Involvement of mast cells in basal and neurotensin-induced intestinal absorption of taurocholate in rats

Xianyong Gui and Robert E. Carraway

Department of Physiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0127

Submitted 17 April 2003 ; accepted in final form 15 December 2003


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Neurotensin (NT), a hormone released from intestine by ingested fat, facilitates lipid digestion by stimulating pancreatic secretion and slowing the movement of chyme. In addition, NT can contract the gall bladder and enhance the enterohepatic circulation (EHC) of bile acids to promote micelle formation. Our recent finding that NT enhanced and an NT antagonist inhibited [3H]taurocholate ([3H]TC) absorption from proximal rat small intestine indicated a role for endogenous NT in the regulation of EHC. Here, we postulate the involvement of intestinal mast cells in the TC uptake process and in the stimulatory effect of NT. In anesthetized rats with the bile duct cannulated for bile collection, infusion of NT (10 pmol·kg–1·min–1) enhanced the [3H]TC recovery rate from duodenojejunum by 2.2-fold. This response was abolished by pretreatment with mast cell stabilizers (cromoglycate, doxantrazole) and inhibitors of mast cell mediators (diphenhydramine, metergoline, zileuton). In contrast, mast cell degranulators (compound 48/80, substance P) and mast cell mediators (histamine, leukotriene C4) reproduced the effect of NT. NG-nitro-L-arginine methyl ester enhanced and L-arginine inhibited basal and NT-induced TC uptake, consistent with the known inhibitory effect of nitric oxide (NO) on mast cell reactivity. These results argue that basal and NT-stimulated TC uptake in rat jejunum are similarly dependent on mast cells, are largely mediated by release of mast cell mediators, and are subject to regulation by NO.

enterohepatic circulation; nitric oxide; histamine; leukotriene


BILE ACIDS (BA) play a central role in lipid digestion and absorption in animals and humans by forming mixed micelles with monoglycerides and fatty acids (29). The enterohepatic circulation (EHC) enhances the efficiency of fat digestion in animals and humans by recycling the conjugated BA pool 5–15 times per day, depending on the fat content of the diet (30). The uptake of conjugated BAs in the intestine is the rate-determining step for this process, because hepatic uptake and secretion occur ~10 times faster (31). The gall bladder serves primarily to store the BA pool between meals, and it empties rather rapidly with the ingestion of food. Presumably, BA cycling occurs most readily when the demand for BA exceeds that present in the gall bladder. Thus it seems likely that the EHC is regulated at the level of the intestine, especially in animals lacking a gall bladder (e.g., rats and horses) but also in those possessing one (e.g., cats, dogs, and humans).

The regulation of intestinal BA uptake is not well understood. Not only is there controversy regarding the primary site (41) and the mechanism (2, 58) of conjugated BA absorption, but there is uncertainty as to whether hormonal factors control this process (31). However, our recent studies in the rat (25, 26) argue that neurotensin (NT), a peptide stored in mucosal enteroendocrine cells (60) and released by the ingestion of fat (21, 22), is a strong candidate regulator of intestinal BA uptake. It is well accepted that NT operates hormonally to optimize the digestion of fat by slowing the movement of chyme (28) and stimulating pancreatic enzyme secretion (3). A role for NT in the regulation of BA cycling would be a logical extension of these findings. Support for the notion that NT relates to BA function derives from our finding that intestinal NT mRNA expression and blood levels of NT are acutely affected by altering intestinal BA concentration during feeding and by changing the distribution of the BA pool in models of cholestasis and biliary diversion (26). Also consistent is the pharmacological similarity between NT and cholescystokinin, which includes effects on pancreatic secretion, gall bladder contractility, gut motility, anorexia, and neuroleptic activity (4, 7).

