Interaction between neurokinin A, VIP, prostanoids, and enteric nerves in regulation of duodenal function

Anneli Hällgren, Gunnar Flemström, and Olof Nylander

Department of Physiology, Biomedical Center, Uppsala University, S-751 23 Uppsala, Sweden

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Neurokinin A (NKA) induces duodenal motility and increases mucosal permeability and bicarbonate secretion in the in situ perfused duodenum in anesthetized rats. In the present study, the NKA-induced increase in mucosal permeability was potentiated by luminal perfusion with lidocaine and diminished by vasoactive intestinal peptide (VIP) but unaltered by elevated intraluminal pressure. Elevation of intraluminal pressure, however, potentiated the stimulatory effect of NKA on bicarbonate secretion. In contrast, the tachykinin decreased the rate of alkalinization in rats subjected to elevated intraluminal pressure and treated with indomethacin. Similarly, NKA partially inhibited the VIP-stimulated bicarbonate secretion. Luminal lidocaine did not affect the secretory response to NKA. The motility induced by NKA was unaffected by VIP or lidocaine but decreased by elevated intraluminal pressure. It is concluded that the NKA-induced increase in duodenal mucosal bicarbonate secretion is independent of neurons and possibly mediated by prostanoids. The increase in mucosal permeability in response to NKA may be suppressed by mucosal nerves, perhaps utilizing VIP as one of the transmitters.

bicarbonate; duodenum; motility; permeability; tachykinin

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

GASTROINTESTINAL secretomotor reflexes, activated by chemical or mechanical stimuli such as distension, have been demonstrated in different regions of the gut and in several species (4). We have previously suggested (11, 32, 33) that a secretory reflex is involved in the regulation of duodenal alkalinization. This suggestion is based on the findings that elevation of intraluminal pressure, either by distension or induction of duodenal motility, increases duodenal mucosal bicarbonate secretion by a neural mechanism involving nicotinic transmission.

The tachykinin neurokinin A (NKA), which is present in excitatory motoneurons in the enteric nervous system (9), is a known stimulant of duodenal motility (14, 18, 21, 25). Furthermore, the tachykinins, of which substance P is the most extensively studied, stimulate intestinal secretion of H2O and electrolytes from the mucosa of the small intestine as well as of the colon (26). In a recent study, we demonstrated that the tachykinin NKA, while inducing intense duodenal motility, moderately increased duodenal mucosal bicarbonate secretion (10). These results support the notion of a motility-activated secretory reflex. However, an inhibitory mechanism seemed to be concealed in the response, because the indomethacin-induced increase in alkalinization was diminished by NKA. One of the objectives of the present study was therefore to further investigate the inhibitory and stimulatory mechanisms involved in the effect of NKA on basal and stimulated duodenal alkalinization. Duodenal bicarbonate secretion was stimulated by vasoactive intestinal peptide (VIP) or distension of the duodenal segment.

One of the major roles of the intestinal mucosa is to limit passive passage of luminal contents across the epithelium. The capacity to impede such movement is generally referred to as the barrier function of the mucosa. The tight junctions are the main structures that restrict the passage of solutes and ions by the paracellular route (28). It has been demonstrated in vitro that the tight junctions between epithelial cells are highly dynamic, enabling regulation of paracellular permeability in response to various stimuli (19, 20). We have previously demonstrated (10) that NKA increases mucosal permeability. This effect was independent of nicotinic transmission but required the activation of NK-2 receptors. NKA is one of the neuropeptides suggested to mediate neurogenic inflammation (34), and the effect on mucosal permeability may thus be important in the inflammatory process. A major aim of the present study was to further elucidate the mechanism behind the increase in mucosal paracellular permeability obtained in response to NKA. The question as to whether the paracellular permeability is physiologically regulated in the intact intestinal epithelium in vivo was also addressed.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Surgical Procedures

Male Sprague-Dawley rats (Møllegaard Breeding Center, Ejby, Denmark), weighing 200-300 g, were fasted overnight with free access to drinking water. The animals were kept under constant conditions (temperature 21°C; 12:12-h light-dark cycle) in groups of at least two. The rats were anesthetized by an intraperitoneal injection of 120 mg/kg body wt Na-5-ethyl-1-(1'-methyl-propyl)-2-thiobarbituric acid (Inactin). The surgical procedure was similar to that previously described by Hällgren et al. (11). Briefly, the rats were tracheotomized to facilitate spontaneous breathing, and a PE-90 cannula containing heparin (12.5 IU/ml dissolved in isotonic saline) was inserted into the left common carotid artery for blood sampling. The right femoral artery was catheterized and connected to a pressure transducer (P23 ID, Gould Electronics, Bilthoven, The Netherlands) and a polyrecorder (Polygraph model 7D, Grass Instruments, Quincy, MA) to monitor arterial blood pressure. Both femoral veins, and in some experiments also the left jugular vein, were catheterized for the infusion of fluid, electrolytes, and the radioactive isotope Cr-EDTA and the administration of drugs.

The abdominal cavity was opened by a midline incision. To exteriorize pancreaticobiliary secretions, which would otherwise interfere with the determination of mucosal bicarbonate output, a small catheter (PE-10) was inserted into the common bile duct very close to its entrance into the duodenum (16). A soft plastic tubing (1 mm ID; Silastic, Dow Corning) was introduced via the mouth and gently pushed through the esophagus into the stomach and then through the pylorus into the duodenum. This tubing was secured by a ligature 2-4 mm distal to the pylorus. Another cannula (PE-320) was inserted into the duodenum through an incision ~3 cm distal to the pylorus and secured by ligatures. The orally introduced tubing was connected to a peristaltic pump (Gilson Miniplus 3, Villiers, Le Bel, France) for perfusion of the duodenal segment. The abdominal cavity was closed with sutures, and the body temperature of the animal was maintained at ~37.5°C with a heating pad controlled by an intrarectal thermistor. All experiments were approved by the Uppsala Ethics Committee for Animal Experiments.

