Perception and gut reflexes induced by stimulation of gastrointestinal thermoreceptors in humans

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1 Keywords: Visceral pain, Temperature, Gut 6209 Journal of Physiology (1997), 502.1, pp Perception and gut reflexes induced by stimulation of gastrointestinal thermoreceptors in humans Nicola Villanova, Fernando Azpiroz * and Juan-R. Malagelada Digestive System Research Unit, Hospital General Vall d Hebron, Barcelona, Spain 1. Experimental studies in animals suggest the existence of thermoreceptors in the gastrointestinal tract. Our aim was to investigate the distribution and specificity of upper gut thermoreceptors in humans. 2. In healthy subjects, thermal stimulation of the stomach (n = 8) and the small intestine (n = 6) was produced by means of a thermostat, which recirculates water at adjusted temperatures through an ultrathin intraluminal bag. Progressively warm (42, 47 and 52 C) and cold (32, 22 and 12 C) stimuli of 3 min duration were alternately applied at 13 min intervals. Perception was scored on a scale of 0 6 and gastric tone responses were measured with a barostat. 3. Thermal stimuli induced specific responses: cold stimuli induced abdominal cold sensation and a reflex contraction of the stomach, whereas warm stimuli induced warm sensation and a reflex gastric relaxation. 4 Thermal stimuli induced similar stimulus-related perception in the stomach and small intestine (temperatures between 12 and 49 5 ± 0 5 C were tolerated). 5. The reflex responses were site specific. Warm and cold stimulation of the stomach induced gastric reflexes (76 ± 26 ml isobaric expansion at 47 C, and 68 ± 10 ml contraction at 12 C; P < 0 05 for both). However, only warm, not cold, stimulation of the intestine induced enterogastric reflexes. 6. These results indicate that in humans, warm and cold receptors are distributed along the gastrointestinal tract and project afferent input both into perception and reflex circuits with specific topographic organization. A variety of receptors sensitive to different stimuli have been described in the gastrointestinal tract. Conceivably, the vast majority of gastrointestinal receptors are involved in the reflex regulation of gut function. Indeed, acid-sensitive, osmotic and nutrient-specific chemoreceptors detect the characteristics of chyme and elicit specific responses to accomplish the digestive function (Grundy & Scratcherd, 1989; Malagelada & Azpiroz, 1989). Normally, this process is not perceived by the person, but under some circumstances gut stimuli may induce the conscious perception of abdominal sensations, suggesting that afferent pathways project to both the reflex nuclei and the brain cortex (Grundy & Scratcherd, 1989; Azpiroz & Malagelada, 1990a; Sengupta & Gebhart, 1994). However, the different types of receptors and the relation between reflex and sensory responses is not completely understood. Experimental studies in animals suggest the existence of thermal receptors in the gastrointestinal tract (El Ouazzani & Mei, 1979; Gupta, Nier & Hensel, 1979; Delbro, Lisander & Andersson, 1982; El Ouazzani & Mei, 1982; Rosza, Mattila & Jacobson, 1988; Sengupta & Gebhart, 1994). However, the responses to thermal stimuli have not been systematically investigated in humans, and most data available relate to relatively old studies (Bisgard & Nye, 1940; Ray & Neill, 1947). We hypothesized that specific warm- and cold-sensitive receptors are distributed along the human gut. Our aim was to investigate perception and reflex responses in gastric tone induced by warm and cold stimulation of the stomach and small intestine. To explore the responses to thermoreceptor stimulation we used a similar experimental approach to that we previously employed to characterize gut mechanoreceptors. Basically, we developed a method to produce graded thermal stimuli at different sites of the gut and we measured the responses using a perception questionnaire and gastric barostat, both previously validated and extensively used in our laboratory (Azpiroz & Malagelada, 1985, 1987, 1990a; Azpiroz, 1995). * To whom correspondence should be addressed.

