Transport of galactose and sodium across lizard duodenum

Camp. Eioehem. Physiol. Vol. 85A, No. I, pp. 103407, 1986

03~9629/86

Printed in Great Britain

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OF GALACTOSE AND SODIUM LIZARD DUODENUM

ACROSS

T. G~MEZ, P. BADfA, A. BOLA~~OSand A. LORENZO Department of Animal Physiology, University of La Laguna, Tenerife, Spain (Received 3 Januury 1986)

Abstract-l. Electrical parameters and transep~~elial Nat and galactose transport were determined in vitro across isolated duodenum of Lacertu golroti lizard. 2. Electrical potential difference (PD) and short-circuit current (Z,) were dependent on the presence of Nat in the bathing solutions. 3. PD and Z, were affected by addition of glactose to the mucosal solution, 4. Isotopic flux of Na+ measurements across short-circuited duodenum showed a net active Na+

absorption. 5. The net flux of Na+ (Zz+) accounted for the observed Z,. Both (Zz::‘) and I, were increased by the addition of galactose 5 mM to the mucosal solution. 6. Isotopic flux galactose measurements in open-circuit conditions showed a net active galactose absorption. 7. The net transport of galactose was decreased to zero in the absence of Na+ in mucosal and serosal reservoirs. 8. Galactose has been used to induce changes in short-circuit current (AZ=) across intestine. AZ, was a hyperbolic function of galactose concentration characterized by the parameters V_ (rn~rn~ change in AZ=) and &,, (con~ntration needed to attain a velocity equal to half the V,).

INTRODUCTION

Active transport of sugars and electrolytes by the intestine has been well studied in mammals (Curran and Schultz, 1968; Carte&u and Kimmich, 1979; Kaunitz et al., 1982; Kimmich, 1981). Few references are found, however, to reptile intestinal absorption, where the transport capacity for monosaccharides has been studied only in some species of tortoises and lizards. Crysemys pkta (Fox, 1961) and Testudo grueca (Wright, 1966) actively absorb D-galactose; the latter species also shows active transport of L-methyIglucoside and 3-O-methyl pyranose. The intestine of the tortoise (Testudo hermanni robertmertensi Vermuth) exhibits ability for the active transport of glucose but not for that of galactose (Lamsfus et al., 1976). Lacerta galjoti (Lorenzo et al., 1980) shows active transport of glucose fully

dependent on sodium. Little is known about electrical parameters and ion transport in reptile intestine. Recently (Kirk and Dawson, 1985; Halmam and Dawson, 1984a,b) Na+ and K+ transport and electrical parameters have been studied in turtle colon. The experiments presented here were designed to examine electrical parameters and transepithelial Na+ and galactose transport across isolated duodenum of Lacerta galloti, a lizard. MATRRIALS AND METHODS Animals, softrtions nit lizards were captured in their natural medium (Tenerife) and kept in a terrarium until use. After the lizards were sacrificed by decapitation, the abdomens were opened and the duodenum removed. This was rinsed free of faeces with IO-20ml of cold bathing solution, and then put into ice-cold bathing solution. Once rinsed, the intestine was cut

longitudinally and placed between the two halves of an Ussing chamber with an exposure surface of 0.21 cm*. A continuous flow of water thermostated at 30°C circled through the chamber. The tissue was bathed on both sides with 4 ml of Ringer solution continuously gassed with Or at 95%. The bathing solution contained (in mM): NaCl, 107; KCI, 4.5; NaHCO,, 25; Na,HPO,, 1.8; NaHsPOI, 0.2; CaCl,, 1.25; MgSO,, 1.0 and glucose, SmM. The glucose was only in the serosal solution. In some experiments, choline was used to substitute partially or totally for Na+ in solutions used to bathe both sides of the intestine. Electrical measurements

Agar bridges, 4% w/v made with bathing solution, were positioned near each surface of the tissue and at opposite ends of the chamber calomel electrodes and Ag/AgCl electrodes in saturated KC1 were connected via the agar bridges to measure the transmural potential difference fpD) and to pass direct current, respectively. Electrical measurements were continously obtained with the aid of an automatic computer-controlled voltage-clamp device (AC-microclamp, Aachen, FRG). The offset potential and solution resistance were obtained before mounting the tissue and were automatically corrected for. The tissue was first incubated under open-circuit conditions for some time, determined by the experimental requirements, and then short circuited. Every 5 set the tissue was alternately pulsed with a (+) or (-) 10 PA pulse of 1 set duration. After a 0.5 set delay the displacement in potential difference caused by the pulse was measured and from the change in potential difference and pulse amplitude, tissue conductance (G,), was obtained. This procedure was used for both open and short-circuited conditions. Thus, under open-circuit conditions the open-circuit PD and G, were measured and from these values a calculated short-circuit current (i,) was obtained. Under short-circuit conditions the I, and G, were measured and from these values a calculated PD was obtained. All three parameters PD, G, and Z,, were recorded

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Fig. I. Electrical parameters across isolated duodenum lizard. (a) Normal Ringer solution. (b) Sodium-free Ringer

solution. PD (I)), Z, (II), G, CA).

