AMERICAN JOURNAL Vol. 228, No.
OF
Ih~smLoc~
1,January
1975.
PrinIrd
in U.S.A.
by rabbit
Ion transport DAVID Defartment
jejunum
FROMM, RICHARD P. DIBALA, AND HENRY W. SULLIVAN, JR. of Surgery, Harvard kfedical School and Beth Israel Husfiital, Boston, Massachusetts
and Division
of Surgery,
FROMM,DAVID,RICI-XARD
P.
Walter
Reed
Army
DIBALA,AND HENRY
W.
Institute
of Research,
SULLIVAP~,
je$num in uivo. Am. J. Physiol. 228( 1) : 160465. 1975.--Net ion and Hz0 transport by jejunum adjacent to the ligament of Treitz (proximal jejunum) and midjejunum were measured in vivo by continuous perfusion with HCQ~-Ringer solution containing a volume marker. Proximal jejunum secreted Na and HgO, whereas midjejunum absorbed Na and H20. Both segments secreted CO2 and absorbed K and Cl. ~-Glucose stimulated absorption of Na and Hz0 and the transmural electrical potential difference (I’D) in both segments, but these changes were not accompanied by alterations in Cl, Cog, or K fluxes. However, the increase in Na absorption caused by 3-G methylglucose was matched by an increase in Cl absorption. This, in addition to increased tissue lactate concentration after addition suggests that organic anion maintains electroof D-glucose, neutrality for Na transport enhanced by D-glucose. Cholera toxin had no effect on ion transport or P3I) in proximal jejunum, but cholera toxin stimulated secretion and increased the PD in more distal jejunum. Although proximal jejunum shows spontaneous secretory activity, its capacity for secretion is not as great as more distal small intestine. JR.
hl
absorption;
transport
by rabbit
secretion;
cyclic
AMP;
cholera
toxin
RESULTS OF SEVERAL STUDIES (9, IO, 16, 18) suggest that ion transport by rabbit jejunum in vivo differs substantially from that observed in vitro (5). However, segments of jejunum in vivo of various lengths and distances from the ligament of Treitz were studied, whereas in vitro a specific segment ofjejunum, that adjacent to the ligament of Treitz, jejunum absorbs Na and was used. In vivo, “proximal” Hz0 (9, 10, 16, 18); h owever, in vitro, jejunum obtained adjacent to the ligament of Treitz secretes Na (5). Another difference between “proximal” jejunum and the most proximal portion of jejunum concerns the effects of agents that increase the concentration of cyclic AMP. Cholera toxin stimulates ion and fluid secretion by proximal jejunum in vivo (9, lo), but theophylline does not affect net ion transport by isolated jejunum obtained adjacent to the ligament of Treitz (5). These differences may be due to the possibility that ion transport across isolated jejunum is altered by conditions in vitro. For example, the absence of an electrochemical gradient can markedly influence net ion transport, as has been shown for guinea pig ileum (11) Transport in vitro may also reflect the absence of humoral agents, which influence ion transport in vivo. It is also possible that ion transport across the most proximal portion of jejunum in vitro does reflect the situation that occurs in vivo and that this proximal segment, differing from slightly THE
in vivo
Washington,
DC
02215;
XXX?
more distal jejunum, functionally behaves as duodenum. Spontaneous ion and fluid secretion in vivo has been reported to occur in rabbit duodenum excluded from biliary and presumably all pancreatic secretion (18). This study examines net ion and fluid transport by the most proximal and a more distal segment of je-junum in vivo in the absence and presence of luminal glucose and after exposure to cholera toxin. METHODS
Laparotomy under local anesthesia1 (lidocaine, 0.5 %) was performed on New Zealand white rabbits (3-4 kg) that previously had been allowed free access to a standard diet. The ligament of Treitz was incised in order to mobilize the most proximal portion ofjejunum. A soft plastic cannula (inflow) was inserted into the lumen 0.5-1.0 cm distal to the end of the ligament of Treitz, and an identical cannula (outflow) was inserted approximately 8-9 cm distally (6). This cannulated segment of intestine, which is referred to as proximal jejunum, corresponds to that used in vitro (5). Encircling ligatures holding the cannulas in place were passed between the parallel end vessels supplying the jejunum. This maneuver avoids macroscopic necrosis adIn order to avoid kinking or jacent to these ligatures. twisting, the proximal end of the isolated je.junal segment was tacked to the abdominal wall through which the cannulas were led. The cannulated segment was gently flushed with HCOa-Ringer solution in order to clean the segment and verify proper placement of the cannulas. The abdomen was then closed with sutures. In a separate series of experiments, a segment of jejunum, beginning at the sixth arterial (which corresponds to midjebranch2 in the mesentery junum), was prepared in an identical manner and is referred to as midjejunum. In order to minimize hormonal influences that “ay follow distention of the duodenum ( 17) during perfusion of proximal or midjejunum, a cannula was also placed in the distal duodenum and its effluent was drained by gravity. At the conclusion of each experiment the jejunal segment was excised and its length measured. The transmural electrical potential difference (PD) was l This procedure, as shown in this and previous studies (3 6), is tolerated providing gentle technique is used. 2 An anatomic landmark rather than a measured distance from the ligament of Treitz was chosen for the location of midjejunum. Reasonably accurate measurements of jejunal segments longer than 10 cm are difficult because of the short mesentery. When the jejunum was excised from its mesentery, the distal segments lay 35-40 cm distal to the ligament of Treitz. well
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ION
TRANSPORT
BY
RABBIT
JEJUNUM
161
measured by plicating the serosa over an end-beveled HCO a-Ringer-agar-filled PE-90 tubing with fine (6-O Tevdek) sutures so that the agar surface of the tip of the bridge was in contact with the serosa, and an identical bridge was passed into the lumen alongside the distal (outflow) cannula of the isolated segment so that the tips of both bridges were placed opposite one another (6). The PD (serosa positive) was monitored continuously. Immediately after closure of the abdomen, the jejunal loop was perfused with HCO 3-Ringer solution equilibrated with 5 % CO2 in oxygen and containing polyethylene glycol (PEG 4000), 1 g/liter, as a volume marker and the following, in mM; Na, 141; K, 10; Ca, 1.3; Mg, 1.1; Cl, 127; HC03, 25; HaPOd, 0.3; HPOJ, 1.7, Mannitol, 10 mM, was added to make the solution approximately isotonic with plasma. When D-glucose, 5 mM, or 3-O-methylglucose, 5 mM, was added to the perfusate, the concentration of mannitol was reduced to 5 mM. Blood, 5 ml, was collected from a femoral vein catheter whose tip lay approximately at the level of the right atrium. A comparison of the concentrations of the major constituents of the perfusate to those of plasma from 24 rabbits used in this study is shown in Table 1. The concentration gradients for Na and CO2 across the mucosa were negligible, whereas the concentration gradients for Cl and K favor the absorption of these ions. The perfusate, which, excluding PEG, is identical to that used previously in vitro (5), was infused continuously with a syringe pump (Harvard Apparatus model 990 with servo control) calibrated to deliver 0.25 ml/min. The infusion circuit was water jacketed so that the perfusate was 37°C as it entered the jejunum (6). A 6O-min period was allowed to pass before the jejunal effluent was collected for analysis, since blood pressire and rectal temperature are stabilized byI the end of this period (6). Thereafter, the jejunal effluent was collected by gravity over 30-min intervals. When the effects of cholera toxin were measured, a base-line, or control, interval of perfusion was interrupted and either crude toxin (National Institutes of Health lot no. 002, Inaba 569B), 5-l 0 mg/ml, dialyzed as described previously (3), or purified toxin (generous gift of Dr. R. A. Finkelstein) (4), 2 pg/ml, in HCOa-Ringer was placed in the jejunal segment for 30 min. Thereafter, perfusion was continued after gentle flushing of the segment with perfusate. Concentrations of Na and K were determined by flame photometry (Instrumentation Laboratory model 143), Cl by the method of Schales and Schales (14), CO2 by Nat&on microgasometry or bY AutoAnalyzer (Technicon co me thou poration), glucose by a gl ucose oxidase-peroxidase TABLE