Evidence linking NT with intestinal BA cycling can be summarized as follows. NT secretion and conjugated BA absorption follow a similar pattern, increasing with eating and decreasing with fasting (17). Infusion of NT into fasted rats, at doses giving near physiological blood levels, specifically enhanced intestinal absorption of taurocholate (TC), the primary conjugated BA, without altering that for cholic acid, its unconjugated counterpart (25). More importantly, infusion of NT antagonist SR-48692 inhibited intestinal TC absorption, suggesting a role for endogenous NT in this process (26). The effect of NT was more evident in proximal than in distal intestine, and it appeared to involve carrier-mediated absorption rather than active transport. Although the distal intestine, which is endowed with a specific Na+-BA cotransporter, has been classically regarded as the primary site of BA absorption (31), work indicates that >60% of the TC secreted into rat (41) and pig (33) intestine is absorbed before it reaches the ileum. Thus the work on NT is consistent with this new school of thought that contends that proximal intestine plays a major role in BA cycling and that distal intestine functions mostly to ensure that any remaining BA is not lost into the colon. With this model in mind, NT appears to be a good candidate regulator for BA cycling.

A detailed study regarding the effects of NT on intestinal uptake of model compounds known to permeate the epithelium via specific routes gave results consistent with an effect of NT on paracellular permeability and/or vascular permeability (25). Various physiological and pathological conditions, such as food digestion (13), food allergy (52), and inflammation (23), are known to alter epithelial and vascular permeability. Mast cell activation occurs in many of these conditions, and mediators released from mast cells play a prominent role in initiating the permeability change (39). Given that NT is a potent activator of mast cells (8, 55) and that mast cells contribute to its effects on vascular permeability (9) and its involvement in stress-induced reactions (11) and gut inflammation (12), it seems reasonable to propose that the effect of NT on intestinal TC absorption involves mast cells.

The present study aimed to determine whether the enhancing effect of NT on intestinal TC absorption is mast cell dependent and whether it can be reproduced by mast cell mediators. We tested the effects of mast cell stabilizers and stimulants as well as antagonists that specifically block the formation of or actions of histamine, serotonin, leukotriene C4 (LTC4), and nitric oxide (NO). Our results implicate mast cells not only in the response to NT, but also in the normal TC uptake process.


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Materials. Sodium chromoglycate, doxantrazole, diphenhydramine, cymetidine, metergoline, indomethacin, NT, TC (sodium salt), PGE2, substance P, L-arginine, NG-nitro-L-arginine methyl ester (L-NAME) hydrochloride, and buffer salts were purchased from Sigma-Aldrich (St Louis, MO). [3H]TC was obtained from New England Nuclear (Boston, MA). LTC4 was from Cayman Chemical (Ann Arbor, MI). Sanofi Recherche (Montpellier, France) supplied SR-48692. Stocks of NT (1 mM) and SR-48692 (1 mM; DMSO) were at –20°C, and dilutions were made daily in 0.85% NaCl and 0.1% BSA. Solutions of TC (5.0 mM) were prepared daily in PBS (in mM: 138 NaCl, 3 KCl, 6 Na2HPO4, and 6 KH2PO4) and [3H]TC (2.5 µCi/ml) was added. Sodium cromoglycate, cimetidine, metergoline, and indomethacin were dissolved in DMSO and diluted into saline and 0.1% BSA, giving a final DMSO concentration of <5%. Doxantrazole was dissolved in 0.5% NaHCO3 and 0.85% NaCl and was diluted into saline-BSA before use. Zileuton, obtained from Abbott Labs (Chicago IL), was suspended in 1% methylcellulose, which served as the control.

Animals. Male Sprague-Dawley rats (200–300 g, Taconic Farms, Germantown, NY) were housed in the University of Massachusetts Medical School (UMMS) animal facility and given rat chow and water ad libitum. All protocols were approved by the UMMS animal care and use committee.

