Acta Physiol Scand 1984, 120:311-316

The involvement of the enteric nervous system in the intestinal secretion evoked by cyclic adenosine 3’,5’-monophosphate

STEFAN EKLUND, JEAN CASSUTO, MATS JODAL and OVE LUNDGREN Department of Physiology, University of Goteborg, Sweden

EKLUND, S., CASSUTO, J., JODAL, M. & LUNDGREN, 0.: The involvement of the enteric nervous system in the intestinal secretion evoked by cyclic adenosine 3’3’- monophosphate. Acta Physiol Scand 1984, 120: 31 1-316. Received I 1 July 1983. Depart- ment of Physiology, University of Goteborg, Sweden Intestinal fluid secretion w a s evoked in vivo in rats and cats by introducing dibutyryl- cyclic adenosine 3.5-monophosphate (db-CAMP) or theophylline, a phosphodiesterase inhibitor, in the intestinal lumen. The intestines were denervated periarterially. It was demonstrated that three compounds of varying chemical structure and with different modes of action on nerves (tetrodotoxin, lidocaine, hexamethonium) decreased the secre- tory response 60-70%. It is concluded that the secretion induced by increasing the intracellular cAMP concentrations is in part evoked via the enteric nervous system

Key uwrds: Intestinal secretion, CAMP, hexamethonium, lidocaine, tetrodotoxin, cholera

Cyclic adenosine 3’3’-monophosphate (CAMP) is considered to be the mediator of the secretion in the small intestine induced by cholera toxin via a direct action of the toxin on the intestinal epithelial cells (Field 1981, Holmgren 1981). However, it has recently been suggested that the enteric nervous system may mediate part of the cholera secretion (Cassuto et al. 1981), infemng that the toxin elicits nervous secretory reflexes confined to the intesti- nal wall. This makes the role of cAMP somewhat less clear. The present study is an attempt to exam- ine the interrelations between intestinal secretion, cAMP and the enteric nervous system. The intra- cellular concentration of cAMP in the intestinal epithelium was increased by introducing the cAMP analogue dibutyryl-CAMP (db-CAMP) or theophyl- line, a phosphodiesterase inhibitor, into the lumen of periarterially denervated rat or cat jejunal seg- ments in vivo. This led to decreased net fluid ab- sorption or, in most experiments, to a net fluid secretion. The aim of this study was to test the effect on this intestinal secretion of drugs known to interfere with autonomic nervous activity (tetrodo- toxin, lidocaine, hexamethonium).

METHODS Anesthesia and operative procedures Rat expeimenrs. The experiments were performed on Sprague-Dawley rats (Anticimex, Stockholm, Sweden) of

both sexes weighing 200-300 g. They amved to the animal quarters at least 7 days prior to experiments and were kept under standardized environmental conditions: 25°C 5 5 4 % relative humidity, artificial lighting 06.W18.00.

Anesthesia was induced with a shortacting barbiturate (Brietal, Lilly) intraperitoneally, and continued with inter- mittent injections of chloralose (10 m g h l ) in the dissected and cannulated femoral vein.

Arterial blood pressure w a s recorded via a catheter in the femoral artery, connected to a pressure transducer (Statham P23AC) and monitored continuously on a Grass polygraph. The catheter was via a T-tube connected to a cannula through which a continuous infusion of glucose (556 mM) and bicarbonate ( I 0 0 mM) at a rate of approxi- mately 0.03 ml per min was given. This arrangement served to keep the catheter open and also to maintain acid-base balance at a normal level (Cassuto et al. 1982 b).

A midline abdominal incision was performed and a segment of mid-jejunum, 7-10 cm in length, was isolated with its blood supply intact, while the remainder of the small intestine and colon was removed. Nerves around the superior mesenteric artery were cut to eliminate ex- trinsic nervous influence.

Cat experiments. The cats weighed 3-5 kg and were anesthetized intravenously with chloralose (50 mg/kg b.wt.) after induction with ether. The cats were deprived of food for 24 h but had free access to water.

The operative procedures and recordings of blood flow were similar to those described by Jodal et al. (1975). In short, after a midline laparotomy a 10 to 15 cm long segment from the mid-jejunum was isolated with intact vascular supply. The remainder of the small intestine, the colon, the spleen, the greater omentum, and a major portion of the pancreas were extirpated. The left adrenal gland was ligated and the right one denervated. The

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312 S. Eklund et al .

