`With 8 text figures
`Printed in Great Britain
`
`101
`
`SUGARS AND SUGAR DERIVATIVES
`WHICH INHIBIT THE SHORT-CIRCUIT CURRENT OF THE
`EVERTED SMALL INTESTINE OF THE RAT
`
`BY I. W. MUFLIH* AND W. F. WIDDAS
`From the Department of Physiology, Bedford College,
`Regent's Park, London NW 1 4NS
`
`(Received 29 December 1975)
`
`SUMMARY
`1. The short-circuit current of everted rat intestine supported on a
`perforated cannula proved to be stable for up to 3 hr and has been used
`to study competition between transportable and non-transportable sugars.
`2. 4,6-O-Ethylidene-a-D-glucopyranose (ethylidene glucose) and 4,6-O-
`benzylidene-a-D-glucopyranose (benzylidene glucose), two non-trans-
`portable inhibitors of the hexose transfer system in human erythrocytes,
`were found to reduce the short-curcuit current generated by transportable
`sugars such as galactose or 3-0-methyl glucose.
`3. These compounds were also found to reduce the basal short-circuit
`current established by the everted intestine in a sugar-free Krebs solution.
`Both types of inhibition approached saturation at the higher concentra-
`tions used.
`4. Similar inhibitory properties were shown by mannose, a non-actively
`accumulated monosaccharide, and by the fl-disaccharides lactose and
`cellobiose.
`5. It is suggested that this common pattern of behaviour is due to the
`ability of these compounds to react with the sites for active hexose trans-
`fer but without translocation by the system. The significance of the in-
`hibition of the basal short-circuit current is briefly discussed in this
`context.
`
`INTRODUCTION
`Soon after the publication of the hypothesis by Crane (1960), Crane,
`Miller & Bihler (1961) regarding Na÷ dependent sugar transport across the
`mucosal membrane of the intestine, it was considered, e.g. by Schultz &
`Zalusky (1963a, b) that the increments in the short-circuit current
`induced by the actively transported sugars were in support of this
`* Present address: Physiology Department, College of Medicine, Al-Mustansiriyah
`University, Baghdad, Iraq.
`
`MYLAN EXHIBIT - 1041
`Mylan Pharmaceuticals, Inc. v. Bausch Health Ireland, Ltd. - IPR2022-00722
`
`
`
`I. W. MUFLIH AND W. F. WIDDAS
`
`102
`hypothesis. During the last 15 years many studies have been made to
`determine the underlying process of active sugar accumulation on the basis
`of short-circuit current and potential difference measurements using in vitro
`preparations (e.g. Barry, Dickstein, Matthews & Smyth (1961), Barry,
`Smyth & Wright (1965), Clarkson, Cross & Tool (1961), Quay & Armstrong
`(1969) and Field, Fromm & McColl (1971) but the short-circuit current
`techniques had not so far been used to study competitive inhibition
`between sugars. Nevertheless a few reports have demonstrated that
`fructose (Asano, 1964; Lyon & Crane, 1966) and 2-deoxyglucose (Lyon &
`Crane, 1966) inhibited the basal electrical parameters recorded in sugar-
`free solutions. Barry, Eggenton & Smyth (1969) showed that mannose
`(28 mm) inhibited the basal transepithelial potential, an effect which they
`ascribed to osmotic gradients.
`Baker & Widdas (1973) showed that 4,6-O-ethylidene-a-D-gluco-
`pyranose (ethylidene glucose) could react strongly with the hexose trans-
`fer system in red cells and competitively inhibit glucose exit from the cell
`but could not enter red cells on the hexose system. Similar observations
`with a related compound, 4,6-O-benzylidene-x-D-glucopyranose (benzyl-
`idene glucose) have been reported on the hexose transfer system of the
`red cells by Novak & Lefevre (1974).
`In the present work, therefore, we set out to investigate the effects of
`known non-transportable competitive inhibitors of the hexose system in
`human red cells on the short-circuit current of everted rat intestine in-
`duced by the actively transported sugars. It was considered that if the
`compound, e.g. (ethylidene glucose, benzylidene glucose) could react with
`the intestinal hexose transport system without being transported by it,
`then it should inhibit competitively those increments in electrical para-
`meters which were dependent upon a small concentration of the actively
`transported sugars.
