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Sugar absorption in the intestine of the chiton, Cryptochiton stelleri
Corap. Biochem. Physiol., 1967, Vol. 22, pp. 341 to 357. pergamon Press. Printed in Great Britain
SUGAR ABSORPTION IN THE INTESTINE OF THE CHITON, CR Y P T O C H I T O N S T E L L E R I * A. L. LAWRENCE and D. C. LAWRENCE Biology Department, University of Houston, Houston, Texas 77004, U.S.A., and Hopkins Marine Station, Pacific Grove, California, U.S.A. (Received 7 February 1967)
Abstract--1. Intestinal absorption of various monosaccharides was studied
in the chiton, Cryptochiton stelleri, using an everted sac technique. 2. D-Glucose and 3-o-methylglucose were actively transported by the anterior intestine but D-galactose was actively transported only by the posterior intestine. 3. D-Mannose and D-fructose were not actively absorbed by any region of the chiton intestine. 4. D-Fructose, D-marmoseand D-galactosewere not absorbed by the anterior intestine by being converted to D-glucose. 5. Duration studies showed that the everted sac preparation supported active transport of D-glucose, D-galactose and 3-o-methylglucose for 12 hr. 6. Active transport of D-glucose and 3-o-methylglucose was reversibly inhibited by anaerobic conditions. 7. There was no significant inhibition of active transport of D-galactose by anaerobic conditions. INTRODUCTION T~E ABSORPTIONof nutrients is an important physiological function of the gut of animals. Active transport, as a mechanism for increasing the rate of absorption and of concentrating a substance, is a significant characteristic of vertebrate guts. Although active transport for many organic molecules has been studied extensively in vertebrate intestines, it has not been demonstrated in the gut of many invertebrate phyla including the Mollusca. In fact, the principal site of absorption of nutrients in molluscs has been thought not to be in the gut but in the digestive gland. This conclusion has been reached in studies on bivalves (Potts, 1923; Vonk, 1924; Yonge, 1926 a, b; Morton, 1956; Allen, 1962), chitons (Fretter, 1937) and on gastropods (Peczenik, 1925 ; Hirsch, 1925 ; Graham, 1932; Millott, 1937; Graham, 1938; Fretter, 1939; Howells, 1942; Carriker, 1946; van Weel, 1961; Martoja, 1961 ; Morse, 1966). Only Bidder (1950) has suggested that the absorption of nutrients occurred in the caecal sac and intestine and not in the liver or pancreas in four species of cephalopods. * This investigation was supported in part by USPH Grant 2-F2-GM-8812 and NSF Grant GB-3256 to A. L. Lawrence. 341
342
A.L. LAWRENCEAND D. C. LAWRENCE
This study demonstrates the absorption of several monosaccharides by active transport mechanisms in the intestine of the chiton, Cryptochiton stelleri. MATERIALS AND METHODS Chitons (Cryptochiton stellen) of both sexes and weighing between 650 and 1200 g were obtained from the Pacific Ocean near Monterey, California (37 ° 11' N., 122 ° 24' W.). The animals were maintained in the laboratory at 5°C in aerated seawater for at least 1 week before they were used. The procedure used for the in vitro study of sugar transport was a modification of the method described by Crane & Wilson (1958). The apparatus consisted of a glass test tube with an enlarged top and a twoholed rubber stopper with a glass cannula. A hypodermic needle was forced through the stopper at a slight angle and a length of polyethylene tubing was attached to the needle. The anterior and posterior intestine and the associated digestive gland were removed from the animal and placed in chiton Ringer solution. The anterior and posterior intestine was then isolated, washed out with chiton Ringer at 150C and everted over a glass rod. Five segments were then taken from the intestine which represented proximal anterior intestine, distal anterior intestine, proximal posterior intestine, medial posterior intestine and distal posterior intestine (Fig. 1). All segments were approximately 6-10 cm in length. One end of the everted segment was tied to the cannula and the other end to a glass weight. The cannula with the attached everted segment was placed in the glass test tube which contained 32 ml of chiton Ringer (15°C) and a monosaccharide. The cannula was adjusted so that the entire everted segment was immersed. A solution which was identical to the solution contained by the glass test tube was pipetted into the everted segment until the everted segment was filled and the cannula contained a column of fluid not more than 2 cm in height above the level of the fluid contained by the glass test tube. The initial concentration of the monosaccharide on both sides of the intestine was 1/zmole/ml. The inner solution contained in the everted segment and the cannula will be referred to as the serosal solution and the outer solution as the mucosal solution. Unless otherwise indicated, between 10 and 30 ml of atmospheric air/min was passed through the mucosal solution entering by way of the hypodermic needle and leaving by way of the hole in the stopper. The glass test tube containing the intestinal preparation was placed in a constant temperature water bath at 15°C. After a preliminary incubation period of 1 hr the serosal fluid was removed and weighed to determine its volume. Immediately, the cannula with the attached everted intestinal segment was transferred to a new glass chamber containing 32 ml of fresh solution which was identical in composition to the original mucosal solution. A new aliquot of solution, which was identical in composition to the original serosal solution , was pipetted into the cannula and the everted sac for the succeeding experimental period. The experimental periods lasted for 3 or 4 hr,
FIG. 1. Isolated anterior and posterior intestine of an adult Cryptochiton steki 2-3, valve separating anterior intestine (weight = 960 g). 1-2, anterior intestine; from posterior intestine; 3-4, posterior intestine; 4-S rectum. The regions whicfi . A-A‘, proximal anterior intestme; B-B , were used in this study are as follows. distal anterior intestine; C-C’, proximal posterior intestine; D-D’, medial POSterior intestine ; E-E’, distal posterior intestine.
SUGAR ABSORPTION IN CRYPTOCHITON STELLERI
343
with one, two or three such time periods being run back to back on the same gut segments. In anaerobic studies, the first time period was a normal aerobic experimental period, the second experimental period was made anaerobic by replacing aeration with atmospheric air with nitrogen, and the third experimental period was identical to the first. At the end of each experimental period the preceding procedure was followed. The initial serosal and mucosal volumes were identical for all three experimental periods. The formula for the chiton Ringer solution used was NaCI, 0.462 M; MgC12.6H20 , 0.00983 M; KC1, 0.0121 M; MgSO4.7H~O 0.00243 M; CaC12, 0.01135 M; NaHCOs, 0.00238 M. For some experiments estimation of the amount of D-glucose and total carbohydrate present in the serosal and mueosal fluid was made at the end of each time period using chemical methods. The amount of D-glucose was determined colorimetrically by means of the glucose oxidase method. The amount of total carbohydrate was determined by the phenol method of Dubois et al. (1956). All determinations were done in duplicate. All solutions were deproteinized using zinc sulfate and barium hydroxide before the carbohydrate analyses were done. The movement of monosaccharides across the intestine for the remaining experiments was followed using the following labeled compounds: D-glucose-U-t4C obtained from California Corporation for Biochemical Research; 3-O-I4CHa-D glucose, D-galactose-l-14C, D-mannose-1-14C and D-fructose-UJ4C were obtained from New England Nuclear Corporation. The initial level of 14C activity on both sides of the intestine was 0.019/~c/ml. Terminal 100-1ambda samples were taken for each time period of both serosal and mucosal fluids. The samples were evaporated to dryness and activity levels determined using a gas flow GeigerMueller counter. All terminal serosal contents were weighed to determine their final volumes. RESULTS Initial studies included measurement of loss of D-glucose and other carbohydrates by the gut segment into sugar-free incubation media. Only very small quantities of D-glucose or other carbohydrates appeared in the medium at the end of the experimental periods (Table 1). Thus the results obtained using chemical methods were not significantly affected by loss of endogenous sugars from the isolated gut segments. There was no indication of net movement of water between serosal and mucosal compartments in any of the experiments. Mapping Mapping experiments using D-glucose indicate that S/M ratios > 1.0 were established in the proximal anterior intestine (Table 2). These gradients were not observed in any region of the posterior intestine. On the contrary, terminal serosal concentrations were much lower than terminal mueosal concentrations, causing S/M ratios to be surprisingly small fractions. Also, no significant amounts of D-glucose were converted to other carbohydrates during the movement of D-glucose
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A . L . LAWRENCEAND D. C. LAWRENCE
TABLE 1--APPEARANCE OF SUGARS IN THE SEROSAL AND MUCOSAL COMPARTMENTS WHEN INITIALLY NO SUGARSWERE PRESENT IN THE INCUBATIONFLUID Terminal D-glucose concentration (/zmole/ml) Time period
Terminal total carbohydrate concentration (/zmole/ml)
Serosal
Mucosal
Serosal
Mucosal
<0"04
6-9
0'10 +0"06 0"09 + 0-07 <0.07
0"05 +0"02 0"04 + 0.02 <0.02
9-12
<0.07
<0.04
0"18 +0"10 0"12 +_0.08 0.07 + 0.03 <0.05
0-3 3-6
<0"04 <0.04
<0.02
Samples were taken for four consecutive 3-hr experimental periods. Segments represented the proximal anterior intestine. Values represent means + S.E.M. for five observations. Values were obtained using chemical methods. T A B L E 2 - - A B s o R P T I O N OF D-GLUCOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal D-glucose concentration (/~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Distal posterior intestine
Terminal total carbohydrate concentration (~mole/ml)
Serosal
Mucosal
S/M ratio
Serosal
Mucosal
S/M ratio
1-35 +0"13 0.88 + 0.02 0"33 + 0.07 0'77 _+0"06
0"84 +0"13 0"97 + 0.01 0"96 + 0"01 0"95 + 0"02
1"60 _+0"25 0"09 _+0.02 0"34 + 0-07 0"81 + 0"07
1"60 +0"10 0-93 + 0"03 0'48 +_0"09 1 '05 + 0'10
0"93 +0-03 1"07 + 0.03 1 "07 + 0"03 1 '06 + 0'01
1 '72 +0"03 0"87 + 0"05 0"39 + 0"09 0"99 + 0.09
Values represent means +_S.E.M. for six observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1-0/~mole/ml. Incubation period was 3 hr. S / M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. across t h e g u t as t h e t e r m i n a l serosal a n d m u c o s a l D-glucose c o n c e n t r a t i o n s are n o t significantly different from the terminal total carbohydrate concentrations. To d e m o n s t r a t e t h a t t h e D-glucose m o l e c u l e s a c c u m u l a t e d o n t h e serosal side d i d indeed come from the mucosal compartment, the preceding experiments were r e p e a t e d u s i n g D - g l u c o s e - U - l ~ C . A s s e e n in T a b l e 3, l a b e l e d D-glucose-U-~4C d i d a c c u m u l a t e o n t h e serosal side of p r o x i m a l a n t e r i o r s e g m e n t s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . S i g n i f i c a n t a m o u n t s o f r a d i o a c t i v e D-glucose w e r e lost f r o m t h e i n c u b a t i o n fluids d u r i n g t h e 3 - h r e x p e r i m e n t a l p e r i o d .
