Substance P mediates a gastrointestinal thermoreflex in rats

GASTROENTEROLOGY 1988:95:265-76

ALIMENTARY

TRACT

Substance P Mediates a Gastrointestinal Thermoreflex in Rats ZSUZSANNA

ROZSA, JYRKI MATTILA,

Departments of Physiology and Pharmacology, Medical Center, Kansas City, Kansas

We have characterized a viscerocirculatory thermoreflex, quantified its responses, and identified the major neurotransmitter. Application of fluid at 45°C to mucosas or serosas of the stomach, jejunum, or ileum of anesthetized rats promptly evoked consistent cardiovascular responses, namely arterial hypotension, tachycardia, and a diminished intestinal blood flow (latency 5’5 s with a duration of 2-4 min). Thus, for example, warming of the stomach caused blood pressure to decrease 40%, heart rate to increase 15%, and mesenteric blood flow to decline 50%. These responses were inhibited totally or mostly by capsaicin, administered neonatally, topically, or perineurally, by topical lidocaine, and by parenteral administration of substance P antiserum, somatostatin, or hexamethonium. Epidural anesthesia also inhibited the cardiovascular responses to visceral warming. Pretreatment with reserpine or splanchnic ganglionectomy converted the thermally induced decline in mesenteric blood flow into a vasodilator response, which could then be blocked by an antiserum to substance P. Visceral warming also stimulated afferent, preganglionic sympathetic neural activity. Our conclusions are (a) application of fluid at 4S’C to gastrointestinal organs of anesthetized rats initiates a viscerocirculatory reflex that involves primary sensory, afferent C fibers; (b) the major neurotransmitter of this reflex appears to be substance P; and (c) visceral afferent C fibers have central and peripheral vasomotor functions.

and EUGENE

School of Medicine,

D. JACOBSON

University

of Kansas

ity is associated with mechanical, chemical, or thermal stimuli (1,2). Abdominal, visceral sensory endings can be activated by a variety of chemical and mechanical stimuli to induce reflex regulation of the cardiovascular system (3,4). Electrophysiologic data indicate that visceral afferent A and C fibers probably initiate viscerocardiac reflexes by way of different populations of spinal neurons (5). The nature and functional significance of thermoreceptors in the mucosa of the gastrointestinal tract is not clear. Visceral thermoreflexes were first suspected when evidence was noted of thermoregulatory reactions initiated from the abdominal cavity (6,7). Later, visceral warming was found to elicit unitary discharges from mesenteric nerves (81, and cold receptors were described associated with splanchnic nerves (9). Warm, cold, and mixed receptors seem to be widespread in the digestive system (10,ll). These various thermoreceptors are apparently associated with the unmyelinated afferent C fibers. The aims of the present work were to investigate a visceral cardiovascular reflex, which is stimulated by a moderate elevation in temperature, and to quantify its responses. We also explored the possibility that visceral, peptide-containing, afferent neurons may have a vasomotor function, and we attempted to identify the major sensory neurotransmitter substance of the reflex.

Materials and Methods

V

arious studies of visceral nerves have emphasized the importance and diversity of afferent fibers, which are far more numerous than had been previously believed and outnumber the efferent fibers in many sympathetic and parasympathetic nerves (l,2). Afferent terminals of visceral nerves are present in the mucosa, muscularis, and serosa of the stomach and gut. Visceral afferent units have been classified according to whether their principal activ-

Animal Preparations Two groups of anesthetized rats were prepared. Group 1. Sprague-Dawley rats (Sasco) of either sex, weighing 280-320 g, were fasted for 24 h before the experiments.

Abbreviation

but received

drinking

water

ad libitum.

used in this paper: SP, substance P. 0 1988 by the American Gastroenterological Association 0016-5085/88/$3.50

An-

266

ROZSA ET AL.