on a digital printer every minute. For determining concentration-dependent gaiactose-induced current (called Al,) small volumes of gaiactose-containing solutions were added to the solution bathing the mucosai surface of each preparation. The final con~nt~tion of gaiactose in the mucosal solution varied from 5 to 50mM. A standard concentration of sugar was added at regular intervals throughout an experiment. Determination of unidirectional juxes

Forty minutes after the tissue was mounted, isotopes (“Na and galactose “(2) were added to the bathing solution on one side of the tissue. After an additional 30min, by

which time isotope fluxes had reached a steady state, samples of 200~1 were taken from the unlabelled side at regular 20-min intervals for 1 hr. Samples were taken in duplicate to avoid errors, the same volume being replaced with unmarked Ringer solution. Unidirectional fluxes were determined from the standard equations (Schultz and Zalusky, 1964)s RESULTS Electrical measurements

The electrical parameters in the duodenum from lizards are given in Fig. 1. The data shown were obtained from pieces of tissue which were first incubated for 60 min (tm - t,,) under open-circuit conditions. In duodenum epithelium all three electrical parameters remained stable after 60 min for several hours (4 hr) under both open and short-circuit conditions. In addition, under open-circuit conditions the

measured PD and G, and the caicualted Z, were equal to the respective calculated PD and measured G, and Z, under short-circuiting of the duodenum from lizards appears to be without effect on the tissue. In normal Ringer solution (Fig. la) in all experiments (IV = 15) the serosal side of the epithelium was found to be electrically positive compared to the luminal side. The transepithelial PD varied from 2.6 to 3.3 mV. The transepithelial conductance ranged from 8 to 15 mS cme2 and the Z, from 20 to 28 pA.crn12. In the absence of Na+ (Fig. lb), both PD and Z, progressed towards serosal negativity with respect to mucosa (PD = -0.4fO.O8mV, Z, = -6.96 & 1.5 pA.crne2). The G, remained unchanged. Figure 2 shows the results in which galactose (5,15,30,45,60 and 75 mM) was added to the mucosal bathing solution. In the presence of Na+ (Fig. 2a) addition of galactose produced an increase in Z, and PD. Thus the PD was increased from 2.6 f 0.25 to 7.3 &-1.25 mV and Z, from 28.1 + 3.1 to 75 k 8.2 ~A*crn-~ when the concentration of galactose was of 45 mM. Addition of higher doses (60, 70mM) did not alter either parameter. In contrast, when Na+ is replaced by choline (Fig. 2b) addition of galactose to the mucosal reservoir had no effect on electrical values. Transmural fluxes

Unidirectional and net galactose fluxes across lizard duodenum under standard conditions and in the absence of Na+ are given in Table 1. Under control conditions, the unidirectional galactose flux J,,,$ from the luminal to serosal side (0.247 pmol*cm-*.hr-‘) is about three times larger than the flux .Z,, from the serosal to the iuminal side (0.096 pmolscm-2. hr-I). The transepitheiiai galacIn the tose net flux, J& was 0.151 ~moi~cn-2~hr-‘. absence of Na+, net transport of galactose was decreased to zero and resulted from a decrease in the mucosal to serosal flux of galactose. Table 2 shows the transepithelial Na+ fluxes of the different experiments compared with simultaneously measured Z,. The value for net Na+ absorption, Jtl’, under control conditions, was very similar to the observed value for the total current generated. After addition of galactose, the mucosal to serosal flux of Nat, Jz’ increased significantly, causing an increase in the net flux of Na+, Jtz’. Galactose increases Z, by 0.6 peq.cm-2.hr-‘. This increase was very similar to the observed increase for net Na+ absorption. Use of AI, (IS a measure

of gatactose entry into tissue

Initial criteria which need to be satisfied before adopting the AZ, method to measure kinetic constants for galactose entry through a Na+-dependent high affinity system are: (1) that the response is shown be Na+-dependent; (2) that the response should show saturation kinetics. Two points are summarized in Fig. 3. The Lineweaver-Burk plot of the transport data from galactose-induced current allows us to calculate the kinetic parameters of the transport system for galactose. The influence of Na + on these parameters

Transport in the lizard intestine

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Fig. 2. EfTects of galactose (5, 15, 30, 45, 60 and 75 mM) on PD (@), Is (I) and G, (A) in: (a) normal Ringer solution. (b) sodium-free Ringer solution. Galactose was added to the mucosal solution.

Table 1. Effect of Na+ substitution on galactose transmural fluxes across hxard duodenum Control (11) Na+-free

Jd 0.2472 4 0.03

Jg 0.0962 4 0.02

JYI;: 0.151 f0.03

0.1518 +0.02*

0.1752 & 0.03

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is shown in Fig. 4. Increasing Na” concentration has no consistent effect on V_, whilst K, decreases as Na+ concentration increases: 7.71, 20.93 and 31.05 mM for 100, 50 and 25%, respectively. DISCU!SSION

Lizard intestine was mounted in a flux chamber and bathed with Ringer solution. “C o-galactose. (5 mM) was added to one face of the tissue and the rate of the appearance of the isotope in the opposite solution was monitored. Ion fluxes are in amol.cn-‘.hr-‘. The values are the mean* SEM of the number of the diierent animals given in parentheses. l, t, t Significant differences P