345
SUGAR ABSORPTION IN C R Y P T O C H I T O N S T E L L E R 1 T A B L E 3 - - A B S O R P T I O N OF D-GLUCOSE BY DIFFERENT REGIONS OF THE CHITON OUT
Terminal concentration ~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
Serosal
Mucosal
1 "35 __0"33 0"98 _+0"10 0"17 +_0.01 0"15 +_0"01 0"55 _+0" 18
0"82 + 0"04 0-91 _+0"02 0"97 +_0.02 0"92 _+0"00 0"87 +_0"00
S / M ratio 1 "74 + 0-44 1"09 + 0"05 0-18 +_0.01 0"17 _+0"01 0-63 + 0"21
Values represent means +_S.E.M. for two observations. Values were obtained using 14C-labeled D-glucose. Initial serosal and mucosal D-glucose concentrations were 1 "0/~mole/ ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. A s s e e n in T a b l e 4, 3 - o - m e t h y l g l u c o s e was a b s o r b e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t in t h e a n t e r i o r i n t e s t i n e , w i t h t h e o p t i m a l r e g i o n of a b s o r p t i o n b e i n g t h e p r o x i m a l half. O n c e again, active a b s o r p t i o n d i d n o t o c c u r in t h e p o s t e r i o r i n t e s tine. T h e r e was no loss o f 3 - o - m e t h y l g l u c o s e d u e to m e t a b o l i s m as no significant TABLE 4
A B S O R P T I O N OF 3-O-METHYLGLUCOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration ~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
(6) (5) (4) (4) (4)
Serosal
Mucosal
1-71 + 0.28 1-60 + 0"17 0"92 + 0"05 0.98 + 0.01 0.81 + 0"04
0-90 + 0"02 0.96 + 0.04 0.97 + 0.05 0.99 + 0.04 0.92 + 0.04
S / M ratio 1"91 + 0-10 1 "64 + 0"18 0"94 + 0-02 0.99 + 0"05 0'88 _+0"01
Values represent means _+S.E.M. Numbers in parentheses represent number of observations. Values were obtained using 14C-labeled-3-o-methylglucose. Initial serosal and mucosal 3-o-methylglucose concentrations were 1-0/~mole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of 3-o-methylglucose in serosal and mucosal media.
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A. L. LAWRENCEAND D. C. LAWRENCE
amount of radioactive 3-o-methylglucose was lost from the incubation fluids during the 3-hr experimental period. The results obtained using n-galactose as a test compound differed from those using D-glucose and 3-o-methylglucose. In the mapping studies for D-galactose (Table 5), there were no S/M ratios > 1.0 in the anterior intestine. S/M ratios > 1.0 were observed, however, in the posterior intestine. Optimal absorption T A B L E 5 - - A B s o R P T I O N OF D-GALACTOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration (/zmole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
(4) (3) (6) (6) (6)
Serosal
Mucosal
0.77 + 0"03 0-69 +_0"04 1.91 _+0.20 1'48 + 0'13 1"52 _+0.08
0"99 + 0"03 1"03 + 0-01 0.96 _+0.02 0"99 + 0-01 0-99 +_0-03
S/M ratio 0.78 +_0.04 0"67 _+0"04 2.00 _+0.24 1-50 _+0"13 1"55 _+0"10
Values represent means _+S.E.M. Numbers in parentheses represent the number of observations. Values were obtained using 1'C-labelled n-galactose. Initial serosal and mucosal v-galactose concentrations were 1.0/~mole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media.
against an apparent concentration gradient occurred in the proximal portion of the posterior intestine. The data in Table 6, based on chemical methods, also indicate that D-galactose is not actively transported or converted to D-glucose T A B L E 6 - - A B s o R P T I O N OF D-GALACTOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration (prnole/ml)
Terminal total carbohydrate concentration (/xmole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
< 0"12
< 0.06
--
6-9
<0-12
<0-06
--
9-12
<0-12
<0.06
--
Serosal
Musocal
S/M ratio
0"93 + 0"08 0.87 + 0.04. 0.98 + 0.04
0'98 + 0"05 0.99 + 0.05 1.05 + 0.01
0.95 +_0.05 0.91 + 0.05 0.91 + 0.03
Values represent means + S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-galactoae concentrations were 1"0 pmole/ ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media.
SUGAR ABSORPTION IN CRYPTOCHITON STELLERI
347
during three consecutive 3-hr periods by the proximal anterior intestine. As the percentage recovery of D-galactose from the incubation fluids was much greater than the percentage recovery for D-glucose after a 3-hr incubation, D-galactose is not metabolized by the chiton intestine as well as D-glucose is. D-fructose and D-mannose are not accumulated against an apparent concentration gradient in any portion of the chiton intestine (Tables 7 and 8). As seen by TABLE 7--ABsoRPTION
OF D-FRUCTOSE BY DIFFERENT REGIONS OF THE C H I T O N OUT
Terminal concentration (gmole/ml) Region tested
Serosal
Mucosal
Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
0-59 + 0"01 0"61 +_0.05 0"59 + 0"01 0"53 _+0.01 0"54 _+0.03
0.99 __0-02 0-99 + 0-01 0.99 _+0.02 0.98 +_0.03 1.04 + 0.03
S / M ratio 0-60 _+0.00 0"61 _+0-04 0-59 _+0"01 0-54 _+0.00 0"54 _+0-04
Values represent means + S.E.M. for two observations. Values were obtained using 14C-labelled D-fructose. Initial serosal and mucosal v-fructose concentrations were 1"0 /xmole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-fructose in serosal and mucosal media. TABLE 8~ABsoRPTION
OF D-MANNOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration (ftmole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
Serosal
Mucosal
0"45 + 0"20 0.49 +0"19 0-36 + 0"06 0"18 + 0.05 0"39 + 0" 15
0.95 + 0.03 0.93 +0-01 0"97 + 0"01 0"96 + 0.02 1"10 + 0-01
S / M ratio 0.49 + 0"22 0"63 +0"13 0"38 + 0"07 0"18 + 0.03 0"38 + 0-15
Values represent means + S.E.M. for two observations. Values were obtained using 14C-labeled D-mannose. Initial serosal and mucosal D-mannose concentrations were 1.0 pmole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-mannose in serosal and mucosal media.
348
A. L. LAWRENCE AND D. C. LAWRENCE
comparing terminal D-glucose concentrations in Tables 9 and 10 with those in Table 1, D-fructose and D-mannose are not converted to D-glucose by the proximal region of the anterior intestine. T h e terminal total carbohydrate concentrations in Tables 9 and 10 indicate that neither D-fructose or D-mannose is actively transported in any of the three consecutive 3-hr periods. T h e r e was a significant loss of D-fructose and D-mannose from the incubation fluids for all regions of the intestine (Tables 7 and 8). This suggests that D-fructose and D-mannose can be metabolized by chiton intestinal tissue. T A B L E 9 ~ A B S O R P T I O N OF D-FRUCTOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration (ttmole/ml)
Terminal total carbohydrate concentration (#mole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
<0"12
<0"06
--
6-9
<0"12
<0.06
--
9-12
<0"12
<0.06
--
Serosal
Mucosal
S/M ratio
0"78 +_0"05 0.72 + 0.07 0.93 + 0"03
0"89 _+0.14 0"85 _+0"07 0"94 + 0.04
0.93 _+0"17 0.85 _+0.09 0"98 -+0"03
Values represent means _+S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-fructose concentrations were 1"0 pmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-fructose in serosal and mucosal media. T A B L E 10----ABgORPTION OF D-MANNOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration ~mole/ml)
Terminal total carbohydrate concentration (/~mole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
<0"12
<0.06
--
6-9
<0-12
<0.06
--
9-12
<0"12
<0-06
--
Serosal
Musocal
S/M ratio
0.66 _+0"07 0"78 -2-_0.21 0'66 + 0.06
0"88 _+0"08 0-85 _+0.09 0.93 _+0.03
0"76 +0.13 0.93 _+0.18 0-70 _+0"05
Values represent means + S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-mannose concentrations were 1.0/~mole/ ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of Dmannose in serosal and mucosal media.
Duration T h e proximal anterior intestine, singled out for further D-glucose studies, was capable of in vitro active absorption for periods of 12 hr (Table 11). F u r t h e r
SUGARABSORPTIONIN C R Y P T O C H I T O N
349
STELLERI
TABLE 11--ABSORPTIONOF D-GLUCOSEBY THE PROXIMALREGIONOF THE ANTERIORINTESTINE
Terminal D-glucose concentration (/zmole/rnl) Time period 0-3
(2)
3-6
(14)
6-9
(14)
9-12 (11)
Terminal total carbohydrate concentration (p.mole/ml)
Serosal
Mucosal
S/M ratio
Serosal
Mucosal
S/M ratio
0-79 + 0"05 1"52 +0-15 1.65 +0"12 1"59 + 0.27
0"84 + 0"01 0"77 +0"01 0-76 +0"01 0"77 + 0"01
0-93 + 0"05 2"00 +0-19 2"19 +0"20 2"11 + 0"26
1-08 + 0"06 1"70 +0"12 1-95 +0"14 1"99 + 0"20
0-95 + 0"01 0"91 +0"01 0-85 +0"01 0"91 + 0"01
1"10 + 0-08 1"90 +0"15 2"31 +0"18 2"21 + 0"25
Values represent means + S.E.M. The numbers in parentheses represent the number of observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1"0 ftmole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. duration studies in the anterior gut were done using 3-o-methylglucose, so that results could be obtained which would not be complicated by the effects of metabolism. I n the 12-hr period of the duration studies, serial samples demonstrated that S / M ratios developed in the absorption of 3-o-methylglucose increased linearly for at least 12 hr. It will be noted (Fig. 2) that since no preincubation
~.o-
j
,~.