glucox
1. Plasma andperfusata Na, K, Cl, COZ, and concentrations _ --~_ and osmolality ” __---- .--. ---_--_ I _.____.-~ Plasma _” - .--..-
Concentration, Na K Cl CO2 Glucose Osmolality,
- --~ Perfusate
-------
--
mM
mosmol/kg
Hz0
140.0 3.5 100.0 24.0 10.0 300.0
+ * + zt + It
0.5 0.1 I.6 0.5 0.4 2.0
141 10 127 26 0 302
designed for the AutoAnalyzer (J. David and C. Angel, manuscript in preparation), amylase by the method of Caraway (I), and PEG by the turbidimetric method of Hyden (8). Preliminary experiments (n = 5) indicated that PEG recovery (mean =t: 1 SE) from the perfusate of proximal and distal jejunum was 99.9 + 1.4 %. Net ion and Hz0 fluxes were calculated as described previously (6). Osmolality was measured with a Fiske osmoxneter. In a few experiments, tissue concentrations of lactate were measured after perfusion of either proximal jejunum or midjejunum. At the completion of the perfusion, the jejunal segment was excised and opened and its mucosa stripped from the underlying muscularis as described by Schultz and his associates ( 15). The rnucosal strip was quickly blotted and placed in a preweighed vial containing HN03, 0.5 M. Approximately 48 h later, after periodic shaking, the concentration of lactate was measured in the extraction medium by an enzymatic method (Sigma, Bull. No. 826-UV). Results in the text are given as means & 1 SE or as ind ividual values. S tatistical an .alyses were perfor med with the Studen t t test for paired a nd unpai red variates. RESULTS
LooF lengh and duodenal drainage. The mean length of the proximal jejunal segments, 8.6 + 0.4 cm (n = SO), was not significantly different (a > 0.05) from that of the distal segments, 9.6 & 0.5 cm (n = 32). In five experiments each of proximal and distal jejunal perfusion, the duodenal catheter was allowed to drain freely for 90 min and then was clamped for an additional 30 min. In each of another five experiments this sequence was reversed. Significant differences in net ion or water fluxes were not observed when the duodenal catheter was either open or closed. The rate of duodenal drainage, 6.7 =t 0.2 ml/30 min (n = 23), approximated the rate of jejunal perfusion, 7.5 ml/30 min, and did not change measurably during perfusion of proximal or distal jejunum with glucosefree solution or after addition of glucose to the perfusate or after exposure to cholera toxin. Sjmntaneous ion tranx/mrt (Fig. 1). Net secretion of Na, COZ, and Hz0 and absorption of Cl occurred in proximal jejunum. Although the absorptive flux of K was significantly different from zero, the magnitude of this flux was less than 1 pmol/cm per 30 min. LJ four experiments, the plasma osmolality was measured before perfusion of proximal jejunum was begun, and the osmolality of the perfusate was adjusted to that of the plasma by altering the concentration of mannitol. After a base-line flux measurement, the base-line osmolality of the perfusate was increased by 10 mosmol, by increasing the concentration of mannitol, and then lowered by 10 mosmol, by lowering the concentrations of mannitol and KCl. These alterations in perfusate osmolality were done in alternating sequences and detectable differences in net Na or Hz0 transport were not observed. When the perfusion rate of proximal jejunum was increased from 7.5 ml/30 min to 90 ml/30 min (n = 5), the mean results were identical to those observed with the slower rate of perfusion. However, considerable variability in flux
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162
FROMM, PROXIMAL (N= IO)
01 STAL (N =I01
60~ 50i z 4o 5 308 202 1" IO3. 0
H20:
t-t-i-
-IO -20 !
2:
H-t
:
DIBALA,
AND
SULLIVAN
effluent from proximal jejunum was virtually undetectable in 5 experiments. EJectsof glucose (pig. 2). After the addition of glucose to the perfusate of proximal jejunum, the mean Auxes of Na and Hz0 changed from secretion to absorption. Although these changes were statistically significant, the mean rates of Na and Hz0 transport were not significantly different from zero (P > 0.05). This was due to a decrease in secretion of Na and Hz0 (but not enough to result in absorption) by three segments after exposure to glucose. Reversal of secretion to absorption occurred in the other segments. An increase in PD occurred after the addition of glucose to all segments in which this parameter was measured (n = 9). Glucose caused similar changes in the midjejunum, but the magnitudes of the increases in absorption of Na, HZO, and PD were greater.
PROXIMAL N = IO
DISTAL N=8
[ if t : :* :ry/--“30
60 90
120150
180 MINUTES
30 60
90
120150180
FIG. 1. Spontaneous water and ion transport and PD by proximal jejunum and midjejunum (distal). Each point represents mean value for a 30-min interval for measurement for n animals. Brackets indicate 2 SEM.
measurements for each rabbit was observed and therefore the results gave no assurance of steady-state values. This variability most likely was related to a high perfusion rate of a relatively short (approximately 8 cm) intestinal segment. In contrast to proximal jejunum, absorption of Na and Hz0 occurred in midjejunum. However, this segment is similar to proximal jejunum in that secretion of COP and absorption of Cl and K were also observed. The presence of glucose in the effluent was undetectable in five experiments. The initial 30-min flux values for Na, Cog, Cl, and K of either proximal jejunum or midjejunum did not differ significantly from each of the subsequent 30-min values shown in Fig. I. For each 30-min flux interval, of either jejunal segment, net cation movement did not differ significantly from net anion movement. The maximal difference between the sums of net cation and net anion movement was 1.9 =t 0.6 pmoI/cm per 30 min (J’ > 0.05). The PD of either jejunal segment also did not change significantly with time and was of greater magnitude distally. In three rabbits, net fluxes were also measured for perfused duodenum distal to the entrance of the main pancreatic duct. High rates of Na secretion (390-410 pmol/cm per 30 min), Hz0 secretion (2,621-2,763 &‘cxn per 30 min), and CO2 secretion (44-208 pmol/cm per 30 min) were observed. However, at the end of a Z- to 3-h perfusion, the amylase content of the duodenal effluent was between 216 and 538 U/100 ml, whereas the amylase content of
GLUCOSE
GLUCOSE MINUTES
2. Effects of luminal D-glucose, 5 mM, on water and ion transm port and PD by proximal and midjejunum (distal). Each point represents mean value for a 30-min interval for n animals. Measurement of PD were made in 9 of 10 proximal jejunal segments. Brackets represent 2 SEM. FIG.