imals were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The cervical trachea was cannulated and ventilated with a pressure-controlled rat ventilator (Rodent Ventilator model 683; Harvard Apparatus, South Natick, Mass.). Systemic arterial pressure was monitored continuously from the right carotid artery via a polyethylene catheter (PE-50) attached to a strain gauge transducer (P-50; Gould-Statham) that was connected to a polygraphic recorder (Sensor Medics Dynograph R611). Heart rate was calculated from recordings of phasic arterial pressure. The left jugular vein was cannulated (PE-50) for the injection of drugs. Core temperature was maintained at 37°C with a small heating pad regulated by a rectal thermistor and controller (model 74; Yellow Springs Instrument). After performing a midline laparotomy, we implanted a flow probe (1.0-1.3 mm) on the trunk of the superior mesenteric artery and recorded blood ilow with a directional, pulsed Doppler flowmeter (Bioengineering, The University of Iowa, 545C-4). The signals were monitored continuously as the pulsatile and mean flow velocity of a Doppler shift (in volts) on the recorder. Previous studies have documented close agreement between changes (magnitude and direction) in blood flow determined with pulsed Doppler and electromagnetic flow probes in vivo (12). In each of our experiments, electronic zero flow was equal to mechanical zero flow obtained by arterial occlusion distal to the probe. Intraluminal application of either a thermal stimulus or drugs was made via silicone rubber cannulas (0.4 x 0.85 mm, silastic; Dow Corning Corporation, Midland, Mich.) inserted via small incisions for a distance of 2-3 cm into the gastric cardia, jejunum, and proximal ileum. A number of holes were placed in the terminal centimeter of the cannulas to facilitate the delivery of the warm fluid or solutions containing drugs onto the mucosal surface. Injection of Evans blue dye through these cannulas colored a 12-cm segment of the small intestine, indicating the extent to which the mucosal surface was affected by these applications. Before opening the abdomen, a similar cannula was introduced into the ventrolateral abdominal cavity for delivery of warm fluid to the serosal surface of the viscera. After surgery, the preparation was allowed to stabilize for 25 min. The thermal stimulus was produced by slow injection (0.2 ml/s) of normal saline (up to 1.0 ml total volume) warmed to 40”-55°C. In most experiments, the thermal stimulus involved injecting fluid at 45°C. For control purposes, fluid at 37°C was injected. The sites into which warmed fluid was injected to elicit a thermal reflex included the lumens of the stomach, jejunum, and ileum, and the ventrolateral abdominal peritoneum. When repetition of the thermal stimuli was performed, a 15-min waiting period was allowed between separate series of injections. No more than three applications of warm fluid were used at any one site. The experiments were divided into four subgroups, classified by their state of denervation. In the first subgroup of anesthetized rats, after the previously described procedures, vagotomy was performed bilaterally in the midcervical portion of the nerves 30 min before subjecting the rats to thermal stimulation of the viscera.

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In the second subgroup, acute ganglionectomy was performed. The celiac-superior mesenteric ganglion complex was gently freed from the surrounding vasculature and removed, and its nerves were stripped from the blood vessels 1 h before studying the effects of thermal stimulation. In a third subgroup of rats, laminectomy was performed at the level of the 10th thoracic vertebra, and a catheter (PE-10) was inserted into the epidural space. After completion of surgery, the local anesthetic lidocaine (1 mg/kg, 0.5 ml, including catheter volume) was injected into the epidural space of these animals. Control animals received an equal volume of saline. Epidural blockade was considered to be present at a dose that was sufficient to reduce arterial pressure. In the fourth subgroup, the animals were injected subcutaneously with 50 mg/kg of capsaicin daily on the second and third days of neonatal life. Paired control rats were injected with equal volumes of the vehicle for capsaicin. These neonatal injections were performed after anesthetizing the rats with ether. Experiments were subsequently conducted in these animals when they had reached the age of 3-4 mo, at which time body weights ranged from 280 to 320 g. Group 2. These rats were prepared for the recording of multiunit sympathetic nerve activity. All rats were fasted, anesthetized, and subjected to tracheostomy and laparotomy as described previously. Catheters were inserted into the lower abdominal aorta for recording of blood pressure and into a femoral vein for injection of drugs. Phasic arterial pressure was recorded by connecting the aortic catheter through tubing (Tygon) to a low-volume displacement pressure transducer (Statham P23 Gb). Heart rates were monitored simultaneously by triggering a biotachometer with the phasic signal from the transducer. Frequency of spike potentials was recorded from a splanchnic sympathetic nerve that was identified as the major nerve bundle entering the celiac ganglion. Nerve segments isolated under a dissecting microscope were placed over a bipolar stainless steel electrode and immersed in mineral oil. Spike potentials were amplified (P15 AG preamplifier; Grass Instrument Co., Quincy, Mass.), recorded on magnetic tapes, and passed through an amplitude analyzer (F. Haer & Co., Brunswick, Me.) to convert individual spikes into uniform pulses. The number of individual pulses per second was counted with a rate analyzer whose output was continuously recorded as a histogram on a thermal-writing recorder (13). Neural activity recorded from the splanchnic nerve was expressed as spikes per second and averaged for 30 s before and 5 s during thermal stimulation. Latency of response was determined as the time from onset of the thermal stimulus to onset of the increase in firing frequency. Drugs Capsaicin (Biomedicals, Inc.) was prepared as a 1% (wtivol) solution containing 10% ethanol, 10% Tween 80, and 80% physiologic saline. Other drugs included sodium pentobarbital (Veterinary Laboratories, Inc.), 1% solutions