`In addition to ethylidene glucose (EG) and benzylidene glucose (BG),
`the effects of mannose and lactose on the short-circuit current generated
`by the actively transported sugars were investigated. In a continuation of
`this study the effects of cellobiose and phloridzin on the basal short-
`circuit current recorded in sugar-free Krebs solution were examined.
`A preliminary account was presented to the Physiological Society
`(Muflih & Widdas, 1976).
`
`METHODS
`Male Albino rats of Wistar strain were used in this work. The rats were adult and
`usually in the weight range 350-450 g. They were maintained on a standard com-
`mercial diet, food and water being available ad libitum. Under Nembutal (pento-
`barbitone sodium) anaesthesia, the peritoneal cavity was opened by mid-line
`incision and the exposed viscera was rinsed continuously with Krebs saline solution
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 103
`
`at laboratory temperature. Two openings were made through the intestinal wall,
`one at each end of the small intestine. The proximal opening was connected to a
`calibrated saline (Krebs) reservoir via a polyethylene tube. Intestinal contents
`were gently washed out by administering 25 ml. Krebs saline solution (at laboratory
`temperature) from the reservoir. A length of jejunum (8-10 cm long) was excised
`from the region in between 25-35 or 35-45 cm from the end of the duodenum. The
`excised segment was then immersed in the Krebs saline solution and a length of
`thin glass rod was passed through the lumen. The intestine was ligatured near to
`the end of the segment which was rapidly everted and passed over a perforated
`
`Agar p.d.
`electrode \, 4,
`
`Ag, AgCl
`
`Current
`electrode
`/AgiAgC1
`
`O
`
`/C O 2
`
`• 0
`
`0
`
`0
`
`0
`
`B
`
`•
`0
`I
`
`A
`
`Fig. 1. A, supporting cannula for everted segment of intestine. B, cannula
`and segment in electrode assembly in water-jacketed bath.
`
`supporting cannula made from a disposable needle container (Fig. 1). Lightly tied
`ligatures secured the segment above and below the perforations. The effective area
`of the mucosa between the two ligatures (without making any allowance for the
`macroscopic and microscopic infoldings of the mucosa) was 10 cm2. The time that
`elapsed between excision of the segment and the onset of incubation was less than
`3 min. The supporting cannula (with the gut) was mounted in an electrode assembly
`similar to that described by Barry et al. (1965). Current was supplied by a series of
`12 V dry batteries via a potentiometer which was driven by a low-inertia motor
`energized by an amplifier connected to the output of the potential measuring device
`(a Vibron Electrometer 33B) so as to form a simple feed-back servo which short-
`circuited the preparation. The current was monitored by a Kipp recording micro-
`graph BD2 (Kipp & Zonen).
`
`12
`
`P H Y 263
`
`
`
`104
`
`I. W. MUFLIH AND W. F. WIDDAS
`Solutions were made in Krebs-Henseleit (1932) saline of composition (mm):
`Na+ 144, K+ 5.9, Ca2+ 3.4, Mg2+ 1.2, Cl- 127.1, POi 1.2, HCO 25 and gassed with
`95 % O2-5 % CO2. Sugars were Analar grade.
`Ethylidene glucose was purified by preparative paper chromatography before use.
`Benzylidene glucose was prepared for us by D. A. Basketter according to the
`method followed by Novak & Lefevre (1974).
`Sugar-free Krebs solution (5 ml.) was recirculated at the serosal side of the seg-
`ment throughout the experiment, glass dropping chambers in the tube lines provided
`air gaps to maintain the electrical isolation of the serosal fluid.
`In the presence and absence of the actively transported sugars (galactose or
`3-O-methyl glucose), addition of different concentrations of the non-transportable
`sugars or phloridzin was in stepwise increments each for 6 min by which time the
`short-circuit current achieved a steady rate. In some experiments ethylidene glucose
`was present on the serosal side of the segment while actively transported sugar
`(3-O-methyl glucose) solution was on the mucosal side.
`
`200
`
`150
`
`50
`
`0
`0
`
`I.
