Effect of Serotonin on Water and Electrolyte Transport in the In Vivo Rabbit Small Intestine

Vol 71. So. 6 Prmted In " ..'1 A .

G.STROENTEROLOGY 71 :1033- 10:18. 1976

Copyright © 1976 by The William. & Wilkins Co.

EFFECT OF SEROTONIN ON WATER AND ELECTROLYTE TRANSPORT IN THE IN VIVO RABBIT SMALL INTESTINE BARRV KISLOFF, M.D., AND EDWARD

W.

MOORE, M.D .

Department 0/ Medicine, Medical Col/ege 0/ Virginia , Virginia Commonwf'alth University. Richmond. Virginia

The influence of intravenously administered serotonin on water and electrolyte fluxes in the in vivo rabbit jejunum and ileum was examined. Animals were divided into four groups: (1) those receiving saline intravenously while a glucose-free isotonic saline solution perfused the jejunum and ileum; (2) serotonin given intravenously while glucose-free intestinal perfusate was used as in group I; (3) intravenous saline given while a 10 mM glucose-isotonic saline solution perfused the jejunum and ileum; and (4) intravenous serotonin given while the intestinal perfusate was as in group 3. Serotonin administration resulted in highly significant net secretion of H 2 0 and sodium in both jejunum and ileum in the groups with a glucose-free perfusate. In jejunum, serotonin evoked net water and sodium secretion. whereas controls absorbed water and sodium. In ileum. serotonin significantly enhanced secretion. The addition of glucose to the perfusate completely abolished the serotonin effect. Unidirectional 22Na flux analysis revealed a marked diminution in both mucosal to serosal and serosal to mucosal fluxes in serotonintreated animals. The decrease in mucosal to serosal flux was greater than the decrease in serosal to mucosal flux. thus explaining the enhanced net secretion observed with serotonin in the groups receiving glucose-free perfusate. In spite of its pronounced effect on water and electrolyte transport. serotonin failed to produce any detectable histological alterations in small bowel mucosa. either by light or electron microscopy. We postulate that serotonin may be an important mediator of the diarrhea so frequently noted in the carcinoid syndrome by virtue of its effects on small intestinal H 20 and electrolyte transport. Diarrhea is a frequent manifestation of the carcinoid syndrome. ,-3 The diarrhea is typically watery and unac companied by steatorrhea or other evidence of malabsorption. Although carcinoid tumors have been found to produce a variety of substances such as histamine. bradykinin. adrenocorticotropin, and prostaglandins. the substance most universally produced in excess by carcinoid tumors is serotonin (5-hydroxytryptamine) . <-T Despite the fact that serotonin is normally found in abundance in the intestinal mucosa.' the central nervous system.· and platelets, '0 the physiological function of this amine remains unknown. Serotonin is most often implicated as a pathogenetic agent in producing the diarrhea seen in carcinoid syndrome. This is based on: (1) its known ability to stimulate intestinal smooth musculature, especially that of the small intestine. and Received March 19, 1976. Accepted May 17. 1976. Address reguests for reprints to: Dr. Edward W. Moore. Box 908. MeV Station. Medical College of Virginia, Richmond. Virginia 23298. Supported by Grants 15393 and 18887 from the National Institute of Arthritis, Metabolism and Digestive Diseases. The authors gratefully acknowledge the assistance of Dr. Gordon E . Madge, Department of Pathology, for his help in preparing and interpreting light and electron-microscopic material. In addition. we wish to thank Frances B. Keith and Donna e . Robbins for their able technical assisance. and Brenda B. Powell for her secretarial assist ance.

thus produce a state of intestinal hypermotility; II·" (2) the efficacy of antagonists of serotonin action"'" or inhibitors of serotonin production ,6 in ameliorating the diarrhea of the carcinoid syndrome; and (3) the correlation that exists between the intensity of diarrhea and of serotonin secretion. \1 However. the relationship between intestinal hypermotility and diarrhea is far from clear, " ...· n with intestinal hypermotility being considered etiological for both constipation ,. and diarrhea.' Thus it seems necessary to examine other possible means by which serotonin might mediate this symptom. The possible effects of serotonin on intestinal H 20 and electrolyte transport have not been previously defined. Serotonin administration is known to evoke secretion in salivary glands of dog. cat.23 and blowfly (Calliphora erythrocephala).24. 2' and in the pyloric mucosa of dogs and cats. 21 . 27 In addition, the secretory effects of serotonin in the blowfly salivary gland seem clearly mediated by the activation of the adenyl cyclase system. H. U a sys~em implicated in the secretory process induced by several bacterial and hormonal small intestinal secretagogues. 2I Recently. Donowitz and Binder" demonstrated jejunal net water and sodium secretion in 2 of 3 carcinoid tumor patients with diarrhea. This secretion was reversed in 1 of these patients when given the serotonin antagonist methysergide. Because of these known associations between serotonin and secretory systems, we