Bicarbonate Secretion

The rate of luminal alkalinization was determined by titration of the effluent to pH 6 with 50 mM HCl (pH-stat equipment, Autoburette ABU 12, TTT 80 Titrator, and PHM 64 pH meter, Radiometer, Copenhagen, Denmark), under continuous gassing with 100% N2. The pH electrode was routinely calibrated with standard buffers (pH 7 and pH 4) before titration. The rate of luminal alkalinization was expressed as the amount of base (micromoles) secreted per centimeter of intestine per hour. After each experiment, the duodenal segment was excised and the length was measured under a standardized stretch, by attachment of a fixed weight (2.7 g) to one of the ends of the segment. The net increase in secretion was calculated by subtracting the mean basal bicarbonate secretion from each sample collected during or after treatment. The values given are mean increases calculated as the average increase during the plasma steady state of NKA (thus the first 10-min period of the infusion was omitted).

Motility

Duodenal contractions (motility) were monitored as changes in intraluminal pressure. A pressure transducer (Gould P23 ID) was connected to the inlet cannula of the perfusion system via a T-tube. Via a digitizer, changes in intraluminal pressure were recorded on a computer using Superscope software (GW Instruments, Somerville, MA). Duodenal motility was assessed in sample intervals of 10 min in the following ways: 1) by planimetry, i.e., the total area under the pressure curve (area under curve; AUC) during the sample period; 2) by calculating the fraction of time occupied by contractions (fractional contraction time; FCT); and 3) frequency of contractions, i.e., the number of contractions per minute during the sampling interval. The values given are means of the sample intervals before or during the infusion of NKA.

Mucosal Permeability

After completion of surgery, 51Cr-EDTA was administered intravenously as a bolus of ~75 µCi, followed by a continuous infusion at a rate of ~50 µCi/h. The radioactive isotope was diluted in a Ringer-bicarbonate solution and infused at a rate of 1 ml/h (infusion pump 22, Harvard Apparatus, Edenbridge, UK). One hour was permitted for tissue equilibration of 51Cr-EDTA and for the animal to recover from surgery. Three to four blood samples (0.2 ml) were collected at regular time intervals during the experiment, and the blood volume loss was compensated for by the injection of a 5% albumin solution. After centrifugation of the samples, we removed 50 µl of the plasma for measurement of radioactivity. The luminal perfusate and blood plasma were analyzed for 51Cr activity (gamma counter 1282, Compugamma CS, Pharmacia, Uppsala, Sweden). The plasma samples were extrapolated by linear regression to obtain a corresponding plasma value for each effluent sample. After each experiment, the duodenal segment was excised, rinsed, and weighed on an electric precision balance for determination of wet tissue weight. The clearance of 51Cr-EDTA from blood to lumen was calculated according to the following formula
<SUP> 51</SUP>Cr-EDTA clearance 
= <FR><NU>effluent (cpm/ml) × perfusion rate (ml/min)</NU><DE>plasma (cpm/ml) × tissue wt (g)</DE></FR> × 100 g
51Cr-EDTA clearance was expressed as milliliters per minute per 100 g wet tissue weight. The net increase was calculated by subtracting the mean basal permeability from each sample collected during or after treatment. The mean increases in response to NKA are calculated as the average increase during the steady-state plasma concentration of NKA (thus the first 10-min period of the infusion was omitted).

Experimental Design

The duodenal segment under study was perfused with isotonic saline at a rate of ~0.4 ml/min both during the hour of recovery and subsequently throughout the experiments. During the experiments, the effluent was collected in 10-min samples. All protocols began with a 20- or 30-min control period to assess basal conditions.

Infusion of NKA. After a 30-min control period, NKA was infused for 60 min at a rate of 400 pmol · kg-1 · min-1 (n = 11). The experiments were continued for an additional 30 min after the infusion had been stopped.

Pretreatment with VIP. VIP is an established stimulant of duodenal mucosal bicarbonate secretion. This set of experiments was performed to investigate the effect of NKA on VIP-stimulated bicarbonate secretion. Twenty minutes after the start of effluent collection, the animals (n = 6) were given an intravenous injection of 9 µg/kg VIP followed by continuous infusion of VIP at a rate of 9 µg · kg-1 · h-1. After an additional 40 min, an NKA infusion was started (400 pmol · kg-1 · min-1) and maintained throughout the experiment. After 60 min of NKA, the VIP infusion was turned off while the infusion of NKA was continued for an additional 30 min.

VIP controls. This group of rats (n = 5) received VIP in the same manner as the above-described group, i.e., 9 µg/kg as a bolus injection followed by 9 µg · kg-1 · h-1 as an intravenous infusion. The infusion was stopped 30 min before termination of the experiment. This group served as controls to the VIP-NKA group.

Pretreatment with lidocaine. To examine whether any of the effects of NKA are mediated via enteric neurons, we perfused the duodenal segment with the local anesthetic lidocaine. Ten minutes before the start of effluent collection, the luminal perfusion was switched from isotonic saline to an isotonic 0.3% lidocaine solution (n = 6). After a 30-min control period, NKA was administered as an intravenous infusion (400 pmol · kg-1 · min-1) for 1 h. Thirty minutes after the offset of the infusion the experiments were terminated.