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ION
TRANSPORT
BY
RABBIT
163
JEJUNWM
in either proximal The presence of glucose, 5 mM, longed exposure (2 h) to the toxin also did not alter ion or Hz0 fluxes or PD in the proximal jejunum of two rabbits. jejunum or midjejunum did not appreciably alter the fluxes of COZ, Cl, or K. However, measurement of the concentraFurthermore, purified cholera toxin did not appreciably tion of lactate in the mucosa showed an increase after the affect transport or PD in the proximal jejunum of three rabbits. presence of glucose in the perfusate. Lactate concentrations of proximal jejunal mucosa not exposed to glucose in two In contrast to proximal jejunum, secretion of Xa, CI, experiments were I.63 and 1.66 mg/g wet wt. After perand H20 and a slight increase in secretion of CO2 occurred fusion of proximal jejunum with glucose in the perfusate in in distal jejunum about 90 min after exposure to dialyzed two experiments, the concentrations of lactate were 1.92 toxin. These parameters tended to plateau 120-150 min after exposure to the toxin. Statistically significant secretion and 1.81 mg/g wet wt. In the absence of glucose, the lactate concentrations of midjejunum from two rabbits were 1.64 of K was observed about 180 min after exposure to the and 1.61 mg/g wet wt. In the presence of glucose, the lactate toxin, but the magnitude of this change was small, and therefore an earlier change could have been missed. The concentrations of midjejunum from another two rabbits were 2.18 and 2.24 mg/g wet wt. PD also increased and tended to plateau 150 min after After perfusion of midjejunum from five rabbits with exposure to the toxin. Prior to and after the addition of the sugar-free solution, 3-O-methylglucose, 5 mM, was added toxin, the maximal difference between the sums of steadyto the perfusate. Sodium absorption increased by 8.6 ZIZ state net cation and net anion movement was 2.9 =t I.4 2.1 pmol/cm per 30 min in the presence of this sugar and pmol/cm per min (P > 0.05). Purified cholera toxin added was accompanied by an increase in Cl absorption that was to the midjejunum of two rabbits caused changes identical not significantly different (P > 0.05) from the increment in to those observed for dialyzed crude toxin. Na flux. No statistically significant changes occurred in the fluxes of CO2 or K. DISCUSSION Effects of cholera toxin (Fig. 3). Dialyzed, crude choXera toxin did not significantly alter ion or H20 transport or Despite the presence of an electrical gradient, and for PD in proximal jejunum over a 4-h interval beginning 30 some ions a concentration gradient, across the proximal min after exposure to this agent. The maximal difference jejunal mucosa of rabbits in vivo, several similarities exist between the sums of net cation and net anion movement between net ion transport measured in vivo and that was 0.4 Z/X 0.2 pmol/cm per 30 min (P > 0.1). More pro, measured in the absence of an electrochemical gradient in vitro. Spontaneous secretion of Na occurs both in vivo and in vitro (6) in the absence of major concentration gradients across the mucosa. The secretion of CO2 in the absence of a major concentration gradient in vivo parallels the in vitro observation of an unmeasured net ion flux that \yas consistent with the net luminal appearance of HCO 3 (5). Although the luminal gain of CO2 can be equated with the 3, we have avoided use of the term luminal alkali gain due to true HC03
" CHOLERA
38
90 TOXIN
150
210
270
R
90
150
210
270
CHOLEFt?TOXIN
FIG. 3. Effect of cholera toxin on water and ion transport proximal and midjejunum (distal). Each point represents for a 30-min interval of measure for n rabbits. Brackets SEti.
and PD by mean value indicate 2
C1 absorption differs from that observed where net Cl transport was negligible (5). However, absorption of Cl in vivo occurred along a favorable electrochemical gradient. Potassium ‘absorption also occurred along its concentration gradient in vivo, but the magnitude of the flux was small. Although K fluxes have not been measured along proximal jejunum in vitro, it is unlikely that significant rates of net K transport occur in the absence of an electrochemical gradient (7). Ion transport in vivo by proximal jejunum of the rabbit is similar to that by jejunurn of the guinea pig, since both segments secrete Na and CO2 and absorb Cl (13). However, it is possible that these segments are dissimilar in vitro, since Powell and his associates ( 11, 13) showed substantial dXerences between guinea pig ileum in vivo and in the absence of an electrochemical gradient in vitro. Ion transport by proximal jejunum in vivo shares some characteristics with that observed for more distal jejunurn, but important differences also exist. In contrast to more proximal intestine, midjejunurn spontaneously absorbs Na. The spontaneous net fluxes of the other measured ions have the same direction of transport in both intestinal segments.