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Vol. 71, No . 6

KISLOFF AND MOORE

have evaluated the influence of intravenous serotonin on simultaneous jejunal and ileal water and electrolyte transport in the rabbit in vivo to answer the following questions: (1) Does serotonin administration result in an increase in net small "intestinal secretion? (2) If so, is net secretion induced by the inhibition of H,O and electrolyte absorption, or by stimulation of secretion, or both? and (3) Does the addition of glucose to the intestinal perfusate alter the action of serotonin? Materials and Methods Acute experiments were performed in male New Zealand white rabbits weighing 2 .0 to 3.0 kg and given food and water ad libitum until the day of study. Anesthesia was achieved by using intramuscular ketamine hydrochloride (Ketalar, 50 mg per ml, Parke-Davis & Co., Detroit, Mich.), 50 mg per kg, plus intramuscular paraldehyde (paraldehyde USP, Elkins-Sinn. Inc., Cherry Hill, N. J .), 0.5 ml per kg. This combination of drugs has been found to be a safe and effective means of inducing and maintaining anesthesia in rabbits for prolonged periods. I . Isolated jejulClal and ileal loops, approximately 20 cm in length, with their blood supplies intact, were prepared as described previously. II. so Loops were rinsed for 30 min with either of the two test solutions described below and then continuously perfused for 3 hr (six 30-min periods) at 37°C with isosmotic solutions containing (mM) either: (l) Na, 155; K, 5; CI, 135; HCO., 25; phenolsulfonphthalein (PSP), 0.3; and isotopic UNa; or (2) Na, 150; K, 5: CI, 130; HCO .. 25; PSP, 0.3; glucose, 10; and isotopic UNa. These fluids were perfused at a rate of 2 ml per min using a Sage 275-A roller pump (Sage Instruments, Cambridge. Mass.). Animals were divided into four groups: (1) group 1 consisted of 10 rabbits whose loops were perfused with glucose-free solution (solution 1) while hydration was maintained by intravenous (i.v.) infusion of isotonic NaCl (1.5 ml per kg per hr); (2) group 2 consisted of 10 rabbits whose loops were perfused with solution 1 but whose i.v. infusate, administered at a similar rate, consisted of isotonic saline plus 5 hydroxytryptamine (serotonin) creatinine sulfate complex (Sigma Chemical Co., S1. Louis, Mo.) delivered at a rate of 20 lig per kg per min; (3) group 3 was composed of 7 rabbits whose loops were perfused with glucose-containing solution (solution 2) while hydration was maintained with isotonic saline as described above; (4) group 4, consisting of 7 rabbits, had their intestinal loops perfused with solution 2, while hydration plus serotonin were administered intravenously as per group 2. Blood was drawn from the inferior vena cava at time zero and at 180 min, and was analyzed for pH, electrolytes, and osmolality. Intestinal perfusates were sampled at time zero, just before beginning the serotonin infusion, and subsequently at 30-min intervals for analysis. Phenolsulfonphthalein (PSP) was used as the volume marker. The pH was determined with a Corning capillary blood pH electrode at 37°C, and a digital electrometer (Orion Research, Inc. Cambridge, Mass.). Na and K were determined by flame photometry (KLiNa Flame System. Beckman Instruments, Fullerton, Calif.). Chloride determinations were made by amperometric titration (Fiske Chlor-Q-Counter, Fiske Associates, Inc., Uxbridge, Mass.). Bicarbonate concentration was calculated from observed Na, K, and Cl concentrations: [HCO.) = [Na) + [K) - (Cl). The validity of this calculation has been previously demonstrated." Osmolality was determined by freezing point depression (Model 3D, Advanced Instruments, Inc., Needham Heights, Mass.). PSP concentration was determined colorimetrically in a

Zeiss spectrophotometer (Carl Zeiss, Inc •• New York, N. Y.) with PSP concentration determined by the following formula:· ....

0.0.