GASTROINTESTINAL THERMOREFLEX

August 1988

of heparin and lidocaine (Elkins-Sinn, Inc.), 2% Evans blue dye (Eastman Organic-Chemicals, Rochester, N.Y.), atropine sulfate, hexamethonium bromide, somatostatin, reserpine, propranolol (Sigma Chemical Co., St. Louis, MO.), and pentolinium tartrate (Wyeth Laboratories, Philadelphia, Pa.). Reserpine was injected intraperitoneally 24 h before the experiment. Antiserum to substance P (SP) was raised in rabbits (L:83) against synthetic undecapeptide SP and has been characterized previously (14). When tested in radioimmunoassay, this antiserum reacted only with SP in extracts of spinal cord and gut. Close intraarterial injection of SP resulted in a dose-dependent increase in mesenteric blood flow that could be inhibited in a dose-dependent manner by the respective antisera for SP (15). All drugs were dissolved in isotonic saline except reserpine, which was dissolved in a minimum volume of acetic acid before dilution in distilled water. Control animals were given equal volumes of vehicle for the respective drug.

Statistical

Analysis

All data in the text are presented as mean f standard error of the mean (SE). Statistical analyses were performed using Student’s t-test, analysis of variance, and Dunnett’s multiple comparison (18). A p value of CO.05 was required for statistical significance.

Results Control Experiments

(Group 1 Preparation)

In control experiments we studied the hemodynamic effects of local warming of the viscera using gradually increasing temperatures. These results were as follows. When fluid at 37°C was applied to either the mucosal or serosal linings of the stomach, jejunum, or ileum, we failed to detect any significant changes in either systemic arterial blood pressure, heart rate,

Table 1. Cardiovascular Responses Surfaces Site of application of 45’C fluid Gastric mucosa Jejunal mucosa Ileal mucosa Abdominal serosa

Gastric mucosa Jejunal mucosa Ileal mucosa Abdominal serosa

of Gastrointestinal

or mesenteric blood flow (n = 6). When fluid at 40°C was applied to the gastrointestinal serosas after a short latency period (3-5 s), there was a transient drop in blood pressure, accompanied by an increase in heart rate, and a decrease in perfusion of the superior mesenteric artery (n = 6). However, application of fluid at 4VC to the visceral mucosas elicited inconsistent changes (n = 6). In 48 rat experiments per anatomic site of warming, the application of fluid at 45’C to either mucosal or serosal surfaces of the viscera evoked a transient but significant hypotensive response, tachycardia, and a decrease in mesenteric blood flow. The onset of cardiovascular changes began 3-5 s after the start of warming and persisted for 2-4 min (Figures la and lb and Table 1.). A repeat stimulus of 45°C 15 min later evoked similar results. Thus, in 20 rats there were no significant differences between the changes in any of the hemodynamic variables comparing the first to the second warming stimulus (Table 1). When fluid was warmed to 50°C and applied intraperitoneally or to the gastric mucosa, there was an increase in the heart rate and biphasic changes in arterial blood pressure and mesenteric blood flow (n = 6). Typically, a transient depressor response and decline in mesenteric blood flow were succeeded by a moderate rise in blood pressure and simultaneous increase in mesenteric blood flow. Application of fluid at 50°C into the lumen of the jejunal or ileal segments stimulated moderate elevation of blood pressure and heart rate without changing mesenteric blood flow. The delivery of fluid warmed to a temperature of 55°C to mucosal or serosal surfaces prompted transient increases in all three circulatory measurements (n = 6).

to a First and a Repeat Application Visceraa

of Fluid at 45°C to the Mucosal

Mean arterial blood pressure C 118 2 118 k 120 k 116 "

Heart rate (beatslmin)

A 7 4 5 8

71 f 88 f 81 f 70 f

C 8= 3b 4c 5c

C

B

124 z!z 6 120 r 5 118 k 8 120 k 6

74 zz4c 76 f 6c 64 k 4' 60 k P

284 2 306 2 304 f 296 +

A 12 7 8 10

C 308 f 302 f 284 + 306 +

336 5 346 f 344 2 358 f B

16 8 12 14

Mean blood flow (V] C

7' 6' 8' 9'

354 k 18= 348 zt14c 338 + 16= 364 Z 12’

267

A

3.0f 0.2 2.82 0.6 3.2f.0.4 2.8t 0.4 C 3.2f 3.02 3.2f 3.0 f

1.4f 1.4f 1.22 0.92

Latency of onset of depressor change (s)

and Serosal

Duration of depressor change (min)

0.6' 0.4' 0.6' 0.4'

4 4 5 3

a t r t

1.8 1.6 1.4 1.2

3 2 2 4

+ 1.2 -t1.6 +-1.4 t 1.8

0.2' 0.2c 0.4' 0.4’

3 3 4 3

z!z 1.4 C 1.8 I?1.0 ” 1.2

3 2 2 4

2 1.0 k 1.2 ?I1.6 2 1.2

B 0.4 0.2 0.4 0.4

1.6" 1.42 1.2+ 0.8 +

' Numbers represent mean values -t SE in 48 (A] and 20 (B) experiments using recorded data for each measurement. C = control period before application of warm stimulus. A = the first and B = the second (15 min later) maximal response to the thermal stimulus. Comparison is made between C and A or B. b p < 0.05. ’ p < 0.01.