2 216 N. Villanova, F. Azpiroz and J.-R. Malagelada J. Physiol METHODS Participants Twenty-two healthy individuals without abdominal symptoms (17 men and 5 women; age range, years) participated in the study after giving their written, informed consent. The protocol for the study had been previously approved by the Institutional Review Board of the Hospital General Vall d Hebron. Thermal stimulation To produce warm and cold stimuli we used a thermostat, which consisted of an intraluminal bag maintained at the desired temperature by recirculating temperature-adjusted water. The bag was made of ultrathin polyethylene 16 cm long and 11 cm perimeter (150 ml capacity). Both ends of the bag were attached with an air-tight seal to an infusion aspiration polyvinyl tube assembly. Water was recirculated through the bag by infusion at the caudad end of the bag via a 1 0 mm i.d., 2 0 mm o.d. tube and aspiration at the orad end of the bag was via a 1 5 mm i.d., 2 5 mm o.d. tube. The tube assembly proximal to the bag was thermally insulated by a 0 5 mm thick polyvinyl sheet. The intrabag temperature was monitored by a temperature probe (model 427, Yellow Springs Instruments, Yellow Springs, OH, USA) located adjacent to the aspiration port. Once positioned intraluminally, the perfusion and aspiration tubes of the bag were connected in a close circuit by semi-rigid tubes (7 mm i.d., 11 mm o.d.) to a demand pump (model , Flojet Corporation, Irvine, CA, USA). A 4 5 m long copper tube (8 mm i.d.) forming a twenty-coil spiral (7 cm diameter, 20 cm long) was located in series in the infusion path of the circuit. The temperature probe was connected to a telethermometer (model 44TA, Yellow Springs Instruments). Rapid temperature changes in the intraluminal bag were produced by immersing the copper spiral in a 5 l water reservoir at temperature levels that were previously established by preliminary validation studies. To achieve intrabag temperatures of 12, 22, 32, 42, 47 and 52 C, the copper spiral was maintained at temperature levels of 5, 15, 30, 50, 55 and 60 C, respectively. After each stimulus the copper spiral was immersed in a water reservoir at 37 C to return intrabag temperature to baseline between stimuli. Gastric tone responses We used a gastric barostat to measure reflex responses in gastric tone as the changes in isobaric volume. The barostat maintained a constant pressure within an air-filled bag by means of a feedback mechanism. The feedback mechanism consisted of a strain gauge linked by an electronic relay to an air injection aspiration system. Both the strain gauge and the injection aspiration system were independently connected by a double lumen polyvinyl tube (French no. 12, Argyle, Sherwood Medical, St Louis, MO, USA) to an ultrathin polyethylene bag (900 ml capacity, 17 cm maximal diameter). A dial in the electronic system allowed selection of the desired pressure level. The operating pressure level was individually adjusted 2 mmhg above intra-abdominal pressure. Intra-abdominal pressure was determined by increasing intrabag pressure in 1 mmhg steps every 2 min until intrabag volume exceeded 60 ml (minimal distending pressure). With this operating pressure the barostat measured variations in gastric tone as volume changed: a volume increase reflected a gastric relaxation and a volume decrease a contraction. A detailed description of the barostat and validation studies have been published previously (Azpiroz & Malagelada, 1985, 1987; Azpiroz, 1995). Perception measurement To measure the intensity of the perception induced by different stimuli we used a graded questionnaire. The following abdominal sensations were graded by the questionnaire: warmth, cold, pressure, fullness, distension, hunger and nausea. Participants were instructed to specify other perceived sensations in an open choice box in the questionnaire. Each sensation was scored on an independent graphic rating scale that combined verbal descriptors on a visual analog scale graded from 0 to 6 (Gracely, 1994; Azpiroz, 1995). All participants received standard instructions, specifying that score 0 represented no perception at all, score 5 represented discomfort and score 6 represented a painful sensation, which was not intended and was to be instantaneously reported for immediate discontinuation of the stimulus. Any sensation had to be scored on the scale based on its perceived intensity, and orientative descriptors were provided, indicating that score 1 represented vague perception of mild sensation; score 2 represented definite perception of mild sensations; scores 3 and 4 represented vague and definite perception of moderate sensation, respectively. Participants were also told that, if appropriate, they