 125nmol/kg b.wt),
387
after pretreatment with TTX, a significantly higher frequency of contractions was observed
388
compared to that observed in the control group (C). This indicates that in duodenum the
389
obestatin may influences on the tonically active inhibitory intramural nonadrenergic
390
noncholinergic (NANC) nerves.
391
In conclusion, our study demonstrated that enteral stomach administration of obestatin
392
to suckling rats influences the small intestinal contractility. Enteral administration of obestatin
393
to suckling rats effects on cholinergic neurotransmission in the small intestine rather than the
394
network of peacemaker cells of Cajal. In the duodenum, also nonadrenergic noncholinergic
395
(NANC) nerve fibres could possibly be affected by obestatin, but further studies in this field
396
are required.
397 398 399
Grants
400
This research was supported by National Science Center Grant no. 2011/03/D/NZ9/03697.
16
401 402 403 404 405 406
Author contribution M.S. designed the research study, performed the research, analyzed the data and wrote the paper; P.G, J.K and M.G performed the research; I.K. and A.K. contributed essential reagents; J.W. performed the research, analyzed the data and revised the article
407
Acknowledges
408
The authors wish to thank Żaneta Dzięgelewska for her support in the immunoblotting of M2
409
receptors.
410 411
References
412 413 414
Ataka, K., Inui, A., Asakawa, A., Kato, I., Fujimiya, M., 2008. Obestatin inhibits motor activity in the antrum and duodenum in the fed state of conscious rats. Am J Physiol Gastrointest. Liver Physiol. 294 (5), G1210-1218.
415 416 417
Bassil, A.K., Haglund, Y., Brown, J., Rudholm, T., Hellstrom, P.M., Naslund, E., Lee, K., Sanger, G., 2007. Little or no ability of obestatin to interact with ghrelin or modify motility in the rat gastrointestinal tract. Br. J. Pharmacol. 150 (1), 58-64.
418 419 420 421 422 423 424 425 426 427
Bertoni, S., Gabella, G., 2001. Hypertrophy of mucosa and serosa in the obstructed intestine of rats. J. Anat. 199 (6), 725-734. Boass, A., Lovdal, J.A., Toverud S.U., 1992. Pregnancy- and lactation-induced changes in active intestinal calcium transport in rats. Am. J. Physiol. 263 (1), G127-134.
428 429 430
Chen, H., Redelman, D., Ro, S., Ward, S.M., Ordog, T., Sanders, K.M., 2007. Selective labeling and isolation of functional classes of interstitial cells of Cajal of human and murine small intestine. Am. J. Physiol. Cell. Physiol. 292 (1), C497-507.
431 432 433
De Smet, B., Thijs, T., Peeters, T.L., Depoortere, I., 2007. Effect of peripheral obestatin on gastric emptying and intestinal contractility in rodents. Neurogastroenterol Motil. 19 (3), 211217.
434 435 436
Gourcerol, G., Million, M., Adelson, D.W., Wang, Y., Wang, L., Rivier, J., St-Pierre, D.H., Tache, Y., 2006. Lack of interaction between peripheral injection of CCK and obestatin in the regulation of gastric satiety signaling in rodents. Peptides 27 (11), 2811-2819.
437 438
Grasa, L., Rebollar, E., Arruebo, M.P., Plaza, M.A., Murillo, M.D., 2004. The role of Ca2+ in the contractility of rabbit small intestine in vitro. J. Physiol. Pharmacol. 55 (3), 639-650.
Chartrel, N., Alvear-Perez, R., Leprince, J., Alvear-Perez, R., Leprince, J., Iturrioz, X., Reaux-Le Goazigo, A., Audinot, V., Chomarat, P., Coge, F., Nosjean, O., Rodriguez, M., Galizzi, J.P., Boutin, J.A., Vaudry, H., Llorens-Cortes, C., 2007. Comment on "Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake". Science 315 (5813), author reply 766.