10.785 +0"145 (T)
1.8
<>-
L.6!
/
1.2 I'OIf 0.8 0.6 0-4 0.2
0
I I I I I I I I I I11 4
6 T,
8
12
hr
FIG. 2. Regression curve (solid line) for the increment of S/M ratio with time using 3-o-methylglucose in the proximal anterior intestine. Initial serosal and mucosal 3-o-methylglueose concentrations were 1 pmole/mL Each solid circle and vertical bar represent a mean _+S.E.M. for five observations.
A. L. LAWRENCE AND D. C. LAWRENCE
350
period was used, the increase in S/M ratio went through a lag phase. This caused the regression line (Y = 0.783 + 0.145t) to have aY-intercept < 1.0 and an X-intercept of approximately 1.5 hr. As with 3-o-methylglucose, the S/M ratios for D-galactose in the proximal posterior intestine increased linearly with time for at least 12 hr (Fig. 3). The 4.0 m
~B
~48 +0"226 (T) 3.0
-
~
2.0
<>"
1.8
/
1.6 1.4 1.2 1.0
0.8 0.6 0.4-0.2-0
] 1 1 1 14 1 1 161 1 18 T,
12
hr
FIG. 3. Regression curve (soEd Ene) for the increment of S / M ratio with time using D-galactose in the proximal posterior intestine. Initial serosal and mucosal D-galactose concentrations were | pmole/ml. Each solid circle and vertical bar represents a mean +_S.E.M. for five observations.
Y-intercept < 1.0 and X-intercept of approximately 2 hr, indicative of omission of the preincubation period, are seen for the regression curve (Y = 0.48+0"226t). Anaerobic conditions Anaerobic conditions with D-glucose as the test monosaccharide and using proximal anterior intestinal segments resulted in a 57 per cent reduction of the S/M ratios (Table 12). The S/M ratios recovered almost completely when aerobic conditions were restored. The S/M ratios for 3-o-methylglucose were reduced by 28 per cent by anaerobic conditions (Table 13). Aerobic conditions again increased the S/M ratio to 92 per cent of its initial value. When segments of proximal posterior intestine were removed from aerated mucosal solution and placed in nitrogenated mucoaal solution, there was only
3 51
SUGAR ABSORPTION I N CR Y P T O C H I T O N S T E L L E R I
TABLE 12--EFFECT
OF ANAEROBIC CONDITIONS ON ACTIVE TRANSPORT OF D-GLUCOSE
Terminal D-glucose concentration (/~mole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
1-68 +0"08 1-07 +0.09 1"65 +0"13
0"84' +0"01 0.92 +0.02 0"86 +0"02
Terminal total carbohydrate concentration (prnole/ml) S/M ratio 2"01 +0'06 1-16" +0"10 1-93" +0"12
Serosal
Mucosal
S/M ratio
1-82 +0"14 1"06 +0"17 1"68 +0-13
0'93 +0"07 0"93 +0-03 0-88 +0"03
2"01 +0"10 1"07" ___0"15 1"91 * _+0"12
Values represent means _+S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1"0 pmole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. * P < 0"05 (t calculated using paired determinations). T A B L E 1 3 - - E F F E C T OF ANAEROBIC CONDITIONS ON ACTIVE TRANSPORT OF
3-O-METHYLGLUCOSE
Terminal concentration (pmole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
S/M ratio
1"82 +_0"08 1"32 + 0"06 1"64 + 0'07
0"99 + 0"04 0"97 + 0"02 0"98 + 0"01
1"86 + 0"10 1"35 * + 0"06 1"67" + 0"07
Values represent means _+S.E.M. for nineteen observations. Values were obtained using 14C-labeled 3-o-methylglucose. Initial serosal and mucosal 3-o-methylglucose concentrations were 1"0 ftmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of 3-o-methylglucose in serosal and mucosal media. * P < 0.05 for change in condition (t calculated using paired determinations). 8 per cent reduction of the S/M ratio using D-galactose as the test monosaccharide (Table 14). Moreover, this reduction was not reversed by return .to aerobia. Instead, a further reduction of about 3 per cent followed. DISCUSSION T h e development of S / M ratios > 1.0 which are due to net accumulation in the serosal comparUt,ent must be taken as conclusive evidence that active transport occurred. T h u s , D-glucose and 3-o-methylglucose are both actively transported
352
A.L. LAWRENCEAND D. C. LAWRENCE
TABLE 14----EFFECTOF ANAEROBICCONDITIONSON ACTIVETRANSPORTOF D-GALACTOSE Terminal concentration (/~mole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
1.94 + 0.09 1'89 +0'08 1"79 +_0"10
0.93 + 0.02 0"98 +0"02 0"93 +0"02
S/M ratio 2"16 + 0.14 1"98 +0"11 1-95 +0"13
Values represent means + S.E.M. for twenty-two observations. Values were obtained using 14C-labeled D-galactose. Initial serosal and mucosal D-galactose concentrations w e r e 1"0 pmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media. by proximal anterior intestinal segments. It is possible that D-glucose and 3-o-methylglucose are transported by the same carrier mechanism as occurs in vertebrate intestines (Cs~y, 1958). Further, as 3-o-methylglucose is actively transported by distal anterior intestinal segments one has the choice of either assuming that D-glucose also is actively transported by this region or that there are two distinct carrier molecules in the intestine with one being more specific for 3-0methylglucose and located in both regions of the anterior intestine, while the other is more specific for D-glucose and located only in the proximal region of the anterior intestine. Of these two possibilities, the assumption that there is only one carrier in the anterior intestine and that D-glucose also is transported by the distal anterior intestinal segments is the most acceptable. The basis for this is the following. With the in vitro method used, the lesser the rate of movement of the test substance across the gut and the greater it is metabolized by the gut, the smaller the final S/M ratio. The rate of metabolism of D-galactose by distal anterior segments is less than that for D-glucose because the percentage recovery of radioactive D-galactose is greater than that for radioactive D-glucose. Thus, one would explain the significantly smaller S/M ratio obtained for D-galactose than that obtained for D-glucose because the rate of movement of D-galactose into the serosal compartment is less than that of D-glucose in the distal anterior intestinal segments. Because of the structural similarity of D-glucose and D-galactose, this difference in the rate of movement is probably not due to a greater rate of diffusion of D-glucose than D-galactose across the gut, but rather that D-glucose is probably transported by active or facilitative mechanisms by this region of the gut and D-galactose is not. The lack of S/M D-glucose ratios > 1.0 in the distal portion of the anterior intestine may therefore be due to the rate of metabolism being greater than the rate of transport of D-glucose. Whether or not D-glucose is definitely transported by the distal anterior intestinal segments will have to be decided by doing
SUGAR ABSORPTION I N C R Y P T O C H 1 T O N S T E L L E R I
353
kinetic and competitive inhibition studies and using a tissue accumulation technique. However, the startling fact is that D-galactose is actively absorbed by an entirely different region of the chiton intestine; D-galactose is actively absorbed throughout the posterior intestine, with the highest rate in the proximal portion. The regions where large S/M ratios are observed for D-galactose are the same regions which appear to be only slightly permeable to o-glucose. S/M ratios < 1.0 indicate that movement of n-glucose into the serosal compartment lags behind its removal from the serosal compartment due to metabolism. These results alone strongly suggest that there are two different carrier molecules for the transport of o-glucose or 3-o-methylglucose and D-galactose. In contrast to this, all three of these sugars are reported to share the same carrier mechanism in mammalian intestine (Fisher & Parsons, 1953; Riklis et al., 1958; Crane, 1960; Jorgensen et al., 1961; Annegers, 1964). The significance of the existence of more than one active transport mechanism for the absorption of monosaccharides by the chiton gut is great. Inferences which have been made as to the nature and specificity of the absorption mechanism in vertebrates have been made on the basis of the assumption that only one common pathway exists for the active transport of all monosaccharides. The two mechanisms for the active transport of monosaccharides by different regions of the chiton gut indicate that this assumption is not valid for all phyla and offer a rich research opportunity for a fresh viewpoint as to the nature of monosaccharide active transport. The question of how long would be necessary for a maximum S/M ratio to be reached in a mounted gut segment was not answered. The ratios of 3-o-methylglucose and D-galactose with the proximal anterior intestine and proximal posterior intestine respectively were still increasing linearly at the end of 12 hr. However, since in many cases the mounted segments were beginning to appear physically worn at the end of 12 hr, it is probable that the gradients possible in the living organism are beyond the physical limits of this technique. The duration studies did demonstrate the need of a preincubation period of 1-2 hr. In addition, they indicated that the present technique was sufficient for long anaerobic studies and provided a statistical basis for linear extrapolation of S/M or accumulation values from one length of incubation time to another. The primary interest in the anaerobic studies was to establish whether or not anaerobic conditions had a specific and reversible effect upon the active mechanisms due to removal of oxidative energy sources. Anaerobic conditions had such an effect upon the net accumulation of o-glucose and 3-o-methylglucose, in the proximal anterior intestine. Transport mechanisms for monosaccharides in vertebrate guts also depend on an oxidative supply of energy (Darlington & Quastel, 1953; Wilson & Wiseman, 1954; Wilson & Vincent, 1955; Cordier & Chanel, 1961; Lluch & Ponz, 1962; Musacchia et al., 1966). Lack of total recovery was very likely due to general, irreversible effects suffered during anaerobic conditions (e.g. tissue death, cellular damage, etc.).