Lidocaine controls. After a 20-min control period, the saline perfusion was substituted by 0.3% lidocaine solution (n = 4). The lidocaine perfusion was continued throughout the experiment (130 min of luminal exposure). The experiments were designed to match the lidocaine-NKA group described above and also to assess the effects of local anesthesia on basal parameters. The lidocaine solution had a slightly higher pH than isotonic saline. To obtain correct values of bicarbonate secretion, we titrated two blanks of the lidocaine solution to pH 6 and subtracted the mean from each effluent sample.

Elevated intraluminal pressure. These experiments were performed for two reasons. First, we were interested in studying the effect of the tachykinin on duodenal alkalinization induced by distension. Second, we wanted to investigate the possible mechanism of a hydrostatic pressure gradient moving Cr-EDTA from interstitium to lumen, and therefore we applied an opposing intraluminal pressure. Twenty minutes before the start of effluent collection, the intraluminal pressure was elevated 10 cmH2O by raising the outlet cannula of the perfusion system (n = 6). After a 30-min control period the rats received an intravenous infusion of NKA (400 pmol · kg-1 · min-1) for 1 h. Thirty minutes after cessation of the infusion the experiments were terminated.

Elevated intraluminal pressure and indomethacin pretreatment. The intraluminal pressure was elevated in the same manner as in the protocol described above (i.e., 10 cmH2O 20 min before the start of effluent collection). In addition, indomethacin was administered in a dose of 5 mg/kg given intravenously 10 min before the start of effluent sampling. As described above, after a 30-min control period, the rats received a 1-h infusion of NKA administered intravenously (400 pmol · kg-1 · min-1). The collection of effluent and registration of motility continued for an additional 30 min after termination of the NKA infusion.

Controls. One group of rats (n = 6) served as time controls (120 min) and did not receive any of the tested drugs.

Chemicals

Inactin was obtained from Research Biochemicals (Natick, MA). NKA was purchased from Peninsula Laboratories (Merseyside, UK). Lidocaine hydrochloride (10 mg/ml Xylocaine for injection, without preservatives) was from Astra (Södertälje, Sweden). Indomethacin (Confortid for injection) was obtained from Dumex (Copenhagen, Denmark). VIP, rabbit albumin, and NaCl were purchased from Sigma Chemical (St. Louis, MO). Heparin was from Pharmacia and 51-EDTA from NEN (Boston, MA).

Statistics

Values are expressed as means ± SE. Data were tested for statistical significance by ANOVA followed by Fishers protected least-significant difference test, comparing results before and after drug treatment (repeated measures) and comparing differences between groups of animals (nonrepeated measures). Unpaired Student's t-test was also used where appropriate. Two-way ANOVA was used to compare the effects of NKA on mucosal permeability in VIP-treated and "untreated" animals. The values given in RESULTS are pooled from the measurements taken during the basal or treatment period. All statistical analyses (except the 2-way ANOVA) were performed on a Macintosh computer using Statview software (Abacus Concepts, Berkeley, CA). P < 0.05 was considered significant.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Bicarbonate Secretion

NKA significantly increased bicarbonate secretion, and the mean increase in bicarbonate output was 2.3 ± 0.7 µmol · cm-1 · h-1. After cessation of infusion, bicarbonate secretion returned to basal level and did not differ from controls (Fig. 1A).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 1.   Net effect of intravenously infused neurokinin A (NKA) (400 pmol · kg-1 · min-1) on duodenal mucosal bicarbonate secretion. Net rate of alkalinization was calculated by subtracting mean basal secretion from each subsequent value. Arrows indicate onset and offset of infusion. Values are means ± SE. A: 1 group of animals received NKA alone (bullet ; n = 11), whereas a second group was perfused with 0.3% lidocaine before, during, and after NKA infusion (star ; n = 6). The third group served as time controls and did not receive NKA or lidocaine (open circle ; n = 6). B: as in A, 1 group received NKA alone (bullet ; n = 11) and the control group did not receive any drugs or other treatment (open circle ; n = 6). The 2 remaining groups were subjected to elevated intraluminal pressure (10 cmH2O) before NKA infusion and throughout the experiment, in the absence (black-triangle; n = 6) or in the presence of indomethacin (5 mg/kg; down-triangle; n = 7).

Lidocaine perfusion decreased basal bicarbonate secretion from 9.4 ± 1.2 to 5.3 ± 1.0 µmol · cm-1 · h-1 (P < 0.001). However, the net response to NKA (mean increase 2.1 ± 0.9 µmol · cm-1 · h-1) was similar to that in saline-perfused rats (Fig. 1A).

Elevation of intraluminal pressure has previously been shown to increase duodenal bicarbonate secretion (33). Thus the objective was to test whether NKA affected this response. Basal secretion (i.e., during the control period) was doubled (16.5 ± 3.5 µmol · cm-1 · h-1) in rats subjected to elevated intraluminal pressure compared with those with normal luminal pressure (8.0 ± 1.2 µmol · cm-1 · h-1; P < 0.05). Furthermore, in the group subjected to elevated intraluminal pressure, the stimulatory effect of NKA on duodenal bicarbonate secretion was significantly augmented (P < 0.01), and the mean increase was 5.7 ± 0.8 µmol · cm-1 · h-1 (Fig. 1B). In contrast, in animals subjected to elevated intraluminal pressure in combination with indomethacin, NKA decreased the rate of luminal alkalinization (Fig. 1B). The mean decrease was 10.0 ± 1.5 µmol · cm-1 · h-1 (P < 0.001). Basal secretion (20.5 ± 2.7 µmol · cm-1 · h-1) did not differ significantly from the level in the group exposed to elevated intraluminal pressure alone.

