Department of Physiology and Biophysics, Mayo Clinic and Mayo
Foundation, Rochester, Minnesota 55905
The effect of intracerebroventricular (ICV)
perfusion of bombesin (BBS) on the interdigestive migrating myoelectric
complex (MMC) actvity was examined in conscious dogs with electrodes
implanted on the stomach and small intestine. Cannulas and a catheter
were chronically positioned in the lateral and fourth cerebral
ventricles, respectively. ICV perfusion of BBS, which failed to
increase plasma BBS levels, replaced phase I activity in the stomach
and duodenum by intense irregular spike activity and decreased the
occurrence rate of MMCs, whereas intravenous infusion of BBS evoked
phase II-like activity, mainly in the jejunum and ileum, and suppressed phase III activity. These data suggest that the effect of ICV administration of BBS was mediated by direct activation of central brain structures. During ICV perfusion of BBS, cycling in plasma levels
of motilin persisted even when phase III activity was absent and plasma
levels of epinephrine rose significantly. Epinephrine infusion,
however, did not affect myoelectric gastrointestinal activity except
for prolonging phase II. Thus it is unlikely that the central action of
BBS is exerted by motilin or epinephrine.
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INTRODUCTION |
INDEPENDENT LINES of research in the early 1970s led to
the discovery and isolation of bombesin (BBS), a tetradecapeptide, and
other BBS-like (BBL) peptides (7). BBL immunoreactivity is present in
the central nervous system of mammals and mainly in the hypothalamus
(the highest concentrations being in the preoptic area, ventral and
medial part of the paraventricular nuclei, and arcuate nuclei) but also
in the pons and medulla oblongata (solitary tract, trigeminal complex,
vagal motor nucleus, etc.) and other locations (15, 21). In the
gastrointestinal tract, BBL immunoreactivity is found in endocrine
cells, smooth muscle cells, and in myenteric neurons, mainly in the
fundus of the stomach but also throughout the small and large bowel (8,
10). The existence of BBL immunoreactivity and the presence of binding
sites for BBS in both brain and gut raise the possibility that BBS
regulates the myoelectric and contractile pattern of the
gastrointestinal tract. The effect of BBS on myoelectric activity of
the stomach and small bowel has been examined by intravenous (IV)
infusions in dogs (4, 24). Infusion of BBS increased the frequency of
pacesetter potentials, decreased their amplitude (4, 24), abolished
phase III activity, and induced a weak phase II activity (24). The
effect of intracerebroventricular (ICV), intrathecal, and direct
injection of BBS in brain nuclei, performed mainly in rats, has shown
that BBS increases contractions in the stomach (28) and small bowel
(27), delays gastric emptying and small intestinal transit (26, 27),
and inhibits vagally stimulated gastric contractility (13). The
specific effect if any of central administration of BBS on the
migrating myoelectric complex (MMC) has not been studied. The purpose
of the present study was to determine the effect of ICV perfusion of
BBS on MMCs in conscious dogs.
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MATERIALS AND METHODS |
Fourteen healthy female dogs (9.5-15.0 kg) were used in this
study. The results described were obtained from four of these dogs.
Premature occlusion of the ICV drainage system prevented completion of
a full set of experiments in the other 10 dogs. These animals and the
partial results of the experiments performed on them are therefore
excluded from this report. The Mayo Institutional Animal Care and Use
Committee approved the use of the animals and the experimental
procedures.
Surgical procedure.
All operations were performed under general anesthesia induced with
Brevital (12.5 mg/kg iv). An endotracheal tube was inserted for a free
airway, and anesthesia was maintained by assisted respiration with
halothane (Halo-Carbon Laboratory) in oxygen supplied by a mechanical
respirator. A prophylactic dose of 600,000 U of Flo-cillin (Bristol
Laboratory, Syracuse, NY) was given at the beginning of each operation.