17
439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458
Holst, B., Egerod, K.L., Schild, E., Vickers, S.P., Cheetham, S., Gerlach, L.O., Storjohann, L., Stidsen, C.E., Jones, R., Beck Sickinger, A.G., Schwartz, T.W., 2007. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 148 (1), 13-20. Korczynski, W., Ceregrzyn, M., Kato, I., Wolinski, J., Zabielski, R., 2006. The effect of orexins on intestinal motility in vitro in fed and fasted rats. J. Physiol. Pharmacol. 57 (Suppl 6), 43-54. Lauwers, E., Landuyt, B., Arckens, L., Schoofs, L., Luyten, W., 2006. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem. Biophys. Res. Commun. 351 (1), 21-25. Matini, P., Mayer, B., Faussone-Pellegrini, MS., 1997. Neurochemical differentiation of rat enteric neurons during pre- and postnatal life. Cell Tissue Res. 288 (1), 11-23. Mondal, A., Aizawa, S., Sakata, I., Goswami, C., Oda, S., Sakai, T., 2013. Mechanism of ghrelin-induced gastric contractions in Suncus murinus (house musk shrew): involvement of intrinsic primary afferent neurons. PLoS One 8 (4), e60365. Postorino, A., Mancinelli, R., Racanicchi, C., Adamo, E.B., Marini, R., 1990. Spontaneous electromechanical activity in the rat duodenum in vitro. Arch. Int. Physio.l Biochim. 98 (1), 35-40.
459 460 461 462 463 464 465
Slupecka, M., Pierzynowski, S.G., Kuwahara, A., Kato, I., Wolinski, J., 2014. Age-dependent effect of obestatin on intestinal contractility in Wistar rats. Gen. Comp. Endocrinol. 208, 109115.
466 467 468 469 470 471 472 473 474 475 476 477 478 479 480
Tanahashi, Y., Waki, N., Unno, T., Matsuyama, H., Iino, S., Kitazawa, T., Yamada, M., Komori, S., 2013. Roles of M2 and M3 muscarinic receptors in the generation of rhythmic motor activity in mouse small intestine.Neurogastroenterol Motil. 25 (10), e687-97.
Streutker, C.J., Huizinga, J.D., Driman, D.K., Riddell, R.H., 2007. Interstitial cells of Cajal in health and disease. Part I: normal ICC structure and function with associated motility disorders. Histopathology 50 (2), 176-189.
Wurner, L., Diener, M,. Receptors and mechanisms mediating the biphasic response evoked by bradykinin in rat colonic smooth muscle. Neurogastroenterol. Motil. 25 (9), e581-590. Zhang, J.V., Ren, P.G., Avsian-Kretchmer, O., Luo, C.W., Rauch, R., Klein, C., Hsueh, A.J., 2005. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 310 (5750), 996-999. Zhao, C.M., Furnes, M.W., Stenstrom, B., Kulseng, B., Chen, D, 2008. Characterization of obestatin- and ghrelin- producing cells in the gastrointestinal tract and pancreas of rats: an immunohistochemical and electron-microscopic study. Cell. Tissue Res. 331 (3), 575-587. .
481 482 483
18
484 485 486
List of tables
487 488 489 490 491
Table 1. Effect of TTX-6M and ATR-6M on the amplitude (mm) (A), and frequency (number of contractions per 1s) (B) of spontaneous contraction according to the small intestinal segment examined and treatments A Duodenum -6 TTX
Basal
ATR
-6
Middle jejunum -6 TTX
Basal **
ATR
-6
C LO
0.64 ± 0.11 0.46 ± 0.20ab
a
0.40 ± 0.22 0.27 ± 0.09
*
0.33 ± 0.13 0.30 ± 0.06
0.51 ± 0.18 0.25 ± 0.04b
a
0.15 ± 0.09 0.15 ± 0.10
***
0.30 ± 0.18 0.19 ± 0.09
HO
0.37 ± 0.17
b
0.36 ± 0.19
0.31 ± 0.12
0.26 ± 0.16
b
0.12 ± 0.09
*
0.24 ± 0.08
P
0.021
0.494
0.919
0.031
0.780
Duodenum -6 TTX
-6
Basal
Middle jejunum -6 TTX
0.386
492 493
B Basal
494 495 496 497 498 499 500
ATR a**
C LO HO
0.86 ± 0.06 1.03 ± 0.07 1.17 ± 0.16
1.34 ± 0.04 * 2.29 ± 0.55b ab* 1.99 ± 0.18
P
0.173
0.011
***
2.34 ± 0.15 * 3.15 ± 0.55 ** 2.63 ± 0.17 0.304
a
ATR
-6
1.07 ± 0.08 ab 1.61 ± 0.17 b 1.76 ± 0.18
3.04 ± 0.54 * 3.74 ± 0.62 3.42 ± 0.85
3.29 ± 0.74 ** 3.94 ± 0.56 * 3.56 ± 0.72
0.034
0.889
0.801
C—saline solution (twice a day), LO- intra stomach obestatin (125 nmol/kg BW, twice a day); HO-intra stomach obestatin (250 nmol/kg BW twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups within type of contraction; *- indicates statistical differences between basal and TTX or ATRblocked contraction, (* P ≤ 0.05, ** P ≤ 0.001, ***P ≤ 0.0001).
