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Serotonin action on short-circuit current and ion transport across isolated rabbit ileal mucosa
Life Sciences, Vol . 24, pp . 1609-1616 Printed in the U.S .A .
Perganon Press
SEROTONIN ACTION ON BHORT-CIRCUIT CURRHYT AHD ION TRANSPORT ACROSS ISOLATED RABBIT ILÉAL MUCOSA H.E . 8heeria Department of Biology University of: Massachusetts - Bonton Boétoa, Massachuaetta 02],25 (Received in final fprm March 19, 1979) Summary 8erotonin has previously been implicated as the cause of the diarrhea associated with carciaoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops in vivo . In the present paper the action of aerotonin on isolated segments of rabbit deal muco e stripped of muscle layers was studied is vitro. 8erotonin (10-~) caused an abrupt significant rise is short-circuit current (I~c) across the mucoeal epithelial cell layer but this effect was transient . Ho change was observed in ti~sue conductance . Ia this preparation, aerotonin did not alter ~Na, 3°C1 or residual ion fluxes across the mucosa . High blood aerotonin levels for a period of several days also did not alter ion fluxes or Iac in isolated rabbit ileum. Therefore, it is concluded that aerotonin must cause its secretory activity observed _ia vivo by some mechanism other than a direct action on epithelial cell transport mechanisms . Malignant carcinoid tore of enterochromaffin cells of the gut are characterized by excessive synthesis, storage and release of 5-hydro~grtryptamine (aerotonin) . Blood levels of the amine may reach several times above normal (1-4) . Symptoms of the disease include broachoconstrictioa, tachypaea, tachycardia, hypotensioa, flushing, colic and satery diarrhea . Several studies have suggested that the diarrhea is caused by the high levels of aerotonin (1-6) . For example, treatment with a peripheral antagonist of aerotonin, metl~ysergide, often alleviates the diarrhea (5) " In addition, treatment with pare-chloropheaylalaniae, which causes depletion of aerotonin by inhibiting tryptophane hydroxylase, can control the diarrhea of the carcinoid ayndra~me (4-6) . Many of the earlier studies on aerotonin and the gut concentrated on the ability of aerotonin to stimulate smooth muscle of the small intestine causing it to contract (i-12) . However, the action of aerotonin on smooth musculature of the gut appears to be very camplez. In the smell intestine aerotonin appears to affect muscle directly as well ae indirectly by an action on intramural cholinergic nerve elements (8,11) . It bas been pointed out that other areas of the gut, for example stomach and colon, may react differently to aerotonin. The amine causes hypomotility of the human colon (q,12) . The smooth muscle reaction to aerotonin also appears to be apeciea specific and often highly variable within apeciea (3) . It has also been emphasized previously that the relationship between hypermotility and hypamotility to diarrhea and constipation is still far from clear (10,11,13) " 0025-3205/79/171609-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1610
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Recently, the results of two _in vivo perfusion studies have suggested that serotonin is an intestinal secretagogue causing water and salt secretion in rabbit small intestine (13,14) . Kieloff and Moors found that intravenous ad ministration of serotonin resulted in a significant net secretion of water and sodium in both jejunum and ileum without altering the histology of the small intestinal mucosa (13) . Donowitz, _et . _al ., subjected rabbits to two weeks of daily subcutaneous injections of serotonin resulting in ileel secretion and decreased mid-j~junal absorption of water and electrolytes, again without any alterations in intestinal histology (14) . In both studies, serotonin did not alter the transport of nonelectrolyte nutrients such as glucose and amino acids . Since the effect of serotonin on the release of hormones, on smooth muscle activity and on mucosal blood flow was not controlled in the _in v_ivo studies, the present _in vitro work was undertaken in order to define more precisely any action serotonin may exert directly on intestinal mucosal cell ion transport . Methods Male white New Zealand rabbits weighing between 2 and 4 kg and given standard rabbit chow end water ad l ibitum were used . One group of rabbits was given daily subcutaneous injections of 5-hydroxytryptemine-creatinine sulfate (Sigma Chem . Co ., St . Louis) suspended in peanut oil . A second group of enimala was injected similarly with creatinine sulfate (Sigma) . Both groups received injections for 14 days . Concentrations of serotonin and creatinine sulfate were both 52 umol/kg . Immediately prior to sacrifice, a few cc of blood was obtained from the ear vein, placed in tubes containing EDTA and ascorbic acid and frozen for later measurement of blood serotonin levels . A third group of rabbits was used for the experiments in which serotonin was administered in vitro only . Much of the procedure, apparatus and methods of flux measurements have been described previously by Field, _et . _al . (15) . The rabbits were killed by a blow to the neck, the distal ileum removed, stripped of both longitudinal end circu lar muscle layers, and mounted in modified Ussing chambers . Preparative rinse solutions end reservior solutions were standard Kreba-Ringers bicarbonate buffers at pH ~ .4 . The were kept at 3i ° C and constantly gassed with 95x 02 5x Cot . The tissues were maintained in the short-circuited condition using a Netronics AVC-2 Automatic Voltage Clamp (Netronics, Inc ., Hudson, Mass .) . Periodic measurements of mucosal electrical resistance were performed, especially immediately before and after isotope flux periods . Mucosa (m) to, serosa (s) a~d (s) to (m) ion fluxes were determined on paired tissues using 22Na and 36C1 (New England Nuclear, Boston, Mass .) . Flux periods were between 30 end 45 minutes duration . Resistances of paired tissues did not deviate more that + 20x . Tissue from ~nimals receiving daily serotonin injections were maintained in contact with 10 M serotonin throughout their preparation . The serosal solution also contained 10 -1+ serotonin throughout the experiments with these tissues . Tissues from serotonin-injected and control-injected animals were taken from the same area of terminal ileum . In experiments where serotonin was added _in vitro alone, it was added 5-7 minutes prior to flux measurement at a final concentration of 10 - 4M . Serotonin-treated tissues and controls were obtained from adjacent areas in the terminal ileum . In all experiments, glucose (10 umol/ml) was added to the serosal reservior and mannitol (10 umol/ml) to the mucosal reservior . In some experiments, theophylline (5 umol/ml seroeal solution),
Vol . 24, No . 17, 1979
Serotonin and Ion Fluxes in Rabbit Ileum
1611
epinephrine (10~M, serosal solution) or glucose (10 umol/ml mucosal solution) (all Sigma) was added either prior to or after Serotonin addition as indicated below. Whole blood aerotonin was measured fluorometrically by the OPD method of Maickel (16) . Statistics were performed using Student's t-test for paired variatea . Results are expressed as means + 1SE. Result s Effect of aerotonin on short-circuit current . Addition of aerotonin - creatinine sulfate to the serosal bathing solution caused an immediate, rapid increase in short-circuit current (Isc) that peaked in a few minutes . The Isc declined rapidly to baseline sad sometimes below baseline . This transitory rise in Isc is depicted in Fig. 1 . Further addition of aerotonin at any time after this peak caused no change in Isc . Addition of creatinine sulfate alone caused no change in Inc . Tissue resistance did not change significantly after serotonin application.
140-
10~
120100-
^, 800
~ 604
4020-
6
8
TIME (min .)
Fig . 1 Effect of serotonin addition to aerosal solution, given at time zero, oa Concentration of short-circuit current in isolated rabbit ileal mucosa . serotonin shown in moles/liter.
161 2
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Serotonin did not seem to affect the action of other agents known to exert ezcitatory or inhibitory influences on rabbit ileum Iac . Theophylline, by its action as e phosphodiesterase inhibitor, causes a rise in Iac due to an increase in intra-cellular cyclic AMP levels in the epithelial cell (17-19) . In 3 exPeriments, prior addition of aerotoain did not affect the stimulatory effect of theophylliae on Iac . Epinephrine causes a decline in Iac in rabbit ileum (20,21) . Prior addition of aerotonin did not affect this action of epinephrine (3 experiments) . Glucose added to the mucosal solution causes a rise in SCC by stimulating active sodium transport (22 ) . Serotonin did not affect the glucoaestimulated Esc . Glucose was added routinely at the end of each experiment to teat viability of the tissue . Prior addition of théophy111ne, epinephrine or glucose did not affect the transitory rise in Iac caused by Serotonin (3 experiments each) . Effect of aerotonin on ion fluxes . Results of aerotonin addition to the serosal fluid immediately prior to measurement of steady state ion fluxes are shown in Table 1 . Also shown is the average Iac during the Plux periods which were begun after the peak in Iac observed with aerotoain addition . Residual Flux (Jr ) calculations were performed Prom the resulting data . The residual flux Table 1 Effect of aerotonin addition to serosal solution on ion fluxes across rabbit ileal mucosa . Control Serotonin 10~
M 10
J~a
14 .62+0 .86
14 .16+1 .08
14 .67+1 .92
JN ~
12 .30±0 .89
12 .80±1.36
12 .01+1.15
Jnet
+2 .32±0.62
+2 .60±0 .73
+2 .66±1.23
JmB
l0 .50±0.49
l0 .60±0 .62
9.49±1.13
J~
9 .52±0.66
10 .03±0.53
7 .77±0 .94
JC1
+0 .98±0.66
+0 .57±0 .72
+1 .72+1 .34
I ac
2.20±0.22
1 .82±0.29
2 .62±0.16
Jr
0.86±0.71
-0 .21±0.96
1 .68±0.42
net
35 .0±2 .9 33 .1±0 .83 33 .7±2 " 7 t Ion fluxes, including short-circuit current Iac) are is pequiv/hr .em . and conductance (Gt) is in mmhos/em 2 . J~ refers to unidirectional mucosa to aeroeal flux . J~ refers to unidirectional serosa to_mucosa flu:, and Jaet is the difference between J~ sad J~ (+ indicates absorptC ion, - indicates accretion) . Jr refers to the residual ion Plux (Isc - Jâé + J~3~t) . Values are means 1SE . Numbers of animals used was : control, ~4 ; aerotoain (10-6M, 6; Serotonin (10- 4), 8. G