268

Figure

ROZSA

ET AL.

GASTROENTEROLOGY

Vol. 95, No. 2

1. Original records of cardiovascular responses to warm stimuli (45°C) applied topically (at vertical lines on the graph) to the gastric mucosa and to the abdominal serosa (o), and to the mucosas of the ileum and jejunum (b). Records from the top down: time signal at l-min intervals, phasic arterial blood pressure, mean arterial blood pressure, heart rate, phasic mesenteric blood flow, and mean mesenteric blood flow. Chart speed 0.5 mm/s.

GASTROINTESTINAL

August 1988

For purposes of studying the mechanism of this thermocirculatory reflex, we opted to use fluid at a temperature of 45’C in the remaining experiments to be described because application of fluid at this temperature to either serosal or mucosal surfaces stimulated similar hemodynamic alterations in a consistent manner. Furthermore, solutions at this temperature are ingested into the stomach of humans as a normal dietary component. Neural Mechanisms

(Group 1 Preparation)

After pretreatment of mucosal or serosal surfaces with a solution of 1% lidocaine, both serosal and mucosal applications of 45°C fluid caused no significant changes in arterial blood pressure, heart rate, or mesenteric blood flow (Table 2, group A). Pretreatment of anesthetized rats with hexamethonium (25 mg/lOO g) lowered both the blood pressure and blood flow before application of warm fluid and reduced the magnitude of the subsequent hypotensive and mesenteric vascular responses to visceral warming (Table 2, group B). This dose of hexamethonium in rats will effectively block all cardiovascular responses to spinal stimulation (17). Atropine (1 mg/lOO g) failed to alter subsequent circulatory responses to visceral warming (Table 2, group C), although the alkaloid stimulated an increase in heart rate before warming. Pretreatment with propranolol [initial dose of 1.5 mgikg, followed by 0.7 mgikg . h) did not significantly affect the thermally stimulated cardiovascular responses (Table 2, group D). Bilateral cervical vagotomy did not alter the cardiovascular responses to warming of the serosal or mucosal surfaces of the viscera (Table 3, group A]. Furthermore, hemodynamic measurements were not significantly different from basal values by 15 min after vagotomy and just before warming. Epidural anesthesia almost completely blocked the thermally stimulated hypotensive response and greatly inhibited the changes in heart rate and mesenteric blood flow (Table 3, group B). Neonatal capsaicin totally prevented all adult circulatory responses to both serosal and mucosal warming (Table 4, group A). Perineural application of capsaicin to the mesenteric trunk, to affect periarterial afferent C fibers, markedly inhibted subsequent circulatory responses to both mucosal and serosal warming (Table 4, group B). Topical capsaicin (intraluminal application, 1.0mg/ 100 g, 15 min before warming) almost abolished the hypotensive, tachycardic, and mesenteric circulatory effects of 45°C fluid applied to the mucosal surfaces of the viscera but did not affect the circula-

,

THERMOREFLEX

269

3. Epidural

36 ? 8 34 k 8

32 k 6 5 * 5b

36 2 2 8 t 6b

26 t 6 5 xi 5b

32 + 7 30 2 6 38 t 2 16 k 2b

38 t 4 30 ? 6 28 2 4 10 5 2"

44 5 3 38 -c 5

(beatslmin) Ileum

29 2 7 14 -+ 2"

36 2 4 32 2 6

response

Jejunum

Tachycardic

Warmi&

Stomach

to Visceral

44 + 5 6 ?I 4b

42 2 6 36 k 4

Serosa

Responses (mmHg)

Ileum

response

Circulatory

Jejunum

Hypotensive

Affects

52 f 3 46 2 8

Stomach

Anesthesia

54 k 6 28 2 4b

40 + 2 34 + 8

Serosa

2.4 k 0.8 0.8 2 0.2b

2.2 % 0.4 2.0 5 0.2

Stomach

(V]

1.4 + 0.4 0.4 2 0.2b

1.6 2 0.4 1.4 2 0.6

Ileum

flow decrease

1.6 f 0.2 0.6 k 0.2b

1.8 2 0.2 1.8 t 0.4

Jejunum

Mesenteric

2.4 2 0.4 1.6 2 0.4c

1.4 2 0.4 1.2 k 0.4

Serosa

42 2 4 3 -c 2b

capsaicin Group C Control Intraluminal

36 t 5 2 It lb

36 If6 2 + 2b

40 2 6 2 5 2b

Jejunum

34 " 2 2 I? Zb

38 k 2 4 k lb

36 2 4 2 2 2b

Ileum

48 t 6 44 e 4

62 2 4 4 -r-2b

46 t 4 2 2 2b

Serosa

to Visceral

32 2 8 4 r 2b

46 2 2 3 2 lb

38 2 4 2 " 2b

28 ? 4 4 4 2b

32 2 8 4 4 lb

32 k 8 2 2 2b

22 * 8 2 2 2b

38 k 4 2 t 2b

40 k 4 2 + 2b

Ileum

(beatslmin)