Table 1 . The effect on intestinal net fluid transport of varying db-CAMP concentration in the intestinal lumen of the rat

db-CAMP Rate of fluid transport concen- plxmin-'x100 cm-' serosa No. of tration obser- (mM) Control db-CAMP vations

20 4 8 f 9 - 175 I3 6 80 53+8 -47 f9 14

320 47+17 -61+15 6

A minus sign denotes a net fluid transport from the tissue into the lumen.

splanchnic nerve fibers to the small intestine were divided in most experiments and placed on ring electrodes for efferent stimulation. The nerves were stimulated with a Grass stimulator (Model S5) at 8 Hz, 5 V for 5 msec. In some experiments all nerves surrounding the mesenteric artery and vein were divided and the distal ends placed on ring electrodes. The stimulation characteristics in these experiments were 8 Hz, 12 V, and 5 msec.

After the animal had been heparinized (3-5 mg/kg b.wt.) blood pressure was measured by a pressure transducer (Statham P23AC) connected to the femoral artery. For venous outflow recordings the superior mesenteric vein draining the intestinal segment and its lymph nodes was cannulated and connected to a drop recorder unit operat- ing an ordinate writer. Local intra-arterial injections were given by means of a catheter in a small branch of the superior mesenteric artery.

Recording of net jluid transport Rat experiments. The determination of net fluid transport was made with the gravimetric method described by Cas- suto et al. (1982 b ) . The intestinal segment was placed in a

plastic chamber specially designed for this purpose. The lumen of the intestinal segment was connected through its distal end to a basin on the chamber, allowing free pas- sage of fluid in and out of the segment. Net fluid transport across the intestinal epithelium, as reflected by the changes in the weight of the plastic plate including its basins and intestinal segment, was continuously recorded with a force displacement transducer and monitored via a Grass polygraph.

In some experiments the proximal end of the intestine and the outflow tube of the basin were connected with each other by plastic tubing via a reservoir. The solution was recirculated by means of a roller pump at a rate of 0. I ml xmin.-l

The intestinal segment was carefully covered by a plas- tic film to avoid evaporation. However, evaporation from the surface of the plastic chamber and from its basin may lead to a decrease of registered weight that would be misinterpreted as fluid absorption. Thus, to minimize this artifact and to have a good control of the temperature of the intestinal segment, the rat was placed in a small plastic cage, in which the air temperature was kept constant at 363°C giving the intestinal segment a temperature about 38°C. By this arrangement the air surrounding the rat became saturated with water vapour, which minimizes futher evaporation. The head of the rat was placed through an opening in the cage wall, the animal thereby being able to breath air of room temperature. Body tem- perature was registrered by a thermometer in the esopha- gus, and kept at 38°C.

Cat experiments. To record intestinal net fluid transport in the cat principially the same weighing method as de- scribed above for rat was used without the recirculation of the luminal perfusate (Sjovall et al. 1983).

Drugs and solutions The following drugs were used: lidocaine (AB Astra, SO- dertalje, Sweden), hexamethonium bromide (Sigma Chemicals), tetrodotoxin (TTX; Sigma Chemicals), dibu-

Table 2. Arterial blood pressure (BP; mmHg) and intestinal net fluid transport (NFT; mgxmin- 'x100 serosa) at "resting" control conditions and during intestinal secretion, induced by exposing the

intestinal mucosa to a solution of db-CAMP (80 mM) or theophylline (30 mM), before and after giving hexamethonium i.v. or placing lidocaine on the serosal surface

db-CAMP- or theophylline-induced secretion

"Resting" control Before drug After drug

BP NFT BP NFT BP NFT

db-CAMP Hexamethonium (6) 102f6 5 2 f 9 100+4 -47f12 7 3 f 7 29+ 10 Lidocaine (6) 104+8 6859 94+5 -28+ 1 I 95 f5 35f5

Theophy lline Hexamethonium (8) 102f6 92+8 92+3 - 5 8 f 3 2 66k5 52+19 Lidocaine (5) 91 +6 68 + 26 94+7 -59f21 73+9 3 8 f 2 1

Mean values f SE. Numbers within parenthesis denote number of animals. A minus sign denotes net fluid transport from tissue to lumen.

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CAMP and intestinal secretion 313

100-

80-

60-

40-

MEAN ARTERIAL BLOOD

PRESSURE, mm Hg

1 2 0 1

- -100-

-

-

T

0 'Resting' control

Theophylline

tyryl c-AMP (Sigma Chemicals) and theophylline (Sigma Chemicals).

During periods of control absorption, a NaCl solution of around, 320 mM, was used. db-CAMP of different concen- trations (20, 80 and 320 mM) was dissolved in saline, the osmolality of the solution being adjusted to isotonicity. The theophylline solution contained theophylline 30 mM and NaCl 135 mM.