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164
FROMM,
The magnitudes of CO2 secretion are similar for both segments, but the rates of Cl and K absorption are greater distally. The greater distal absorption of C1 carries no implication as to involvement of an active transport process, since the electrical gradient favoring Cl absorption is greater distally. With the exception of CO:! transport, spontaneous ion transport by the midjejunuxn of the rabbit is qualitatively similar to that by the jejunum of the rat ( 12). The presence of luminal D-glucose stimulates Na absorption and increases the PD in both proximal jejunum and midjejunum, but the effects of sugar are greater distally. The effect of D-glucose on Na transport in both jejunal segments is not accompanied by appreciable alterations in K, Cl, or CO2 transport. This is in contrast to the study of Powell and his associates (12) showing that en .hanced Na absorption by rat jejunum exposed to glucose is accol nPa nied by an increase in Cl absorption. However, the study of rat jejunum is not stric tly compara .ble to the present one, since the glucose solution used to perfuse rat jejunum had ion concentration gradients different from the glucose-free solution and the mucosal-to-serosal glucose concentration gradient was approximately 7.7 to 1. In the present study the concentration of electrolytes in the infusate was not altered and the mucosa1-to-serosal glucose concentration gradient was only 0.5 to 1. Measurement of mucosal lactate concentrations of rabbit jejunum after exposure to glucose suggests that electroneutrality under open-circuit conditions may be maintained bv an increase in the cell-to-serosal flux of lactate. Support for this concept is provided by the observation that the increase in Na absorption after perfusion with the sugar 3-O-methylglucose, which is not converted to lactate by the intestine (I 9)? is accompanied by an increase in Cl absorption. Markedly enhanced lactate production is associated with glucose transport by full-thickness segments of rat in vitro (19), but the significance of this observawjejunum tion for transport by rat intestine in vivo is unclear. Lactate production by f&-thickness segments of rabbit intestine in the presence of sugar has been shown to occur and to be considerably less than the rat in vitro ( 19), but submucosal accumulation of lactate has not been excluded. It is unlikely that the increase in mucosal lactate concentration of ejunal. mucosa of the rabbit in the presence of D-gIucose is due to hypoxia or a change in pH. With the same experimental preparation as that in the present study (6), arterial
DIBALA,
AND
SULLIVAN
blood gases and pH have been shown not to change significan tlv over a Z-h interval. Another difference between proximal jejunum and midjejunum of the rabbit is demonstrated by the effect of cholera toxin, which in midjejunum is qualitatively similar to that observed for rabbit ileum in vivo (10). Moritz and coworkers (9, 10) also observed a change in the PD of cholera toxin-treated segments of rabbit jejunum located between the proximal and more distal segment ofjejunum used in the present study. The flux values of the presen t: study before m to c holera toxin a re verv and after exposure of midjejunu similar to those observed by these invest igators. The effects of agents that increase the concentrati on of c y&c AMP indicate that the proximal jejunum in vivo and in vitrc does not have the same capacity for secretion as does more distal jejunum or ileum (2, 3, 9, 10). The lack of an appreciable effect of cholera toxin on net ion or net fluid transport by proximal jejunum in vivo could have been anticipated . on the basis of observations in vitro. Theophylline has been shown to have no statistically appreciable effect on net Na, C1, and unmeasured ion fluxes of the same intestinal segment in vitro (5). The ineffectiveness of these agents on net ion in cyclic AMP do tran sport does not imply that alterations not occur in the proxim al jejunum, because the ophyll me has dis tin cl effects on unidirecti .onal Na and Cl fluxes, PD, fl uxes and total tissue condu ctance in vitro ‘. u nidirectional in vivo, and a change in tissue conwere not measured ductance does not necessarily have to result in a change in net ion transport or PD. Althoug-h the factor(s) accounting for the differences in ion transbort between the most proximal and a more distal are unclear, it has been suggested that segment of jejunum the most proximal segment of j qlunum ma y functionally behave as distal duodenum (5). However, the finding of relatively high values of amylase in duodenal effluent excluded from the main (but not minor ones) pancreatic duct casts some doubt on the validity of observations suggesting ion secretion by rabbit duodenal mucosa in vivo
(18).
In conducting the research described adhered to the “Guide for Laboratory prepared by the National Academy Council.
in this report, the Animal Facilities of Sciences-National
Received
1973.
for
publication
25 October
investigators and Care” Research
REFERENCES 1. CARAWAY, W. T. A stable starch substrate for the determination of amylase in serum and other body fluids. Am. J. Clin. Pat/id. 32: 97-99, 1959. 2. FIELD, M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3’) 5’-AMP. Am. J. Physiol. 22 1 : 992-997, 197 1. 3. FIELD, hf., D. FROMM, Qa AL-AWQATI, AND Mr. I3. GREENOUGH III. Effect of cholera enterotoxin on ion transport across isolated ileal mucosa. J. Clin. Inaest. 5 1 : 796-804, 1972. 4. FINKELSTEIN, R. A., AND J.J. LOSPALLUTO. Production, purification and assay of cholera toxin. J. Infect. Diseases 12 1, Suppl. : 563-572, 1970. 5. FROMM, D. Na and Cl transport across isolated proximal small intestine of the rabbit. Am. J. Physiol. 224: 110-l 16, 1973. 6. FROMM, D. Intestinal absorption during hypovolemic shock. Surgery 177 : 448-452, 1973. 7. FROMM, D., AND W. &LEN. Effects of secretin and pancreozymin on ion transport across small intestine. In : Current Topics in Surgical
Research, Academic,
edited by G. 31). Zuideman and D. B. Skinner. New York: 1969, vol. I, p. 249-261. 8. HYDEN, S. A turbidimetric rnethod for the determination of higher polyethylene glycols in biological materials. Kungl. Lad. Ann. 22: 139-145, 1955. Rabbit cholera: 9. MORITZ, M., F. L. I BER, AND E. W. MOORE. relation of transmural potentials to water and electrolyte fluxes. Am. J. Physd. 22 1 : 19-24, 1971. 10. MORITZ, M., F. 1;. IBER, AND E. W. MOORE, Rabbit cholera: effects of cycloheximide on net water and ion fluxes and transmural electric potentials. Gastrocnterology 63 : 76-82, 1972. 11. POWELL, D. W., H. J. BINDER, AND P. F. CURRAN. Electrolyte secretion by the guinea pig ileum in vitro. Am. J. Physiol. 223 : 531-537, 1972. 12. POWELL, D. W., AND S. J. MALAWER. Relationship between water and solute transport from isosmotic solutions by rat intestine in vivo. Am. J. Physid. 215 : 49-55, 1968.
Downloaded from www.physiology.org/journal/ajplegacy at Tulane University (129.081.226.078) on February 15, 2019.
ION
TRANSPORT
13. POWELL,
BY
RABBIT
165
JEJUNUM
D. W.,S.J. MALAWER, AND G. K. PL~TKIN. Secretion of and water by the guinea pig small intestine in viva, Am. Jb Physiol. 215 : 1226-1233, 1968. 14. SCHALES, 0, AND S. S. SCHALES. A simple and accurate method for the determination of chloride in biological fluids. J. Biol. Chem. 140: 879-884, 1941. 15. SCHULTZ, S. G., K. E. FUISZ, AND P. F. CURRAN. Amino acid and sugar transport in rabbit ileum. J. Gen. PhysioZ. 49: 849-866, 1966. 16. SEREBRO, H. A., T. M. BAYLISS, T. Ii. HENDRIX, F. L. IBER, AND T. MCGONAGLE. Absorption of d-glucose by the rabbit jejunum during cholera toxin-induced diarrhoea. ivature 2 17 : 1272-l 273,
17.
electrolytes
SUM, P. T., H. L. SCHIPPER,
AND
R.
M.
PRESHAW.
and pancreatic secretion during intestinal testinal perfusion with choline derivatives. col. 47: 115-l 18, 1969. 18.
VOGEL,
G.,
Flussigkeitsverschiedenen 19.
I.