could mark half-unit scores on the scale, so that twelve intensity grades were actually provided. Before the study the questionnaire was fully explained to the participants. This type of questionnaire has been extensively used and validated in our laboratory (Azpiroz, 1995; see Discussion). Procedure Participants were intubated after an 8 h fast. The bags of the thermostat and the barostat, carefully folded, were introduced throughthemouthintothestomach.thebagofthethermostatwas positioned under fluoroscopic control in the stomach along the smaller curvature, in the descending duodenum, or in the proximal jejunum. For five subjects, in whom the bag was located in the stomach, an additional double-lumen polyvinyl tube (2 mm o.d., 0 78 mm i.d. each) with an inflatable tip balloon was passed into the jejunum for nutrient infusion (see Experimental design). To unfold the bag of the barostat, one lumen of the connecting tube was connected to a pressure transducer and the bag was slowly inflated through the other lumen of the tube under controlled pressure (< 20 mmhg) with 300 ml of air. The bag was then completely deflated and connected to the barostat. The bag of the thermostat was filled with 100 ml of water at 37 C when located in the stomach, and with 30 ml when located in the small intestine. The bag was then connected to the pump after carefully clearing any air bubbles from the circuit. The temperature probe was connected to the telethermometer, the copper spiral was immersed into a 37 C bath and the pump was turned on. Pressure and volume in the barostat bag and temperature in the thermostat bag were continuously recorded on a paper polygraph (model 1600; MFE Corporation, Salem, NH, USA). The experiments were performed with the subjects standing with the arms and upper trunk reclined (without compressing the abdomen) on a specially designed 1 5 m high table slanted at a 30 deg angle. Once the minimal distending pressure in the stomach was established, the barostat was set at a pressure level 2 mmhg above it, and was ready to measure gastric tone during the study. After allowing 10 min for stabilization we applied stimuli of 3 min duration at 13 min intervals; warm and cold stimuli were alternately applied, and during the 10 min periods between stimuli, intrabag temperature was maintained at 37 C. Warm stimuli (42, 47 and 52 C) and cold stimuli (32, 22 and 12 C) were applied without surpassing the respective threshold for discomfort

3 J. Physiol Gut thermoreceptors in humans 217 (perception score 5). The 3 min stimulation period included the time for the temperature to change from baseline to the test level. The time required for the temperature change was 27 ± 3 s for cold stimuli and 22 ± 2 s for warm stimuli, and it was not significantly different for the various levels tested. Measurements were performed during the last 90 s of the stimuli (see Data analysis). Experimental design Gastric and intestinal stimuli were tested in different groups of subjects. In eight subjects we tested gastric stimuli. In five of them we repeated the stimuli during a continuous nutrient infusion in the jejunum (Osmolite, M & R Laboratories, Zwolle, The Netherlands) at 37 C and a rate of 4 ml min using an infusion pump (model pp50-300, Lubrationics GmbH, Boeblingen, Germany). In six subjects we tested intestinal stimuli: series of duodenal and jejunal stimuli were performed in random order by relocating the thermostat bag during the study; in one subject the second part of the study in the jejunum could not be performed. Ancillary study Since thermal stimuli applied to the abdominal surface have been reported to change gut motor activity (Bisgard & Nye, 1940), we performed an ancillary study to compare these somatovisceral effects with those produced by gut stimuli in the main experiments. Hence, in eight healthy subjects we measured the effects on gastric tone of thermal stimuli applied over the anterior abdominal wall via an 18 cm ² 12 cm flat plastic bag in close contact with the skin and filled with 150 ml of water. We tested warm stimuli in 3 C increments up to 49 C, and cold stimuli in 6 C decrements down to 13 C, otherwise following the procedure described above. Data analysis For each stimulation episode we measured the putative reflex response. We measured the difference between the volume geometrically averaged during the 2 min period preceding the stimulus (reference volume), and during the last half of the stimulus. To check the volume recovery we also compared the volume averaged during the 2 min period starting 1 min after the stimulus with the reference volume before the stimulus. The reflex response in gastric tone was measured as the isobaric volume