Vol . 24, No . 17, 1979
Serotonin and Ion Fluxes in Rabbit Ileum
1613
has previously been determined to be a good estimate of net bicarbonate secreSerotonin did not have a statistically signifition in rabbit ileum (23,24) " cant effect oa unidirectional Ha and C1 flume, net Ha and Cl Hazes, Isc (during the fluz period), residual inn Plums or total d-c tissue ca~nductance (Gt ) determined frem tissue resistance measurements at the beginning and end of the fluz period . In order to determine if long-term ezposure to high concentrations of serotonin can cause alterations in intestinal transport mechanisms, a group of 6 rabbits were injected daily for tw weeks with serotonia, sacrificed and ion fluz measurements were performed . 14 days has been reported to be sufficie~ time for elevated blood levels of serotoaia to cause a secretary effect is in viva loops of rabbit intestine (14) . At the time of sacrifice the serotoaiaControl injected rabbits had blood serotonin values of 7.79*0.58 pg/liter . rabbits had blood serotoain values of 3 .91±0" 53 u8/liter " Tie~ues from the serotonin-injected rabbits were maintained in contact with 10~M serotonin throughout the ezperimeata . The results of chronic aerotonin administration on Ha and C1 flume, SCC, residual ion flux and tissue conductance are shown in Table 2. Again, serotonin does not have a statistically significant effect on any of these pareaeters when measured in vitro. Table
2
Tffect of chronic serotonin treatment on ion flume across rabbit deal aucosa. Control
Serotonia
JHa ms
15 .69±0 .75
14 .88±0.91
~a am
11 .34±0 .87
10 .26±0 .94
~.Aa net
+4 .35±1 .33
+4 .62±0.068
9" 90±0 .87
g.0~o .91
sm
7.55±0.77
7 .28±0.72
JC1 net
+2 .35±0 .61
+1 .78±0.63
2 .41±0.2T
1.68±0.34
0.41±0.80
-1 .04±0.70
~:C1 me JCl
Isc J
r
G t
31 .2±2 .8
29 .7±2 .1
Aumber of animals used vas 6 for each group. 8eroto~aia-creatinine sulfate vsa injected daily for 14 days (52 1~1/kg) and vas includednia rz ero ~e~~aad Control serosal solution at a concentration of 10 with creatiniae-sulfate (52 l~ol/àg) for 14 days . See legend to Table 1 for ezplanation of symbols and units .
1614
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Discussion The results herein indicate that serotonin does not cause intestinal secretion of electrolytes, and presumably water, by means of a direct action on the small intestinal mucosal cells . Serotonin, either administered acutely in vitro , or chronically over a period of two weeks to the intact animal, causes no change in fta, C1 and presumably HC03 movement into or out of isolated rabbit small intestinal mucosa . There is also no long-term change in shortcircuit current across intestinal mucosa, a good indicator of total active transport . Unidirectional ion fluxes, which are predominantly pensive diffusion via intercellular shunt pathways (25), and tissue conductances are also not changed indicating that serotonin is not altering physical characteristics of diffusional pathways in the absence of muscle layers and blood circulation . Serotonin does cause an immediate transient rise in Isc following its addition to the serosal bathing media, but this effect disappears promptly and cannot be reactivated by further addition of serotonin . A similar serotonin induced rise in SCC was reported by Bolton and Field (26) . They observed that omission of calcium from the serosal bathing solution diminished but did not entirely block the serotonin action on potential difference across isolated rabbit ileum . Neither theophylline, epinephrine nor glucose addition prior to serotonin seem to affect the tissue's response to the amine . Likewise, prior addition of serotonin does not affect the action of these agents on the SCC . Thin suggests that the rise in SCC due to serotonin is not related to cyclic nucleotide metabolism or glucose-coupled sodium active transport across the intestine . Thus, the nature and significance of the serotonin-activated transitory rise in SCC remains unknown . The results of two recent _in vivo studies in rabbit small intestine showed that serotonin stimulates water and electrolyte transport in the secretioy direction (13,14) . Serotonin has also been shown to decrease cholera toxin induced fluid secretion in both rabbit and rat small intestine (14,27) . The present results indicate that these fluid transport effects of the amine are probably not the result of e direct effect on the intestinal mucosal cells . Therefore, further studies are required on the action of serotonin on mucosal blood flow and intestinal smooth muscle motility, and the relationship these effects have on intestinal secretion . The results of such studies may not only help answer the questions of the site on action of serotonin on intestinal secretion but also aid in further understanding the mechanisms involved in fluid and electrolyte movements which do not involve biochemical, biophysical and/or histological changes in the intestinal epithelial cell layer . References 1. 2. 3. 4. 5" 6. 7. 8.