Experimental procedures. Intestinal uptake of [3H]TC was measured in biliary fistula rats as described by us (25, 26). The rate of appearance of [3H]TC in bile was equated with the rate of intestinal uptake, because this is the rate-determining step (26). Briefly, rats were fasted 24 h and anesthetized with ketamine-xylazine (60:10 mg/kg ip). The right jugular vein was catheterized for NT infusion, the bile duct was cannulated for bile collection, and polyethylene tubing (PE-50) was cemented into the duodenum for infusion of [3H]TC into the duodenojejunum. After surgery, the animals were kept warm with a heat lamp and bile flow was stabilized for 20 min before experiments. Bile was collected into Eppendorf tubes using a fraction collector at 10- to 20-min intervals for 120–180 min as indicated. Bile volume was determined gravimetrically, assuming specific gravity of 1.0. To assess the [3H]TC uptake rate, 1.0 ml of [3H]TC (5 mM) was infused (40 µl/s) into the duodenojejunum and biliary recovery of [3H]TC was quantified by determining radioactivity in collected bile (50 µl) using a Beckman LS 3801 liquid scintillation counter with quench correction.

Saline or NT (10 pmol·kg–1·min–1) was given by constant intravenous infusion via the cannulated jugular vein at 40 µl/min. This dose of NT was chosen to mimic plasma levels of NT observed in animals and humans after ingestion of fatty foods (25). Hepatic portal plasma levels of NT in rats infused with this dose of NT rose from ~25 pM to plateau at ~55 pM within 20 min (25). In our experiments, NT infusion began 20 min before injection of [3H]TC to allow blood levels of NT to plateau. To test the effects of the following drugs on the response to NT, animals were pretreated by an initial intravenous injection 10–20 min before NT infusion, followed by an intraperitoneal injection 40 min later at the following doses: cromoglycate (1 mg/kg iv; 10 mg/kg ip), doxantrasole (10 mg/kg), diphenhydramine (5 mg/kg iv; 10 mg/kg ip), cimetidine (10 mg/kg), metergoline (2 mg/kg), and indomethacin (10 mg/kg). Zileuton (100 mg/kg) was given orally by gavage 1 h before NT infusion. SR-48692 (1 mg/kg) was given intraperitoneally 20 min in advance when the endogenous NT is expected to be antagonized. For each drug, the dose and method of delivery were determined by choosing from the literature conditions shown to be most effective.

Data expression and statistical analysis. TC uptake was plotted in time as the cumulative [3H]TC output expressed as percentage of the administered dose. Data were plotted as means ± SE, and the Student's t-test was used for statistical comparisons with P < 0.05 indicating a significant difference.


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Stabilizers of mast cells blocked the effect of NT on [3H]TC uptake. In agreement with our earlier findings (25, 26), intravenous infusion of biliary fistula rats with a near-physiological dose of NT (10 pmol·kg–1·min–1) caused a 2.4-fold increase in the rate of recovery of [3H]TC from the jejunum (Fig. 1A). TC recovery (%dose) was linear over the 3-h time period, and the uptake rate (Table 1) increased from ~0.10%/min (control) to ~0.24%/min (NT). To test the involvement of mast cells in this response, we examined effects of the mast cell stabilizers sodium cromoglycate and doxantrazole on the response to NT. Pretreatment of rats with cromoglycate (1 mg/kg) or with doxantrazole (10 mg/kg) 10 min before testing abolished the effect of NT, giving TC recovery rates similar to that for saline-injected controls (Fig. 1A).



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Fig. 1. Rate of TC uptake from proximal intestine was enhanced by neurotensin (NT) (A), compound 48/80 (B), and substance P (SP; C and D), and these effects were inhibited by mast cell (MC) stabilizers. Infusion of stimuli began at –20 min, [3H]taurocholate ([3H]TC) was injected at 0 min (arrow), and MC stabilizers were given at times shown (arrows). Cumulative recovery (%) of the administered dose of [3H]TC is plotted as a function of time (means ± SE; n = 5). A: enhancing effect of NT (10 pmol·kg–1·min–1) on TC uptake was abolished (P < 0.01) by pretreatment with cromoglycate (1 mg/kg) or doxantrasole (10 mg/kg). B: compound 48/80 (1 µg·kg–1·min–1) enhanced TC uptake (P < 0.01). C: SP (400 pmol·kg–1·min–1) enhanced TC uptake (P < 0.01), but SP (10 pmol·kg–1·min–1) had no effect. D: response to SP (400 pmol·kg–1·min–1) was inhibited by cromoglycate (P < 0.05) and doxantrazole (P < 0.05).