change recorded by the barostat, that is, relaxation as isobaric expansion, andcontractionasavolumereduction. For each stimulation we also measured the perception score of the sensation elicited. The threshold for discomfort was defined as the stimulus that produced a perception score of 5 or greater. When more than one sensation was scored, only the maximal score, instead of the cumulative score, was computed for comparisons. In each subject we counted the total number of warm and cold stimuli that were perceived, and the number of stimuli that produced sensations other than warmth or cold; the proportion was expressed as a percentage. Statistical analysis In each group of subjects we calculated the mean values (± s.e.m.) of the parameters measured. Statistical comparisons were performed using ANOVA; the level of significance was P < Student s paired t test was used for post hoc comparisons of normally distributed data. Comparisons of perception scores were performed by the Wilcoxon signed-rank test. The proportion of symptoms other than warm and cold were compared by Fisher s exact test. RESULTS Thermal stimulation of the stomach In the stomach thermal stimuli induced both perception and gastric reflex responses, but the responses were specific for the type of stimulation. Cold stimuli induced abdominal cold sensation and a reflex contraction of the stomach (Fig. 1), whereas warm stimuli induced warmth sensation and a reflex relaxation (Fig. 2). Perception and reflex responses were related to the temperature levels, both for warm and cold stimuli (Table 1 and Fig. 3). Temperature levels in the range between 32 and 42 C were barely perceived (Fig. 3) and did not induce significant reflex responses (Table 1). Cold temperatures Figure 1. Reflex gastric contraction induced by cold stimulation of the stomach The desired intragastric temperature was maintained by a thermostat, and gastric tone was measured with a barostat as the change in isobaric volume. Note volume decrease (contraction) during gastric cooling. The subject scored mild cold sensation (perception score 2).

4 218 N. Villanova, F. Azpiroz and J.-R. Malagelada J. Physiol Table 1. Gut thermoreflexes Gastric tone response to stimulation (ml) Stomach Duodenum Jejunum Cold stimuli ( C) 32 7±17 13±39 9± ±9* 40±30 0± ±10* 15±41 4±9 Warm stimuli ( C) 42 24±17 3±22 10± ±26* 35±26 19± ± 25 * 145 ± 41 * 23 ± 18 Values are means ± s.e.m. Gastric tone responses measured by the barostat as the change in intragastric volume. * Gastric tone change; P < 0 05 (level during the stimulus versus reference level before the stimulus). Threshold for discomfort (average of individual data at 47 and 52 C). below this range were relatively well tolerated and induced significant gastric contraction. The threshold for discomfort could not be established, because only two out of the eight subjects experienced discomfort at the lowest temperature level tested (12 C). In contrast, the range of warm temperatures that were tolerated was narrower, and the threshold for discomfort was established at 49 5 ± 0 9 C (four subjects reported discomfort at 47 C and the remaining four at 52 C). Nevertheless, 47 C in the stomach, only 10 C above body temperature, induced a significant reflex relaxation. The responses to warm and cold stimulation reverted after discontinuation of the stimuli, so that 1 min after returning gastric temperature to 37 C (post-stimulation period) gastric tone was similar to that measured before the stimulus (data not shown). Besides warmth and cold, 43 % of gastric stimuli (cold and warm alike) induced perception of other sensations, and one-third of the stimuli induced a predominant non-specific sensation. Similar types of non-specific sensations were induced by warm and cold stimuli and were distributed as follows (pooled data for warm and cold stimuli): 7 % abdominal pressure (mean perception score, 3 5), 19 % fullness (mean score, 1), 44 % distension (mean score, 2 2), 19 % hunger (mean score, 1), and 11 % nausea (mean score, 5 6). Three subjects reported non-specific symptoms in response to warm stimuli, and in them the reflex gastric relaxation was larger than in the other five, who only experienced warmth sensation, e. g. at 47 C gastric relaxation was 142±49ml (n =3) versus 36±11ml (n = 5), respectively (P < 0 05). The gastric responses to Figure 2. Reflex gastric relaxation induced by warm stimulation of the stomach The desired intragastric temperature was maintained by a thermostat, and gastric tone was measured with a barostat as the change in isobaric volume. Note volume increase (relaxation) during gastric warming. The subject scored warm sensation at the discomfort threshold (perception score 5).