354
A. L. LAWRENCEAND D. C. LAWRENCE
Surprisingly, however, D-galactose transport did not suffer this reversible reduction by removal of oxygen from the mucosal fluid. There is the possibility that anaerobic conditions were not established sufficiently to affect the mechanism. This, however, is improbable for a number of reasons. Metabolism of D-galactose was reduced (note terminal mucosal concentrations in Table 14). In addition, the segments which were used were from the same animals as those used in the anaerobic studies with 3-o-methylglucose (where reversible inhibition occurred), and the experiments were simultaneous. Anaerobic conditions resulted in inhibition of amino acid transport (Greer & Lawrence, unpublished data) and membrane potential (Mailman & Lawrence, unpublished data) in the identical segments which were used for the studies with D-galactose. The results with D-galactose were highly reproducible ( P < 0"01) over all twenty-two animals tested. The 8 per cent decrease of the S/M ratio with D-galactose under anaerobic conditions was not of a reversible nature, but was probably due to general non-specific causes. In view of the present data, the only hypothesis which can be drawn is that active absorption of D-galactose does not derive its energy from oxidative phosphorylation in the chiton intestine. Wilson & Lin (1960) noted that in contrast to the adult, the fetal and new-born rabbit intestine transported sugars and amino acids against a concentration gradient under anaerobic conditions. Bullhead catfish intestinal segments under anaerobic conditions have also been reported to actively transport sugars across the intestinal wall (Musacchia et al., 1964). It is interesting that D-fructose and D-mannose were not converted to D-glucose for absorption by segments from the chiton anterior intestine which actively transports D-glucose. Conversion of D-fructose to D-glucose within the intestinal mucosa has been demonstrated in the guinea pig (Riklis & Quastel, 1958), hamster (Wilson & Vincent, 1955) and frog (Lawrence, 1963). Conversion of D-mannose to D-glucose has been reported for frog intestine (Lawrence, 1963). On the other hand, very little conversion of D-fructose to D-glucose occurs in the intestine of rat (Kiyasu & Chaikoff, 1957) and man (Holdsworth & Dawson, 1965). As this inability to convert D-fructose to D-glucose has been associated with the absence of glucose-6-phosphatase in the rat intestine (Kiyasu & Chaikoff, 1957), it is possible that this enzyme is also lacking in the chiton intestine. Another difference between the chiton gut and the vertebrate gut was the absence of net water movement from the mucosal to serosal compartments with any region of the chiton gut in the presence of D-glucose. T h e presence of D-glucose affects the net movement of water by some regions of vertebrate guts (Fisher & Parsons, 1953; Fullerton & Parsons, 1956; Smyth & Taylor, 1957; Parsons et al., 1958; Barry et al., 1961; Detheridge et al., 1966). T h e degree to which monosaccharides are actively transported by the chiton intestine indicates that the chiton intestine is important in the absorption of monosaccharides and quite possibly organic nutrients in general. Though Fretter (1937) concludes that the chiton digestive gland rather than the intestine is the primary site of absorption, her data do not disagree with the data reported in this paper. This is because the data in Fretter's paper are based upon the ability of the chiton
SUGAR ABSORPTION I N C R Y P T O C H I T O N
STELLERI
355
digestive system to absorb insoluble iron saccharate, soluble iron oxide, blood corpuscles, carmine, emulsion of olive oil stained with Nile Blue sulphate, etc., which does not deal with the problem of active transport. Consequently, both the chiton digestive gland and intestine are important in the absorption of nutrients. T h e question which now arises is what is the relative importance of the digestive gland and intestine in the absorption of nutrients ? In addition, as the data from which other investigators (Potts, 1923; Vonk, 1924; Yonge, 1926a, b; Graham, 1932; Millott, 1937; Graham, 1938; Fretter, 1939; Howells, 1942; Carriker, 1946; Morton, 1956; van W e d , 1961; Martoja, 1961; Allen, 1962; Morse, 1966)have concluded that the digestive gland is the primary organ of absorption in various gastropods and bivalves is based upon similar experimental procedures as Fretter (1937) used, it is quite possible that active transport mechanisms also exist for monosaeeharides in the intestines of the animals of other major molluscan groups. It is surprising that no other direct studies on active transport of nutrients by molluscan guts exist.
REFERENCES ALLENJ. A. (1962) Preliminary experiments on the feeding and excretion of bivalves using Phaeodactylura labelled with sap. ~7. mar. Biol. Ass. U.K. 42, 609-623. ANNECFatSJ. H. (1964) Intestinal absorption of hexoses in the dog. Am. aT. Physiol. 206, 1095-1098. BALLANTINED. & MORTONJ. E. (1956) Filtering, feeding and digestion in the lamellibrancb, Lasaea rubra. J. mar. Biol. Ass. 35, 241-274. BARRYB. A., MATTHEWSJ. & SMYTHD. H. (1961) Transfer of glucose and fluid by different parts of the small intestine of the rat. J. Physiol. 157, 279-288. BiI~V~a A. M. (1950) The digestive mechanism of the European squids Loligo vulgaris, Loligo forbesii, Alloteuthis media, and Alloteuthis subulata. Quart.~Y. microsc. Sci. 91, 1--43. CAmaIK~ M. R. (1946) Observations on the functioning of the alimentary system of the snail Lymnaea stagnalis appressa Say. Biol. Bull., Woods Hole 91, 88-111. CoaDxm~ D. & CHANELJ. (1961) Influence de la tension d'oxyg~ne dans l'air inspirg sur l'absorption intestinale des solutions de pentoses et d'hexoses chez le rat. J. Physiol., Paris 43, 695-698. C~NE R. K. (1960) Mutual inhibition, in vitro, between some actively transported sugars. Biochim. biophys. Acta 45, 477--482. C R ~ R. K. & WILSONT. H. (1958) In vitro method for the study of the rate of intestinal absorption of sugars, a~. appl. Physiol. 12, 145-146. CS/LKY T. Z. (1958) Active intestinal transport of 3-o-methylglucose. Proc. Fourth int. Congr. Biochem., Vienna, p. 80, Pergamon Press, Oxford. DA~LINGTONW. A. & QtTASTELJ. H. (1953) Absorption of sugars from isolated surviving intestine. Archs Biochem. Biophys. 43, 194-207. DETHERIDGEJ. F., MATTH~WSJ. & SM~erHD. H. (1966) The effect of inhibitors on intestinal transfer of glucose and fluid. ~. Physiol. 183, 369-377. DtmoIs M., GILES K. A., H~mTON J. K., REBELSP. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356. FISH~ R. B. & PARSONSD. S. (1953) Galactose absorption from the surving small intestine of the rat. ~7. Physiol. 119, 224-232. Fmrra'ma V. (1937) The structure and function of the alimentary canal of some species of polyplacophora (Mollusea). Trans. R. Soc. Edinb. 59, 119-163.
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FRETTER V. (1939) T h e structure and function of the alimentary canal of some tectibranch molluscs, with note on excretion. Trans. R. Soc. Edinb. 59, 599-646. FULLERTON P. M. & PARSONS D. S. (1956) T h e absorption of sugars and water from rat intestine in vivo. Quart. J. exp. Physiol. 41, 387-397. GRAHAM A. (1932) On the structure and function of the alimentary canal of the limpet. Trans. R. Soc. Edinb. 57, 287-308. GRAHAM A. (1938) T h e structure and function of the alimentary canal of aeoliid molluscs, with a discussion on their nematocysts. Trans. R. Soc. Edinb. 59, 267-307. HIRSCH G. C. (1925) Der Weg des resorbierzen Eisens und des phagocytierten Carmins bei Murex trunculus. Z. vergl. Physiol. 2, 1-22. HOLDSWORTH C. D. & DAWSONA. M. (1965) Absorption of fructose in man. Proc. Soc. exp. Biol. Med. 118, 142-145. HOWELLS H. H. (1942) T h e structure and function of the alimentary canal of Aplysia punctata. Quart. ft. microsc. Sci. 83, 357-397. JORGENSEN C. R., LANDAU B. R. & WILSON T. H. (1961) A common pathway for sugar transport in hamster intestine. Am. ft. Physiol. 200, 111-116. KIYASU J. Y. & Chaikoff I. L. (1957) On the manner of transport of absorbed fructose. ft. biol. Chem. 224, 935-939. LAWRENCE A. L. (1963) Specificity of sugar transport by the small intestine of the bullfrog, Rana catesbeiana. Comp. Biochem. Physiol. 9, 69-73. LLUCH M. & PONZ F. (1962) Influencia de la anoxia sobre la absorci6n activa de azucares por el intestino. Revta. esp. Fisiol. 18, 157-162. MARTOJA M. (1961) Signification fonctionnelle de la glande digestive (htpatopancrtas) de Nassa reticulata L. (Mollusque prosobranche). C. r. Aead. Sci., Paris 252, 1664-1666. MILLOTT N. (1937) On the morphology of the alimentary canal, process of feeding, and physiology of digestion of the nudibranch mollusc ~orunna tomentosa (Cuvier). Phil. Trans. R. Soc. B 228, 173-217. MORSE M. P. (1966) On the structure and function of the digestive system of the nudibranch mollusc Acanthodoris pilosa. Ph.D. Thesis, University of New Hampshire. MORTON J. E. (1956) T h e tidal rhythm and action of the digestive system of the lamellibranch Lasaea rubra, ft. mar. Biol. Ass. U.K. 35, 563-586. MOSACCHIA X. J., WESTHOFF D. D. & NEFF S. S. (1964) Active transport of D-glucose by intestinal segments, in vitro, of Ietalurus nebulosus. Biol. Bull., Woods Hole 126, 291-301. MUSACCHIAX. J., WESTHOFF D. D. & VAN HAAm~ R. (1966) Active transport of D-glucose in intestinal segments, in vitro, of the scup, Stenotomus versicolor, and the puffer, Sphero/des maculatus. Comp. Biochem. Physiol. 17, 93-106. PARSONS B. J., SMYTH D. H. & TAYLOR C. B. (1958) T h e action of phlorrhizin on the intestinal transfer of glucose and water in vitro, ft. Physiol. 144, 387-402. PECZENIK O. (1925) (3ber intracellulare EiweissVerdauung in der Mitteldarmdrtise yon Limnea. Z. vergl. Physiol. 2, 215-225. POTTS F. A. (1923) T h e structure and function of the liver of Teredo, the shipworm. Proc. Camb. phil. Soc. biol. Sci. 1, 1-17. RIKLIS E., HABER B. & QUASTEL J. H. (1958) Absorption of mixtures of sugars by isolated surviving guinea pig intestine. Can. ft. Biochem. Physiol. 36, 373-380. RIKLIS E. & QUASTELJ. H. (1958) Effects of cations on sugar absorption by isolated surviving guinea pig intestine. Can. ft. Biochem. Physiol. 36, 347-362. SMYTH D. H. & TAYLOR C. B. (1957) Transfer of water and solutes b y a n in vitro intestinal preparation, ft. Physiol. 136, 632--648. vAN WELL P. B. (1961) T h e comparative physiology of digestion in molluscs. Am. Zoologist l, 245-252. VONK H. J. (1924) Verdauungsphagocytose bei den Austern. Z. vergl. Physiol. 1, 607-623.