Warmin&’ response

Jejunum

Tachycardic Stomach

Responses

40 k 6 42 k 2

58 f 4 2 + 2b

44 ? 6 2 5 2b

Serosa

2.6 t 0.6 0.2 * 0.2b

2.8 f 0.4 0.2 f 0.2b

3.0 * 0.4 0.2 +- 0.2b

Stomach

1.6 + O.Zb 0.2 -c O.Zb

1.6 2 0.2 0.2 + O.Zb

(V)

1.8 2 0.4 0.2 * 0.2b

1.8 T!Y 0.4 0.2 * 0.2b

1.6 +- 0.2 0.2 I 0.2b

Ileum

flow decrease

2.4 z!z 0.2 0.2 k 0.2b

Jejunum

Mesenteric

2.8 2 0.4 2.0 2 0.4

2.4 ?I 0.6 0.2 2 0.2b

2.0 " 0.6 0.2 " 0.2b

Serosa

a Numbers represent calculated maximal changes from six experiments each, in which effects of applying warm fluid were compared between capsaicin and vehicle (control) groups. The basal values for blood pressure, heart rate, and mesenteric blood flow were not significantly different from control values. Group A control values were not significantly different from B and C. Group A control values were: blood pressure, 112 + 18 mmHg; heart rate, 318 + 12 beats/min; mean blood flow, 3.4 f 0.6 V.“ p < 0.01.

cansaicin

50 f 6 6 f 2b

capsaicin Group B Control Perineural

Stomach

(mmHg]

the Cardiovascular

response

Prevent

Hypotensive

Pretreatments

40 -c 8 2 k 2b

4. Cansaicin

Group A Control Neonatal

Table

a Numbers represent calculated maximal changes from six experiments each, in which effects of applying warm fluid were compared before (control) and 15 min after surgical vagotomy or epidural anesthesia. Epidural anesthesia reduced resting arterial pressure by 38 * 6 mmHg, blood flow by 0.4 -t 0.2 V, and heart rate by 42 -t 4 beats/min. Resting circulatory parameters before epidural anesthesia and after vagotomy were not significantly different from those shown in Table 1. b p < 0.01. ’ p < 0.05.

Control Vagotomy Group B Control Epidural anesthesia

Group A

Table

GASTROINTESTINAL THERMOREFLEX

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271

Table 5. Chemical or Surgical Interruption of the Splanchnic Sympathetic Pathway Prevents the Decrease in Mesenteric Blood Flow in Response to Visceral Warming’ Mesenteric

Resting values after reserpine (n = 18) Postwarming values after reserpine Resting values after ganglionectomy (n = 6) Postwarming values after ganglionectomy

blood flow (V)

Stomach

Jejunum

Ileum

Serosa

3.2 ? 0.4

3.2 2 0.4

3.2 + 0.4

3.2 * 0.6

5.0 2 0.6b

4.6 +- O.Zb

4.8 k 0.4"

5.4 5 0.4b

3.8 f 0.2 5.8 ? 0.4b

3.4 t 0.4 5.4 * 0.2b

3.6 + 0.2 5.2 t 0.4b

4.0 2 0.4 6.2 C 0.4b

’ By contrast with findings shown in Table 1, exposure of mucosal and serosal surfaces to fluid at 45°C in rats receiving reserpine a vasodilator response in each case. b p < 0.01.

tory responses to warming the serosas (Table 4, group C). In six separate experiments, the vehicle for solubilizing capsaicin, when applied topically to the mesenteric trunk and mucosa, did not alter subsequent circulatory responses to 45%. Pretreatment with reserpine (injected intraperitoneally 24 h before experiments at a dose of 5 mg/lOO g) failed to prevent either the fall in arterial blood pressure or the increase in heart rate stimulated by visceral warming to 45°C; however, in this group of animals, we found an increase in mesenteric blood flow upon exposure of the viscera to warm fluid (Table 5). After splanchnic ganglionectomy, warming the mucosas to 45°C also resulted in an increase in mesenteric blood flow, but the procedure abolished the blood pressure and heart rate effects of intraluminal warming. However, the hypotensive effect of serosal warming was not entirely abolished by prior ganglionectomy (Table 5). The splanchnic nerves in rats consist of anterior and posterior portions (17). It is possible that a fraction of the sensory nerve population was not interrupted by the ganglionectomy. Splanchnic Nerve Activity Preparation)

(Group 2

Multiunit splanchnic nerve activity was monitored before and during exposure of gastrointestinal mucosal and serosal surfaces to 45°C liquid. After a latency of 3-4 s, the frequency of neural firing increased at approximately the same time as the circulatory change described previously (Figures 2A and 2B and group A of Table 6). As shown in group B of Table 6, the temperature-stimulated increase in neural activity was not inhibited by pretreatment with pentolinium in a dose (0.5 mg/lOO g) reported to block sympathetic ganglia (18).