In the first operation, through a midline laparotomy, nine Ag-AgCl
electrodes were sutured to the stomach and small intestine. Two
electrodes were sutured to the distal antrum: one 3 cm and the other 1 cm orad to the pylorus. Two electrodes were sutured to the duodenum:
one between the upper and middle thirds and the other between the
middle and bottom thirds. The other five electrodes were sutured to the
remaining small bowel at intervals of one-sixth the distance between
the ligament of Treitz and the ileocecal junction. A stainless steel
cannula containing the socket connector was positioned in the right
anterior abdominal wall and anchored in place by 2-O stainless
sutures.
One week later, by a stereotaxic technique previously described (18),
cannulas were inserted into the lateral ventricles of the brain and a
polyethylene (PE-100) catheter with a Silastic tube was placed into the
fourth ventricle to be used for drainage during perfusion experiments.
Experimental procedures.
Experiments were carried out 1 wk after the dogs recovered from the
second operation. None of the dogs showed any sign of discomfort or
neurological damage. Dogs received one daily meal, at a regular hour,
which consisted of 822 g of Alpo Beef Chunks (Allen Products,
Allentown, PA) and received water ad libitum.
Recordings of myoelectric activity were made at the same time of the
day on nonconsecutive days. The dogs were fasted for 20 h before each
recording session. Recordings were made on an eight-channel rectilinear
penwriting recorder (Gould 2800S, Gould, Cleveland, OH) using
preamplifiers with low- and high-frequency filters set at 0.05 and 10 Hz, respectively. The recordings also were digitalized and stored on
VHS, 3M T120 professional videocassettes using a pulse code
modulator (PCM-8, Medical Science, Greenvale, NY). During recording
sessions, the animals rested quietly in a supporting canvas sling.
Perfusion of the cerebral ventricular system was done by inserting
needles into the lateral ventricles through the cannulas, which were
placed during the second operation. One of these needles was used to
monitor the ventricular pressure by a Tektronix 5113 Oscilloscope
(Tektronix, Beaverton, OR), with a Gould pressure transducer. The other
was used for perfusion of the ventricular system, using a Harvard pump,
model 975 (Harvard Apparatus, South Natick, MA). Perfusions were at a
constant rate of 0.15 ml/min, and the outflow was simultaneously
collected from the fourth ventricle, thereby preventing increases in
pressure in the ventricular system and escape of the cerebrospinal
fluid (CSF) from the fourth ventricle into the subarachnoid space (18).
Artificial CSF with or without BBS was used to perfuse the cerebral
ventricular system. Artificial CSF was prepared daily by mixing three
solutions: 7.46 g NaCl, 0.19 g KCl, 0.14 g
CaCl2, 0.19 g
MgCl2, and 182.11 ml distilled water (solution A); 1.7 g
NaHCO3 and 50 ml distilled water
(solution B); and 1.7 g
Na2HPO4
and 50 ml distilled water (solution
C). The mixing ratio was 20:5:5, to which 70 ml of
distilled water and 1 mg of bovine serum albumin (Sigma Chemical, St.
Louis, MO) were added. It was filtered through an Acrodisc, 25-mm
filter assembly (Gelman Sciences, Ann Arbor, MI). IV infusions were
made through a 19-gauge butterfly needle positioned into one of the superficial veins in the limbs, with normal saline solution, at a rate
of 0.15 ml/min, using the same pump as for the cerebral ventricular
perfusions. Perfusions and infusions were initiated after at least two
complete MMCs were observed and while phase I activity was present in
the stomach.
Four sets of experiments were conducted. Each experiment was performed
on three dogs and repeated in each dog at least three times. The first
set (group A) consisted of ICV perfusions of BBS (n = 10). BBS (mol wt 1,618.8, Peninsula Laboratories, Belmont, CA) was dissolved in CSF and
administered for 2 h at a rate of 1.2 pmol · kg
1 · min
1,
followed by a further 1-h perfusion of CSF alone. BBS was
administered only once a week to prevent any possible cumulative
effect. The second set (group B), which
served as a control study, consisted of ICV perfusions of CSF of 3-h
duration (n = 9). The third set (group C) consisted of IV infusions of BBS
(n = 9). BBS was dissolved in normal
saline and infused for 2 h at a rate of 1.2 pmol · kg
1 · min
1,
followed by a further 1-h infusion of normal saline only.