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WILSON T . H. & L I N E . C. C. (1960) Active transport of intestines of fetal and newborn rabbits. A m . ft. Physiol. 199, 1030-1032. WILSON T . H. & VINCENT T. N. (1955) Absorption of sugars in vitro by the intestine of the golden hamster, ft. biol. Chem. 216, 851-866. WILSON T. H. & W l s ~ m ~ G. (1954) T h e use of sacs of everted small intestine for the study of the transference of substances from the mucosal to the serosal surface. J . Physiol. 123, 116-125. YONCE C. M. (1926a) T h e digestive diverticula in the lamellibranchs. Trans. R . Soc. Edinb. 54, 703-718. YONCE C. M. (1926b) Structure and physiology of the organs of feeding and digestion in Ostrea edulis, ft. mar. Biol. Ass. U.K. 14, 295-386.
SUGAR ABSORPTION IN THE INTESTINE OF THE CHITON, CR Y P T O C H I T O N S T E L L E R I * A. L. LAWRENCE and D. C. LAWRENCE Biology Department, University of Houston, Houston, Texas 77004, U.S.A., and Hopkins Marine Station, Pacific Grove, California, U.S.A. (Received 7 February 1967)
Abstract--1. Intestinal absorption of various monosaccharides was studied
in the chiton, Cryptochiton stelleri, using an everted sac technique. 2. D-Glucose and 3-o-methylglucose were actively transported by the anterior intestine but D-galactose was actively transported only by the posterior intestine. 3. D-Mannose and D-fructose were not actively absorbed by any region of the chiton intestine. 4. D-Fructose, D-marmoseand D-galactosewere not absorbed by the anterior intestine by being converted to D-glucose. 5. Duration studies showed that the everted sac preparation supported active transport of D-glucose, D-galactose and 3-o-methylglucose for 12 hr. 6. Active transport of D-glucose and 3-o-methylglucose was reversibly inhibited by anaerobic conditions. 7. There was no significant inhibition of active transport of D-galactose by anaerobic conditions. INTRODUCTION T~E ABSORPTIONof nutrients is an important physiological function of the gut of animals. Active transport, as a mechanism for increasing the rate of absorption and of concentrating a substance, is a significant characteristic of vertebrate guts. Although active transport for many organic molecules has been studied extensively in vertebrate intestines, it has not been demonstrated in the gut of many invertebrate phyla including the Mollusca. In fact, the principal site of absorption of nutrients in molluscs has been thought not to be in the gut but in the digestive gland. This conclusion has been reached in studies on bivalves (Potts, 1923; Vonk, 1924; Yonge, 1926 a, b; Morton, 1956; Allen, 1962), chitons (Fretter, 1937) and on gastropods (Peczenik, 1925 ; Hirsch, 1925 ; Graham, 1932; Millott, 1937; Graham, 1938; Fretter, 1939; Howells, 1942; Carriker, 1946; van Weel, 1961; Martoja, 1961 ; Morse, 1966). Only Bidder (1950) has suggested that the absorption of nutrients occurred in the caecal sac and intestine and not in the liver or pancreas in four species of cephalopods. * This investigation was supported in part by USPH Grant 2-F2-GM-8812 and NSF Grant GB-3256 to A. L. Lawrence. 341
342
A.L. LAWRENCEAND D. C. LAWRENCE
This study demonstrates the absorption of several monosaccharides by active transport mechanisms in the intestine of the chiton, Cryptochiton stelleri. MATERIALS AND METHODS Chitons (Cryptochiton stellen) of both sexes and weighing between 650 and 1200 g were obtained from the Pacific Ocean near Monterey, California (37 ° 11' N., 122 ° 24' W.). The animals were maintained in the laboratory at 5°C in aerated seawater for at least 1 week before they were used. The procedure used for the in vitro study of sugar transport was a modification of the method described by Crane & Wilson (1958). The apparatus consisted of a glass test tube with an enlarged top and a twoholed rubber stopper with a glass cannula. A hypodermic needle was forced through the stopper at a slight angle and a length of polyethylene tubing was attached to the needle. The anterior and posterior intestine and the associated digestive gland were removed from the animal and placed in chiton Ringer solution. The anterior and posterior intestine was then isolated, washed out with chiton Ringer at 150C and everted over a glass rod. Five segments were then taken from the intestine which represented proximal anterior intestine, distal anterior intestine, proximal posterior intestine, medial posterior intestine and distal posterior intestine (Fig. 1). All segments were approximately 6-10 cm in length. One end of the everted segment was tied to the cannula and the other end to a glass weight. The cannula with the attached everted segment was placed in the glass test tube which contained 32 ml of chiton Ringer (15°C) and a monosaccharide. The cannula was adjusted so that the entire everted segment was immersed. A solution which was identical to the solution contained by the glass test tube was pipetted into the everted segment until the everted segment was filled and the cannula contained a column of fluid not more than 2 cm in height above the level of the fluid contained by the glass test tube. The initial concentration of the monosaccharide on both sides of the intestine was 1/zmole/ml. The inner solution contained in the everted segment and the cannula will be referred to as the serosal solution and the outer solution as the mucosal solution. Unless otherwise indicated, between 10 and 30 ml of atmospheric air/min was passed through the mucosal solution entering by way of the hypodermic needle and leaving by way of the hole in the stopper. The glass test tube containing the intestinal preparation was placed in a constant temperature water bath at 15°C. After a preliminary incubation period of 1 hr the serosal fluid was removed and weighed to determine its volume. Immediately, the cannula with the attached everted intestinal segment was transferred to a new glass chamber containing 32 ml of fresh solution which was identical in composition to the original mucosal solution. A new aliquot of solution, which was identical in composition to the original serosal solution , was pipetted into the cannula and the everted sac for the succeeding experimental period. The experimental periods lasted for 3 or 4 hr,
FIG. 1. Isolated anterior and posterior intestine of an adult Cryptochiton steki 2-3, valve separating anterior intestine (weight = 960 g). 1-2, anterior intestine; from posterior intestine; 3-4, posterior intestine; 4-S rectum. The regions whicfi . A-A‘, proximal anterior intestme; B-B , were used in this study are as follows. distal anterior intestine; C-C’, proximal posterior intestine; D-D’, medial POSterior intestine ; E-E’, distal posterior intestine.