Enteric Peptides

evoked

[Group 1 Preparation)

Pretreatment with the antiserum to SP (diluted 1:lOO and administered in two intravenous doses for 10 + 4 min) inhibited the hypotensive and mesenteric circulatory responses to 45°C fluid. The larger dose of antiserum against SP was more effective in antagonizing hemodynamic responses to warm fluid applied to the serosal and mucosal surfaces, although neither dose significantly affected the tachycardia (Table 7). In six experiments, there was no significant effect of control rabbit serum on the

Table 6. SpIanchnic Nerve Responses to Visceral Warming Are Not Affected by Pentolinium” Splanchnic

Group A (n = 15) Control Intraluminal warming (A% of control) Latency (s) Group B (n = 6) Control Intraluminal warming before pentolinium (A% of control) Control after pentolinium Intraluminal warming after pentolinium (A% of control)

nerve firing rate (spikes/s)

Gastric warming

Jejunal warming

Ileal warming

42 2 9 180 ” 9

48 + 5 187 + 5

50 +- 6 152 ? 2

321

422

422

48 t 9 183 ” 4

52 + 6 211 It; 5

46 5 7 273 ? 2

40 t 4

44 ? 8

42 rt 6

155 t 6

231 t 8

266 ? 6

O1Numbers represent multiunit splanchnic nerve firing rates in two groups of rats. In group A, resting values (control) are compared with maximal values (A% of control) obtained after warming of the mucosal surfaces of stomach, jejunum, and ileum in 15 rats. In group B, 6 rats were subjected to the same procedures as in group A, after which pentolinium was administered and exposure of viscera to warming was repeated.

272

ROZSA ET AL.

GASTROENTEROLOGY Vol. 95. No. z

aastric

Figure

2. Original records of splanchnic nerve impulse activity to warm stimuli (WC) applied topically (at vertical lines) to m ucosal surfaces of the stomach (a), and ileum and jejunum (b). Records from the top down: time signal at 10-s intervals, mean aortic pressure, heart rate, and multiunit splanchnic nerve impulse activity. Chart speed 0.5 m/s.

GASTROINTESTINAL THERMOIUFLEX

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273

Table 7. Substance P Antiserum Inhibits the Magnitude of Hemodynamic Responses to Visceral Warming’ Hypotensive Stomach Control responses Responses after 8 ~1 SP antiserum Responses after 16 ~1 SP antiserum

34 +

a

16 + 10b

10 f 4”

response

Mesenteric

(mmHg) Stomach

Jejunum

Ileum

Serosa

20 + 4

22 ? 8

42 + 12

12 t a

14 + 6 a 2 6'

29 + 14b 12 2 6"

6 k 4”

flow decrease (V)

Jejunum

2.2 2 6 1.2 t 0.6 1.4 t 0.4b 0.6 t_ 0.4b 0.4 t 0.2': 0.2 f 0.2"

Ileum

Serosa

1.6 k 0.4 0.6 f 0.2b 0.2 -r-0.2"

2.8 iI 0.4 1.6 '- 0.2b 0.6 + 0.2'

SP, substance P. ’ Numbers represent calculated maximal changes after application of fluid at 45°C before intravenous injection of SP antiserum (control, n = 12 experiments], and 1 min after two doses of antiserum (n = 6 experiments each). Values obtained after injection of antiserum were compared for significant difference with control responses. Values for heart rate are not shown because there was no significant difference between control and antiserum responses to warm fluid. The basal values for blood pressure, heart rate, and mesenteric blood flow did not differ significantly from those shown in Table 1. b p < 0.05.' p < 0.01.

responses to application of 45°C fluid and also no effect of either SP antiserum or control rabbit serum at the same volume on basal hemodynamic parameters. Pretreatment with somatostatin temporarily inhibited the postwarming declines in blood pressure and mesenteric blood flow, but had no significant effect on the increase in heart rate. The administered doses of somatostatin did not have any significant influence on the control circulatory parameters. Circulatory responses to warming were reduced by two intravenous doses (25 and 50 ng/lOO g) of somatostatin (6 rats for each dose] as follows: the hypotensive response was diminished 32% _t 6% and 74% _t 2%, respectively; the mesenteric circulatory response was diminished 36% -+ 5% and 76% ? Sob, respectively. In separate groups of rats after pretreatment with reserpine, the antiserum to SP prevented the subsequent mesenteric vasodilator response to visceral warming (Table 8). The doses of antisera to SP that were employed did not alter basal hemodynamic parameters.