STOECKERT, and
abschnitte
Pjuegers WILSON,
intestinal Arch. 325 : 247-261, 197 1, T. H. The role of lactic acid
sorption
from
the
intestine.
des
durch traktas production
gastric and
Can. J. Physiol.
AND E. MEYERING.
substanzbewegungen
Canine
distention
Unterschiedliche die mucosa beim in
glucose 1956.
Downloaded from www.physiology.org/journal/ajplegacy at Tulane University (129.081.226.078) on February 15, 2019.
der
kankhen.
J. Biol. Chem. 222 : 75 l-563,
1968.
in-
Pharma-
ab-
OF
Ih~smLoc~
1,January
1975.
PrinIrd
in U.S.A.
by rabbit
Ion transport DAVID Defartment
jejunum
FROMM, RICHARD P. DIBALA, AND HENRY W. SULLIVAN, JR. of Surgery, Harvard kfedical School and Beth Israel Husfiital, Boston, Massachusetts
and Division
of Surgery,
FROMM,DAVID,RICI-XARD
P.
Walter
Reed
Army
DIBALA,AND HENRY
W.
Institute
of Research,
SULLIVAP~,
je$num in uivo. Am. J. Physiol. 228( 1) : 160465. 1975.--Net ion and Hz0 transport by jejunum adjacent to the ligament of Treitz (proximal jejunum) and midjejunum were measured in vivo by continuous perfusion with HCQ~-Ringer solution containing a volume marker. Proximal jejunum secreted Na and HgO, whereas midjejunum absorbed Na and H20. Both segments secreted CO2 and absorbed K and Cl. ~-Glucose stimulated absorption of Na and Hz0 and the transmural electrical potential difference (I’D) in both segments, but these changes were not accompanied by alterations in Cl, Cog, or K fluxes. However, the increase in Na absorption caused by 3-G methylglucose was matched by an increase in Cl absorption. This, in addition to increased tissue lactate concentration after addition suggests that organic anion maintains electroof D-glucose, neutrality for Na transport enhanced by D-glucose. Cholera toxin had no effect on ion transport or P3I) in proximal jejunum, but cholera toxin stimulated secretion and increased the PD in more distal jejunum. Although proximal jejunum shows spontaneous secretory activity, its capacity for secretion is not as great as more distal small intestine. JR.
hl
absorption;
transport
by rabbit
secretion;
cyclic
AMP;
cholera
toxin
RESULTS OF SEVERAL STUDIES (9, IO, 16, 18) suggest that ion transport by rabbit jejunum in vivo differs substantially from that observed in vitro (5). However, segments of jejunum in vivo of various lengths and distances from the ligament of Treitz were studied, whereas in vitro a specific segment ofjejunum, that adjacent to the ligament of Treitz, jejunum absorbs Na and was used. In vivo, “proximal” Hz0 (9, 10, 16, 18); h owever, in vitro, jejunum obtained adjacent to the ligament of Treitz secretes Na (5). Another difference between “proximal” jejunum and the most proximal portion of jejunum concerns the effects of agents that increase the concentration of cyclic AMP. Cholera toxin stimulates ion and fluid secretion by proximal jejunum in vivo (9, lo), but theophylline does not affect net ion transport by isolated jejunum obtained adjacent to the ligament of Treitz (5). These differences may be due to the possibility that ion transport across isolated jejunum is altered by conditions in vitro. For example, the absence of an electrochemical gradient can markedly influence net ion transport, as has been shown for guinea pig ileum (11) Transport in vitro may also reflect the absence of humoral agents, which influence ion transport in vivo. It is also possible that ion transport across the most proximal portion of jejunum in vitro does reflect the situation that occurs in vivo and that this proximal segment, differing from slightly THE
in vivo
Washington,
DC
02215;
XXX?
more distal jejunum, functionally behaves as duodenum. Spontaneous ion and fluid secretion in vivo has been reported to occur in rabbit duodenum excluded from biliary and presumably all pancreatic secretion (18). This study examines net ion and fluid transport by the most proximal and a more distal segment of je-junum in vivo in the absence and presence of luminal glucose and after exposure to cholera toxin. METHODS
Laparotomy under local anesthesia1 (lidocaine, 0.5 %) was performed on New Zealand white rabbits (3-4 kg) that previously had been allowed free access to a standard diet. The ligament of Treitz was incised in order to mobilize the most proximal portion ofjejunum. A soft plastic cannula (inflow) was inserted into the lumen 0.5-1.0 cm distal to the end of the ligament of Treitz, and an identical cannula (outflow) was inserted approximately 8-9 cm distally (6). This cannulated segment of intestine, which is referred to as proximal jejunum, corresponds to that used in vitro (5). Encircling ligatures holding the cannulas in place were passed between the parallel end vessels supplying the jejunum. This maneuver avoids macroscopic necrosis adIn order to avoid kinking or jacent to these ligatures. twisting, the proximal end of the isolated je.junal segment was tacked to the abdominal wall through which the cannulas were led. The cannulated segment was gently flushed with HCOa-Ringer solution in order to clean the segment and verify proper placement of the cannulas. The abdomen was then closed with sutures. In a separate series of experiments, a segment of jejunum, beginning at the sixth arterial (which corresponds to midjebranch2 in the mesentery junum), was prepared in an identical manner and is referred to as midjejunum. In order to minimize hormonal influences that “ay follow distention of the duodenum ( 17) during perfusion of proximal or midjejunum, a cannula was also placed in the distal duodenum and its effluent was drained by gravity. At the conclusion of each experiment the jejunal segment was excised and its length measured. The transmural electrical potential difference (PD) was l This procedure, as shown in this and previous studies (3 6), is tolerated providing gentle technique is used. 2 An anatomic landmark rather than a measured distance from the ligament of Treitz was chosen for the location of midjejunum. Reasonably accurate measurements of jejunal segments longer than 10 cm are difficult because of the short mesentery. When the jejunum was excised from its mesentery, the distal segments lay 35-40 cm distal to the ligament of Treitz. well
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ION
TRANSPORT
BY
RABBIT
JEJUNUM
161