 

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Table 1. Effect of mast cell-directed agents on rate of [3H]TC uptake from rat duodenojejunum in vivo

 
Activators of mast cells mimicked the effect of NT on [3H]TC uptake. Intravenous infusion of the mast cell degranulator compound 48/80 (500 pmol·kg–1·min–1) into biliary fistula rats enhanced the [3H]TC uptake rate by 2.5-fold (Fig. 1B; Table 1). A similar response (Fig. 1C; Table 1) was obtained by infusing substance P (400 pmol·kg–1·min–1), an inflammatory neuropeptide shown to increase mucosal permeability (23). However, substance P was less potent than NT, because it was ineffective at a dose of 10 pmol·kg–1·min–1 (Fig. 1C). Pretreating the rats with mast cell stabilizer cromoglycate or doxantrazole greatly inhibited the response to substance P (Fig. 1D), showing that it was mast cell mediated.

Diphenhydramine blocked the effect of NT on [3H]TC uptake. Because rat mast cells release histamine in response to NT in vitro (8) and in vivo (9), we tested the effects of antihistamines on the [3H]TC response to NT. Pretreatment of rats with H1-histamine receptor antagonist diphenhydramine (5 mg/kg) given 40 min before testing abolished the TC uptake response to NT (Fig. 2A). In contrast, the H2-histamine receptor antagonist cimetidine (10 mg/kg) given similarly did not alter the effect of NT (Fig. 2A). These data indicated that histamine, acting via H1-histamine receptors, was a potential mediator of the response to NT.



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Fig. 2. Antagonists of MC mediators inhibited NT-induced [3H]TC uptake (A, C, and D), whereas histamine mimicked the effect of NT (B). NT infusion (10 pmol·kg–1·min–1) started at –20 min, [3H]TC was given at 0 min (arrow), and antagonists were given at times shown (arrows). Cumulative recovery (%) of the administered dose of [3H]TC is plotted as a function of time (means ± SE; n = 5). A: effect of NT was abolished by diphenhydramine (10 mg/kg; P < 0.01) but was unaffected by cymetidine (10 mg/kg). B: intra-arterial infusion of histamine (275 nmol·kg–1·min–1) enhanced [3H]TC uptake (P < 0.01). C: metergoline (2 mg/kg) inhibited the [3H]TC uptake response to NT by ~50% (P < 0.05). D: zileuton (100 mg/kg) abolished the effect of NT on [3H]TC uptake (P < 0.01).

 
Histamine mimicked the effect of NT on [3H]TC uptake. Histamine appears to be rapidly destroyed in the blood circulation of rats, because its half-life is estimated at <1 min (49). When infused into the femoral vein of bile fistula rats, histamine (275 nmol·kg–1·min–1) did not alter [3H]TC uptake (data not shown). However, when given into the mesenteric artery of the intestine, histamine (275 nmol·kg–1·min–1) enhanced the rate of [3H]TC uptake by 2.5-fold (Fig. 2B; Table 1), reproducing the effect of NT. These data indicated that histamine was a potential mediator of the effect of NT, but only when given locally or released locally (i.e., from mast cells within the intestine, not from distant mast cells). Consistent with this, blood levels of histamine were not elevated during NT infusion into these rats, in that histamine concentration was <50 nM before and after 5, 15, and 60 min of 10 pmol·kg–1·min–1 NT infusion.