The last set (group D) consisted of IV
infusion of epinephrine (n = 9).
Epinephrine (Abbott Laboratories, Chicago, IL) was dissolved in normal
saline with 1 mg/ml ascorbic acid (Baxter Healthcare, Deerfield, IL).
Epinephrine was administered at a rate of 0.05 µg · kg
1 · min
1
in a volume of 0.15 ml/min and infused for 2 h, followed by a 1-h
infusion of normal saline only.
Blood samples and CSF samples from the fourth ventricle were collected
before the initiation and at the end of each perfusion-infusion to
determine BBS concentrations in the CSF and peripheral circulation, respectively. Plasma levels of motilin also were examined every 20 min.
Blood samples to measure free catecholamine (i.e., norepinephrine, epinephrine, and dopamine) plasma levels were collected at 0, 10, 20, 40, 60, 90, 120, 150, and 180 min of each perfusion-infusion. Blood was
sampled through a 19-gauge butterfly needle positioned in a superficial
vein in one of the limbs. To determine the concentrations of BBS and
motilin, 2 ml of blood were collected in sterile Vacutainer tubes
(Becton Dickinson, Rutherford, NJ), containing 0.04 ml of liquid 7.5%
EDTA (K3) solution (3 mg). The tubes were immediately placed on crushed
ice. At the end of the experiments, the blood samples were spun for 30 min in a refrigerated centrifuge (4°C) at 2,500 rpm and the
supernatants were transferred to other empty tubes and stored at
20°C. The radioimmunoassay procedures used to determine the
concentration of BBL immunoreactivity were similar to the technique
described previously (31). The BBS antibody used in this study was
rabbit N388. The sensitivity of the assay was 16-1,000 pg/ml with
intra-assay and interassay variations of 7% and 12%, respectively.
The radioimmunoassay procedures used in this study to determine the
concentration of motilin were similar to the technique described
previously (6). The antibody used was guinea pig 71 (gift of Dr. J. C. Brown). The sensitivity of the assay was 50 pg/ml with intra-assay and
interassay variations of 11.4% and 8%, respectively. When the
concentrations of free catecholamines were determined, 10 ml of blood
were collected into sterile Venglet blood collection tubes (Terumo
Medical, Elkton, MD) containing 143 IU sodium heparin. The tubes were
placed on crushed ice, and the blood samples were immediately spun for
30 min in a refrigerated centrifuge (4°C) at 2,500 rpm. Five
milliliters of the supernatant plasma were transferred to other tubes
containing 40 µl of sodium metabisulfite 5% solution and stored at
20°C. The catecholamine assay procedure used in this study
was described previously (5).
The general condition of the experimental animals during the
experiments was monitored by recording heart rate and temperature every
20 min. Observations were also made on their general behavior.
Electrical activity was analyzed by identifying MMCs at each recording
electrode. The activity was divided into three phases as previously
described (29). In the study described herein, an MMC was considered to
be present at each recording site when phases I, II, and III were
present in sequence, when phase III activity lasted >3 min, and when
all three phases migrated past at least two consecutive electrode
sites. The duration of each cycle was the time measured between the
beginning of two consecutive phase I activities of complete MMCs at the
same electrode site.
Data are expressed as means ± SD. The appropriate means between
control and experimental data were compared using Student's t-test for paired data. Values of
P
0.01 were considered significant.
 |
RESULTS |
In general, the data obtained in the various stages of the study were
consistent between each trial in the same dog and between the trials of
each dog in the same set of experiments. In the two exceptional
instances in which inconsistent data were obtained within one set of
experiments, the group was subdivided to examine each type of results
separately.
Effects of ICV perfusion of BBS on MMCs.