M . DONOWITZ and H .J . BINDER, Dig . Dis . _20, 1115-1121 (1975) " C .E . MENGEL and R .D . SHAFFER, in : Cancer Meidcine, ed . by J .F . Holland and E . Fris, 1584-1594, Lea and Febiger, Philadelphia (1973) . A . SIREK and O .V . SIREK, C .M .A . Journal _102, 846-849 (1970) . A . SJOERDSMA, W . LOVENBERG, K . ENGELMAft, W .T . CARPERTER, R .J . WYATT and G .L . GESSA, Ann . Int . Med . 33, 607-629 (1970) " K . L . MELMOft, A . SJOERDSMA, J .A . GATES and L . LASTER, Gastroenterology, 48, 18-24 (1965) " W .G . SATFER~~~ , A . SERPICK and J .R . BIANCHINE, Ann . Int . Med . ~, 919-921 (1970) . D .F . FISHLOCK and A .G . PARKE, Br . J . Pharmae, Chemother . _28, 164-171 (1966) . M .D . GERSHON, Gastroenterology, ~, 453-465 (1968) "
Vol . 24, No . 17, 1979
9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 " 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 " 26 . 27 .
Serotonin and Ion Fluxes in Rabbit Ilea
1615
T .R . HENDRIX, M . ATKIN80N, J .A . CLIFTON and F .J . INGELFINGER, Am . J . Med . _22, 886-893 (1957) " J .A . HIGGINS, C .F . CODE and A .L . ORVIS, Geatrcenterology, 31, 708-715 (1956) . D .K . KENNEDY, M .N . GRUBB and T .F . BURKS, Gastroenterology, 66, 39~-402 (1974) . J .J . MISIEWICZ, B .L . WALLER and M . EISNER, Gut ., Z, 208-216 (1966) . B . KISIAFF and E .W . MDORE, Gastroenterology, 31, 1033-1038 (1976) . M . DCQIOWITZ, A .N . CHARNEY and J .M . HEFFERNAN, Am . J . P}~raiol . 2 2, E85-E94 . M . FIELD, D . FROMM and I . McCOLL, Am . J . Phyeiol . 220, 1388-13~ (1971) . R .P . MAICKEL ia : Methode of Neurochemiat , Yol . 2, ed . by R . Fried, 101 129, Marcel Deàker, New York 1922 . M . FIELD . Am . J . Phyaiol . _221, 992-997 (1971) . M. FIELD, D . FROMM, Q . AL-AWgpTI and W .B . GREENOUGH, J . Clin . Inveat . ~, 796-804 (1972) . H .E . SHEERIN sad M . FIELD, Am . J . Physiol . 232, E210-E215 (1977) . M . FIELD and I . McCOLL, Am . J . Physiol . 2~~52-857 (1973) . M . FIELD, H .E . SHEERIN, A . HENDERSON and P .L . SMITH, Am . J . Physiol . 22~, 86-92 (1975) " S .G . SCHULTZ and P .F . CURRNAN, Phyaiol . Rev . 0, 637 (1970) . J . DIETZ and M . FIELD, Am . J . Physiol . ~, 85~-861 (19'j3) . H .E . SHEERIN aad M. FIELD, Am . J . Physiol . _228, 1965-1974 (1975) " S .G . SCHULTZ, R .A . FRIZZELL and H . NELLANS, Ann . Rev . Phyaiol . ~6, 51-91 (1975) . ' J .E . BOLTO~i and M . FIELD, J . Membrane Biol . ~ 159-173 (1977) . D .R . STROM9ECK, Life Sci . 12, 211-318 (1973) .