5 J. Physiol Gut thermoreceptors in humans 219 Figure 3. Gut thermosensitivity in humans The intensity of perception, scored from 0 (no perception) to 6 (pain), was temperature related and similar along the upper gastrointestinal tract. Most stimuli were specifically perceived as warmth or cold; only 33 % of gastric and 10 % of intestinal stimuli (warm and cold alike) induced a predominant non-specific sensation, such as abdominal distension, fullness, pressure, hunger or nausea. 0, stomach; þ, duodenum; 8, jejunum. cold stimuli were similar regardless of the type of sensation perceived. Effect of intestinal nutrients on the responses to thermal stimuli Intestinal nutrient infusion induced a profound gastric relaxation. Before administration of nutrients, baseline intragastric volume measured by the barostat was 214 ± 32 ml, and the volume increased to 504 ± 43 ml 4 min after starting the nutrient infusion (n = 5;P < 0 01). This volume level was maintained throughout the infusion period; at the end of the experiment baseline volume in the same subjects was554±35ml(n = 5). Gastric responses to thermal stimuli were no longer apparent during intestinal infusion of nutrients. Thus, the relaxation of the stomach induced by intestinal nutrients was neither enhanced by warm stimuli nor antagonized by cold stimuli. Perception of thermal stimuli was not significantly affected by the nutrients. For instance, in the same five subjects, the perception score at 47 C was 4 0 ± 0 7 without and 4 6 ± 0 2 with intestinal nutrient infusion. Thermal stimulation of the small intestine Thermal stimuli, whether applied in the duodenum or in the jejunum, induced perception and overall the perception scores were not significantly different from those with gastric stimulation (Fig. 3). Cold stimuli induced abdominal cold sensation, which was tolerated by all subjects down to 12 C. Warm stimuli induced a warmth sensation that was perceived as uncomfortable between 47 and 52 C. Thermal stimuli applied in the intestine induced a smaller proportion of non-specific symptoms than when applied in the stomach. Only 11 % of warm and cold stimuli, applied either in the Figure 4. Reflex gastric relaxation induced by warm stimulation of the duodenum The desired intraduodenal temperature was maintained by a thermostat, and gastric tone was measured with a barostat as the change in isobaric volume. Note volume increase (gastric relaxation) during duodenal warming. The subject scored warm sensation at the discomfort threshold (perception score 5).

6 220 N. Villanova, F. Azpiroz and J.-R. Malagelada J. Physiol duodenum or in the jejunum, induced sensations other than warmth and cold (P < 0 05 versus gastric stimuli). Reflex gastric responses to intestinal stimuli depended on the type of stimulation (Table 1). Warm stimuli in the duodenum induced a gastric relaxatory reflex (Fig. 4). There were no consistent differences between the gastric relaxatory responses induced by gastric and duodenal warm stimuli; the response to duodenal stimuli was somewhat smaller at 47 C, but it was greater at the threshold for discomfort (Table 1). This reflex response faded with more distal stimulation and warm stimuli in the jejunum had no consistent effects on gastric tone at any level tested, even though perception was similar to that in the duodenum. Cold stimuli, applied either in the duodenum or in the jejunum, did not elicit any consistent reflex response in the stomach (Table 1). Ancillary study: effect of cutaneous thermal stimuli All subjects tolerated the whole range of temperatures tested on their abdominal surface. Warm stimuli applied over a wide area of the anterior abdominal wall had no apparent effects on gastric tone. Likewise, cold stimuli had no consistent effects; only at the lowest temperature tested (13 C) we observed a small but significant decrease in the intragastric air volume measured by the barostat ( 39 ± 14 ml change; P < 0 05 vs. reference volume before the stimulus). DISCUSSION Our study provides evidence that warm and cold stimulation of the stomach and small intestine induces specific sensory and reflex responses. These data indicate the existence in humans of gut thermosensitive receptors with afferent input both into sensory and reflex