measured by plicating the serosa over an end-beveled HCO a-Ringer-agar-filled PE-90 tubing with fine (6-O Tevdek) sutures so that the agar surface of the tip of the bridge was in contact with the serosa, and an identical bridge was passed into the lumen alongside the distal (outflow) cannula of the isolated segment so that the tips of both bridges were placed opposite one another (6). The PD (serosa positive) was monitored continuously. Immediately after closure of the abdomen, the jejunal loop was perfused with HCO 3-Ringer solution equilibrated with 5 % CO2 in oxygen and containing polyethylene glycol (PEG 4000), 1 g/liter, as a volume marker and the following, in mM; Na, 141; K, 10; Ca, 1.3; Mg, 1.1; Cl, 127; HC03, 25; HaPOd, 0.3; HPOJ, 1.7, Mannitol, 10 mM, was added to make the solution approximately isotonic with plasma. When D-glucose, 5 mM, or 3-O-methylglucose, 5 mM, was added to the perfusate, the concentration of mannitol was reduced to 5 mM. Blood, 5 ml, was collected from a femoral vein catheter whose tip lay approximately at the level of the right atrium. A comparison of the concentrations of the major constituents of the perfusate to those of plasma from 24 rabbits used in this study is shown in Table 1. The concentration gradients for Na and CO2 across the mucosa were negligible, whereas the concentration gradients for Cl and K favor the absorption of these ions. The perfusate, which, excluding PEG, is identical to that used previously in vitro (5), was infused continuously with a syringe pump (Harvard Apparatus model 990 with servo control) calibrated to deliver 0.25 ml/min. The infusion circuit was water jacketed so that the perfusate was 37°C as it entered the jejunum (6). A 6O-min period was allowed to pass before the jejunal effluent was collected for analysis, since blood pressire and rectal temperature are stabilized byI the end of this period (6). Thereafter, the jejunal effluent was collected by gravity over 30-min intervals. When the effects of cholera toxin were measured, a base-line, or control, interval of perfusion was interrupted and either crude toxin (National Institutes of Health lot no. 002, Inaba 569B), 5-l 0 mg/ml, dialyzed as described previously (3), or purified toxin (generous gift of Dr. R. A. Finkelstein) (4), 2 pg/ml, in HCOa-Ringer was placed in the jejunal segment for 30 min. Thereafter, perfusion was continued after gentle flushing of the segment with perfusate. Concentrations of Na and K were determined by flame photometry (Instrumentation Laboratory model 143), Cl by the method of Schales and Schales (14), CO2 by Nat&on microgasometry or bY AutoAnalyzer (Technicon co me thou poration), glucose by a gl ucose oxidase-peroxidase TABLE
glucox
1. Plasma andperfusata Na, K, Cl, COZ, and concentrations _ --~_ and osmolality ” __---- .--. ---_--_ I _.____.-~ Plasma _” - .--..-
Concentration, Na K Cl CO2 Glucose Osmolality,
- --~ Perfusate
-------
--
mM
mosmol/kg
Hz0
140.0 3.5 100.0 24.0 10.0 300.0
+ * + zt + It
0.5 0.1 I.6 0.5 0.4 2.0
141 10 127 26 0 302
designed for the AutoAnalyzer (J. David and C. Angel, manuscript in preparation), amylase by the method of Caraway (I), and PEG by the turbidimetric method of Hyden (8). Preliminary experiments (n = 5) indicated that PEG recovery (mean =t: 1 SE) from the perfusate of proximal and distal jejunum was 99.9 + 1.4 %. Net ion and Hz0 fluxes were calculated as described previously (6). Osmolality was measured with a Fiske osmoxneter. In a few experiments, tissue concentrations of lactate were measured after perfusion of either proximal jejunum or midjejunum. At the completion of the perfusion, the jejunal segment was excised and opened and its mucosa stripped from the underlying muscularis as described by Schultz and his associates ( 15). The rnucosal strip was quickly blotted and placed in a preweighed vial containing HN03, 0.5 M. Approximately 48 h later, after periodic shaking, the concentration of lactate was measured in the extraction medium by an enzymatic method (Sigma, Bull. No. 826-UV). Results in the text are given as means & 1 SE or as ind ividual values. S tatistical an .alyses were perfor med with the Studen t t test for paired a nd unpai red variates. RESULTS
LooF lengh and duodenal drainage. The mean length of the proximal jejunal segments, 8.6 + 0.4 cm (n = SO), was not significantly different (a > 0.05) from that of the distal segments, 9.6 & 0.5 cm (n = 32). In five experiments each of proximal and distal jejunal perfusion, the duodenal catheter was allowed to drain freely for 90 min and then was clamped for an additional 30 min. In each of another five experiments this sequence was reversed. Significant differences in net ion or water fluxes were not observed when the duodenal catheter was either open or closed. The rate of duodenal drainage, 6.7 =t 0.2 ml/30 min (n = 23), approximated the rate of jejunal perfusion, 7.5 ml/30 min, and did not change measurably during perfusion of proximal or distal jejunum with glucosefree solution or after addition of glucose to the perfusate or after exposure to cholera toxin. Sjmntaneous ion tranx/mrt (Fig. 1). Net secretion of Na, COZ, and Hz0 and absorption of Cl occurred in proximal jejunum. Although the absorptive flux of K was significantly different from zero, the magnitude of this flux was less than 1 pmol/cm per 30 min. LJ four experiments, the plasma osmolality was measured before perfusion of proximal jejunum was begun, and the osmolality of the perfusate was adjusted to that of the plasma by altering the concentration of mannitol. After a base-line flux measurement, the base-line osmolality of the perfusate was increased by 10 mosmol, by increasing the concentration of mannitol, and then lowered by 10 mosmol, by lowering the concentrations of mannitol and KCl. These alterations in perfusate osmolality were done in alternating sequences and detectable differences in net Na or Hz0 transport were not observed. When the perfusion rate of proximal jejunum was increased from 7.5 ml/30 min to 90 ml/30 min (n = 5), the mean results were identical to those observed with the slower rate of perfusion. However, considerable variability in flux
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162
FROMM, PROXIMAL (N= IO)
01 STAL (N =I01
60~ 50i z 4o 5 308 202 1" IO3. 0
H20:
t-t-i-
-IO -20 !
2:
H-t
:
DIBALA,
AND
SULLIVAN
effluent from proximal jejunum was virtually undetectable in 5 experiments. EJectsof glucose (pig. 2). After the addition of glucose to the perfusate of proximal jejunum, the mean Auxes of Na and Hz0 changed from secretion to absorption. Although these changes were statistically significant, the mean rates of Na and Hz0 transport were not significantly different from zero (P > 0.05). This was due to a decrease in secretion of Na and Hz0 (but not enough to result in absorption) by three segments after exposure to glucose. Reversal of secretion to absorption occurred in the other segments. An increase in PD occurred after the addition of glucose to all segments in which this parameter was measured (n = 9). Glucose caused similar changes in the midjejunum, but the magnitudes of the increases in absorption of Na, HZO, and PD were greater.