Discussion This study has demonstrated the presence of a thermoreflex that originates in the gastrointestinal tract of the anesthetized rat and evokes distant changes in the circulatory system. Within several seconds after the application of fluid warmed to 45’C

to the mucosal or serosal surfaces of the rat stomach, jejunum, or ileum, there was a reproducible pattern of responses consisting of systemic arterial hypotension, tachycardia, and a diminution of blood flow through the superior mesenteric artery. Maximal circulatory changes were dramatic: for example, the magnitude of the hypotensive change amounted to about one-third of the resting arterial pressure and occurred within 5 s after the onset of visceral warming. These cardiovascular responses could be elicited without significant tachyphylaxis upon exposure to repeated thermal stimulation. The rapid onset of distant circulatory responses to warming of the viscera suggests a neurally mediated reflex rather than a hormonally regulated event. Further support for this conclusion stems from our findings that the responses to visceral warming were prevented by anesthetization of mucosal receptors with lidocaine, by mucosal sensory desensitization with topical capsaicin, by interruption of afferent C fiber conduction with perineurally applied capsaitin, and by pretreatment of rats with parenteral capsaicin during neonatal life. There is considerable evidence from morphologic, electrophysiologic, and biochemical studies that capsaicin selectively affects primary sensory neurons (19,20). The desensitizing effect of intraluminal capsaicin suggests that thermal stimulation is detected by mucosal receptors. However, this pretreatment did not influence the reflex response evoked by intraperitoneal warming of serosal receptors.

Table 8. Substance P Antiserum Inhibits the Mesenteric Vasodilator Response to Visceral

Warming

in Rats Receiving

Reseruine’ Mesenteric

Postwarming values after reserpine Postwarming values after reserpine SP antiserum (16 ~1)

alone and

vasodilator

response

(V]

Stomach

Jejunum

Ileum

Serosa

2.8 + 0.2 0.6 t 0.2b

2.4 f 0.2 0.4 f 0.2b

2.2 k 0.4 0.2 k 0.2b

3.0 + 0.4 0.8 t 0.4"

SP, substance P. ” Data represent calculated maximal vasodilator changes from 6 reserpine-treated rats after application of visceral warming. Basal value before warming was 3.6 f 0.4 V. Substance P antiserum was administered to the same reserpine-treated rats and visceral warming was repeated. ” p i 0.01.

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Our direct measurements indicated an increase in splanchnic neural activity coincident with the onset of circulatory changes. The greater splanchnic nerve of the rat contains both preganglionic and postganglionic axons, and these fibers are homogeneously distributed throughout the nerve. Splanchnic postganglionic responses can be estimated by subtracting preganglionic responses from responses recorded before ganglionic blockade (17). We demonstrated that the thermally stimulated increase in splanchnic neural activity was not inhibited with ganglionic blockade. Moreover, we found that either neonatal or perineural capsaicin entirely inhibited the cardiovascular responses to warming. It was reported that these capsaicin pretreatments do not affect the efferent fibers of the autonomic nervous system (21). Based on the foregoing considerations, we have concluded that preganglionic splanchnic afferent nerve activity was increased in our experiments. In addition, the results of bilateral vagotomy and celiac-superior mesenteric ganglionectomy suggest that the afferent fibers conducting the reflex travel in the sympathetic nerves and not in the vagi. It has been proposed that capsaicin-sensitive neurons not only possess the afferent function of primary sensory nerves, but also have an efferent function, which is mediated by the release of peptides at their peripheral endings. This latter capacity involves such nerves in local regulation of visceral organs, and recent investigations implicate SP in primary sensory, afferent nerve transmission (22,23). We found that the cardiovascular thermoreflex was prevented by three types of pretreatments with capsaicin, and that both somatostatin and specific immunoblockade by the antiserum to SP prevented the hypotensive and mesenteric circulatory responses to visceral warming. It has been reported that somatostatin inhibits the release of SP from primary sensory neurons (24). These findings support the concept that the thermoreflex is transmitted by primary sensory neurons involving the release of SP-like peptides from afferent C fibers of the viscera. There is evidence that many vascular beds are affected by primary sensory neurons in which the neurotransmitter is SP (25). The release of this peptide may be prompted by antidromic stimulation through an axon reflex that results in vasodilation (26). However, in the only vasculature where flow was monitored, there was a decrease in mesenteric perfusion after visceral warming. There is morphologic evidence that primary afferent nerves also synapse with sympathetic neurons in prevertebral ganglia (27). The physiologic significance of noncholinergic, sensory transmission in sympathetic ganglia is unknown. Our studies also suggest that the mesenteric vas-