SUGAR ABSORPTION IN CRYPTOCHITON STELLERI
343
with one, two or three such time periods being run back to back on the same gut segments. In anaerobic studies, the first time period was a normal aerobic experimental period, the second experimental period was made anaerobic by replacing aeration with atmospheric air with nitrogen, and the third experimental period was identical to the first. At the end of each experimental period the preceding procedure was followed. The initial serosal and mucosal volumes were identical for all three experimental periods. The formula for the chiton Ringer solution used was NaCI, 0.462 M; MgC12.6H20 , 0.00983 M; KC1, 0.0121 M; MgSO4.7H~O 0.00243 M; CaC12, 0.01135 M; NaHCOs, 0.00238 M. For some experiments estimation of the amount of D-glucose and total carbohydrate present in the serosal and mueosal fluid was made at the end of each time period using chemical methods. The amount of D-glucose was determined colorimetrically by means of the glucose oxidase method. The amount of total carbohydrate was determined by the phenol method of Dubois et al. (1956). All determinations were done in duplicate. All solutions were deproteinized using zinc sulfate and barium hydroxide before the carbohydrate analyses were done. The movement of monosaccharides across the intestine for the remaining experiments was followed using the following labeled compounds: D-glucose-U-t4C obtained from California Corporation for Biochemical Research; 3-O-I4CHa-D glucose, D-galactose-l-14C, D-mannose-1-14C and D-fructose-UJ4C were obtained from New England Nuclear Corporation. The initial level of 14C activity on both sides of the intestine was 0.019/~c/ml. Terminal 100-1ambda samples were taken for each time period of both serosal and mucosal fluids. The samples were evaporated to dryness and activity levels determined using a gas flow GeigerMueller counter. All terminal serosal contents were weighed to determine their final volumes. RESULTS Initial studies included measurement of loss of D-glucose and other carbohydrates by the gut segment into sugar-free incubation media. Only very small quantities of D-glucose or other carbohydrates appeared in the medium at the end of the experimental periods (Table 1). Thus the results obtained using chemical methods were not significantly affected by loss of endogenous sugars from the isolated gut segments. There was no indication of net movement of water between serosal and mucosal compartments in any of the experiments. Mapping Mapping experiments using D-glucose indicate that S/M ratios > 1.0 were established in the proximal anterior intestine (Table 2). These gradients were not observed in any region of the posterior intestine. On the contrary, terminal serosal concentrations were much lower than terminal mueosal concentrations, causing S/M ratios to be surprisingly small fractions. Also, no significant amounts of D-glucose were converted to other carbohydrates during the movement of D-glucose
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A . L . LAWRENCEAND D. C. LAWRENCE
TABLE 1--APPEARANCE OF SUGARS IN THE SEROSAL AND MUCOSAL COMPARTMENTS WHEN INITIALLY NO SUGARSWERE PRESENT IN THE INCUBATIONFLUID Terminal D-glucose concentration (/zmole/ml) Time period
Terminal total carbohydrate concentration (/zmole/ml)
Serosal
Mucosal
Serosal
Mucosal
<0"04
6-9
0'10 +0"06 0"09 + 0-07 <0.07
0"05 +0"02 0"04 + 0.02 <0.02
9-12
<0.07
<0.04
0"18 +0"10 0"12 +_0.08 0.07 + 0.03 <0.05
0-3 3-6
<0"04 <0.04
<0.02
Samples were taken for four consecutive 3-hr experimental periods. Segments represented the proximal anterior intestine. Values represent means + S.E.M. for five observations. Values were obtained using chemical methods. T A B L E 2 - - A B s o R P T I O N OF D-GLUCOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal D-glucose concentration (/~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Distal posterior intestine
Terminal total carbohydrate concentration (~mole/ml)
Serosal
Mucosal
S/M ratio
Serosal
Mucosal
S/M ratio
1-35 +0"13 0.88 + 0.02 0"33 + 0.07 0'77 _+0"06
0"84 +0"13 0"97 + 0.01 0"96 + 0"01 0"95 + 0"02
1"60 _+0"25 0"09 _+0.02 0"34 + 0-07 0"81 + 0"07
1"60 +0"10 0-93 + 0"03 0'48 +_0"09 1 '05 + 0'10
0"93 +0-03 1"07 + 0.03 1 "07 + 0"03 1 '06 + 0'01
1 '72 +0"03 0"87 + 0"05 0"39 + 0"09 0"99 + 0.09
Values represent means +_S.E.M. for six observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1-0/~mole/ml. Incubation period was 3 hr. S / M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. across t h e g u t as t h e t e r m i n a l serosal a n d m u c o s a l D-glucose c o n c e n t r a t i o n s are n o t significantly different from the terminal total carbohydrate concentrations. To d e m o n s t r a t e t h a t t h e D-glucose m o l e c u l e s a c c u m u l a t e d o n t h e serosal side d i d indeed come from the mucosal compartment, the preceding experiments were r e p e a t e d u s i n g D - g l u c o s e - U - l ~ C . A s s e e n in T a b l e 3, l a b e l e d D-glucose-U-~4C d i d a c c u m u l a t e o n t h e serosal side of p r o x i m a l a n t e r i o r s e g m e n t s a g a i n s t a c o n c e n t r a t i o n g r a d i e n t . S i g n i f i c a n t a m o u n t s o f r a d i o a c t i v e D-glucose w e r e lost f r o m t h e i n c u b a t i o n fluids d u r i n g t h e 3 - h r e x p e r i m e n t a l p e r i o d .
345
SUGAR ABSORPTION IN C R Y P T O C H I T O N S T E L L E R 1 T A B L E 3 - - A B S O R P T I O N OF D-GLUCOSE BY DIFFERENT REGIONS OF THE CHITON OUT
Terminal concentration ~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
Serosal
Mucosal
1 "35 __0"33 0"98 _+0"10 0"17 +_0.01 0"15 +_0"01 0"55 _+0" 18
0"82 + 0"04 0-91 _+0"02 0"97 +_0.02 0"92 _+0"00 0"87 +_0"00
S / M ratio 1 "74 + 0-44 1"09 + 0"05 0-18 +_0.01 0"17 _+0"01 0-63 + 0"21
Values represent means +_S.E.M. for two observations. Values were obtained using 14C-labeled D-glucose. Initial serosal and mucosal D-glucose concentrations were 1 "0/~mole/ ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. A s s e e n in T a b l e 4, 3 - o - m e t h y l g l u c o s e was a b s o r b e d a g a i n s t a c o n c e n t r a t i o n g r a d i e n t in t h e a n t e r i o r i n t e s t i n e , w i t h t h e o p t i m a l r e g i o n of a b s o r p t i o n b e i n g t h e p r o x i m a l half. O n c e again, active a b s o r p t i o n d i d n o t o c c u r in t h e p o s t e r i o r i n t e s tine. T h e r e was no loss o f 3 - o - m e t h y l g l u c o s e d u e to m e t a b o l i s m as no significant TABLE 4
A B S O R P T I O N OF 3-O-METHYLGLUCOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration ~mole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
(6) (5) (4) (4) (4)
Serosal
Mucosal
1-71 + 0.28 1-60 + 0"17 0"92 + 0"05 0.98 + 0.01 0.81 + 0"04
0-90 + 0"02 0.96 + 0.04 0.97 + 0.05 0.99 + 0.04 0.92 + 0.04
S / M ratio 1"91 + 0-10 1 "64 + 0"18 0"94 + 0-02 0.99 + 0"05 0'88 _+0"01
Values represent means _+S.E.M. Numbers in parentheses represent number of observations. Values were obtained using 14C-labeled-3-o-methylglucose. Initial serosal and mucosal 3-o-methylglucose concentrations were 1-0/~mole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of 3-o-methylglucose in serosal and mucosal media.
346
A. L. LAWRENCEAND D. C. LAWRENCE
amount of radioactive 3-o-methylglucose was lost from the incubation fluids during the 3-hr experimental period. The results obtained using n-galactose as a test compound differed from those using D-glucose and 3-o-methylglucose. In the mapping studies for D-galactose (Table 5), there were no S/M ratios > 1.0 in the anterior intestine. S/M ratios > 1.0 were observed, however, in the posterior intestine. Optimal absorption T A B L E 5 - - A B s o R P T I O N OF D-GALACTOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration (/zmole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
(4) (3) (6) (6) (6)
Serosal
Mucosal
0.77 + 0"03 0-69 +_0"04 1.91 _+0.20 1'48 + 0'13 1"52 _+0.08
0"99 + 0"03 1"03 + 0-01 0.96 _+0.02 0"99 + 0-01 0-99 +_0-03
S/M ratio 0.78 +_0.04 0"67 _+0"04 2.00 _+0.24 1-50 _+0"13 1"55 _+0"10
Values represent means _+S.E.M. Numbers in parentheses represent the number of observations. Values were obtained using 1'C-labelled n-galactose. Initial serosal and mucosal v-galactose concentrations were 1.0/~mole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media.
against an apparent concentration gradient occurred in the proximal portion of the posterior intestine. The data in Table 6, based on chemical methods, also indicate that D-galactose is not actively transported or converted to D-glucose T A B L E 6 - - A B s o R P T I O N OF D-GALACTOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration (prnole/ml)
Terminal total carbohydrate concentration (/xmole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
< 0"12
< 0.06
--
6-9
<0-12
<0-06
--
9-12
<0-12
<0.06
--
Serosal
Musocal
S/M ratio
0"93 + 0"08 0.87 + 0.04. 0.98 + 0.04
0'98 + 0"05 0.99 + 0.05 1.05 + 0.01
0.95 +_0.05 0.91 + 0.05 0.91 + 0.03
Values represent means + S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-galactoae concentrations were 1"0 pmole/ ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media.
SUGAR ABSORPTION IN CRYPTOCHITON STELLERI
347
during three consecutive 3-hr periods by the proximal anterior intestine. As the percentage recovery of D-galactose from the incubation fluids was much greater than the percentage recovery for D-glucose after a 3-hr incubation, D-galactose is not metabolized by the chiton intestine as well as D-glucose is. D-fructose and D-mannose are not accumulated against an apparent concentration gradient in any portion of the chiton intestine (Tables 7 and 8). As seen by TABLE 7--ABsoRPTION
OF D-FRUCTOSE BY DIFFERENT REGIONS OF THE C H I T O N OUT
Terminal concentration (gmole/ml) Region tested
Serosal
Mucosal
Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
0-59 + 0"01 0"61 +_0.05 0"59 + 0"01 0"53 _+0.01 0"54 _+0.03
0.99 __0-02 0-99 + 0-01 0.99 _+0.02 0.98 +_0.03 1.04 + 0.03
S / M ratio 0-60 _+0.00 0"61 _+0-04 0-59 _+0"01 0-54 _+0.00 0"54 _+0-04
Values represent means + S.E.M. for two observations. Values were obtained using 14C-labelled D-fructose. Initial serosal and mucosal v-fructose concentrations were 1"0 /xmole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-fructose in serosal and mucosal media. TABLE 8~ABsoRPTION
OF D-MANNOSE BY DIFFERENT REGIONS OF THE CHITON GUT
Terminal concentration (ftmole/ml) Region tested Proximal anterior intestine Distal anterior intestine Proximal posterior intestine Medial posterior intestine Distal posterior intestine
Serosal
Mucosal
0"45 + 0"20 0.49 +0"19 0-36 + 0"06 0"18 + 0.05 0"39 + 0" 15
0.95 + 0.03 0.93 +0-01 0"97 + 0"01 0"96 + 0.02 1"10 + 0-01
S / M ratio 0.49 + 0"22 0"63 +0"13 0"38 + 0"07 0"18 + 0.03 0"38 + 0-15
Values represent means + S.E.M. for two observations. Values were obtained using 14C-labeled D-mannose. Initial serosal and mucosal D-mannose concentrations were 1.0 pmole/ml. Incubation period was 4 hr. S / M ratio calculated from terminal concentrations of D-mannose in serosal and mucosal media.