Experimental procedures After completing the operation, “resting” absorption from a saline solution was registered for about one hour, whereafter the intestine was rinsed with 10 ml bodywarm control solution. The test solution was then instilled in, or perfused through the intestinal lumen. The secretory re- sponse was followed for at least 20 min after which one of the following agents were given: hexamethonium (i.v.),

lidocaine (on the serosal surface), TTX (i.a.) or atropine (i.v.). Net fluid transport was then recorded for at least another 30 min.

In the cat experiments tetrodotoxin ( l T X ; 5-10 pg) was given intra-arterially, while the superior mesenteric artery was clamped for 2 min. When the arterial occlusion was released, mesenteric venous blood was collected and sub- stituted with dextran for about 1 min to avoid generalized TTX effects on the animal. With this procedure a revers- ible nerve-blocking effect was accomplished, as judged by the absence of vasoconstrictor effects on electrical nerve stimulation.

Statistical analyses Statistical analyses were performed using the sign test (Siege1 1956). Differences resulting in P values less than 0.05 were considered significant.

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314 S . Eklund et al.

Table 3. The effect of hexamethonium (20 mglkg i .u . ) and lidocaine (10 mglml placed on the serosal surface) on arterial blood pressure (BP; mmHg) and net fluid transport (NFT; mgxmin-'x100 cm-' serosa) in the normal small intestine of the rat

Before drug After drug

BP NFT BP NFT

Hexamethonium (7) 125f3 47+ I 1 8959 64f 12 Lidocaine (5) 126+8 42+7 12758 44f 10

Mean values + SE. Numbers within parentheses denote number of animals.

RESULTS

Rat experiments In a first series of experiments dibutyryl-cyclic adenosine 3',5'-monophosphate (db-CAMP), dis- solved in a saline solution, was administered into the intestinal lumen at different concentrations (20, 80 and 320 mM). The secretory response, recorded for at least 20 min, showed a dose related response as shown in Table 1, but the effect of 320 mM db- CAMP may in part have been due to the absence of sodium chloride in the luminal fluid. No consistent effect of db-CAMP on arterial pressure was noted. In the following rat experiments a 80 mM db-CAMP solution was used. This concentration is higher than that utilized in most in vitro experiments but it was used to ensure a constant secretion for at least one hour.

Hexamethonium is a receptor blocker for acetyl- choline in autonomic nerve synapses. The adminis- tration of this drug i.v. (10 or 20 mglkg) turned the secretion evoked by db-CAMP into absorption (Ta- ble 2; p<0.02). Expressed in relative terms, hexa- methonium inhibited 62+12 (SE) % (n=6) of the total change of fluid transport rate evoked by db-

CAMP. A small effect of hexamethonium was ob- served on "resting" fluid absorption from normal intestines (Table 3; p=0.06). We have ealier dem- onstrated that in the concentration used, hexameth- onium inhibits the effect of efferent vagal stimula- tion on heart rate in the rat for 30-60 min (Cassuto et al. 1982a).

If the enteric nervous system is involved in the db-CAMP evoked intestinal secretion, it would be possible to inhibit the fluid secretion by anesthetiz- ing the myenteric and submucosal plexuses in the intestinal wall. For this reason a saline solution containing lidocaine (10 mglml), a local anesthetic, was placed on the serosal surface of the rat intes- tine. This experimental procedure turned db-CAMP induced fluid secretion into absorption @<0.02) while there was no effect on control animals @=0.5; Tables 2 and 3). Expressed in relative terms the effect of lidocaine was 66+ 12% (n=6). We have earlier shown by autoradiography that l id~caine- '~C, applied to the serosal surface in vivo as described above, seems not to reach beyond the muscular layer (Cassuto et al. 1983). In two other series of rat experiments the intesti-

nal lumen was perfused with an isotonic solution

Table 4. Arterial blood pressure, intestinal blood flow and intestinal fluid transport in the cat during "resting" control conditions and during db-CAMP evoked secretion before and after giving tetrodotoxin (TTX) i.a.

db-CAMP induced secretion

Control Before TTX After TTX

Arterial blood pressure, mmHg 97+9 99+8 92+7 Intestinal blood flow, mlxmin-'xIW g-l 28+4 37+7 41f7

Net fluid transport, mgx min-' x 100 serosa 104+ 18 -46+20 46f I4

Mean values f SE. n=7. A minus sign denotes a net fluid secretion from tissue to lumen.