circuits. Previous experimental studies have evidenced several types of intra-abdominal receptors selectively activated by temperature changes, specifically, receptors that only respond to warmth, others that respond to cold, and a third type of mixed thermoreceptors activated both by high and low temperatures (El Ouazzani & Mei, 1979). Animal studies further suggest that thermoreceptors are located at different levelsalongthegutand,infact,thermalstimulationofthe oesophagus, stomach, antroduodenal junction, duodenum, jejunum and ileum elicits specific responses (El Ouazzani & Mei, 1979; Gupta et al. 1979; El Ouazzani & Mei, 1982; Delbro et al. 1982; Rosza et al. 1988). Some data indicate that thermoreceptors may be located on the serosal as well as on the mucosal side of the gut wall (Rosza et al. 1988). Conceivably, the responses we have observed are related to stimulation of mucosal thermoreceptors. However, our experimental design does not allow us to discern the precise depth location of the receptors activated, or to exclude activation of other intra-abdominal thermoreceptors (Rawson & Quick, 1972; Riedel, 1976). Nevertheless, cutaneous stimuli did not elicit detectable reflexes, except for a small response at the lowest temperature tested. Our study was designed to examine responses to thermal stimuli at different sites in the upper gut. We observed that cold and warmth perception induced by thermal stimuli was uniform from the stomach down to the jejunum. Thus, human thermoreceptors appear to be evenly distributed in the regions tested. In addition to specific warmth and cold perception, thermal stimuli, particularly in the stomach, also induced perception of non-specific sensations, such as abdominal distension and fullness, which are similar to those induced by distension (Coffin, Azpiroz, Guarner & Malagelada, 1994; Azpiroz, 1995). Polymodal receptors, which respond both to mechanical and thermal stimuli, have been described in the gut (Cottrell, 1984), and hence, nonspecific symptoms associated with warmth and cold sensations could be related to simultaneous activation of a subpopulation of polymodal receptors present in the stomach, but scarce in the intestinal wall. There is no gold standard to measure visceral perception, but we selected a scaling method that has been proposed before (Gracely, 1994), and we carefully validated by a series of studies in our laboratory the reproducibility and the sensitivity of the measurements (Azpiroz, 1995). Specifically, we have shown that repeated stimuli in the same subjects induce reproducible perception scores (Notivol, Coffin, Azpiroz, Mearin, Serra & Malagelada, 1995). Different types of stimulation, mechanical, electrical, and in the present study thermal, applied in stepwise increments induce perception scores which are related to the magnitude of the stimuli (Azpiroz & Malagelada, 1990a, b; Rouillon, Azpiroz & Malagelada, 1991a, b; Accarino, Azpiroz & Malagelada, 1992, 1995). When the same stimuli are applied under different experimental conditions, significant and reproducible changes in perception can be detected (Coffin, Azpiroz & Malagelada, 1994b; Iovino, Azpiroz, Domingo & Malagelada, 1995; Notivol et al. 1995). Additional evidence was obtained in selected groups of patients which showed increased perception of a specific stimulus, but normal perception of other stimuli, even though the symptoms elicited could not be distinguished (Coffin et al. 1994a; Accarino et al. 1995). In the present study we did not test the reproducibility of the responses within individuals on repeated occasions, but we found that in different groups of subjects thermal stimulation applied at various sites of the upper gut induced similar perception. In all subjects, the threshold for discomfort ranged between 47 and 52 C, both in the stomach and the small intestine. Likewise, all subjects tolerated temperatures down to 12 C, except one which reported discomfort at 22 C in the duodenum. Thermal stimuli also induced reflex responses, which depended on the type of stimulation: the stomach contracted in response to cold stimuli, and relaxed in response to warm stimuli. Previous studies in animals under various experimental conditions have shown similar motor responses of the gut to thermal stimulation, namely, contractile responses to cold and relaxatory responses to