=

PSP... -

PSP' II + PSP ••• 2

where 0 .0. s colorimetric reading of PSP, directly proportional to its concentration as determined by a linear standard curve, and PSP = optical density at the given wavelength. Net water flux (J H.O), expressed as microliters per centimeter of intestine per hour, was determined from the following formula, corrected for 2.0-ml aliquots serially removed, using an Olivetti 1'652 computer:"

~_ 1-

Q. - 2.0

(~I

(PSPJ)

(PSPL

where V, is the volume at any given time and Q. is initial amount of PSP in the loop. Mean PSP recovery from jejunum in groups 1 to 4 respectively was 97.0 ± 1.5% (sE), 94.8 ± 1.9%, 96.6 ± 1.0%, and 94.6 ± 1.2%, and ileal recovery was 96.9 ± 1.3%,94.5 ± 2.1 %,94.4 ± 1.1%, and 92.8 ± 1.0%, respectively. Unidirectional sodium flux studies were performed using UNa (New England Nuclear, Boston, Mass.) added to intestinal perfusion solutions. Unidirectional Na flux from mucosal to serosal (J':::) flux was calculated from the rate of UNa disappearance from the perfusate, and unidirectional serosal to mucosal (J~) flux was obtained from the difference in net Na flux and J!:. Regression analyses were made by the method of least SQuares. Probability values for water and electrolyte fluxes were determined by analysis of variance, whereas pH and osmolality results were compared by Student's t-test." At the termination of each experiment, portions of jejunum and ileum from both control and serotonin-treated animals were fixed in 10% neutral buffered f9rmalin. Sections were stained with hematoxylin and eosin, Mayer's mucicarmine, Movat, and Verhoeffstains. For electron microscopy. multiple samples of tissue from jejunum and ileum, obtained from both control or serotonin-treated animals, were fixed for 2 hr in 4% glutaraldehyde buffered to pH 7.5 with phosphate buffer. After washing in buffer these samples were fixed in buffered osmium tetroxide (1 %r) for 1 hr. After postfixation, the tissues were dehydrated in graded alcohols and propylene oxide and then embedded in Epon-Araldite.

Results Intestinal Net Water and Electrolyte Movement Glucose-free perfusate (groups 1 and 2). table 1. Jejunum. Serotonin resulted in a highly significant reversal of net water movement from absorption (- 16.1 "I per cm per hr) in control animals to secretion (+42.0 "I per cm per hr) in animals receiving the compound (P < 0.(01) . Net Na flux was similarly reversed from absorption (-3.4 ~Eq per cm per hr) in control animals to secretion (+5.6 "Eq per cm per hr) in the serotonintreated group (P < 0'()()1) . Net K and CI movement into the lumen significantly increased (P < 0.001, P < 0_(01), and net HCO. flux remained unchanged. Ileum. In the ileum, serotonin resulted in a significant increase in net water secretion (from 59.4 to 127.4 ,,1 per cm per hr, P < 0.001), and net Na secretion (from 1.7 to 8.0 ~Eq per cm per hr, P < 0.001). K and CI secretion

were also increased (P < 0.05, P < 0.001), and HCO s secretion declined (P < 0.02). Glucose-containing perfusate (groups 3 and 4), table 2. Jejunum. Glucose (10 mM) in the perfusate completely abolished the net secretion induced by serotonin observed with a glucose-free perfusate. Water was absorbed in both control (-10.9 ~l per cm per hr) and serotonintreated animals (-9.3 ~l per cm per hr, not significant (NS» when glucose was present in the perfusate. Na absorption, observed in control loops, was slightly increased in the serotonin-glucose group ( - 0.5 versus -1.3 ~Eq per em per hr, P < 0.05). K, Cl, and HCO, transport were not influenced by serotonin when glucose was present in perfusate. Jejunal glucose absorption was Net fluxes of water and electrolytes in control and serotonin·treated rabbit jejunum and ileum; glucose·free perfusate (mean = sEl·

TABLE 1.

Jejunum

H.O Na K CI HCO. Ileum H.O Na K CI HCO.