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cAMP and intestinal secretion 3 15

containing theophylline (30 mM) and sodium chlo- ride (135 mM). This evoked a net fluid secretion, which was not influenced by atropine i.v. (0.25 mg/kg; p = 0 . 7 ) . Giving hexamethonium (i.v.) and lidocaine (applied on the serosal surface) as de- scribed above for the db-CAMP experiments, inhib- ited the secretory response in a manner similar to that described above in connection with db-CAMP (Table 3; p<O.OS). Fig. 1 shows the time course of the experiments in which hexamethonium was giv- en. In three of these experiments net fluid transport was recorded for another half an hour and secretion then returned towards the control level seen before giving hexamethonium. The relative effect was 65fIO (n=8) and 61f10 (n=S) % for hexametho- nium and lidocaine, respectively.

Cat experiments Tetrodotoxin (TTX), a nerve conduction blocking agent, was administered close i.a. to cat intestinal segments secreting in response to db-CAMP (20 mM) placed in the intestinal lumen. These experi- ments were performed on cats since it is very diffi- cult to administer TTX to rats intravascularly with- out killing the animals. The results are given in Table 4. The secretory response to db-CAMP was markedly diminished after giving TTX @<0.01), the averge decrease amounting to 67*8% (n=7) of the response on fluid transport induced by db- CAMP. Control experiments have been reported elsewhere (Cassuto et al. 1981) showing no signifi- cant effect of TTX on absorbing intestines (glucose- free luminal solution).

DISCUSSION

In this study the intracellular concentration of cAMP was increased in the intestinal epithelium by exposing the intestinal mucosa to db-CAMP or to theophylline, a phosphodiesterase inhibitor. This evoked in most experiments net secretion of fluid as demonstrated by other authors (Field 1981). The results of this study strongly suggest that the intes- tinal secretion is partly mediated via nerves, since three compounds of varying chemical structure and with different modes of action on the nerves (TTX, lidocaine, hexamethonium) inhibited the cAMP evoked net fluid secretion from the small intestine in vivo. The results furthermore indicate that the nervously induced secretion is mediated via the enteric nervous system since the intestinal seg-

ments used were denervated periarterially. This conclusion is also corroborated by the observed inhibitory effect on secretion of hexamethonium, a cholinergic ganglionic blocking agent, an observa- tion that also seems to exlude that an axon reflex arrangement is involved in the secretory response.

The "nervous" mode of action of cAMP cannot be established from our observations. At least two mechanisms seem possible. First, a direct action of an increased intracellular cAMP concentration in nerves is possible. In other systems cAMP seems to evoke a hyperpolarization of nerves (Cooper et al. 1978) suggesting that its effect on the enteric ner- vous system may be an inhibition of nerves enhanc- ing fluid uptake. Second, an increase in intracellu- lar cAMP concentration in some cell type(s) pres- ent in the epithelium (e.g. amine precursor uptake and decarboxylation (APUD) cells) may secondar- ily activate nerves in its vicinity. Thus, we have proposed that cholera toxin adheres to the enter- ochromaffin (EC) cells and triggers the release of 5- hydroxytryptamine (5-HT) from these cells (Nils- son et al. 1983). 5-HT. in turn, activates adjacent dendrites from nerve cells in the enteric nervous system. The elicited nervous reflex(es) contained a cholinergic synapse and a noncholinergic, nonad- renergic neuron at the effector cell (Cassuto et al. 1981, 1982a, h , 1983). A similar chain of events may have occurred in the present experiments since increasing cAMP in the intestinal epithelium causes the release of 5-HT in vitro, presumably from the EC cells (Forsberg & Miller 1982).

An activation of, for example, the APUD cell system in the intestinal epithelium can also result in a release of peptides and/or amines that via hor- monal and/or paracrine mechanisms may affect transport processes in the enterocytes. If such mechanisms are present and to what extent they are of importance for transport phenomenon in the in- testine in vivo is unknown. However, the present results demonstrate that transport phenomenon elicited by an increase of intracellular cAMP con- centrations nonselectively in the whole intestinal epithelium are not necessarily caused by a direct effect on the enterocytes. Furthermore, determina- tions of cAMP content in tissue samples containing several types of epithelial cells may not reflect the importance of cAMP in a specific transport process in the enterocytes.

Most of the work on secretory mechanisms in cholera and other types of diarrhea have been per-

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316 S. Eklund et al.

formed in vitro on intestines stripped of their mus- cular coat. To what extent the enteric nervous sys- tem participates in such in vitro studies is not known since no control experiments have been re- ported to rule out this possibility. However, the present results clearly suggest that one should be cautious when drawing conclusions regarding the in vivo situation from results obtained on in vitro preparations with regard to the action of CAMP.

This research w a s supported by grants from the Swedish Medical Research Council (2855), Magnus Bergvall's Foundation, Astra Research Foundation, Ake Wiberg's Foundation and the Faculty of Medicine, University of Goteborg.

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