`
`3 (±0.5) mM
`
`10 mm
`
`I
`
`1
`
`I
`
`1
`
`I
`50
`
`1
`
`I
`
`1
`
`1
`
`1
`100
`
`Time (min)
`
`1
`
`I
`
`1
`
`1
`
`1
`150
`
`Fig. 2. Short circuit (s.c.c.) from everted intestine showing the maintained
`response in the absence of sugar and in the presence of 3-O-methyl glucose
`at 3 and 10 mm. The points and lines are the means and s.E. of four
`experiments.
`
`RESULTS
`
`Control experiments
`The use of the supporting cannula for the everted sac of small intestine
`was found to make the responses of the tissue more reproducible and the
`short-circuit current was stable for from 2-3 hr.
`Fig. 2 summarizes the results of four experiments in which a basal level
`of short-circuit current was followed for 40 min and then two increments
`of current induced by 3 and 10 MK 3-0-methyl glucose respectively were
`followed, the whole experiment lasting 2i hr.
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 105
`In the presence of metabolizable sugar such as glucose the preparation
`also showed good recovery properties from the depressive effects of
`anoxia, provided this only produced a partial depression of short-circuit
`current. Table 1 summarizes three experiments where a period of anoxia
`was given at 50 and 150 min from the start of the experiment.
`Even after 150 min the restoration of oxygenation fully restored the
`short-circuit current induced by 4.4 mm glucose and there was no reason to
`fear that oxygenation of the tissue may have deteriorated over the course
`of an experiment in which full oxygenation was continuously maintained.
`
`TABLE 1. The recovery characteristics of the intestinal short-circuit current in-
`duced by 4.4 mm glucose in a preparation subjected to periods of anoxia
`The first and second anoxic periods were started at about 50 and 150 min respec-
`tively from setting up the segment. Means and S.E. of three experiments are given.
`
`Experiment
`
`Gas used to
`aerate the
`medium
`
`1. Initial steady state
`2. First anoxic period
`Recovery
`3. Second anoxic period
`Recovery
`
`95 % Os 5 % CO,
`95 % N2-5 % CO,
`95 % O,-5% CO,
`95% 1•1"2-5 % CO,
`95% O2-5 % CO,
`
`Duration
`(min)
`
`15
`14-17
`10-15
`15
`10
`
`(µA)
`
`128 + 14
`64 + 12
`134 ± 22
`67 + 12
`148 ± 30
`
`Competitive experiments
`In the competitive experiments an increment in short-circuit current
`was first induced by a transportable sugar (galactose or 3-O-methyl
`glucose) at submaximal concentration; then stepwise additions of the in-
`hibitory sugar were made while keeping the concentration of the transport-
`able sugar constant throughout.
`Figs. 3A and 4A show the graded decrease in short-circuit current
`brought about by increasing concentrations of ethylidene glucose and
`benzylidene glucose respectively.
`The relationship in both cases is hyperbolic and similar except that
`benzylidene glucose is effective at lower concentrations than ethylidene
`glucose. In experiments in which 14.7 mm ethylidene glucose was placed
`on the serosal side of the segment, only a small statistically insignificant
`reduction in short-circuit current was observed.
`Figs. 5A and 6A show similar graded reductions in short-circuit
`current produced by mannose and lactose as the inhibitory sugars.
`
`Inhibition of basal short-circuit current
`In addition to producing graded reductions in the short-circuit current
`induced by a transportable sugar the same inhibitory sugars were found to
`I2-2
`
`
`
`106
`
`I. W. MUFLIH AND W. F. WIDDAS
`5 5 mm galactose
`
`80
`
`A
`
`40
`
`el
`
`Basal
`s.c.c.
`
`-40
`
`i
`
`15
`10
`5
`[Ethylidene glucose] (mm)
`
`20
`
`V
`LA
`-**1
`
`—80
`Fig. 3. Effect of ethylidene glucose on the short-circuit current of everted
`rat intestine. (A), means of two experiments showing inhibition of
`induced by 5.5 mm galactose. (B), means of two experiments inhibiting the
`basal short-circuit current. I = 0 represents the steady-state current
`after about 45 min in sugar-free Krebs solution.
`
`12 mm 3-0-methyl-glucose
`
`1
`
`4
`3
`2
`[Benzylidene glucose] (mm)
`
`5
`
`100
`
`80
`
`60
`
`40
`
`20
`
`L.;
`
`A
`
`0
`
`Basal f
`s.c.c.