Group I (control. n ~

Group 2

8)

-16.1=2.7 -3.4 = 0.4 0.0 = 0 .1 -7.8 = 0.7 +4 .5 = 0.9 In

(serotomn. n =- 81

+42.0 ± 5.8 +5.6 ± 1.0 +1.2 ± 0.1 +2.9 ± 1.6 +3 .9 ± 0.7 (n

101

+59.4 ± 6.1 +1.7 ± 1.1 + 1.5 ± 0.1 -11.7±2.2 + 14.8 ± 2.2

p

< O.!lO I <0.001 <0.001 <0.001 NS'

1m

+ 127.4 ± 11.0 +8.9 ± 0.6 +1.9 ± 0.2 +3.9 ± 1.2 +6.9 ± 1.3

<0.001 <0.001 <0.05 <0.001 <0.02

• Water movement in microliters per centimeter per hour. Electro· Iyte movement in milliequivalentR per centimeter per hour. +. net secretion into lumen; minus. net absorption from lumen. • NS. not significant. Net fluxe. of water. electrolytes. and glue~8/! in control and serotonin·treated rabbit jejunum and ileum; glucose·containing perfusate (mean = 91:)·

TABLE 2.

Jejunum

Group:! (contro!. n

7)

H.O Na K CI HCO. Glucose

- 10.9 ± 1.5 -0.5 ± 0.3 -0.2 ± 0.1 -7.6 ± 0.3 +6.9 ± 0.4 -1.1 ±0.1

Ileum

(n .6)

".0 Na K CI HCO. Glucose

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SEROTONIN·INDUCED INTESTINAL SECRETION

December 1976

+59.6 ± 3.9 + 1.6 ± 0.6 + 1.2 ± 0.1 -6.9 ± 2.5 +9.7 ± 2.8 -0.6 ± 0.1

Group 4 (serotonin. n "" 7) -9.3 ± -1.3 ± -0.1 ± -9.5 ± +8.1 ± - 1.3 ± (n .

2.6 0.2 0.1 1.9 2.0 0.1

p

NS' <0.05 NS NS NS <0.05

increased in the serotonin-glucose group as compared with the glucose controls (P < 0.05). Ileum . Serotonin was without effect on water flux when glucose was present in the perfusate. Control and serotonin groups had virtually identical rates of net water secretion (59.6 versus 58.9 ~l per em per hr, respecitvely, NS). Net Na secretion was enhanced with serotonin administration (+ 1.6 versus +4.2 ~Eq per em per hr for glucose control and serotonin-glucose groups, respectively, P < 0.05), as was K (+ 1.2 versus + 1.9 ~Eq per em per hr, P < 0.01). Neither Cl nor HCO. movement was altered in the group receiving serotonin. Glucose absorption was enhanced in serotonin·treated animals ( - 0.6 versus -1.2 ~Eq per em per hr for control and serotonin groups, respectively, P < 0.01) .

Unidirectional Na Fluxe.~ Glucose-free perfusate (groups 1 and 2), figure 1. Jejunum. Serotonin administration diminished both mucosal to serosal (J ~~), P < 0.001) and serosal to mucosal (J:'::. P < 0.01) fluxes. The significant increase in net Na secretion observed in the serotonin·treated groups was due to the greater magnitude of the decrease in J~:: (63%) than in J~~ (39 ':'f ). Ileum . In ileal loops, serotonin produced a significant decrease in J;:" (P < 0.0.')) but did not influence J~~ . Thus, the observed increase in net Na secretion was mediated entirely by a diminution of mucosal to serosal Na fluxes. Glucose-containing perfusate (group.~ 3 and 4), fi!?ure 2. Jejunum. As in groups 1 and 2, serotonin diminished both J~~ and J!,,~ significantly (p < 0.01, P < 0.01). The slightly greater diminution of (32% versus 30% for J~) resulted in a small, but significant (P < 0.05) increase in net jejunal Na absorption in serotonin-treated animals. Ileum. Again, serotonin significantly decreased both J::" (P < 0.001) and J!: (P < 0.000. The greater decrease in Ji: (60%) than in J~;' (51 %) resulted in a small, but significant (p < 0.05), increase in net ileal Na secretion in serotonin-treated groups as compared with glucose controls.

J:.::

JEJUNUM -

ILEUM Con'rotUO) --_ S.,otonln {lei

Cantrol(S)

- - - S.roton,n f8)

150~I

7)

+58 .9 ± 8.8 +4 .2 ± 0.9 +1.9 ± 0.1 -2.6±2.5 +8.7 ± 2.0 - 1.2 ± 0.1

NS <0.05 <0.01 NS NS <0.01

• Water movement in microliters per centimeter per hour. Elec. trolyte movement in millequivalents per centimeter per hour. Glucose movement in micromoles per centimeter per hour. +. net secretion into lumen; -. net absorption (rom lumen. • NS. not significant.