During a total of 2,306 min of recording time before ICV perfusions
with BBS (10 experiments, 3 dogs), 24 MMCs were recorded (an average of
1 MMC/96 min). All MMCs originated in the stomach and propagated to the
ileum. In 10 of 10 experiments (1,200 min total recording time), ICV
perfusion with BBS disrupted the normal pattern of fasting myoelectric
activity. The time lag between onset of ICV perfusion to change in MMC
activity was 20.9 ± 7.4 min as measured in the stomach. Typically,
phase I activity in the stomach and duodenum was replaced by a period
of intense irregular spike activity, more intense than that recorded
during regular phase II activity but less intense and irregular than
that recorded during normal, regular phase III activity. In the jejunum
and ileum, clusters of intense spike activity were separated by short periods of phase I-like activity (Fig. 1).
These results represent an average occurrence rate of 0 MMC/1,200 min
in the stomach, 1 MMC/300 min in the duodenum, and 1 MMC/200 min in the
jejunum. In 4 of 10 experiments, no phase III activity was observed at any electrode site during the ICV perfusion of BBS. In 4 of 10 experiments, phase III activity originated in the duodenum during ICV
perfusion of BBS, and in 2 of 10 experiments, phase III activity originated in the jejunum. All phase III activity periods that originated in the small intestine migrated to the ileum. After ICV
perfusion of BBS was stopped, the duration of intensive irregular spike
activity in the stomach continued for 79.3 ± 72.6 min. MMC activity
resumed in the stomach in 3 of 10 experiments 218.91 ± 61.0 min
after BBS perfusion stopped. Phase III activity occurred in the
duodenum in five experiments and in the jejunum in one experiment, 68.4 ± 44.9 min and 104.6 min, respectively, after ICV perfusion of BBS
stopped. In the remaining experiment, no phase III activity was
observed at any electrode site. ICV perfusion of BBS significantly
elevated the CSF concentration of BBL immunoreactivity. However, there
was no significant change in the level of BBL immunoreactivity in the
peripheral circulation during ICV perfusion of BBS (Table 1).

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Fig. 1.
Effect of intracerebroventricular (ICV) perfusion of bombesin (BBS) on
migrating myoelectric activity in fasted dog. The 3 panels are
consecutive recordings at 4 electrode sites (S, D, J, and I are
stomach, duodenum, jejunum, and ileum, respectively). Intensive spike
activity in stomach and duodenum was recorded during perfusion of BBS.
Arrows indicate initiation ( ) and cessation ( ) of perfusion.
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ICV perfusion of CSF alone had no effect on the MMC cycling
pattern in six of nine experiments (Fig.
2). In the remaining three experiments, the
duration of phase II activity at all electrode sites was prolonged
during the second MMC. Normal MMC activity was resumed immediately
after the end of perfusion. All MMCs that occurred during ICV perfusion
of CSF originated in the stomach and propagated to the ileum. During a
total of 2,197 min of recording time of myoelectric activity before CSF
perfusion (9 experiments, 3 dogs), 20 MMCs were recorded (an average of
1 MMC/109.8 min). During 1,620 min of CSF perfusion time, 12 MMCs were
recorded (an average of 1 MMC/135.0 min).

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Fig. 2.
Effect of ICV perfusion of only cerebrospinal fluid (CSF) on migrating
myoelectric activity in fasted dog. The 3 panels are consecutive
recordings at 4 electrode sites (S, D, J, and I are described in legend
for Fig. 1). Migrating myoelectric complex (MMC) activity remained
unaffected during perfusion of CSF. Periods of gastric arrhythmia were
recorded during phase I activity (2nd and 3rd panels). Arrows indicate
initiation ( ) and cessation ( ) of ICV CSF.
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The mean ± SD maximum ICV pressure recorded before and during ICV
perfusion of BBS was 7.3 ± 3.5 and 5.4 ± 3.5 cmH2O, respectively. There was no
statistically significant (P > 0.04)
difference. ICV pressures measured before and during perfusion of CSF
alone were 4.2 ± 2.3 and 3.7 ± 3.1 cmH2O, respectively (mean ± SD, P > 0.3).
Effect of IV infusion of BBS on MMCs.