501 502 503 504 505 506 507 508
19
509 510 511 512 513
Table 2. The amplitude of EFS-induced off-contraction (mm) (0.5 Hz, 5Hz, 50Hz) in the presence or absence of TTX (10-6M) (A), or ATR (10 6 M) (B) according to small intestinal segment examined and treatments A
C LO HO P
514 515
0.5Hz a 2.18 ± 0.98 a 2.18 ± 1.06 b 1.04 ± 0.76 0.0126
5Hz a 2.48 ± 1.19 ab 2.46 ± 1.06 b 1.62 ± 0.72 0.0233
50Hz a 2.80 ± 1.25 a 2.73 ± 0.78 b 1.20 ± 0.72 0.0036
Duodenum -6 TTX 0.5Hz 5Hz ** ** 0.71 ± 0.40 0.73 ± 0.42 * ** 0.44 ± 0.24 0.52 ± 0.24 * 0.47 ± 0.20 0.52 ± 0.22 0.1740 0.1980
50Hz
Middle jejunum -6 TTX 0.5Hz 5Hz
-6
50Hz a* 1.65 ± 0.64 b*** 0.93 ± 0.50 b 0.63 ± 0.25 0.0010
0.5Hz ** 0.59 ± 0.25 * 0.35 ± 0.10 0.43 ± 0.17 0.0973
ATR 5Hz ** 0.61 ± 0.27 ** 0.43 ± 0.15 * 0.49 ± 0.19 0.3245
0.5Hz
ATR 5Hz
50Hz a* 1.50 ± 0.59 b*** 0.81 ± 0.45 b* 0.57 ± 0.17 0.0026
B 0.5Hz C LO
5Hz
0.95 ± 0.38
a
b
0.25 ± 0.14
b
HO
0.36 ± 0.16
P
0.0005
1.22 ± 0.62
a
b
0.26 ± 0.19
ab
0.59 ± 0.29 0.0013
a
1.80 ± 0.90
b
0.60 ± 0.39
b
0.93 ± 0.33 0.0102
a***
0.29 ± 0.24
0.11 ± 0.06
ab
b***
0.10 ± 0.08 0.0365
0.23 ± 0.16
-6
50Hz ***
0.12 ± 0.07 0.12 ± 0.08 0.1006
***
a*
0.56 ± 0.32
b*
0.15 ± 0.08 0.23 ± 0.15 0.0010
ab**
0.40 ± 0.23
a** b
0.25 ± 0.14 0.33 ± 0.12 0.0490
ab
0.42 ± 0.26
50Hz **
0.16 ± 0.06 0.23 ± 0.10 0.0551
**
1.08 ± 0.50
a
b
0.21 ± 0.07
b***
0.26 ± 0.15 0.0105
516 517 518 519
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups * indicates differences between EFS- induced contraction without blockers and with either TTX or ATR (* P ≤ 0.05, ** P ≤ 0.001, ***P ≤ 0.0001)
20
520 521 522
Table 3. Thickness of muscularis layer (µm) of the small intestine of suckling rats according to small intestinal segment examined and treatments C
523 524 525 526 527 528
LO
HO
P
Duodenum
a
33 ± 7
b
b
38 ± 8
38 ± 9
<0.001
Middle jejunum
30 ± 8
28 ± 7
27 ± 7
0.09
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); different superscript letters indicate statistical difference between the groups
529 530 531 532 533 534 535 536
Table 4. Number of Cajal cells (ICC) distributed across submucosal surface of the circular muscle layer (ICC-SM), or between circular and longitudinal muscle layers (ICC-MY) of the small intestine of suckling rats according to small intestinal segment examined and treatments. C
OL
OH
P
Duodenum ICC-SM
0.50 ± 0.28
0.25 ± 0.25
1.20 ± 0.48
0.2325
ICC-MY
2.16 ± 0.54
2.37 ± 0.32
2.77 ± 0.52
0.6560
P
0.048*
0.002**
0.07
Middle jejunum
537 538 539 540 541 542
ICC-SM
0.0 ± 0.0
1.25 ± 0.75
0.87 ± 0.29
0.2014
ICC-MY
1.33 ± 0.37
1.80 ± 0.80
0.74 ± 0.35
0.3413
P
0.04*
0.639
0.733
C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW, twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given as means ± SEM (n=12); * indicates statistical difference in ICC number between the locations (* P ≤ 0.05, ** P ≤ 0.001).
543 544 545 546 547