50 · UNIDIRECTIONAL ~FWX o~ ' ~--~~~~~ "eq/cm

· 50 '

J~~

-100'

- tSO~_~_p~o~~ I

Z

HOURS

3

HOURS

FIG . !. Unidirectional Na fluxes in jejunum and ileum of glucose·

free perfusate groups.

1036

KlSWFF AND MOORE

Vol. 71. No . 6

influence such transport alterations as have been induced by the test substances-glucagon. and now sero--- Setoton",-,hK:ouC11 tonin. These observations are as yet unexplained and further studies are needed. All control animals in our ~f studies demonstrated net water secretion in ileal loops. oor This differs from several control results 32. u. 3t previously sof noted in the literature that show absorption in the basal UNIDIRECTIONAL i state. We believe this difference can be explained on the 0:(,Ii::---=-.:..::.::=--_=_: ~:~~ 0<,~-~=---basis of the state of hydration of the control animals at , the start of the experiments. In the previously published -so' i reports referred to above. animals were either studied -oo f -'OO ~ with the ligated loop method of De and Chatterje. 4o I which differs from our method of continuous perfusion. -,so,-t_~_~_~ -,sot~_7"._
-

Controt·,..lwcOH (71 - - - Seroton," - t'wCD •• 171

I

ILEUM

-

Corttrat·,hlCow(SI

December 1976

SEROTONIN-INDUCED INTESTINAL SECRETION

rectional fluxes reveals a striking effect of the added glucose on Na transport_ In jejunum the J~ was increased by the addition of glucose to the perfusate from -31.3 ~Eq per cm per hr in glucose-free controls (fig_ 1) to -41.7 ~Eq per cm per hr in glucose containing controls (fig. 2, P < 0.001). In ileum J~~ was increased from -29.3 ~Eq per cm per hr in glucose-free controls (fig. 1) to -43.2 ~Eq per em per hr in glucose-containing controls (fig. 2, P < 0.001). The small effect of this on net flux is the consequence of similar increases in J~:' in glucose perfusate groups. This lack of glucose effect on net Na transport in spite of pronounced increases in Na absorptive flux (J::'~) has previously been noted by Fordtran in human jejunum. ,. The present studies indicate that serotonin's effects on net fluid transport are mediated primarily by a decrease in sodium absorptive flux (J:~). The fact that both J~~ and J ~~ were decreased by serotonin suggests that serotonin may act by reducing intestinal mucosal blood flow ... A diminution in mucosal blood flow as a mediator of the observed effects of serotonin could explain the increase in apparent glucose absorption noted in both jejunum and ileum of serotonin-treated animals. Thus. enhanced glucose absorption might result from enhanced glucose utilization for glycolysis (Pasteur effect) secondary to serotonin-induced mucosal anoxia. Alternatively, a serotonin-induced decrease in mucosal blood flow might result in greater glucose extraction from mucosal capillary blood, thus lowering the final glucose concentration in the mucosal capillaries. This would provide a stimulus for enhanced passive glucose absorption down a steeper concentration gradient. Either or both of these postulated mechanisms could explain the increased glucose absorption noted in serotonin-treated animals. We conclude that serotonin is capable of inducing net secretion in the in vivo rabbit jejunum, and enhancing net secretion in the in vivo rabbit ileum. and that this effect can be entirely abolished by the addition of glucose to the perfusate. If these findings can be confirmed in man, they may provide the basis for a safe and simple means of treating the diarrhea associated with the carcinoid syndrome. Agents currently used to treat this symptom may provoke significant adverse effects.'" 41 The ability of 10 mM glucose to reverse serotinin-induced small intestinal secretion suggests the possibility that diarrhea in patients with carcinoid syndrome can be amliorated with peroral glucose-saline solutions. as is done in the treatment of cholera. , •. ,. Studies in man are now needed to evaluate this possibility. REFERENCES 1. Sjoerdsma A, Melmon KL: The carcinoid spectrum. Gastroenterology 47:104-107, 1964 2. Sauer, WG: Carcinoidosis and the effect of serotonin on the gastrointestinal tract. Am J Oig Ois 6:906-913. 1961 3. Smith AN. Preshaw RM. Sircus W: Clinical features in patients with the carcinoid syndrome. Gastroenterology 48:738-744. 1965 4. Weichert RF: The neural ectodermal origin of the peptide-secret· ing endocrine glands. Am J Med 49:232-241. 1970 5. Oates JA. Pettinger W A. Doctor RB: Evidence for the release of

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