`
`—20
`
`—40
`
`B
`
`Fig. 4. Effect of benzylidene glucose on the short-circuit current of everted
`rat intestine. Means of two experiments: (A) in competition with 12 mm
`3-O-methyl glucose, (B) showing inhibition of the basal short-circuit current.
`= 0 represents the steady current after about 45 min in sugar-free
`Krebs solution.
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 107
`
`6.6 mm galactose
`
`20
`15
`10
`[Mannose] (mm )
`
`25
`
`30
`
`35
`
`100
`
`50
`
`0
`Basal /
`s.c.c.
`
`—50
`
`A
`
`5
`
`U
`- —100
`
`B
`
`—150
`
`Fig. 5. Effect of mannose on the short-circuit current of everted rat intes-
`tine. Means of two experiments (A) in competition with 6.6 ram galactose,
`= 0 repre-
`(B) showing inhibition of the basal short-circuit current.
`sents the steady current after about 45 min in sugar-free Krebs solution.
`
`4.4 m m galactose
`
`5
`
`10
`15
`[Lactose] (m m )
`
`20
`
`25
`
`• 60
`
`40
`
`Le;
`
`20
`
`0
`
`Basal
`s.c.c.
`
`U —20
`
`—40
`
`A
`
`B
`
`Fig. 6. Effect of lactose on the short-circuit current of everted rat intestine.
`Means of two experiments (A) in competition with 4.4 mm galactose, (B)
`showing inhibition of the basal short-circuit current. I,. = 0 represents the
`steady current after about 45 min in sugar-free Krebs solution.
`
`
`
`I. W. MUFLIH AND W. F. WIDDAS
`108
`produce a graded lowering of the basal short-circuit current when added
`to the mucosal bathing fluid in the absence of a transportable sugar. In all
`cases the preparation was left to equilibrate in sugar-free Krebs solution
`for at least 45 min and the short-circuit current had settled down to a
`steady state which was arbitrarily taken as the zero from which experi-
`mentally induced changes occurred.
`Figs. 3B, 4B, 5B and 6B show the reductions in basal short-circuit
`current produced by the same sugars.
`All curves are hyperbolic and if it is assumed that the change in short-
`circuit current is brought about by the occupancy of sites by the inhibitor
`sugar we may write
`
`C
`— ""z. C+KI'
`
`(1)
`
`where A/i,c is the change in short-circuit current, C is the concentration of
`inhibitory sugar, K1 its half-saturation concentration and k a constant.
`This can be linearized as
`
`C1/1„c = -k (C+Ki)
`
`(2)
`
`and a plot of C/A/,c vs. C should give a straight line. This is the equivalent
`of a Hanes (1932) plot and is recommended by Riggs (1972) as more suit-
`able for use when fitting a line to the experimental points by a least-
`squares analysis. The intercept on the abscissa is — ICI, the half-saturation
`concentration of the inhibitory sugar. The same plot can be used for the
`decrements in the short-circuit current in the competitive inhibition type
`of experiments but in this case the intercept would be — K1[I + (S/KM)],
`where S is the concentration of transportable sugar and KM its half-
`saturation concentration. At the low concentrations of transportable sugars
`used, the term in brackets will be of the order 1.5, but estimates of the
`true K1 can be made by dividing the intercept by [1 +(SIKm)].
`In Fig. 7 the above plot has been applied to the data for benzylidene
`glucose both when inhibiting the basal short-circuit current and when
`inhibiting the short-circuit current induced by 3-0-methyl glucose. The
`results from this plot and similar plots for the other sugars yield rough
`values for the half-saturation constants which are collected in Table 2.
`The inhibitory effects of lactose are in contrast with the stimulatory
`effects seen with the disaccharides, maltose and sucrose. However, the
`intestine of adult rats is known to lack a fl-disaccharidase (Alvarez & Sas,
`1961; Doell & Kretchmer, 1962) and the effects of another ft-disaccharide,
`i.e. cellobiose were therefore examined. It was found that this sugar was
`similar to lactose in inhibiting the basal short-circuit current but showed
`less affinity having a higher half-saturation concentration (K1 A 6.9 mm).