21
548
List of figures
549 550
Fig. 1. Effect of ACh -5M on the amplitude (mm) and frequency (number of contractions per
551
1s) of spontaneous contraction according to the small intestinal segment examined and
552
treatments
553
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
554
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day); values are given
555
as means ± SEM (n=12); different superscript letters indicate statistical differences between
556
the groups within type of contraction. *- indicates statistical differences between spontaneous
557
and ACh-evoked contraction.
558 559
Fig. 2. Representative tracing of Ach-evoked contraction in duodenal and middle jejunum
560
segments
561
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
562
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day).
563 564
Fig. 3. Representative tracing of EFS- induced contraction in duodenal and middle jejunum
565
segments
566
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
567
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day).
568 569
Fig. 4. C-kit expression on confocal images in representative cross-sections from the
570
duodenum of suckling rats
22
571
Legend: C—saline solution (twice a day), (A), LO—intra stomach obestatin (125 nmol/kg
572
BW, twice a day), (B); HO—intra stomach obestatin (250 nmol/kg BW, twice a day), (C);
573
Cajal cells (green fluorescence) are pointed by arrows. Nuclei were stained with 7-AAD (red
574
fluorescence)
575 576
Fig. 5. M2 receptors expression on confocal images in representative cross-sections from the
577
duodenum of suckling rats
578
Legend: C—saline solution (twice a day), (A), LO—intra stomach obestatin (125 nmol/kg
579
BW, twice a day), (B); HO—intra stomach obestatin (250 nmol/kg BW, twice a day), (C); M2
580
receptors - green fluorescence.
581 582
Fig. 6. (A) Western blot analysis of M2 receptor expression in the mucosa of middle jejunum
583
segments in suckling rats. (B) Optical density of the M2 receptor β-actin ratio in the study
584
groups.
585
Legend: C—saline solution (twice a day), LO—intra stomach obestatin (125 nmol/kg BW,
586
twice a day); HO—intra stomach obestatin (250 nmol/kg BW, twice a day). Results are
587
presented as means ± SEM from 6 replications.
588 589 590 591
23
592 593 594 595
Fig. 1
596 597
24
598 599 600 601
Fig. 2
602 603
25
604 605 606 607
Fig. 3
608 609
26
610 611 612 613
Fig. 4
614 615
27
616 617 618
Fig. 5
619 620
28
621 622 623 624
Fig. 6
625 626 627
29
628
Highlights:
629
Enteral obestatin administration effects on intestinal spontaneous contractility.
630
In suckling rats obestatin influences on response to ACh and EFS-evoked contractions.
631
Number of Cajal cells was unaffected by obestatin administration.
632
The effect of enteral obestatin on intestine contractility is segment specific.
633
30