Metergoline reduced the effect of NT on [3H]TC uptake. Because NT also stimulates the release of serotonin from rat mast cells, we tested the effect of metergoline, a type 1 serotonin receptor antagonist (9), on the [3H]TC uptake response to NT. Pretreatment of rats with metergoline (2 mg/kg) 20 min before testing did not alter basal [3H]TC uptake; however, it significantly inhibited (by ~50%) the response to NT (Fig. 2C). These data indicated that serotonin participated in the TC uptake response to NT.

Zileuton reduced the effect of NT on [3H]TC uptake. When given intravenously to rats, NT stimulates mast cell-dependent leukotriene formation, presumably by enhancing 5-lipoxygenase (5-LOX) activity (9). To investigate the involvement of 5-LOX in the [3H]TC uptake response to NT, we tested the effect of zileuton, an orally active 5-LOX inhibitor (10). In rats pretreated with zileuton (100 mg/kg) 60 min before testing, the effect of NT on [3H]TC uptake was abolished (Fig. 2D).

LTC4 mimicked the effect of NT on [3H]TC uptake. Bolus injection of LTC4 (60 µg/kg) produced an enhancement of [3H]TC uptake (Fig. 3A) similar to that produced by NT (Table 1). When infused at a lower dose (10 µg·kg–1·min–1), LTC4 did not alter [3H]TC uptake and did not enhance the response to NT (Fig. 3A). These data suggest that the TC uptake response to NT involved the release of LTC4 formed by the action of 5-LOX.



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Fig. 3. Leukotriene C4 (LTC4) mimicked the effect of NT (A) and PGE2 enhanced the response to NT (B), whereas indomethacin did not alter the response to NT (B). Stimuli were given at –20 min, [3H]TC was given at 0 min (arrow), and indomethacin was given as shown (arrows). Cumulative recovery (%) of the administered dose of [3H]TC is plotted as a function of time (means ± SE; n = 5). A: LTC4 was given as a bolus (60 µg/kg) or as an infusion (10 µg·kg–1·min–1) in the presence and absence of NT (10 pmol·kg–1·min–1). The bolus of LTC4 enhanced [3H]TC uptake (P < 0.01), whereas infusion of LTC4 at the lower dose had no effects. B: infusion of PGE2 (2 µg·kg–1·min–1) did not alter [3H]TC uptake but enhanced the response to NT (P < 0.05). Indomethacin (10 mg/kg) did not alter the response to NT.

 
Indomethacin did not alter the effect of NT on [3H]TC uptake. To investigate involvement of prostaglandins in the [3H]TC response to NT, we tested the effect of indomethacin, a cyclooxygenase inhibitor (27). NT was as effective in rats pretreated with indomethacin (10 mg/kg) 30 min before testing as it was in control rats (Fig. 3B), indicating that prostaglandins were not essential participants.

PGE2 enhanced the effect of NT on [3H]TC uptake. By increasing blood flow, vasodilatory prostaglandins such as PGE2 can enhance permeability responses to leukotrienes. Because our results implicated LTC4 in the [3H]TC uptake response to NT, we tested the effect of PGE2 on the response to NT. Although PGE2 (2 µg·kg–1·min–1) by itself had little effect on [3H]TC uptake (Fig. 3B; Table 1), PGE2 enhanced the response to NT (10 pmol·kg–1·min–1) approximately twofold throughout the time course (Fig. 3B). These data were consistent with the participation of LTC4 in the TC uptake response to NT and indicated that the response could be potentiated by PGE2.

L-NAME enhanced [3H]TC uptake by an effect not involving type 1 NT receptor. Because NO synthase (NOS) can participate in reactions involving changes in intestinal permeability (38), we tested the effects of agents known to alter its action. We found that the NOS inhibitor L-NAME (40 mg/kg) given 20 min before testing enhanced [3H]TC uptake, producing an effect equivalent to that of NT (Fig. 4A; Table 1). NT receptor (NTR1) antagonist SR-48692 (1 mg/kg ip) given 20 min before testing inhibited the response to NT (data not shown) but had little effect on the response to L-NAME (Fig. 4B), indicating that L-NAME was not acting via NTR1. These data were consistent with other work showing that L-NAME can enhance intestinal epithelial permeability by disrupting the stabilizing effects of NO on mast cells (36).