PROXIMAL N = IO
DISTAL N=8
[ if t : :* :ry/--“30
60 90
120150
180 MINUTES
30 60
90
120150180
FIG. 1. Spontaneous water and ion transport and PD by proximal jejunum and midjejunum (distal). Each point represents mean value for a 30-min interval for measurement for n animals. Brackets indicate 2 SEM.
measurements for each rabbit was observed and therefore the results gave no assurance of steady-state values. This variability most likely was related to a high perfusion rate of a relatively short (approximately 8 cm) intestinal segment. In contrast to proximal jejunum, absorption of Na and Hz0 occurred in midjejunum. However, this segment is similar to proximal jejunum in that secretion of COP and absorption of Cl and K were also observed. The presence of glucose in the effluent was undetectable in five experiments. The initial 30-min flux values for Na, Cog, Cl, and K of either proximal jejunum or midjejunum did not differ significantly from each of the subsequent 30-min values shown in Fig. I. For each 30-min flux interval, of either jejunal segment, net cation movement did not differ significantly from net anion movement. The maximal difference between the sums of net cation and net anion movement was 1.9 =t 0.6 pmoI/cm per 30 min (J’ > 0.05). The PD of either jejunal segment also did not change significantly with time and was of greater magnitude distally. In three rabbits, net fluxes were also measured for perfused duodenum distal to the entrance of the main pancreatic duct. High rates of Na secretion (390-410 pmol/cm per 30 min), Hz0 secretion (2,621-2,763 &‘cxn per 30 min), and CO2 secretion (44-208 pmol/cm per 30 min) were observed. However, at the end of a Z- to 3-h perfusion, the amylase content of the duodenal effluent was between 216 and 538 U/100 ml, whereas the amylase content of
GLUCOSE
GLUCOSE MINUTES
2. Effects of luminal D-glucose, 5 mM, on water and ion transm port and PD by proximal and midjejunum (distal). Each point represents mean value for a 30-min interval for n animals. Measurement of PD were made in 9 of 10 proximal jejunal segments. Brackets represent 2 SEM. FIG.
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ION
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163
JEJUNWM
in either proximal The presence of glucose, 5 mM, longed exposure (2 h) to the toxin also did not alter ion or Hz0 fluxes or PD in the proximal jejunum of two rabbits. jejunum or midjejunum did not appreciably alter the fluxes of COZ, Cl, or K. However, measurement of the concentraFurthermore, purified cholera toxin did not appreciably tion of lactate in the mucosa showed an increase after the affect transport or PD in the proximal jejunum of three rabbits. presence of glucose in the perfusate. Lactate concentrations of proximal jejunal mucosa not exposed to glucose in two In contrast to proximal jejunum, secretion of Xa, CI, experiments were I.63 and 1.66 mg/g wet wt. After perand H20 and a slight increase in secretion of CO2 occurred fusion of proximal jejunum with glucose in the perfusate in in distal jejunum about 90 min after exposure to dialyzed two experiments, the concentrations of lactate were 1.92 toxin. These parameters tended to plateau 120-150 min after exposure to the toxin. Statistically significant secretion and 1.81 mg/g wet wt. In the absence of glucose, the lactate concentrations of midjejunum from two rabbits were 1.64 of K was observed about 180 min after exposure to the and 1.61 mg/g wet wt. In the presence of glucose, the lactate toxin, but the magnitude of this change was small, and therefore an earlier change could have been missed. The concentrations of midjejunum from another two rabbits were 2.18 and 2.24 mg/g wet wt. PD also increased and tended to plateau 150 min after After perfusion of midjejunum from five rabbits with exposure to the toxin. Prior to and after the addition of the sugar-free solution, 3-O-methylglucose, 5 mM, was added toxin, the maximal difference between the sums of steadyto the perfusate. Sodium absorption increased by 8.6 ZIZ state net cation and net anion movement was 2.9 =t I.4 2.1 pmol/cm per 30 min in the presence of this sugar and pmol/cm per min (P > 0.05). Purified cholera toxin added was accompanied by an increase in Cl absorption that was to the midjejunum of two rabbits caused changes identical not significantly different (P > 0.05) from the increment in to those observed for dialyzed crude toxin. Na flux. No statistically significant changes occurred in the fluxes of CO2 or K. DISCUSSION Effects of cholera toxin (Fig. 3). Dialyzed, crude choXera toxin did not significantly alter ion or H20 transport or Despite the presence of an electrical gradient, and for PD in proximal jejunum over a 4-h interval beginning 30 some ions a concentration gradient, across the proximal min after exposure to this agent. The maximal difference jejunal mucosa of rabbits in vivo, several similarities exist between the sums of net cation and net anion movement between net ion transport measured in vivo and that was 0.4 Z/X 0.2 pmol/cm per 30 min (P > 0.1). More pro, measured in the absence of an electrochemical gradient in vitro. Spontaneous secretion of Na occurs both in vivo and in vitro (6) in the absence of major concentration gradients across the mucosa. The secretion of CO2 in the absence of a major concentration gradient in vivo parallels the in vitro observation of an unmeasured net ion flux that \yas consistent with the net luminal appearance of HCO 3 (5). Although the luminal gain of CO2 can be equated with the 3, we have avoided use of the term luminal alkali gain due to true HC03
" CHOLERA
38
90 TOXIN
150
210
270
R
90
150
210
270
CHOLEFt?TOXIN
FIG. 3. Effect of cholera toxin on water and ion transport proximal and midjejunum (distal). Each point represents for a 30-min interval of measure for n rabbits. Brackets SEti.
and PD by mean value indicate 2
C1 absorption differs from that observed where net Cl transport was negligible (5). However, absorption of Cl in vivo occurred along a favorable electrochemical gradient. Potassium ‘absorption also occurred along its concentration gradient in vivo, but the magnitude of the flux was small. Although K fluxes have not been measured along proximal jejunum in vitro, it is unlikely that significant rates of net K transport occur in the absence of an electrochemical gradient (7). Ion transport in vivo by proximal jejunum of the rabbit is similar to that by jejunurn of the guinea pig, since both segments secrete Na and CO2 and absorb Cl (13). However, it is possible that these segments are dissimilar in vitro, since Powell and his associates ( 11, 13) showed substantial dXerences between guinea pig ileum in vivo and in the absence of an electrochemical gradient in vitro. Ion transport by proximal jejunum in vivo shares some characteristics with that observed for more distal jejunurn, but important differences also exist. In contrast to more proximal intestine, midjejunurn spontaneously absorbs Na. The spontaneous net fluxes of the other measured ions have the same direction of transport in both intestinal segments.