IV infusion of BBS disrupted the normal pattern of myoelectric activity
(Fig. 3). During a total recording time of
2,346 min before IV infusion of BBS (9 experiments, 3 dogs), 19 MMC
cycles were recorded (an average of 1 MMC/123.5 min). During 1,320 min of IV infusion time of BBS, only two periods of phase III activity occurred in the stomach, both of which propagated to the ileum (an
average of 1 MMC/540 min). In seven of nine experiments, no phase III
activity occurred during BBS infusion. Instead, only phase II-like
spike activity was recorded in the jejunum and ileum, whereas only
scant spike activity was observed in the stomach and duodenum. After IV
infusion of BBS was stopped, the first MMCs occurred in the stomach in
seven of nine experiments. In the remaining two experiments, MMC
activity first occurred in the jejunum. IV infusion of BBS
significantly elevated the circulating level of BBL immunoreactivity
(Table 1).

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Fig. 3.
Effect of intravenous (IV) infusion of BBS on migrating myoelectric
activity in fasted dog. The 3 panels are consecutive recordings at 4 electrode sites (S, D, J, and I are described in legend for Fig. 1).
Phase III activity was suppressed and replaced by irregular spike
activity, mainly in jejunum and ileum. Periods of gastric arrhythmia
were recorded during phase I activity (1st and 3rd panels). Arrows
indicate initiation ( ) and cessation ( ) of infusion. NS, normal
saline.
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Effect of ICV perfusion of BBS on motilin.
The maximum and minimum plasma motilin concentrations during ICV
perfusion of BBS are given in Table 2. The
results are given separately for two subgroups:
subgroup A1 included 6 of 10 experiments in which phase III activity occurred in the duodenum or
jejunum and subgroup A2 included 4 of
10 experiments in which no phase III activity occurred at any recording
site. In subgroup A1, the maximum and
minimum plasma motilin concentrations during ICV perfusion of BBS were
not significantly (P > 0.2)
different from respective values observed during the control period.
The corresponding values in subgroup
A2 were significantly
(P = 0.01) higher during ICV perfusion
of BBS than during the control period. An example of the effect of ICV
perfusion of BBS on plasma motilin levels is illustrated in Fig.
4.

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Fig. 4.
Effect of ICV perfusion of BBS on plasma motilin concentration in
fasted dog. Occurrence of MMC activity is shown in schematic form.
Overlapping line represents endogenous plasma motilin concentration.
Cycling of motilin persisted despite suppression of phase III
activity.
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During ICV perfusion of CSF alone, the maximum plasma motilin
concentration when phase III activity of an MMC occurred in the stomach
was 106.0 ± 30.2 pg/ml. The motilin level decreased to 63.4 ± 22.6 pg/ml when phase III activity of the same MMC reached the distal
ileum (mean ± SD, n = 9). This
difference was significant (P < 0.001). The maximum and minimum levels of plasma motilin before ICV
perfusion of CSF alone were 94.0 ± 23.9 and 55.2 ± 18.7 pg/ml,
respectively (mean ± SD, n = 9, P < 0.0001). These values were not
statistically (P > 0.02) different
from the respective values observed during ICV perfusion of CSF. An
example of the effect of ICV perfusion of CSF on plasma motilin levels
is illustrated in Fig. 5.

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Fig. 5.
Effect of ICV perfusion of only CSF on plasma motilin concentration in
fasted dog. Occurrence of MMC activity is shown in schematic form.
Overlapping line represents endogenous plasma motilin concentration.
Peak levels were measured when phase III activity was present in
stomach and duodenum.
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Effect of IV infusion of BBS on motilin.
During IV infusions of BBS, plasma motilin concentration was 110.6 ± 16.5 pg/ml when phase III activity of an MMC occurred in the
stomach, and it decreased to 59.2 ± 20.4 pg/ml (mean ± SD,
n = 9) when the phase III activity of
the same MMC reached the distal ileum. This difference was significant
(P < 0.001). During the control
period before IV infusion of BBS, the values were 96.8 ± 20.1 and 54.9 ± 10.9 pg/ml, respectively (means ± SD,
n = 9, P < 0.001). These values were not
statistically (P > 0.05) different
from the respective values observed during IV infusion of BBS. An
example of the effect of IV infusion of BBS on plasma motilin levels is
illustrated in Fig. 6.