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 109
`Phloridzin, which classically inhibits the short-circuit current generated
`by actively transported sugar such as 3-0-methyl glucose (Schultz &
`Zalusky, 1964), was also found to inhibit the basal short-circuit current
`and results of two experiments with this drug are shown in Fig. 8. Although
`not truly hyperbolic (there may have been non-specific uptake and de-
`pression of the effective bath concentrations at the lowest concentrations)
`
`[Benzylidene glucose] (mM)
`
`—2
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`12 mm 3-0-methyl glucose
`
`Basal S.C.C.
`
`-150
`
`Fig. 7. Linearized plot of results for benzylidene glucose experiment shown
`in Fig. 4. Lines were obtained by a least-squares regression analysis.
`Intercept on the abscissa gives the K, for inhibition of the basal I. but
`not for the competitive experiment (see text).
`
`TABLE 2. Inhibitory constants (Kis) of non-transportable sugars or derivatives in
`competition with transportable sugars and when inhibiting the basal current. Data
`obtained from Hanes plots as in Fig. 7. For competition experiments K,= intercept
`(1 +S/Ku), where S is the concentration of transportable sugar and KM its half-
`saturation constant (for galactose KM = 12.5 mm for 3-O-methyl glucose Km =
`19.1 mM, Muflih, 1975)
`
`Competitive inhibition
`
`Inhibitor
`
`Transported sugar
`
`K, (mm)
`
`Ethylidene glucose
`Benzylidene glucose
`Mannose
`Lactose
`Phloridzin
`
`Galactose (5.5 mM)
`3-O-methyl glucose (12 mM)
`Galactose (6.6 mm)
`Galactose (4.4 mm)
`
`7.2
`1.0
`11
`3.8
`
`Basal
`inhibition:
`K, (mm)
`
`6.7
`1.2
`10
`4.9
`39 x 10-3
`
`
`
`I. W. MUFLIH AND W. F. WIDDAS
`110
`there is evidence of saturation at higher concentrations and the value of
`current maximally inhibited is of the same order as that with the non-
`transportable sugars.
`
`[Phloridzin] (pM)
`40
`60
`
`20
`
`80
`
`100
`
`Basal„,„0 0
`s.c.c.
`
`—10
`
`-20
`
`3 -30
`1)
`
`-40
`
`-50
`
`-60
`
`-70
`
`Fig. 8. Effect of phloridzin on the basal short-circuit current of everted rat
`intestine. Two experiments were carried out;
`= 0 represents the basal
`steady current after about 45 min in a sugar-free medium.
`
`DISCUSSION
`The experiments described in this study necessarily took a considerable
`time; not only was it necessary to allow 6 min for the short-circuit current
`to reach a steady state at each sugar concentration but to attain the basal
`level in sugar-free Krebs solution required an initial settling down period
`of from 45 min to 1 hr usually with at least one change of sugar-free
`medium.
`The everted gut supported by the perforated rigid cannula proved to be
`stable and to give consistent short-circuit current changes for up to 3 hr.
`The reason for the improved stability has not been investigated in detail
`but it was observed that muscular movements (seen in freely hanging
`preparations) were absent and the slight distension of the sac may have
`ensured more uniform oxygenation. The cannula also holds the sac so that
`its position relative to internal and external electrodes does not change
`during an experiment.
`Thus the preparation appeared to avoid oxygenation difficulties such
`as observed by Munk (1972) in everted rat intestine and the interference
`due to muscular contractions observed in everted segments of hamster
`intestine by Baker, Lo & Nunn (1974).
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 111
`Apart from the studies of Quay & Armstrong (1969) with bull frog
`intestine and those of Field et al. (1971) with strips of rabbit ileum, previous
`investigators have usually failed to obtain comparable stability for a
`sufficient length of time to complete such experiments.
`In competitive experiments it was possible to study the changes in short-
`circuit current produced by varying concentrations of non-transportable
`sugars (ethylideneg lucose, benzylidene glucose, mannose and lactose)
`in the presence of constant concentrations of the actively trans-
`ported sugars (galactose or 3-0-methyl glucose). The non-transportable
`sugars all had inhibitory effects in that they produced decrements in the
`short-circuit current. Further, these decrements were graded with con-
`centration in a manner suggesting saturation at high concentration. The
`action was assumed to be a membrane one, whereby the inhibitory sugar
`combined with sites on the active sugar transforming mechanism but in
`such a way that translocation was impossible. The increments in short-
`circuit current which accompanied the translocation of the actively trans-
`ported sugar (which was displaced) were therefore progressively reduced.