7 J. Physiol Gut thermoreceptors in humans 221 warm stimuli (El Ouazzani & Mei, 1979, 1982; Delbro et al. 1982). Our studies further indicate that human thermoreflexes have a specific topographic organization. In contrast to perception, reflex responses depend on the site of stimulation. Both warm and cold stimuli in the stomach induced gastrogastric reflexes, but only warm stimuli in the duodenum induced enterogastric reflexes. Interestingly, the jejunum, which exhibited similar thermosensitivity, did not elicit enterogastric reflexes, suggesting that gastric responses are not mediated by perception. Furthermore, the responses we observed seem not to be related to nociception, because the stimuli were only applied up to the discomfort threshold. Our data indicate that perception and reflex responses to thermal stimuli are induced independently, and conceivably, different pathways are involved in these responses. A classical study in the 1940s showed that the sensation induced by warm and cold stimuli in the human gut is abolished by sympathectomy (Ray & Neill, 1947). The specific pathways involved in the gastrogastric and enterogastric reflexes have not been defined, but based on animal experiments both vagal and splanchnic fibres appear to be activated by thermal gut stimuli (Gupta et al. 1979; El Ouazzani & Mei, 1979, 1982; Delbro et al. 1982; Rosza et al. 1988). We can reasonably exclude the possibility that the responses to thermal stimuli were simply due to expansion or contraction of the circulating water in the thermostat, because the dilatation coefficient of water within the temperature range tested is very small (0 02 % dilatation per C of warming). Conversely, expansion of the air volume in the bag of the gastric barostat by gastric warming (4 8 % expansion from 37 to 52 C) would cause withdrawal of air from the bag to maintain the pressure constant, the opposite effect to that actually observed in the experiments. The same reasoning would apply to the gastric cooling tests. The physiological role of gut thermoreceptors and the effects of meal temperature on digestive function remain poorly understood (Ritschel & Erni, 1977; Weber, Nouri & Bell, 1980). We have previously shown in a canine model that intestinal nutrients induce gastric relaxation by a nonadrenergic, non-cholinergic vagal mechanism (Azpiroz & Malagelada, 1986). This is presumably the same reflex we evidenced in the present study with the intestinal nutrient perfusion; the reflex (both stimulus and response) was not perceived. We further examined whether thermal stimuli modify or interfere with this reflex mechanism, which conceivably participates in the physiological modulation of the postcibal gastric tone and emptying rate (Malagelada & Azpiroz, 1989). Our results show that, in the human gut, nutrient enterogastric reflexes are impervious to a wide range of temperature variations, and hence, thermoreceptors may have a limited role. However, recent pathophysiological studies suggest that unexplained abdominal symptoms in some patients may be related to altered gut sensitivity (Coffin et al. 1994a; Mayer & Gebhart, 1994; Accarino et al. 1995; Azpiroz, 1995). Given the putative importance of gut sensory dysfunctions in relation to some clinical problems, the characterization of sensory afferents, regardless of their physiological relevance, may be important in developing a comprehensive sensory evaluation of the gut in health and disease (Coffin et al. 1994a; Mayer & Gebhart, 1994; Accarino et al. 1995; Azpiroz, 1995). Accarino, A. M., Azpiroz, F. & Malagelada, J.-R. (1992). Symptomatic responses to stimulation of sensory pathways in the jejunum. American Journal of Physiology 263, G Accarino, A. M., Azpiroz, F. & Malagelada, J.-R. (1995). Selective dysfunction of mechanosensitive intestinal afferents in the irritable bowel syndrome. Gastroenterology 108, Azpiroz, F. (1995). Sensitivity of the stomach and the small bowel: human research and clinical relevance. In Progress in Pain Research and Management, vol. 5, Visceral Pain, ed. Gebhart, G. F., pp IASP Press, Seattle, WA, USA. Azpiroz, F. & Malagelada, J.-R. (1985). 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