Perganon Press
SEROTONIN ACTION ON BHORT-CIRCUIT CURRHYT AHD ION TRANSPORT ACROSS ISOLATED RABBIT ILÉAL MUCOSA H.E . 8heeria Department of Biology University of: Massachusetts - Bonton Boétoa, Massachuaetta 02],25 (Received in final fprm March 19, 1979) Summary 8erotonin has previously been implicated as the cause of the diarrhea associated with carciaoid syndrome and the amine has been shown by others to be an intestinal secretagogue in preparations of intestinal loops in vivo . In the present paper the action of aerotonin on isolated segments of rabbit deal muco e stripped of muscle layers was studied is vitro. 8erotonin (10-~) caused an abrupt significant rise is short-circuit current (I~c) across the mucoeal epithelial cell layer but this effect was transient . Ho change was observed in ti~sue conductance . Ia this preparation, aerotonin did not alter ~Na, 3°C1 or residual ion fluxes across the mucosa . High blood aerotonin levels for a period of several days also did not alter ion fluxes or Iac in isolated rabbit ileum. Therefore, it is concluded that aerotonin must cause its secretory activity observed _ia vivo by some mechanism other than a direct action on epithelial cell transport mechanisms . Malignant carcinoid tore of enterochromaffin cells of the gut are characterized by excessive synthesis, storage and release of 5-hydro~grtryptamine (aerotonin) . Blood levels of the amine may reach several times above normal (1-4) . Symptoms of the disease include broachoconstrictioa, tachypaea, tachycardia, hypotensioa, flushing, colic and satery diarrhea . Several studies have suggested that the diarrhea is caused by the high levels of aerotonin (1-6) . For example, treatment with a peripheral antagonist of aerotonin, metl~ysergide, often alleviates the diarrhea (5) " In addition, treatment with pare-chloropheaylalaniae, which causes depletion of aerotonin by inhibiting tryptophane hydroxylase, can control the diarrhea of the carcinoid ayndra~me (4-6) . Many of the earlier studies on aerotonin and the gut concentrated on the ability of aerotonin to stimulate smooth muscle of the small intestine causing it to contract (i-12) . However, the action of aerotonin on smooth musculature of the gut appears to be very camplez. In the smell intestine aerotonin appears to affect muscle directly as well ae indirectly by an action on intramural cholinergic nerve elements (8,11) . It bas been pointed out that other areas of the gut, for example stomach and colon, may react differently to aerotonin. The amine causes hypomotility of the human colon (q,12) . The smooth muscle reaction to aerotonin also appears to be apeciea specific and often highly variable within apeciea (3) . It has also been emphasized previously that the relationship between hypermotility and hypamotility to diarrhea and constipation is still far from clear (10,11,13) " 0025-3205/79/171609-0702 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1610
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Recently, the results of two _in vivo perfusion studies have suggested that serotonin is an intestinal secretagogue causing water and salt secretion in rabbit small intestine (13,14) . Kieloff and Moors found that intravenous ad ministration of serotonin resulted in a significant net secretion of water and sodium in both jejunum and ileum without altering the histology of the small intestinal mucosa (13) . Donowitz, _et . _al ., subjected rabbits to two weeks of daily subcutaneous injections of serotonin resulting in ileel secretion and decreased mid-j~junal absorption of water and electrolytes, again without any alterations in intestinal histology (14) . In both studies, serotonin did not alter the transport of nonelectrolyte nutrients such as glucose and amino acids . Since the effect of serotonin on the release of hormones, on smooth muscle activity and on mucosal blood flow was not controlled in the _in v_ivo studies, the present _in vitro work was undertaken in order to define more precisely any action serotonin may exert directly on intestinal mucosal cell ion transport . Methods Male white New Zealand rabbits weighing between 2 and 4 kg and given standard rabbit chow end water ad l ibitum were used . One group of rabbits was given daily subcutaneous injections of 5-hydroxytryptemine-creatinine sulfate (Sigma Chem . Co ., St . Louis) suspended in peanut oil . A second group of enimala was injected similarly with creatinine sulfate (Sigma) . Both groups received injections for 14 days . Concentrations of serotonin and creatinine sulfate were both 52 umol/kg . Immediately prior to sacrifice, a few cc of blood was obtained from the ear vein, placed in tubes containing EDTA and ascorbic acid and frozen for later measurement of blood serotonin levels . A third group of rabbits was used for the experiments in which serotonin was administered in vitro only . Much of the procedure, apparatus and methods of flux measurements have been described previously by Field, _et . _al . (15) . The rabbits were killed by a blow to the neck, the distal ileum removed, stripped of both