348
A. L. LAWRENCE AND D. C. LAWRENCE
comparing terminal D-glucose concentrations in Tables 9 and 10 with those in Table 1, D-fructose and D-mannose are not converted to D-glucose by the proximal region of the anterior intestine. T h e terminal total carbohydrate concentrations in Tables 9 and 10 indicate that neither D-fructose or D-mannose is actively transported in any of the three consecutive 3-hr periods. T h e r e was a significant loss of D-fructose and D-mannose from the incubation fluids for all regions of the intestine (Tables 7 and 8). This suggests that D-fructose and D-mannose can be metabolized by chiton intestinal tissue. T A B L E 9 ~ A B S O R P T I O N OF D-FRUCTOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration (ttmole/ml)
Terminal total carbohydrate concentration (#mole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
<0"12
<0"06
--
6-9
<0"12
<0.06
--
9-12
<0"12
<0.06
--
Serosal
Mucosal
S/M ratio
0"78 +_0"05 0.72 + 0.07 0.93 + 0"03
0"89 _+0.14 0"85 _+0"07 0"94 + 0.04
0.93 _+0"17 0.85 _+0.09 0"98 -+0"03
Values represent means _+S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-fructose concentrations were 1"0 pmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-fructose in serosal and mucosal media. T A B L E 10----ABgORPTION OF D-MANNOSE BY THE PROXIMAL REGION OF THE ANTERIOR INTESTINE
Terminal D-glucose concentration ~mole/ml)
Terminal total carbohydrate concentration (/~mole/ml)
Time period
Serosal
Mucosal
S/M ratio
3-6
<0"12
<0.06
--
6-9
<0-12
<0.06
--
9-12
<0"12
<0-06
--
Serosal
Musocal
S/M ratio
0.66 _+0"07 0"78 -2-_0.21 0'66 + 0.06
0"88 _+0"08 0-85 _+0.09 0.93 _+0.03
0"76 +0.13 0.93 _+0.18 0-70 _+0"05
Values represent means + S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-mannose concentrations were 1.0/~mole/ ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of Dmannose in serosal and mucosal media.
Duration T h e proximal anterior intestine, singled out for further D-glucose studies, was capable of in vitro active absorption for periods of 12 hr (Table 11). F u r t h e r
SUGARABSORPTIONIN C R Y P T O C H I T O N
349
STELLERI
TABLE 11--ABSORPTIONOF D-GLUCOSEBY THE PROXIMALREGIONOF THE ANTERIORINTESTINE
Terminal D-glucose concentration (/zmole/rnl) Time period 0-3
(2)
3-6
(14)
6-9
(14)
9-12 (11)
Terminal total carbohydrate concentration (p.mole/ml)
Serosal
Mucosal
S/M ratio
Serosal
Mucosal
S/M ratio
0-79 + 0"05 1"52 +0-15 1.65 +0"12 1"59 + 0.27
0"84 + 0"01 0"77 +0"01 0-76 +0"01 0"77 + 0"01
0-93 + 0"05 2"00 +0-19 2"19 +0"20 2"11 + 0"26
1-08 + 0"06 1"70 +0"12 1-95 +0"14 1"99 + 0"20
0-95 + 0"01 0"91 +0"01 0-85 +0"01 0"91 + 0"01
1"10 + 0-08 1"90 +0"15 2"31 +0"18 2"21 + 0"25
Values represent means + S.E.M. The numbers in parentheses represent the number of observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1"0 ftmole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. duration studies in the anterior gut were done using 3-o-methylglucose, so that results could be obtained which would not be complicated by the effects of metabolism. I n the 12-hr period of the duration studies, serial samples demonstrated that S / M ratios developed in the absorption of 3-o-methylglucose increased linearly for at least 12 hr. It will be noted (Fig. 2) that since no preincubation
~.o-
j
,~.
10.785 +0"145 (T)
1.8
<>-
L.6!
/
1.2 I'OIf 0.8 0.6 0-4 0.2
0
I I I I I I I I I I11 4
6 T,
8
12
hr
FIG. 2. Regression curve (solid line) for the increment of S/M ratio with time using 3-o-methylglucose in the proximal anterior intestine. Initial serosal and mucosal 3-o-methylglueose concentrations were 1 pmole/mL Each solid circle and vertical bar represent a mean _+S.E.M. for five observations.
A. L. LAWRENCE AND D. C. LAWRENCE
350
period was used, the increase in S/M ratio went through a lag phase. This caused the regression line (Y = 0.783 + 0.145t) to have aY-intercept < 1.0 and an X-intercept of approximately 1.5 hr. As with 3-o-methylglucose, the S/M ratios for D-galactose in the proximal posterior intestine increased linearly with time for at least 12 hr (Fig. 3). The 4.0 m
~B
~48 +0"226 (T) 3.0
-
~
2.0
<>"
1.8
/
1.6 1.4 1.2 1.0
0.8 0.6 0.4-0.2-0
] 1 1 1 14 1 1 161 1 18 T,
12
hr
FIG. 3. Regression curve (soEd Ene) for the increment of S / M ratio with time using D-galactose in the proximal posterior intestine. Initial serosal and mucosal D-galactose concentrations were | pmole/ml. Each solid circle and vertical bar represents a mean +_S.E.M. for five observations.
Y-intercept < 1.0 and X-intercept of approximately 2 hr, indicative of omission of the preincubation period, are seen for the regression curve (Y = 0.48+0"226t). Anaerobic conditions Anaerobic conditions with D-glucose as the test monosaccharide and using proximal anterior intestinal segments resulted in a 57 per cent reduction of the S/M ratios (Table 12). The S/M ratios recovered almost completely when aerobic conditions were restored. The S/M ratios for 3-o-methylglucose were reduced by 28 per cent by anaerobic conditions (Table 13). Aerobic conditions again increased the S/M ratio to 92 per cent of its initial value. When segments of proximal posterior intestine were removed from aerated mucosal solution and placed in nitrogenated mucoaal solution, there was only
3 51
SUGAR ABSORPTION I N CR Y P T O C H I T O N S T E L L E R I
TABLE 12--EFFECT
OF ANAEROBIC CONDITIONS ON ACTIVE TRANSPORT OF D-GLUCOSE
Terminal D-glucose concentration (/~mole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
1-68 +0"08 1-07 +0.09 1"65 +0"13
0"84' +0"01 0.92 +0.02 0"86 +0"02
Terminal total carbohydrate concentration (prnole/ml) S/M ratio 2"01 +0'06 1-16" +0"10 1-93" +0"12
Serosal
Mucosal
S/M ratio
1-82 +0"14 1"06 +0"17 1"68 +0-13
0'93 +0"07 0"93 +0-03 0-88 +0"03
2"01 +0"10 1"07" ___0"15 1"91 * _+0"12
Values represent means _+S.E.M. for three observations. Values were obtained using chemical methods. Initial serosal and mucosal D-glucose concentrations were 1"0 pmole/ml. Incubation period was 4 hr. S/M ratio calculated from terminal concentrations of D-glucose in serosal and mucosal media. * P < 0"05 (t calculated using paired determinations). T A B L E 1 3 - - E F F E C T OF ANAEROBIC CONDITIONS ON ACTIVE TRANSPORT OF
3-O-METHYLGLUCOSE
Terminal concentration (pmole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
S/M ratio
1"82 +_0"08 1"32 + 0"06 1"64 + 0'07
0"99 + 0"04 0"97 + 0"02 0"98 + 0"01
1"86 + 0"10 1"35 * + 0"06 1"67" + 0"07
Values represent means _+S.E.M. for nineteen observations. Values were obtained using 14C-labeled 3-o-methylglucose. Initial serosal and mucosal 3-o-methylglucose concentrations were 1"0 ftmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of 3-o-methylglucose in serosal and mucosal media. * P < 0.05 for change in condition (t calculated using paired determinations). 8 per cent reduction of the S/M ratio using D-galactose as the test monosaccharide (Table 14). Moreover, this reduction was not reversed by return .to aerobia. Instead, a further reduction of about 3 per cent followed. DISCUSSION T h e development of S / M ratios > 1.0 which are due to net accumulation in the serosal comparUt,ent must be taken as conclusive evidence that active transport occurred. T h u s , D-glucose and 3-o-methylglucose are both actively transported
352
A.L. LAWRENCEAND D. C. LAWRENCE
TABLE 14----EFFECTOF ANAEROBICCONDITIONSON ACTIVETRANSPORTOF D-GALACTOSE Terminal concentration (/~mole/ml) Condition Aerobic Anaerobic Aerobic
Serosal
Mucosal
1.94 + 0.09 1'89 +0'08 1"79 +_0"10
0.93 + 0.02 0"98 +0"02 0"93 +0"02
S/M ratio 2"16 + 0.14 1"98 +0"11 1-95 +0"13
Values represent means + S.E.M. for twenty-two observations. Values were obtained using 14C-labeled D-galactose. Initial serosal and mucosal D-galactose concentrations w e r e 1"0 pmole/ml. Incubation period was 3 hr. S/M ratio calculated from terminal concentrations of D-galactose in serosal and mucosal media. by proximal anterior intestinal segments. It is possible that D-glucose and 3-o-methylglucose are transported by the same carrier mechanism as occurs in vertebrate intestines (Cs~y, 1958). Further, as 3-o-methylglucose is actively transported by distal anterior intestinal segments one has the choice of either assuming that D-glucose also is actively transported by this region or that there are two distinct carrier molecules in the intestine with one being more specific for 3-0methylglucose and located in both regions of the anterior intestine, while the other is more specific for D-glucose and located only in the proximal region of the anterior intestine. Of these two possibilities, the assumption that there is only one carrier in the anterior intestine and that D-glucose also is transported by the distal anterior intestinal segments is the most acceptable. The basis for this is the following. With the in vitro method used, the lesser the rate of movement of the test substance across the gut and the greater it is metabolized by the gut, the smaller the final S/M ratio. The rate of metabolism of D-galactose by distal anterior segments is less than that for D-glucose because the percentage recovery of radioactive D-galactose is greater than that for radioactive D-glucose. Thus, one would explain the significantly smaller S/M ratio obtained for D-galactose than that obtained for D-glucose because the rate of movement of D-galactose into the serosal compartment is less than that of D-glucose in the distal anterior intestinal segments. Because of the structural similarity of D-glucose and D-galactose, this difference in the rate of movement is probably not due to a greater rate of diffusion of D-glucose than D-galactose across the gut, but rather that D-glucose is probably transported by active or facilitative mechanisms by this region of the gut and D-galactose is not. The lack of S/M D-glucose ratios > 1.0 in the distal portion of the anterior intestine may therefore be due to the rate of metabolism being greater than the rate of transport of D-glucose. Whether or not D-glucose is definitely transported by the distal anterior intestinal segments will have to be decided by doing
SUGAR ABSORPTION I N C R Y P T O C H 1 T O N S T E L L E R I
353