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Fig. 4. Nitric oxide synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) increased [3H]TC uptake (A); the effect was independent of NTR1 (B), and it enhanced the response to NT (C); in contrast, NOS substrate (L-arginine) inhibited the effect of NT (D). Stimuli were given at –20 min, and [3H]TC was given at 0 min (arrow). Cumulative recovery (%) of the administered dose of [3H]TC is plotted as a function of time (means ± SE; n = 6). A: L-NAME (40 mg/kg bolus, followed by 25 µg·kg–1·min–1 infusion) enhanced [3H]TC uptake (P < 0.01). B: pretreatment with SR-48692 (1 mg/kg) did not alter the response to L-NAME, although it blocked the response to NT (data not shown). C: L-NAME enhanced the response to 10 pmol·kg–1·min–1 NT (P < 0.05). D: L-arginine (200 mg/kg bolus, followed by 1 mg·kg–1·min–1 infusion) inhibited the response to 10 pmol·kg–1·min–1 NT (P < 0.05).

 
L-NAME enhanced and L-arginine inhibited the effect of NT on [3H]TC uptake. The effect of NT was enhanced by L-NAME (Fig. 4C; 2-fold increase). In contrast, NOS substrate L-arginine (200 mg/kg), given 20 min before testing, inhibited the effect of NT (Fig. 4D; 90% decrease). These results suggested that NO production acted negatively on the response to NT, which is consistent with prior work showing that NO donors inhibit mast cell reactivity in vivo (53).

Mast cell stabilizers and L-arginine reduced basal [3H]TC uptake rate. The basal [3H]TC uptake rate in saline-infused rats was reduced by >50% in animals given cromoglycate, doxantrazole, or L-arginine (Fig. 5; Table 1). Because each of these agents exerted a stabilizing effect on mast cells, these results argue strongly that ≤50% of the basal TC uptake rate was attributable to mast cell-derived activity.



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Fig. 5. Basal [3H]TC uptake was inhibited by MC stabilizers cromoglycate (A), doxantrazole (A), and L-arginine (B). [3H]TC was injected at 0 min (arrow) and MC stabilizers were given at times shown (arrows). Cumulative recovery (%) of the administered dose of [3H]TC is plotted as a function of time (means ± SE; n = 6). A: basal TC uptake was inhibited 48% by doxantrazole (P < 0.01) and 73% by cromoglycate (P < 0.01). B: basal [3H]TC uptake was inhibited 54% by L-arginine (P < 0.05).

 

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This paper extends our earlier finding (25, 26) that physiological doses of NT enhance intestinal absorption of TC, the primary conjugated BA in rats, by demonstrating the involvement of mast cells and mast cell mediators in this response. Mast cell stabilizers not only blocked the stimulatory effect of NT but they also diminished the basal rate of TC uptake, implicating mast cells in the physiological process. In addition, the effect of NT was inhibited by preventing mast cell mediators (histamine, serotonin, LTC4) from acting, and it was reproduced by infusion of these mediators. Because intestinal uptake of BA is rate determining for operation of the EHC, these findings suggest that the NT-mast cell axis could play an important role in regulating the availability of BA to promote lipid digestion and absorption. Although the involvement of mast cells in intestinal inflammation and pathological conditions is well established (27), there is little information on their participation in BA physiology (31). Here, we suggest that intestinal mast cells regulate epithelial and/or vascular permeability to promote the physiological absorption and recycling of BA.