VIP increased duodenal mucosal bicarbonate secretion from 7.0 ± 1.7 to 19.5 ± 4.3 µmol · cm-1 · h-1 at steady state (P < 0.01; Fig. 2). Administration of NKA to animals treated with VIP resulted in a transient increase of bicarbonate output followed by a 60% decrease compared with VIP-treated controls (P < 0.05). Calculations of the change in secretion induced by NKA showed, as mentioned above, a net increase of 2.3 ± 0.7 µmol · cm-1 · h-1 in response to the tachykinin, whereas the net steady-state effect of NKA was negative (-4.4 ± 1.4 µmol · cm-1 · h-1) in VIP-treated rats (P < 0.001; Fig. 3). The transient increase in alkaline secretion observed immediately after the start of the infusion of NKA is most probably due to the induction of intense duodenal motility, expelling already secreted bicarbonate (and bicarbonate-containing mucus) from the segment under study.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 2.   Effect of vasoactive intestinal peptide (VIP) alone (9 µg/kg followed by 9 µg · kg-1 · h-1; square ; n = 5) and NKA (400 pmol · kg-1 · min-1) in rats treated with VIP (black-square; n = 6) on the rate of alkalinization. Infusion of VIP was stopped 30 min before the end of the protocol, whereas infusion of NKA continued throughout the experiments. Arrows depict onset of infusions. A third group served as untreated time controls (open circle ; n = 6). Values are means ± SE.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Net effect of NKA on rate of alkalinization in animals treated with VIP (9 µg/kg followed by 9 µg · kg-1 · h-1; black-square; n = 6) compared with NKA alone (400 pmol · kg-1 · min-1; bullet ; n = 11). Arrow indicates commencement of NKA infusion. A third group served as untreated time controls (open circle ; n = 6). Values are means ± SE.

Motility

During basal conditions, as well as in control animals, postoperative spontaneous duodenal contractions were very rare. Infusion of NKA promptly induced profound duodenal motility (n = 6; Fig. 4A). The mean FCT was 0.33 ± 0.04, and AUC was 1,682 ± 273 mmHg × min. The frequency of contractions was 16 ± 1 contractions/min. The NKA-evoked motility disappeared only minutes after termination of the infusion.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of NKA on duodenal motility determined as the total area under the curve (AUC) of the recorded intraluminal pressure. Values are means ± SE. A: all 3 groups received an intravenous infusion of NKA (400 pmol · kg-1 · min-1), either alone (bullet ; n = 6) or in combination with a 0.3% lidocaine perfusion of the duodenal segment (star ; n = 6) or combined with a VIP infusion (9 µg/kg followed by 9 µg · kg-1 · h-1; black-square; n = 6). Arrows indicate commencement and cessation of NKA infusion. B: 1 group of rats, with normal intraluminal pressure, received NKA (400 pmol · kg-1 · min-1; bullet ; n = 6) as an intravenous infusion. The 2 remaining groups were subjected to elevated intraluminal pressure (10 cmH2O) before NKA infusion and throughout the experiment, either without indomethacin (black-triangle; n = 6) or in combination with indomethacin treatment (5 mg/kg; down-triangle; n = 7).

Perfusion of the duodenal segment with lidocaine did not alter basal conditions. The motility response induced by NKA appeared to be slightly decreased in lidocaine-treated subjects, although the changes did not attain statistical significance (Fig. 4A). Mean FCT was 0.25 ± 0.02, and AUC was 1,199 ± 115 mmHg × min. The frequency was unaltered by lidocaine (14 ± 1 contractions/min).

Infusion of VIP did not significantly alter (although the tendency was toward decreased motility) the response to NKA in any of the investigated parameters (Fig. 4A). Mean FCT was 0.30 ± 0.05, AUC was 1,295 ± 275 mmHg × min, and frequency was 16 ± 1 contractions/min.

NKA also induced duodenal motility in animals subjected to increased intraluminal pressure (Fig. 4B). However, elevation of the intraluminal pressure significantly diminished the motility response to NKA and FCT (0.05 ± 0.01), AUC (145 ± 41 mmHg × min), and the frequency of contractions (4 ± 1 contractions/min) were all substantially decreased compared with animals with unaltered luminal pressure (P < 0.001).

Indomethacin induced duodenal motility with an FCT of 0.10 ± 0.01 and an AUC of 345 ± 56 mmHg × min (n = 12; data not shown). The ability of the cyclooxygenase inhibitor to induce duodenal motility was unaltered by elevation of intraluminal pressure (FCT, 0.10 ± 0.03; AUC, 410 ± 136 mmHg × min; Fig. 4B). In rats subjected to elevated intraluminal pressure, the motility induced by NKA was significantly increased by indomethacin pretreatment (P < 0.05). The AUC (621 ± 166 mmHg × min; P < 0.01; Fig. 4B) and FCT (0.18 ± 0.05; P < 0.05) were, however, still significantly lower than in subjects with normal luminal pressure. The frequency of contractions (13 ± 2 contractions/min) during infusion of the tachykinin was also significantly increased by cyclooxygenase inhibition and no longer differed from the level of the group with normal luminal pressure. The motility pattern induced by indomethacin, characterized by bursts of contractions interrupted by periods of quiescence, differed markedly from the continuous motility induced by NKA. In rats exposed to elevated intraluminal pressure in combination with indomethacin, the motility pattern during infusion of NKA was similar to that normally induced by the neuropeptide and was atypical of indomethacin. Nevertheless, none of the parameters used to assess motility changed significantly in response to NKA in these animals.