longitudinal end circu lar muscle layers, and mounted in modified Ussing chambers . Preparative rinse solutions end reservior solutions were standard Kreba-Ringers bicarbonate buffers at pH ~ .4 . The were kept at 3i ° C and constantly gassed with 95x 02 5x Cot . The tissues were maintained in the short-circuited condition using a Netronics AVC-2 Automatic Voltage Clamp (Netronics, Inc ., Hudson, Mass .) . Periodic measurements of mucosal electrical resistance were performed, especially immediately before and after isotope flux periods . Mucosa (m) to, serosa (s) a~d (s) to (m) ion fluxes were determined on paired tissues using 22Na and 36C1 (New England Nuclear, Boston, Mass .) . Flux periods were between 30 end 45 minutes duration . Resistances of paired tissues did not deviate more that + 20x . Tissue from ~nimals receiving daily serotonin injections were maintained in contact with 10 M serotonin throughout their preparation . The serosal solution also contained 10 -1+ serotonin throughout the experiments with these tissues . Tissues from serotonin-injected and control-injected animals were taken from the same area of terminal ileum . In experiments where serotonin was added _in vitro alone, it was added 5-7 minutes prior to flux measurement at a final concentration of 10 - 4M . Serotonin-treated tissues and controls were obtained from adjacent areas in the terminal ileum . In all experiments, glucose (10 umol/ml) was added to the serosal reservior and mannitol (10 umol/ml) to the mucosal reservior . In some experiments, theophylline (5 umol/ml seroeal solution),
Vol . 24, No . 17, 1979
Serotonin and Ion Fluxes in Rabbit Ileum
1611
epinephrine (10~M, serosal solution) or glucose (10 umol/ml mucosal solution) (all Sigma) was added either prior to or after Serotonin addition as indicated below. Whole blood aerotonin was measured fluorometrically by the OPD method of Maickel (16) . Statistics were performed using Student's t-test for paired variatea . Results are expressed as means + 1SE. Result s Effect of aerotonin on short-circuit current . Addition of aerotonin - creatinine sulfate to the serosal bathing solution caused an immediate, rapid increase in short-circuit current (Isc) that peaked in a few minutes . The Isc declined rapidly to baseline sad sometimes below baseline . This transitory rise in Isc is depicted in Fig. 1 . Further addition of aerotonin at any time after this peak caused no change in Isc . Addition of creatinine sulfate alone caused no change in Inc . Tissue resistance did not change significantly after serotonin application.
140-
10~
120100-
^, 800
~ 604
4020-
6
8
TIME (min .)
Fig . 1 Effect of serotonin addition to aerosal solution, given at time zero, oa Concentration of short-circuit current in isolated rabbit ileal mucosa . serotonin shown in moles/liter.
161 2
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Serotonin did not seem to affect the action of other agents known to exert ezcitatory or inhibitory influences on rabbit ileum Iac . Theophylline, by its action as e phosphodiesterase inhibitor, causes a rise in Iac due to an increase in intra-cellular cyclic AMP levels in the epithelial cell (17-19) . In 3 exPeriments, prior addition of aerotoain did not affect the stimulatory effect of theophylliae on Iac . Epinephrine causes a decline in Iac in rabbit ileum (20,21) . Prior addition of aerotonin did not affect this action of epinephrine (3 experiments) . Glucose added to the mucosal solution causes a rise in SCC by stimulating active sodium transport (22 ) . Serotonin did not affect the glucoaestimulated Esc . Glucose was added routinely at the end of each experiment to teat viability of the tissue . Prior addition of théophy111ne, epinephrine or glucose did not affect the transitory rise in Iac caused by Serotonin (3 experiments each) . Effect of aerotonin on ion fluxes . Results of aerotonin addition to the serosal fluid immediately prior to measurement of steady state ion fluxes are shown in Table 1 . Also shown is the average Iac during the Plux periods which were begun after the peak in Iac observed with aerotoain addition . Residual Flux (Jr ) calculations were performed Prom the resulting data . The residual flux Table 1 Effect of aerotonin addition to serosal solution on ion fluxes across rabbit ileal mucosa . Control Serotonin 10~
M 10
J~a
14 .62+0 .86
14 .16+1 .08
14 .67+1 .92
JN ~
12 .30±0 .89
12 .80±1.36
12 .01+1.15
Jnet
+2 .32±0.62
+2 .60±0 .73
+2 .66±1.23
JmB
l0 .50±0.49
l0 .60±0 .62
9.49±1.13
J~
9 .52±0.66
10 .03±0.53
7 .77±0 .94
JC1
+0 .98±0.66
+0 .57±0 .72
+1 .72+1 .34
I ac
2.20±0.22
1 .82±0.29
2 .62±0.16
Jr
0.86±0.71
-0 .21±0.96
1 .68±0.42
net
35 .0±2 .9 33 .1±0 .83 33 .7±2 " 7 t Ion fluxes, including short-circuit current Iac) are is pequiv/hr .em . and conductance (Gt) is in mmhos/em 2 . J~ refers to unidirectional mucosa to aeroeal flux . J~ refers to unidirectional serosa to_mucosa flu:, and Jaet is the difference between J~ sad J~ (+ indicates absorptC ion, - indicates accretion) . Jr refers to the residual ion Plux (Isc - Jâé + J~3~t) . Values are means 1SE . Numbers of animals used was : control, ~4 ; aerotoain (10-6M, 6; Serotonin (10- 4), 8. G