Some NT effects are mediated by mast cells. The idea that mast cells mediate some of the effects of NT has a strong foundation (7). Bolus injection of a large quantity of NT (≥4 nmol/kg) into rats produces an anaphylactic reaction involving the release of mast cell-derived histamine and leukotrienes (9). The ensuing hypotension, increased vascular permeability, and cyanosis can be prevented by prior treatment with cromoglycate to stabilize mast cells (9) or with compound 48/80 to deplete mast cell mediators (8). Other effects of NT that can be blocked by mast cell stabilizers and antihistaminic agents include vasoconstriction (37), hypothermia (34), and contraction of gastric smooth muscle (43). The ability of NT to bind to and to degranulate isolated mast cells has been demonstrated (8), and the existence of NTR1 on mast cells has been confirmed by mRNA and protein analyses (20, 44). In fact, NT is the only mast cell secretagogue for which a specific G protein-coupled receptor has been identified in mast cells.

NT and mast cells interact in intestinal mucosa. In the small intestine, NT is primarily localized to epithelial endocrine cells, although it is also present in neurons of the mucosa, submucosa, and muscularis (50). Endocrine and/or paracrine release of NT occurs postprandially (51) and is best stimulated by fatty acids (22), BA (16), and hormones (18). The intestinal mucosa is highly enriched with mast cells, which are often closely opposed to and interacting with enteric neurons (59) and vascular elements (38). As an integral part of the nerve-endocrine-immune network, mast cells participate in aspects of inflammation, and they also contribute to physiological regulation. Examples of the latter include the involvement of mast cells in cholecystokinin-induced disruption of the intestinal migrating motor complex (32) and in distension-induced and substance P-induced intestinal secretion (19). Some evidence attests to the importance of NT-mast cell interactions in inflammation. For example, pretreatment of rats with NTR1 antagonist SR-48692 inhibits stress-induced (11) and toxin A-induced (12) intestinal mast cell activation and the associated changes in intestinal permeability and secretion of PGE2 and mucin. These actions could involve both direct and indirect effects of NT on mast cells, because NT stimulates enteric nerves to secrete acetylcholine and substance P (7), which enhance the release of mast cell mediators (40, 42), and mast cell mediators further stimulate enteric neurons (15, 45). The close relationship between NT and mast cells in pathophysiological situations suggests that their interaction may also be utilized physiologically.

Mast cell activation is essential for basal and NT-stimulated TC uptake. Our data show that structurally distinct mast cell degranulators (compound 48/80, substance P, and L-NAME) enhanced the rate of TC absorption, reproducing the effect of NT. In contrast, agents purported to stabilize mast cells (cromoglycate, doxantrasole, L-arginine) reduced basal and NT-stimulated TC uptake. These findings support our contention that mast cells participate in TC uptake and mediate the enhancing effect of NT. Tissue mast cells are heterogeneous, and intestinal mast cells are more sensitive to the inhibitory effects of cromoglycate than are mast cells from other tissues such as lung and skin (46). The fact that cromoglycate totally blocked the effect of NT on TC uptake suggests that this response involves the activation of mast cells within the intestine. There are at least two types of mast cells, mucosal-type mast cells (MMC) and connective tissue-type mast cells (CTMC), in the intestine. Although NT (48) and substance P (56) can stimulate both MMC and CTMC, compound 48/80 is relatively specific for CTMC. Similarly, doxantrasole inhibits the activation of both MMC and CTMC, whereas cromoglycate primarily affects CTMC (47). Our results are most consistent with the involvement of CTMC but do not exclude the participation of MMC in the response to NT. The fact that CTMC are associated with the vasculature suggests that effects on vascular permeability could contribute to NT's effect on [3H]TC uptake. The localization of MMC just below the epithelial layer suggests that effects on epithelial permeability (36, 54) could also be important.