Mucosal Permeability

Mucosal permeability increased in response to NKA, an effect that was rapidly reversed on termination of the infusion. The mean increase during NKA infusion was 0.40 ± 0.05 ml · min-1 · 100 g-1 (Fig. 5A).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 5.   Effect of NKA (400 pmol · kg-1 · min-1) on duodenal mucosal permeability, assessed as the blood-to-lumen clearance of 51Cr-EDTA. A: response to NKA in rats with unaltered luminal pressure (bullet ; n = 11) or increased luminal pressure, either without additional drugs (black-triangle; n = 6) or in combination with indomethacin treatment (5 mg/kg; down-triangle; n = 7). Included for comparison are untreated time controls (open circle ; n = 6). Values are means ± SE. B: NKA-induced response in rats perfused with 0.3% lidocaine (star ; n = 6) or saline (bullet ; n = 11). A third group served as controls and was not given any drugs (open circle ; n = 6).

Basal mucosal permeability was unaffected by lidocaine perfusion (data not shown). The increase in permeability in response to NKA, however, was potentiated (P < 0.01) by local anesthesia, and the mean increase was 0.95 ± 0.21 (Fig. 5B).

The mean increase in mucosal permeability to 51Cr-EDTA in response to NKA was not changed by elevation of intraluminal pressure alone (0.33 ± 0.04 ml · min-1 · 100 g-1) or in combination with cyclooxygenase inhibition (0.30 ± 0.08 ml · min-1 · 100 g-1) (Fig. 5A).

VIP decreased basal mucosal permeability from 0.27 ± 0.06 to 0.09 ± 0.02 ml · min-1 · 100 g-1 (P < 0.05; Fig. 6). Furthermore, the increase in mucosal permeability otherwise obtained in response to NKA was prevented by VIP treatment (P < 0.01; 2-way ANOVA). When the VIP infusion was stopped, basal mucosal permeability increased slowly and slightly in VIP controls, whereas the increase was quick and substantial in animals receiving an ongoing NKA infusion (Fig. 6). The mean increase on termination of the VIP infusion was 0.35 ± 0.13 ml · min-1 · 100 g-1 in subjects infused with NKA. Thus the net increase in mucosal permeability after VIP infusion was stopped was of the same magnitude as seen in animals given NKA alone.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 6.   Effect of NKA (400 pmol · kg-1 · min-1) on duodenal mucosal permeability, assessed as the blood-to-lumen clearance of 51Cr-EDTA, in animals treated with VIP (9 µg/kg followed by 9 µg · kg-1 · h-1; black-square; n = 6) or given NKA alone (bullet ; n = 11). An additional group of animals was treated with VIP alone (square ; n = 5). Finally, an untreated group of rats served as time controls (open circle ; n = 6)

Arterial Blood Pressure

Infusion of NKA had no sustained effect on mean blood pressure (in some animals a transient dip was observed) compared with controls, which had a mean arterial pressure of 98 ± 8 mmHg. None of the treatments, with the exception of VIP, altered arterial pressure compared with controls. During infusion of VIP, arterial pressure decreased ~10-15 mmHg. However, in animals treated with both VIP and NKA, arterial blood pressure was the same as in controls (data not shown).

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

Mucosal Permeability

The rate-limiting barrier to 51Cr-EDTA transport from blood to intestinal lumen is the mucosal epithelium. Because of its size and hydrophilic properties 51Cr-EDTA passes predominantly, by diffusion or convection, through the paracellular shunts (3, 5). Apart from the concentration gradient, which is assumed to be constant in the experimental setup employed, several other factors, such as the permeability of the tight junction, the interstitium-to-lumen hydrostatic pressure gradient, and the length and charge of the paracellular pathway, could influence the transport of 51Cr-EDTA across the intestinal mucosa.

In a recent study, we demonstrated that NKA increases duodenal mucosal permeability by a mechanism dependent on NK-2 receptors but independent of nicotinic receptors (10). We speculated that NKA, by promoting the release of vasoactive substances or by directly affecting the vascular endothelium, increases the filtration of plasma including plasma proteins, subsequently resulting in an increased interstitial fluid pressure. In fact, NKA has been shown to increase duodenal vascular permeability to Evans blue (17). Consequently, an interstitium-to-lumen hydrostatic pressure gradient, driving the isotope across the epithelium, may be created. The interstitial pressure, however, would be counteracted by the increased intraluminal pressure caused by the profound duodenal motility induced by the tachykinin. Because the contractions are interrupted by relaxations more than half of the time (FCT <0.5), theoretically a hydrostatic pressure gradient could still be responsible for the increase in permeation of 51Cr-EDTA across the mucosa. To test this hypothesis, sustained luminal pressure was applied. Interestingly, the response to NKA was practically identical to the response obtained in animals subjected to normal intraluminal pressure. Hence, convection by hydrostatic forces is less likely to be a mechanism involved in NKA-induced increases in mucosal permeability. Instead, we propose that the elevated interstitial pressure increases the dimensions of the paracellular shunts and therefore the pore area available for diffusion of 51Cr-EDTA. Indeed, pressure and/or volume changes in the intercellular space may alter the permeability of the tight junctions (2).