Vol . 24, No . 17, 1979
Serotonin and Ion Fluxes in Rabbit Ileum
1613
has previously been determined to be a good estimate of net bicarbonate secreSerotonin did not have a statistically signifition in rabbit ileum (23,24) " cant effect oa unidirectional Ha and C1 flume, net Ha and Cl Hazes, Isc (during the fluz period), residual inn Plums or total d-c tissue ca~nductance (Gt ) determined frem tissue resistance measurements at the beginning and end of the fluz period . In order to determine if long-term ezposure to high concentrations of serotonin can cause alterations in intestinal transport mechanisms, a group of 6 rabbits were injected daily for tw weeks with serotonia, sacrificed and ion fluz measurements were performed . 14 days has been reported to be sufficie~ time for elevated blood levels of serotoaia to cause a secretary effect is in viva loops of rabbit intestine (14) . At the time of sacrifice the serotoaiaControl injected rabbits had blood serotonin values of 7.79*0.58 pg/liter . rabbits had blood serotoain values of 3 .91±0" 53 u8/liter " Tie~ues from the serotonin-injected rabbits were maintained in contact with 10~M serotonin throughout the ezperimeata . The results of chronic aerotonin administration on Ha and C1 flume, SCC, residual ion flux and tissue conductance are shown in Table 2. Again, serotonin does not have a statistically significant effect on any of these pareaeters when measured in vitro. Table
2
Tffect of chronic serotonin treatment on ion flume across rabbit deal aucosa. Control
Serotonia
JHa ms
15 .69±0 .75
14 .88±0.91
~a am
11 .34±0 .87
10 .26±0 .94
~.Aa net
+4 .35±1 .33
+4 .62±0.068
9" 90±0 .87
g.0~o .91
sm
7.55±0.77
7 .28±0.72
JC1 net
+2 .35±0 .61
+1 .78±0.63
2 .41±0.2T
1.68±0.34
0.41±0.80
-1 .04±0.70
~:C1 me JCl
Isc J
r
G t
31 .2±2 .8
29 .7±2 .1
Aumber of animals used vas 6 for each group. 8eroto~aia-creatinine sulfate vsa injected daily for 14 days (52 1~1/kg) and vas includednia rz ero ~e~~aad Control serosal solution at a concentration of 10 with creatiniae-sulfate (52 l~ol/àg) for 14 days . See legend to Table 1 for ezplanation of symbols and units .
1614
Serotonin and Ion Fluxes in Rabbit Ileum
Vol . 24, No . 17, 1979
Discussion The results herein indicate that serotonin does not cause intestinal secretion of electrolytes, and presumably water, by means of a direct action on the small intestinal mucosal cells . Serotonin, either administered acutely in vitro , or chronically over a period of two weeks to the intact animal, causes no change in fta, C1 and presumably HC03 movement into or out of isolated rabbit small intestinal mucosa . There is also no long-term change in shortcircuit current across intestinal mucosa, a good indicator of total active transport . Unidirectional ion fluxes, which are predominantly pensive diffusion via intercellular shunt pathways (25), and tissue conductances are also not changed indicating that serotonin is not altering physical characteristics of diffusional pathways in the absence of muscle layers and blood circulation . Serotonin does cause an immediate transient rise in Isc following its addition to the serosal bathing media, but this effect disappears promptly and cannot be reactivated by further addition of serotonin . A similar serotonin induced rise in SCC was reported by Bolton and Field (26) . They observed that omission of calcium from the serosal bathing solution diminished but did not entirely block the serotonin action on potential difference across isolated rabbit ileum . Neither theophylline, epinephrine nor glucose addition prior to serotonin seem to affect the tissue's response to the amine . Likewise, prior addition of serotonin does not affect the action of these agents on the SCC . Thin suggests that the rise in SCC due to serotonin is not related to cyclic nucleotide metabolism or glucose-coupled sodium active transport across the intestine . Thus, the nature and significance of the serotonin-activated transitory rise in SCC remains unknown . The results of two recent _in vivo studies in rabbit small intestine showed that serotonin stimulates water and electrolyte transport in the secretioy direction (13,14) . Serotonin has also been shown to decrease cholera toxin induced fluid secretion in both rabbit and rat small intestine (14,27) . The present results indicate that these fluid transport effects of the amine are probably not the result of e direct effect on the intestinal mucosal cells . Therefore, further studies are required on the action of serotonin on mucosal blood flow and intestinal smooth muscle motility, and the relationship these effects have on intestinal secretion . The results of such studies may not only help answer the questions of the site on action of serotonin on intestinal secretion but also aid in further understanding the mechanisms involved in fluid and electrolyte movements which do not involve biochemical, biophysical and/or histological changes in the intestinal epithelial cell layer . References 1. 2. 3. 4. 5" 6. 7. 8.