kinetic and competitive inhibition studies and using a tissue accumulation technique. However, the startling fact is that D-galactose is actively absorbed by an entirely different region of the chiton intestine; D-galactose is actively absorbed throughout the posterior intestine, with the highest rate in the proximal portion. The regions where large S/M ratios are observed for D-galactose are the same regions which appear to be only slightly permeable to o-glucose. S/M ratios < 1.0 indicate that movement of n-glucose into the serosal compartment lags behind its removal from the serosal compartment due to metabolism. These results alone strongly suggest that there are two different carrier molecules for the transport of o-glucose or 3-o-methylglucose and D-galactose. In contrast to this, all three of these sugars are reported to share the same carrier mechanism in mammalian intestine (Fisher & Parsons, 1953; Riklis et al., 1958; Crane, 1960; Jorgensen et al., 1961; Annegers, 1964). The significance of the existence of more than one active transport mechanism for the absorption of monosaccharides by the chiton gut is great. Inferences which have been made as to the nature and specificity of the absorption mechanism in vertebrates have been made on the basis of the assumption that only one common pathway exists for the active transport of all monosaccharides. The two mechanisms for the active transport of monosaccharides by different regions of the chiton gut indicate that this assumption is not valid for all phyla and offer a rich research opportunity for a fresh viewpoint as to the nature of monosaccharide active transport. The question of how long would be necessary for a maximum S/M ratio to be reached in a mounted gut segment was not answered. The ratios of 3-o-methylglucose and D-galactose with the proximal anterior intestine and proximal posterior intestine respectively were still increasing linearly at the end of 12 hr. However, since in many cases the mounted segments were beginning to appear physically worn at the end of 12 hr, it is probable that the gradients possible in the living organism are beyond the physical limits of this technique. The duration studies did demonstrate the need of a preincubation period of 1-2 hr. In addition, they indicated that the present technique was sufficient for long anaerobic studies and provided a statistical basis for linear extrapolation of S/M or accumulation values from one length of incubation time to another. The primary interest in the anaerobic studies was to establish whether or not anaerobic conditions had a specific and reversible effect upon the active mechanisms due to removal of oxidative energy sources. Anaerobic conditions had such an effect upon the net accumulation of o-glucose and 3-o-methylglucose, in the proximal anterior intestine. Transport mechanisms for monosaccharides in vertebrate guts also depend on an oxidative supply of energy (Darlington & Quastel, 1953; Wilson & Wiseman, 1954; Wilson & Vincent, 1955; Cordier & Chanel, 1961; Lluch & Ponz, 1962; Musacchia et al., 1966). Lack of total recovery was very likely due to general, irreversible effects suffered during anaerobic conditions (e.g. tissue death, cellular damage, etc.).
354
A. L. LAWRENCEAND D. C. LAWRENCE
Surprisingly, however, D-galactose transport did not suffer this reversible reduction by removal of oxygen from the mucosal fluid. There is the possibility that anaerobic conditions were not established sufficiently to affect the mechanism. This, however, is improbable for a number of reasons. Metabolism of D-galactose was reduced (note terminal mucosal concentrations in Table 14). In addition, the segments which were used were from the same animals as those used in the anaerobic studies with 3-o-methylglucose (where reversible inhibition occurred), and the experiments were simultaneous. Anaerobic conditions resulted in inhibition of amino acid transport (Greer & Lawrence, unpublished data) and membrane potential (Mailman & Lawrence, unpublished data) in the identical segments which were used for the studies with D-galactose. The results with D-galactose were highly reproducible ( P < 0"01) over all twenty-two animals tested. The 8 per cent decrease of the S/M ratio with D-galactose under anaerobic conditions was not of a reversible nature, but was probably due to general non-specific causes. In view of the present data, the only hypothesis which can be drawn is that active absorption of D-galactose does not derive its energy from oxidative phosphorylation in the chiton intestine. Wilson & Lin (1960) noted that in contrast to the adult, the fetal and new-born rabbit intestine transported sugars and amino acids against a concentration gradient under anaerobic conditions. Bullhead catfish intestinal segments under anaerobic conditions have also been reported to actively transport sugars across the intestinal wall (Musacchia et al., 1964). It is interesting that D-fructose and D-mannose were not converted to D-glucose for absorption by segments from the chiton anterior intestine which actively transports D-glucose. Conversion of D-fructose to D-glucose within the intestinal mucosa has been demonstrated in the guinea pig (Riklis & Quastel, 1958), hamster (Wilson & Vincent, 1955) and frog (Lawrence, 1963). Conversion of D-mannose to D-glucose has been reported for frog intestine (Lawrence, 1963). On the other hand, very little conversion of D-fructose to D-glucose occurs in the intestine of rat (Kiyasu & Chaikoff, 1957) and man (Holdsworth & Dawson, 1965). As this inability to convert D-fructose to D-glucose has been associated with the absence of glucose-6-phosphatase in the rat intestine (Kiyasu & Chaikoff, 1957), it is possible that this enzyme is also lacking in the chiton intestine. Another difference between the chiton gut and the vertebrate gut was the absence of net water movement from the mucosal to serosal compartments with any region of the chiton gut in the presence of D-glucose. T h e presence of D-glucose affects the net movement of water by some regions of vertebrate guts (Fisher & Parsons, 1953; Fullerton & Parsons, 1956; Smyth & Taylor, 1957; Parsons et al., 1958; Barry et al., 1961; Detheridge et al., 1966). T h e degree to which monosaccharides are actively transported by the chiton intestine indicates that the chiton intestine is important in the absorption of monosaccharides and quite possibly organic nutrients in general. Though Fretter (1937) concludes that the chiton digestive gland rather than the intestine is the primary site of absorption, her data do not disagree with the data reported in this paper. This is because the data in Fretter's paper are based upon the ability of the chiton
SUGAR ABSORPTION I N C R Y P T O C H I T O N
STELLERI
355
digestive system to absorb insoluble iron saccharate, soluble iron oxide, blood corpuscles, carmine, emulsion of olive oil stained with Nile Blue sulphate, etc., which does not deal with the problem of active transport. Consequently, both the chiton digestive gland and intestine are important in the absorption of nutrients. T h e question which now arises is what is the relative importance of the digestive gland and intestine in the absorption of nutrients ? In addition, as the data from which other investigators (Potts, 1923; Vonk, 1924; Yonge, 1926a, b; Graham, 1932; Millott, 1937; Graham, 1938; Fretter, 1939; Howells, 1942; Carriker, 1946; Morton, 1956; van W e d , 1961; Martoja, 1961; Allen, 1962; Morse, 1966)have concluded that the digestive gland is the primary organ of absorption in various gastropods and bivalves is based upon similar experimental procedures as Fretter (1937) used, it is quite possible that active transport mechanisms also exist for monosaeeharides in the intestines of the animals of other major molluscan groups. It is surprising that no other direct studies on active transport of nutrients by molluscan guts exist.
REFERENCES ALLENJ. A. (1962) Preliminary experiments on the feeding and excretion of bivalves using Phaeodactylura labelled with sap. ~7. mar. Biol. Ass. U.K. 42, 609-623. ANNECFatSJ. H. (1964) Intestinal absorption of hexoses in the dog. Am. aT. Physiol. 206, 1095-1098. BALLANTINED. & MORTONJ. E. (1956) Filtering, feeding and digestion in the lamellibrancb, Lasaea rubra. J. mar. Biol. Ass. 35, 241-274. BARRYB. A., MATTHEWSJ. & SMYTHD. H. (1961) Transfer of glucose and fluid by different parts of the small intestine of the rat. J. Physiol. 157, 279-288. BiI~V~a A. M. (1950) The digestive mechanism of the European squids Loligo vulgaris, Loligo forbesii, Alloteuthis media, and Alloteuthis subulata. Quart.~Y. microsc. Sci. 91, 1--43. CAmaIK~ M. R. (1946) Observations on the functioning of the alimentary system of the snail Lymnaea stagnalis appressa Say. Biol. Bull., Woods Hole 91, 88-111. CoaDxm~ D. & CHANELJ. (1961) Influence de la tension d'oxyg~ne dans l'air inspirg sur l'absorption intestinale des solutions de pentoses et d'hexoses chez le rat. J. Physiol., Paris 43, 695-698. C~NE R. K. (1960) Mutual inhibition, in vitro, between some actively transported sugars. Biochim. biophys. Acta 45, 477--482. C R ~ R. K. & WILSONT. H. (1958) In vitro method for the study of the rate of intestinal absorption of sugars, a~. appl. Physiol. 12, 145-146. CS/LKY T. Z. (1958) Active intestinal transport of 3-o-methylglucose. Proc. Fourth int. Congr. Biochem., Vienna, p. 80, Pergamon Press, Oxford. DA~LINGTONW. A. & QtTASTELJ. H. (1953) Absorption of sugars from isolated surviving intestine. Archs Biochem. Biophys. 43, 194-207. DETHERIDGEJ. F., MATTH~WSJ. & SM~erHD. H. (1966) The effect of inhibitors on intestinal transfer of glucose and fluid. ~. Physiol. 183, 369-377. DtmoIs M., GILES K. A., H~mTON J. K., REBELSP. A. & SMITH F. (1956) Colorimetric method for determination of sugars and related substances. Analyt. Chem. 28, 350-356. FISH~ R. B. & PARSONSD. S. (1953) Galactose absorption from the surving small intestine of the rat. ~7. Physiol. 119, 224-232. Fmrra'ma V. (1937) The structure and function of the alimentary canal of some species of polyplacophora (Mollusea). Trans. R. Soc. Edinb. 59, 119-163.
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