`That the action was at the outer membrane was suggested by the fact
`that onset of the inhibitory effect developed without delay when the sugar
`was in the mucosal fluid. Ethylidene glucose on the serosal surface had no
`significant inhibitory effect. Lactose was unlikely to penetrate by diffusion
`through intact mucosa and, if it had been hydrolysed, both the sugars
`formed as products of hydrolysis would have stimulated the short-circuit
`current. However, the absence of a fl-glycosidase enzyme (necessary for
`lactose hydrolysis) from the adult rat intestine reported by Alvarez & Sas
`(1961) has been fully confirmed by Doell & Kretchmer (1962), Koldovsky
`& Chytil (1965), and Semenza (1968). Thus the effect of lactose was most
`likely to be exerted extracellularly and this supports the view that the site
`of action was the outer membrane of the absorptive surface in the micro-
`villi of the mucosal cells.
`Lactose and cellobiose, the two fl-glycosides used, were both inhibitory
`whereas maltose and sucrose produced increases in short-circuit current
`(Muflih, 1975).
`An interesting observation of the present study was the inhibitory
`effects of these non-transportable sugars on the short-circuit current
`recorded across the intestine in Krebs solution which was free of trans-
`portable sugar. In these experiments as in the competitive ones, the decre-
`ments obtained in the short-circuit current by the addition of any one of
`the non-transportable sugars were concentration dependent in a manner
`suggesting a saturable effect. The Kis obtained for inhibiting the basal
`short-circuit current were siTilar to those obtained in inhibiting the short-
`circuit current generated by the transported sugars. This finding may
`
`
`
`112
`I. W. MUFLIH AND W. F. WIDDAS
`suggest that reactions were occurring either at the same sites or at sites
`with similar affinities. However, there may be a difference between those
`sugars like mannose which lack the stereo-specificity at carbon atom 2
`and the other non-transportable sugars, ethylidene glucose, benzylidene
`glucose, lactose and cellobiose which have bulky groups at the opposite
`end of the glucose molecule. Indeed phloridzin may belong to this group
`since Newey, Sanford, Smyth & Williams (1963) have suggested that the
`hexosyl unit of the phloridzin plays an important part in the binding of
`this inhibitor.
`In summary, competition for a sugar binding site but without transloca-
`tion is probably the common feature relating to these non-transportable
`sugars in their reactions with the intestinal mucosa. The corollary to
`this view would be the hypothesis that at least part of the in vitro basal
`short-circuit current of the everted gut bathed in sugar-free medium was
`actually being carried (presumably without sugar) on the sugar trans-
`porting system. This would not be inconsistent with the hypothesis of a
`sodium-linked sugar carrier in accordance with Bihler (1965), Crane,
`Forstner & Eichholz (1965), Lyon & Crane (1966) and Goldner, Schultz &
`Curran (1969) but may not exclude some other form of coupling.
`The absence of the actively transported sugar in the medium does not
`exclude the possibility of a small leakage of intracellular sugar into the
`microvillal crypts and reabsorption with Na+ on the transporting system,
`but this explanation would appear to be unlikely. Nevertheless the basal
`short-circuit current's relation to the sugar transporting system may be
`peculiar to the in vitro preparation.
`It was observed that the further depression of the basal short-circuit
`current was greatest with saturating concentrations of mannose, inter-
`mediate with ethylidene glucose and smallest with benzylidene glucose
`and lactose. The reasons for this were not clear but may reflect different
`accessibility of the transport sites since the sizes of the sugars would fall
`in a similar order. Barry et al. (1969) considered that osmotic effects were
`involved in the depression of the potential difference by mannose (at 28
`mm) and the inhibitions by this sugar only saturate at such levels of con-
`centration. However, osmotic effects can only play a minor part since the
`inhibitions by benzylidene glucose and phloridzin are produced by osmoti-
`cally insignificant concentrations.
`During this work I.W.M. was supported by grants from the Calouste Gulbenkian
`Foundation and the University of Baghdad which are gratefully acknowledged. We
`are grateful to Mr D. A. Basketter, &Se., for the synthesis of benzylidene glucose
`and for purifying the ethylidene glucose.
`
`
`
`SUGAR INHIBITORS OF SHORT-CIRCUIT CURRENT 113
`
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