M . DONOWITZ and H .J . BINDER, Dig . Dis . _20, 1115-1121 (1975) " C .E . MENGEL and R .D . SHAFFER, in : Cancer Meidcine, ed . by J .F . Holland and E . Fris, 1584-1594, Lea and Febiger, Philadelphia (1973) . A . SIREK and O .V . SIREK, C .M .A . Journal _102, 846-849 (1970) . A . SJOERDSMA, W . LOVENBERG, K . ENGELMAft, W .T . CARPERTER, R .J . WYATT and G .L . GESSA, Ann . Int . Med . 33, 607-629 (1970) " K . L . MELMOft, A . SJOERDSMA, J .A . GATES and L . LASTER, Gastroenterology, 48, 18-24 (1965) " W .G . SATFER~~~ , A . SERPICK and J .R . BIANCHINE, Ann . Int . Med . ~, 919-921 (1970) . D .F . FISHLOCK and A .G . PARKE, Br . J . Pharmae, Chemother . _28, 164-171 (1966) . M .D . GERSHON, Gastroenterology, ~, 453-465 (1968) "
Vol . 24, No . 17, 1979
9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 " 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 " 26 . 27 .
Serotonin and Ion Fluxes in Rabbit Ilea
1615
T .R . HENDRIX, M . ATKIN80N, J .A . CLIFTON and F .J . INGELFINGER, Am . J . Med . _22, 886-893 (1957) " J .A . HIGGINS, C .F . CODE and A .L . ORVIS, Geatrcenterology, 31, 708-715 (1956) . D .K . KENNEDY, M .N . GRUBB and T .F . BURKS, Gastroenterology, 66, 39~-402 (1974) . J .J . MISIEWICZ, B .L . WALLER and M . EISNER, Gut ., Z, 208-216 (1966) . B . KISIAFF and E .W . MDORE, Gastroenterology, 31, 1033-1038 (1976) . M . DCQIOWITZ, A .N . CHARNEY and J .M . HEFFERNAN, Am . J . P}~raiol . 2 2, E85-E94 . M . FIELD, D . FROMM and I . McCOLL, Am . J . Phyeiol . 220, 1388-13~ (1971) . R .P . MAICKEL ia : Methode of Neurochemiat , Yol . 2, ed . by R . Fried, 101 129, Marcel Deàker, New York 1922 . M . FIELD . Am . J . Phyaiol . _221, 992-997 (1971) . M. FIELD, D . FROMM, Q . AL-AWgpTI and W .B . GREENOUGH, J . Clin . Inveat . ~, 796-804 (1972) . H .E . SHEERIN sad M . FIELD, Am . J . Physiol . 232, E210-E215 (1977) . M . FIELD and I . McCOLL, Am . J . Physiol . 2~~52-857 (1973) . M . FIELD, H .E . SHEERIN, A . HENDERSON and P .L . SMITH, Am . J . Physiol . 22~, 86-92 (1975) " S .G . SCHULTZ and P .F . CURRNAN, Phyaiol . Rev . 0, 637 (1970) . J . DIETZ and M . FIELD, Am . J . Physiol . ~, 85~-861 (19'j3) . H .E . SHEERIN aad M. FIELD, Am . J . Physiol . _228, 1965-1974 (1975) " S .G . SCHULTZ, R .A . FRIZZELL and H . NELLANS, Ann . Rev . Phyaiol . ~6, 51-91 (1975) . ' J .E . BOLTO~i and M . FIELD, J . Membrane Biol . ~ 159-173 (1977) . D .R . STROM9ECK, Life Sci . 12, 211-318 (1973) .