Interestingly, VIP significantly inhibited the effect of NKA on mucosal permeability. VIP has previously been shown to decrease basal mucosal permeability (22, 23), an effect attributed to the decreased arterial blood pressure. However, in the present study VIP only slightly decreased arterial pressure (10-15 mmHg) but significantly reduced mucosal permeability. Hence, a blood pressure-related mechanism is less likely. The discrepancy between the previous (22, 23) and present studies might be explained by the fact that the kidneys were left intact in the present study, whereas the renal pedicles were ligated, thereby excluding renal influence on the response, in the two former investigations. Nylander et al. (23) also showed that the net increase in permeability in response to acid exposure was reduced by VIP. At present, the mechanism by which VIP prevents the NKA- or acid-induced increment in mucosal permeability is unknown. However, VIP receptors are abundant in rat enterocytes (29) and VIP is a potent stimulant of cAMP production in these cells (15). Interestingly, cAMP has been shown to decrease paracellular permeability in Necturus gallbladder epithelium (2, 6). Hence, VIP may diminish the paracellular permeability by a receptor-mediated and cAMP-dependent decrease in tight junctional permeability. However, VIP is also present in nerves closely associated with small blood vessels in rat intestine (13) and the involvement of vascular effects cannot be excluded.

A very interesting observation is that perfusion of the duodenal segment with lidocaine substantially potentiated the NKA-induced increase in mucosal permeability. Accordingly, the existence of a neural mechanism counteracting the effects of NKA on mucosal permeability is indicated. Bearing in mind the ability of VIP to prevent the NKA-induced increase in mucosal permeability, VIP is suggested as a likely transmitter in these neurons.

Bicarbonate Secretion

We have recently shown that, independent of nicotinic transmission, the tachykinin NKA moderately stimulates duodenal mucosal bicarbonate secretion via NK-2 receptor activation (10). In the present study, perfusion of the duodenum with lidocaine in a concentration sufficient to strongly reduce indomethacin-stimulated alkalinization (O. Nylander, A. Hällgren, and M. Sababi, unpublished observation) did not prevent the NKA-induced increase in secretion. These results, together with the previous finding with hexamethonium mentioned above, indicate that the bicarbonate-stimulating action of the tachykinin is mediated by a nonneural mechanism.

In rats subjected to elevated duodenal intraluminal pressure, the effect of NKA on duodenal bicarbonate secretion was potentiated. This effect appears to be mediated via endogenous prostaglandins, because pretreatment with indomethacin transformed the alkaline response to NKA from stimulatory to inhibitory. The most likely candidate is prostaglandin E2 (PGE2), which is a known stimulant of duodenal mucosal bicarbonate secretion (8). In fact, NKA has been shown to increase the output of PGE2 by activation of recombinant NK-2 receptors expressed in Chinese hamster ovary cells (7). Moreover, a brief luminal exposure to capsaicin, a neurotoxin known to release NKA (31) and other neurotransmitters from sensory afferents, increased duodenal mucosal bicarbonate secretion, an effect that was mitigated by indomethacin (36). Substance P-induced ion transport, studied in porcine jejunum and canine colon in vitro, has also been shown to be partly dependent on cyclooxygenase activity (27, 30).

We have previously proposed that the inhibitory effect of NKA may be due to suppression of a mechanoreceptor-activated secretomotor reflex (10). This notion is further supported by our finding that the tachykinin substantially inhibited the combined indomethacin- and distension-induced secretion of bicarbonate. However, NKA did not inhibit the secretion induced by distension alone. Two different mechanoreceptor-activated secretomotor reflexes may exist; one stimulated by distension and one by motility (Fig. 7). A difference between distension- and indomethacin-induced secretion is supported by the previous finding that the former, but not the latter, requires intact vagal nerves (33). We suggest that distension stimulates epithelial secretion of bicarbonate via a reflex involving nicotinic receptor-dependent release of endogenous prostaglandins (Fig. 7).


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 7.   A schematic model of proposed effects of NKA on epithelial bicarbonate secretion. Similar to indomethacin [which acts by preventing prostacyclin (PGI2) formation], NKA induces duodenal motility and thereby stimulates an assumed motility-activated secretory reflex. However, NKA also inhibits transmission to the epithelial cell. Furthermore, NKA, as well as the distension (pressure)-induced reflex, stimulates secretion of bicarbonate via the release of endogenous prostaglandins (PGs). Indomethacin inhibits endogenous prostaglandin synthesis, thereby preventing the positive effects of NKA and distension and revealing the inhibitory effect of the tachykinin. CNS, central nervous system.

Similar to the effect on indomethacin-stimulated bicarbonate secretion (10), NKA also decreased VIP-stimulated alkalinization. In the rabbit duodenum in vitro, VIP has been suggested to exert some of its effect via activation of secretomotor neurons (12). Furthermore, in the conscious rat, the bicarbonate secretion stimulated by exogenous VIP is partly dependent on nicotinic transmission (35). It is possible that the neural mechanisms involved in VIP-stimulated secretion may be suppressed by NKA. Alternatively, PGE2, which we believe to be released by NKA, may decrease VIP-stimulated alkaline secretion as has been shown in guinea pigs (24). Furthermore, the notion that VIP-stimulated duodenal secretion of bicarbonate may be negatively modulated by prostaglandins is supported by the finding that the effect of VIP on luminal alkalinization is potentiated by indomethacin (35).

