Effect of cerebroventricular perfusion of bombesin on gastrointestinal myoelectric activity

M. Hashmonai and J. H. Szurszewski

Department of Physiology and Biophysics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

epinephrine; intravenous infusion of bombesin

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 (up-arrow ) and cessation (down-arrow ) of perfusion.

                              
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Table 1.   CSF and plasma levels of BBS

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 (up-arrow ) and cessation (down-arrow ) of ICV CSF.

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 (up-arrow ) and cessation (down-arrow ) of infusion. NS, normal saline.

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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Table 2.   Plasma levels of motilin during ICV perfusion of BBS


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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.

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.

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.