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cular response to warming was not merely the result of a fall in systemic arterial blood pressure. When reserpine or splanchnic ganglionectomy was used before warming, there was an increase in mesenteric blood flow with application of 45°C fluid, despite the fall in arterial blood pressure, and this vasodilation was inhibited by the antiserum to SP. In additional experiments, the data of which do not appear in this report, we utilized a constant-pressure mesenteric circulatory preparation in rats. Despite the differences between preparations, we found that visceral warming to 45°C still caused mesenteric vasoconstriction in the constant pressure-perfused rat gut. In addition, we found that the hypotensive response to visceral warming was inhibited far more by epidural anesthetization than was the thermally stimulated decline in mesenteric blood flow. Finally, we observed a dissociation between changes in blood pressure and blood flow when viscera were warmed to 50” and 55°C. Taken together with our findings that interference with enteric sensory nerves prevented the thermally induced decrease in mesenteric blood flow, our results are compatible with the idea that splanchnic sensory nerves are capable of mediating mesenteric vasoconstriction. This mediation probably involves a synapse within the sympathetic ganglion between primary sensory neurons and vasomotor sympathetic neurons. The hypotension and tachycardia that followed visceral warming do not appear to be simple baroreceptor responses, because the change in blood pressure was unaffected by pretreatment with vagotomy, atropine, reserpine, or propranolol. Our results are supported by other experimental data indicating that sensory SP has no role in producing the baroreceptor reflex (28). The partial inhibition of hypotensive and mesenteric vascular responses to warming by hexamethonium suggests that sensory SP acts on both cholinergic (nicotinic) receptors and on noncholinergic sites. It has been reported that electrical stimulation of the proximal part of the splanchnic nerve elicited hypotension that could be blocked by pretreatment with capsaicin (29). Furthermore, the hypotension evoked by intravenous capsaicin is of central origin (30). We utilized epidural anesthesia to further localize the reflex center. Epidural anesthesia almost completely blocked the hypotensive response to visceral warming. These data suggest that the hypotensive response to visceral warming involves spinal or supraspinal nerves. Epidural blockade also inhibited thermally prompted responses in heart rate and mesenteric blood flow, but less markedly. Our findings with epidural anesthesia suggest that thermally activated, visceral afferent neurons have both central and peripheral vasomotor functions.

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We observed that hexamethonium, reserpine, and propranolol failed to prevent the tachycardia evoked by visceral warming, suggesting that the increased heart rate was not mediated by cardiac sympathetic nerves. Similarly, it is unlikely that a vagally mediated cardiac response occurred with warming, because of the lack of effect of vagotomy and atropine on the thermoreflex. By contrast, pretreatment with capsaicin (but not the antiserum to SP) prevented the tachycardia. We assume that another sensory neuropeptide is responsible for the tachycardia. Recent data indicate that calcitonin gene-related peptide occurs in and is released from capsaicin-sensitive afferent neurons simultaneously with SP. This peptide exerts noncholinergic, nonadrenergic, positive chronotropic effects under both in vivo and in vitro circumstances. It has also been shown that the desensitization produced by calcitonin gene-related peptide inhibits the positive inotropic effect of capsaicin (31). The gastrointestinal mucosa contains calcitonin generelated peptide in its sensory neurons and, according to recent morphologic data, the immunoreactivity to sensory calcitonin gene-related peptide of the splanchnic nerves is very high (32,33). Reports indicate that different mechanical, chemical, and thermal reflexes that originate in the gut each activate specific enteroreceptors, resulting in a very complex array of afferent information emanating from the viscera (1,2). Other reports describe mechanically and chemically activated reflexes that alter cardiovascular functions, although the physiologic significance of such reflexes is uncertain (3,4). Furthermore, these mechanical and chemical reflexes may evoke pressor or depressor responses, with or without a tachycardia (34-38). For example, gastric distention was followed by increased arterial pressure and mesenteric vasoconstriction (39), whereas jejunal distention was followed by a decrease in arterial pressure in rats that was inhibited by pretreatment with capsaicin (29). Thermal activation of visceral autonomic nerves in anesthetized cats elicited an increase in gastric motility that was blocked by atropine and by pharmacologic antagonism of SP (40). We are unaware of previous reports about a thermal reflex originating in the viscera that alters cardiovascular functions. There is evidence of the presence of capsaicin-sensitive nerves in visceral organs (41,42) and in the regulation of intestinal motor (43) and vascular (44) functions. These primary afferent nerves contain SP among numerous biologically active peptides (45,46). Different populations of peptidergic afferents vary in their contribution to innervation of different organs. Evidence indicates that peptides are synthe-

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sized within small-diameter afferent nerves and are transported to both central and peripheral termini where they are released and contribute to axonal reflexes (47).

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Received November 5, 1966. Accepted October 26, 1967. Address requests for reprints to: Eugene D. Jacobson, M.D.. Dean, School of Medicine, Box C290, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 66262. These investigations were supported in part by National Institutes of Health grant DK37050 entitled “Mesenteric Ischemia” and by an unrestricted grant from The Upjohn Company. The authors thank Dr. Ruben D. Bunag and Dr. Russell T. Dowel1 for scientific advice.