Motility

The effect of NKA on intestinal motility has been reported to be due mostly to direct activation of smooth muscle cells but also to some degree to stimulation of cholinergic neurons (1). A slight but nonsignificant decrease in motility in response to NKA in rats luminally exposed to the local anesthetic lidocaine was noted. The lack of neural involvement, at least of the nicotinic receptor-dependent type, has been implied by one of our previous studies involving the tachykinin (10). VIP, known to relax intestinal smooth muscle, may be anticipated to reduce the contractile response to the tachykinin. Again, a small but nonsignificant decrease in the motor response to NKA was observed in VIP-treated animals. These results indicate that NKA is a very potent stimulant of duodenal motility and that most of its effect is exerted directly on the smooth muscle cell.

The NKA-induced duodenal motility was reduced by distension of the duodenal segment, raising the question of whether the stretched muscle maintains full ability to contract. However, the duodenal motility induced by indomethacin was unaltered by elevation of intraluminal pressure. Furthermore, cyclooxygenase inhibition partly restored the effect of the tachykinin, suggesting that endogenous prostaglandins may reduce smooth muscle activity. Although the motility data of the present study do not enable us to distinguish between indomethacin- and NKA-evoked motility during the infusion of the tachykinin, the pattern was clearly of the NKA type and not of the intermittent kind usually observed in response to indomethacin.

In summary, the results of the present study indicate that epithelial paracellular permeability is under neural control and that VIP is important in maintaining duodenal mucosal integrity. Furthermore, we postulate that NKA stimulates bicarbonate secretion by a nonneural, prostaglandin-dependent mechanism. It may well be that the inhibitory action of NKA on duodenal alkaline secretion is exerted within the enteric nervous system.

    ACKNOWLEDGEMENTS

This study was supported by grants from the Swedish Medical Research Council (Grant 04X-03515), The Swedish National Board of Health and Welfare, and the Swedish Society for Medical Research.

    FOOTNOTES

Address for reprint requests: A. Hällgren, Dept. of Physiology, Biomedical Center, Uppsala Univ., PO Box 572, S-751 23 Uppsala, Sweden.

Received 22 September 1997; accepted in final form 24 March 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bartho, L., and P. Holzer. Search for a physiological role of substance P in gastrointestinal motility. Neuroscience 16: 1-32, 1985[Medline].

2.   Bentzel, C. J., C. E. Palant, and M. Fromm. Physiological and pathological factors affecting the tight junction. In: Tight Junctions, edited by M. Cereijido. Boca Raton, FL: CRC, 1992, p. 151-173.

3.   Bjarnason, I., A. Macpherson, and D. Hollander. Intestinal permeability: an overview. Gastroenterology 108: 1566-1581, 1995[Medline].

4.   Chang, E. B., and M. C. Rao. Intestinal water and electrolyte transport. Mechanisms of physiological and adaptive responses. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 2027-2081.

5.  Crissinger, K. D., P. R. Kvietys, and D. N. Granger. Pathophysiology of gastrointestinal mucosal permeability. J. Intern. Med. 28, Suppl. 1: 145-154, 1990.

6.   Duffey, M. E., B. Hainau, S. Ho, and C. J. Bentzel. Regulation of epithelial tight junction permeability by cyclic AMP. Nature 294: 451-453, 1981[Medline].

7.   Eistetter, H. R., D. J. Church, A. Mills, P. P. Godfrey, A. M. Capponi, R. Brewster, M. F. Schulz, E. Kawashima, and S. J. Arkinstall. Recombinant bovine neurokinin-2 receptor stably expressed in Chinese hamster ovary cells couples to multiple signal transduction pathways. Cell Regul. 2: 767-779, 1991[Medline].

8.   Flemström, G. Gastric and duodenal mucosal secretion of bicarbonate. In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 1285-1310.

9.   Furness, J. B., H. M. Young, S. Pompolo, J. C. Bornstein, W. A. A. Kunze, and K. McConalogue. Plurichemical transmission and chemical coding of neurons in the digestive tract. Gastroenterology 108: 554-563, 1995[Medline].

10.   Hällgren, A., G. Flemström, P. M. Hellström, M. Lördal, S. Hellgren, and O. Nylander. Neurokinin A increases duodenal mucosal permeability, bicarbonate secretion and fluid output in the rat. Am. J. Physiol. 273 (Gastrointest. Liver Physiol. 36): G1077-G1086, 1997[Abstract/Free Full Text].

11.   Hällgren, A., G. Flemström, M. Sababi, and O. Nylander. Effects of nitric oxide inhibition on duodenal function in rat: involvement of neural mechanisms. Am. J. Physiol. 269 (Gastrointest. Liver Physiol. 32): G246-G254, 1995[Abstract/Free Full Text].

12.   Hogan, D. L., B. Yao, J. H. Steinbach, and J. I. Isenberg. The enteric nervous system modulates mammalian duodenal mucosal bicarbonate secretion. Gastroenterology 105: 410-417, 1993[Medline].

13.   Keast, J. R., J. B. Furness, and M. Costa. Distribution of certain peptide-containing nerve fibres and endocrine cells in the gastrointestinal mucosa in five mammalian species. J. Comp. Neurol. 236: 403-422, 1985[Medline].

14.   Kim, Y. C., and P. M. Hellström. Identification of mechanisms for duodenal contraction induced by tachykinins in the rat. J. Gastrointest. Motil. 5: 97-106, 1993.

15.   Laburthe, M., J. C. Prieto, B. Amiranoff, C. Dupont, D. H. B. Hoa, and G. Rosselin. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. II. Characterization and structural requirements of the stimulatory effect of vasoactive intestinal peptide on production of adenosine 3':5'-monophosphate. Eur. J. Biochem. 96: 239-248, 1979[Abstract].