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164
FROMM,
The magnitudes of CO2 secretion are similar for both segments, but the rates of Cl and K absorption are greater distally. The greater distal absorption of C1 carries no implication as to involvement of an active transport process, since the electrical gradient favoring Cl absorption is greater distally. With the exception of CO:! transport, spontaneous ion transport by the midjejunuxn of the rabbit is qualitatively similar to that by the jejunum of the rat ( 12). The presence of luminal D-glucose stimulates Na absorption and increases the PD in both proximal jejunum and midjejunum, but the effects of sugar are greater distally. The effect of D-glucose on Na transport in both jejunal segments is not accompanied by appreciable alterations in K, Cl, or CO2 transport. This is in contrast to the study of Powell and his associates (12) showing that en .hanced Na absorption by rat jejunum exposed to glucose is accol nPa nied by an increase in Cl absorption. However, the study of rat jejunum is not stric tly compara .ble to the present one, since the glucose solution used to perfuse rat jejunum had ion concentration gradients different from the glucose-free solution and the mucosal-to-serosal glucose concentration gradient was approximately 7.7 to 1. In the present study the concentration of electrolytes in the infusate was not altered and the mucosa1-to-serosal glucose concentration gradient was only 0.5 to 1. Measurement of mucosal lactate concentrations of rabbit jejunum after exposure to glucose suggests that electroneutrality under open-circuit conditions may be maintained bv an increase in the cell-to-serosal flux of lactate. Support for this concept is provided by the observation that the increase in Na absorption after perfusion with the sugar 3-O-methylglucose, which is not converted to lactate by the intestine (I 9)? is accompanied by an increase in Cl absorption. Markedly enhanced lactate production is associated with glucose transport by full-thickness segments of rat in vitro (19), but the significance of this observawjejunum tion for transport by rat intestine in vivo is unclear. Lactate production by f&-thickness segments of rabbit intestine in the presence of sugar has been shown to occur and to be considerably less than the rat in vitro ( 19), but submucosal accumulation of lactate has not been excluded. It is unlikely that the increase in mucosal lactate concentration of ejunal. mucosa of the rabbit in the presence of D-gIucose is due to hypoxia or a change in pH. With the same experimental preparation as that in the present study (6), arterial
DIBALA,
AND
SULLIVAN
blood gases and pH have been shown not to change significan tlv over a Z-h interval. Another difference between proximal jejunum and midjejunum of the rabbit is demonstrated by the effect of cholera toxin, which in midjejunum is qualitatively similar to that observed for rabbit ileum in vivo (10). Moritz and coworkers (9, 10) also observed a change in the PD of cholera toxin-treated segments of rabbit jejunum located between the proximal and more distal segment ofjejunum used in the present study. The flux values of the presen t: study before m to c holera toxin a re verv and after exposure of midjejunu similar to those observed by these invest igators. The effects of agents that increase the concentrati on of c y&c AMP indicate that the proximal jejunum in vivo and in vitrc does not have the same capacity for secretion as does more distal jejunum or ileum (2, 3, 9, 10). The lack of an appreciable effect of cholera toxin on net ion or net fluid transport by proximal jejunum in vivo could have been anticipated . on the basis of observations in vitro. Theophylline has been shown to have no statistically appreciable effect on net Na, C1, and unmeasured ion fluxes of the same intestinal segment in vitro (5). The ineffectiveness of these agents on net ion in cyclic AMP do tran sport does not imply that alterations not occur in the proxim al jejunum, because the ophyll me has dis tin cl effects on unidirecti .onal Na and Cl fluxes, PD, fl uxes and total tissue condu ctance in vitro ‘. u nidirectional in vivo, and a change in tissue conwere not measured ductance does not necessarily have to result in a change in net ion transport or PD. Althoug-h the factor(s) accounting for the differences in ion transbort between the most proximal and a more distal are unclear, it has been suggested that segment of jejunum the most proximal segment of j qlunum ma y functionally behave as distal duodenum (5). However, the finding of relatively high values of amylase in duodenal effluent excluded from the main (but not minor ones) pancreatic duct casts some doubt on the validity of observations suggesting ion secretion by rabbit duodenal mucosa in vivo
(18).
In conducting the research described adhered to the “Guide for Laboratory prepared by the National Academy Council.
in this report, the Animal Facilities of Sciences-National
Received
1973.
for
publication
25 October
investigators and Care” Research
REFERENCES 1. CARAWAY, W. T. A stable starch substrate for the determination of amylase in serum and other body fluids. Am. J. Clin. Pat/id. 32: 97-99, 1959. 2. FIELD, M. Ion transport in rabbit ileal mucosa. II. Effects of cyclic 3’) 5’-AMP. Am. J. Physiol. 22 1 : 992-997, 197 1. 3. FIELD, hf., D. FROMM, Qa AL-AWQATI, AND Mr. I3. GREENOUGH III. Effect of cholera enterotoxin on ion transport across isolated ileal mucosa. J. Clin. Inaest. 5 1 : 796-804, 1972. 4. FINKELSTEIN, R. A., AND J.J. LOSPALLUTO. Production, purification and assay of cholera toxin. J. Infect. Diseases 12 1, Suppl. : 563-572, 1970. 5. FROMM, D. Na and Cl transport across isolated proximal small intestine of the rabbit. Am. J. Physiol. 224: 110-l 16, 1973. 6. FROMM, D. Intestinal absorption during hypovolemic shock. Surgery 177 : 448-452, 1973. 7. FROMM, D., AND W. &LEN. Effects of secretin and pancreozymin on ion transport across small intestine. In : Current Topics in Surgical
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edited by G. 31). Zuideman and D. B. Skinner. New York: 1969, vol. I, p. 249-261. 8. HYDEN, S. A turbidimetric rnethod for the determination of higher polyethylene glycols in biological materials. Kungl. Lad. Ann. 22: 139-145, 1955. Rabbit cholera: 9. MORITZ, M., F. L. I BER, AND E. W. MOORE. relation of transmural potentials to water and electrolyte fluxes. Am. J. Physd. 22 1 : 19-24, 1971. 10. MORITZ, M., F. 1;. IBER, AND E. W. MOORE, Rabbit cholera: effects of cycloheximide on net water and ion fluxes and transmural electric potentials. Gastrocnterology 63 : 76-82, 1972. 11. POWELL, D. W., H. J. BINDER, AND P. F. CURRAN. Electrolyte secretion by the guinea pig ileum in vitro. Am. J. Physiol. 223 : 531-537, 1972. 12. POWELL, D. W., AND S. J. MALAWER. Relationship between water and solute transport from isosmotic solutions by rat intestine in vivo. Am. J. Physid. 215 : 49-55, 1968.
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ION
TRANSPORT
13. POWELL,
BY
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JEJUNUM
D. W.,S.J. MALAWER, AND G. K. PL~TKIN. Secretion of and water by the guinea pig small intestine in viva, Am. Jb Physiol. 215 : 1226-1233, 1968. 14. SCHALES, 0, AND S. S. SCHALES. A simple and accurate method for the determination of chloride in biological fluids. J. Biol. Chem. 140: 879-884, 1941. 15. SCHULTZ, S. G., K. E. FUISZ, AND P. F. CURRAN. Amino acid and sugar transport in rabbit ileum. J. Gen. PhysioZ. 49: 849-866, 1966. 16. SEREBRO, H. A., T. M. BAYLISS, T. Ii. HENDRIX, F. L. IBER, AND T. MCGONAGLE. Absorption of d-glucose by the rabbit jejunum during cholera toxin-induced diarrhoea. ivature 2 17 : 1272-l 273,
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AND
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M.
PRESHAW.
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der
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