Mast cell-derived mediators enhance TC uptake. The inhibitory effects of pretreating rats with diphenhydramine and zileuton implicate histamine and leukotriene as obligatory participants in the response to NT. The fact that histamine and LTC4 were capable of enhancing TC uptake supports this contention. Metergoline gave partial inhibition of the NT response, suggesting that serotonin could be a minor contributor. In prior work, we showed that in rats NT causes a rapid release of mast cell histamine, followed by leukotriene generation (9). Because histamine stimulates formation of leukotrienes (7), and because LTC4 is a powerful permeability enhancer (27), it seems likely that NT acts by releasing histamine that generates leukotrienes, the final mediators of the response. Prostaglandins are known to potentiate the effects of leukotrienes, and here we found that PGE2, which was ineffective alone, enhanced the response to NT. The fact that systemic blood levels of histamine were not elevated during NT infusion argues that the histamine involved in NT-induced TC uptake was produced locally.

NO and mast cells in intestinal uptake of BA. Whereas excessive NO released by inflammatory cells can damage the intestinal epithelium (24), a low level produced by the intestinal endothelium plays an important role in maintaining the epithelial barrier (1). Thus NO donors decrease epithelial permeability, whereas inhibitors of NOS have the reverse effect (57). These changes have been attributed to the stabilizing effects of NO on intestinal mast cells (35, 53). Here we found that basal and NT-induced TC uptake were inhibited in animals receiving NO donor L-arginine, whereas they were enhanced in animals that received NOS inhibitor L-NAME. These data support our contention that the enhancement of intestinal TC uptake seen in our experiments is caused by mast cell-mediated increases in intestinal permeability, and they also illustrate that this process can be modulated by NO.

Mechanisms involved in regulation of TC uptake. It is possible that NT altered the luminal [3H]TC during our experiments; however, this would not provide an explanation for the response observed, because NT stimulates secretion of fluid and mucus (21) that would likely act to diminish the rate of [3H]TC uptake. Whereas active transport of TC occurs in ileum, the primary mechanism operating in jejunum is likely to be carrier-mediated transport (41). NT has little effect on TC uptake in ileum, but as shown here it markedly enhances TC uptake in proximal intestine (25). Because NT also enhances intestinal uptake of substrates absorbed primarily via the paracellular route ([3H]mannitol, [51Cr]EDTA) (25), it is conceivable that some of its effect on TC uptake involves increased paracellular permeability. Considerable evidence links mast cell activation and mast cell mediators (5, 54) to increases in paracellular permeability observed during development (14) and in pathological conditions involving stress (62), food allergy (39), enteritis (12), and ischemia (61). On the other hand, some findings are at odds with the hypothesis that NT enhances TC uptake by increasing intestinal paracellular permeability. In the rat, NT secretion (51) and BA uptake (31) are enhanced in the fed state, yet intestinal paracellular permeability is diminished compared with the fasted condition (63). Although it is possible that NT stimulates TC transport at the level of the epithelium, it seems more likely that its effects on vascular permeability enhance the passage of TC into the circulation. It is commonly thought that passage across the intestinal epithelium is rate determining for absorption of conjugated BA (29); however, the dependence on vascular permeability has not been thoroughly examined (29). Because NT and mast cell mediators alter blood flow and dramatically increase vascular permeability to albumin, it seems likely that these effects contribute to the enhancement of TC uptake in the intestine.

In conclusion, basal and NT-stimulated TC uptake in the rat intestine are similarly dependent on mast cells, are largely mediated by release of mast cell mediators, and are subject to regulation by NO. These findings are consistent with the idea that intestinal NT is an endogenous regulator of EHC whose effects are mediated by the activation of intestinal mast cells. Although the mechanism may involve enhanced transport through the epithelium, our data are most consistent with an effect on vascular permeability and blood flow.


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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants 5R01-DK-28565 and 5P30-DK-32520, although the opinions expressed are not necessarily those of the National Institutes of Health.


    FOOTNOTES
 

Address for reprint requests and other correspondence: R. E. Carraway, Dept. of Physiology, Univ. of Massachusetts Medical School, Worcester, MA 01655-0127 (E-mail: Robert.Carraway{at}umassmed.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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