16.   Lambert, R. The pancreatic ducts in the rat. In: Surgery of the Digestive System in the Rat, edited by L. M. Nyhus. Springfield, IL: Thomas, 1965, p. 109-112.

17.   Lördal, M., A. Hällgren, O. Nylander, and P. M. Hellström. Tachykinins increase vascular permeability in the gastrointestinal tract of the rat. Acta Physiol. Scand. 156: 489-494, 1996[Medline].

18.   Lördal, M., C. Johansson, and P. M. Hellström. Myoelectric pattern and effects of small bowel transit induced by the tachykinins neurokinin A, neurokinin B, substance P and neuropeptide K in the rat. J. Gastrointest. Motil. 5: 33-40, 1993.

19.   Madara, J. L. Loosening tight junctions. Lessons from the intestine. J. Clin. Invest. 83: 1089-1094, 1989[Medline].

20.   Madara, J. L., and J. R. Pappenheimer. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membr. Biol. 100: 149-164, 1987[Medline].

21.   Mizrahi, J., S. Dion, P. D'Orleans-Juste, E. Escher, G. Drapeau, and D. Regoli. Tachykinin receptors in smooth muscles: a study with agonists (substance P, neurokinin A) and antagonists. Eur. J. Pharmacol. 118: 25-36, 1985[Medline].

22.   Nylander, O., A. Hällgren, and L. Holm. Duodenal mucosal alkaline secretion, permeability and blood flow. Am. J. Physiol. 265 (Gastrointest. Liver Physiol. 28): G1029-G1038, 1993[Abstract/Free Full Text].

23.   Nylander, O., E. Wilander, G. M. Larson, and L. Holm. Vasoactive intestinal polypeptide reduces hydrochloric acid-induced duodenal mucosal permeability. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G272-G279, 1993[Abstract/Free Full Text].

24.   Odes, H. S., R. Muallem, R. Reimer, M. Schwenk, W. Beil, and K.-F. Sewing. Comparative activities of agonists of active duodenal bicarbonate secretion in the guinea pig. Digestion 55: 410-416, 1994[Medline].

25.   Osakada, F., K. Kubo, K. Goto, I. Kanazawa, and E. Munekata. The contractile activities of neurokinin A, B and related peptides on smooth muscles. Eur. J. Pharmacol. 120: 201-208, 1986[Medline].

26.   Otsuka, M., and K. Yoshioka. Neurotransmitter functions of mammalian tachykinins. Physiol. Rev. 73: 229-308, 1993[Free Full Text].

27.   Parsons, A. M., V. S. Seybold, R. Chandan, J. Vogt, A. A. Larson, C. R. Murray, G. Soldani, and D. R. Brown. Neurokinin receptors and mucosal ion transport in porcine jejunum. J. Pharmacol. Exp. Ther. 261: 1213-1221, 1992[Abstract].

28.   Powell, D. W. Barrier function of epithelia. Am. J. Physiol. 241 (Gastrointest. Liver Physiol. 4): G275-G288, 1981[Abstract/Free Full Text].

29.   Prieto, J. C., M. Laburthe, and G. Rosselin. Interaction of vasoactive intestinal peptide with isolated intestinal epithelial cells from rat. I. Characterization, quantitative aspects and structural requirements of binding sites. Eur. J. Biochem. 96: 229-237, 1979[Medline].

30.   Rangachari, P. K., T. Prior, and D. McWade. Epithelial and mucosal preparations from canine colon: responses to substance P. J. Pharmacol. Exp. Ther. 254: 1076-1083, 1990[Abstract].

31.   Renzi, D., S. Evangelista, P. Mantellini, P. Santicioli, C. A. Maggi, P. Geppetti, and C. Surrenti. Capsaicin-induced release of neurokinin A from muscle and mucosa of gastric corpus: correlation with capsaicin-evoked release of calcitonin gene-related peptide. Neuropeptides 19: 137-145, 1991[Medline].

32.   Sababi, M., A. Hällgren, and O. Nylander. Interaction between prostanoids, NO and VIP in modulation of duodenal alkaline secretion and motility. Am. J. Physiol. 271 (Gastrointest. Liver Physiol. 34): G582-G590, 1996[Abstract/Free Full Text].

33.   Sababi, M., and O. Nylander. Elevation of intraluminal pressure and cyclooxygenase inhibitors increases duodenal alkaline secretion. Am. J. Physiol. 266 (Gastrointest. Liver Physiol. 29): G22-G30, 1994[Abstract/Free Full Text].

34.   Sharkey, K. A. Substance P and calcitonin gene-related peptide (CGRP) in gastrointestinal inflammation. Ann. NY Acad. Sci. 664: 425-442, 1992[Medline].

35.   Smedfors, B., E. Theodorsson, and C. Johansson. HCl-stimulated duodenal HCO-3 secretion in conscious rat. Interactions among VIP, nicotinic receptor mechanisms, and prostaglandins. Dig. Dis. Sci. 39: 2134-2142, 1994[Medline].

36.  Takeuchi, K., T. Ohuchi, J. Matsumoto, and S. Okabe. Regulation of gastroduodenal bicarbonate secretion by capsaicin-sensitive sensory neurons in rats. J. Clin. Gastroenterol. 17, Suppl. 1: S33-S39, 1993.


Am J Physiol Gastroint Liver Physiol 275(1):G95-G103
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society