The uptake and efflux of α-aminoisobutyric acid by the smooth muscle of the guinea pig's taenia...

The uptake and efflux of a-aminoisobutyric acid by the smooth muscle of the guinea pig's taenia coli

J. fP. ~ I D D I C O M B E ' AND D. M. P A T Q N ~

Degartnaent of4dszarmacolog~~, University cPJ' Alberta, Ednzonrom, Alta., Canada

Received January 22, 1979

WIDDICOMBE, J. If., and PATON, D. M. 1979. The uptake and emux of a-aminoisobutyric acid by the snaooth muscle of the guinea pig's taenia coli. Can. J. Physiol. Pharmacol. 57, 1114-1121.

The uptake of a-aminoisobutyric acid (AIR) by guinea pig taenia coli was linear with time over a 5-h period. There was a saturable component with Km of 0.7 rnA4 and a maximal value of 4.75 mmol/kg tissue per hour. A second nonsaturable component of uptake became irn- portant at high concentrations of AIB (> 5 mM). Preincubation in K+-free or Na+-free (high- K+) media greatly reduced uptake. The dependence of AIB uptake on outside [Na+] C[NaTIo) was approximately linear over the range 0-140 mM. With [Na+](, equal to 25 mIW the uptakes with various substitutes were in the order sucrose > Mg2+ > choline+ > Li+ > Gs+ > Wb+ = K.+. Addition of ouabain or removal of K+ during efjlux of AHB led to marked increases in the etfiux rate, ouabain being the more efrective treatment. Changes in tissue Na+ and K+ levels were found to be slightly grcater with ouabain than with K+-free medium. Lanthanum (5 mLW) prevented the uptake of Na+ seen in M+-free medium and also abolished the increase in the rate of loss of AIB. It also reduced the rise in tissue Na4 produced by ouabain; there was a correspondi~lg reduction in the ouabain-induced increase in the rate of loss of AIB. It is con- cluded that both the influx and efBux of AIB in this tissue are Na+ dependent, and that the accumulation of AIB relies on the transmembrane Na+ gradient.

WIDDICOMBE, 9. H., et PATON. D. M. 1979. The uptake and emux of a-arninoisobutyric acid by the smooth muscle of the guinca pig's taenia coli. Can. J. Physisl. Pharrnacsl. 5'7, 1114-1121.

L'absorptisn d'acide a-aminoisobutyrique (AIB) par la bandelette Bongitudinale du c6lon de cobaye est lineaire dans le temps sur une periode de 5 h. On note m e csmposante saturable lorsque le KT,, est de 0.7 n~"kf et une valeur maximale de 4.75 mmol/kg de tissu par heure. Une seconde composante non-saturable de l'absorption devient importante pour de grandes concentrations de AIB (> 5 tnM). Une pr6nc~lbation dans un milieu ne contenant pas de K+ ou de Nab (K+ 6lev6) rkduit l'absorption de facon apprkiable. Ida dependmce de l'absorption de AIB envers [Na+], est approximativement linkaire pour une variation comprise entre 0 et 1140 mhf. Pour 25 mlW de [Na+],, l'absorption de divers substituts se fait dans l'ordre suivant: sucrose > Mg2+ > choline+ > Li+ > Csi- > Rb+ = K+. L'addition de ouabaine, de mCme que la suppression de Ki- durant l'efflux de AIB amkne des augme~ltations marquees du taux d'efnux. le traitement le plus important 6tant celui la ouabaine. On a trouvC que les change- ments dam les niveaux de Na+ et de Ki tissulaires sont leghemetst plus importants avec la ouabalnc qu'avec un milieu ne contenant pas de M '-. Le lanthanum (5 anM) empeche l'absorption de Na qui a @tC notke dans un milieu libre en K+; il abolit aussi l'augmentation du taux de perte de AIB. 1.e lanthanum rkduit Cgalernent l'kl6vation de Na+ tissulaire produite par la ouabaine; il existe une redlactis~l correspondante de l'augmentation induite par la ouaba'ine, du taux de perte de AIB. On coilclut qLae tant l'amux que l'ecoulement de AIB dans le tissu CtudiC sont Na+-dependants et que l'accurnulation de A1B relttvent du gradient transmembranaire de Na4.

[Traduit par le journal]

Introdnetion well known for a number of tissues (Schultz and

Though the prope*ies of alllino acid transpod are Culran 1970), littie attention has been paid to this subiect in smooth nnuscles.

A B B R ~ ~ I ~ I ~ I O N S : AIB, a-aminoisohntyric acid: cpm, counts Amino acid uptake by uteri alld its modification per minute; [Na'],, outside [Na']; [Na'li, inside [Na']: PNI~, Na' permeability; [K'],, inside [K']. by oestrogens has been described (Noall and Allen

'Author to whom reprint requests sho~ild he sent. Present 1 961 ; R@skoski and Stejncr 1 967; Riggs ef al. address: Cardiovascular Research Institute, University of 1968). However, the uterus contains cell types other California Medical School, San Francisco, CA, U.S.A. ban muscle. OsHnan and Paton (1971) 941 43.

'Present address: Department of Pharmacology and Clini- studied the uptake of AIB by rabbit detrusor muscle. csl Pharmacology, University of Auckland, School of Medi- found that ouahain, of K+, and re- cine, Pkvate Bag, Auckland, New Zealand. moval sf Na+ all reduced uptake, thus implicating

0008-42~2/79/lOlll4-08$01.00/0 @ 1979 National Research Council of Canada/Conseil national de recherches du Canada

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WIDDICOMBE AND PATON 1115

the sodium pump in smooth muscle amino acid transport. The possible relationship between the sodium pump, the transmen~brane Na+ gradient, and amino acid transport in smooth muscle is further investigated in this paper.

Guinea pig taenia coli is a pure smooth nluscle preparation which is easy to dissect, and which at 37"C, in a variety of physidlogical salines, is able to maintain high K+ and low Na+ levels for many hours (Axelsson and Holmberg 1971; Burton and Godfraind 1974). For these reasons the taenia coli was selected for this study.

Most workers have concentrated on the uptake of amino acids. Experiments on both the uptake and efflux of AIB are described here.

MateriaIs and Methods

General Male guinea pigs weighing 200-300 g were killed by a

bIow on the head and 8-10 pieces of taenia coli, each weighing about 15 mg, were dissected, their fresh weights deter- nmined, and placed in beakers of Krebs solution at room temperature. They were then mounted on stainless steel holders and allowed to equilibrate in warm (37°C) oxygenated Krebs solution for 1-2 h. At the end of an experiment the tissues were removed from their holders, blotted lightly on Whatman No. 1 filter paper, reweighed for the wet weights, and analyzed for tissue counts or ion content.

Solutions The Krebs solution used was composed as follows (milli-

molar) : Na', 141; K', 5.4; Ca2+, 2.5; Mg2+, 1.2; C1-, 129; HCOtI-, 24; HrPOi-, 1.2; glucose, 11.5. This was kept at 37°C and equilibrated with 95% 0 2 - 5% COs to give a pH of 7.4. Solutions of lower Na' content were prepared by isosmotic replacement of NaCl by the appropriate substitute. In experiments where concentrations of Nat lower than 25mM were used the NaHCO., was replaced by Tris C1. As La"' precipi- tates out in solutions containing HC03- or HrP04-, when this ion was present, the following solution was used (milli- molar) : Na', 140; K+, 5 ; Tris+, 10; Ca2+, 1.5; Mg", 1.2; C1-, 160; glucose, 11.5. It was equilibrated with 100% OOn, and buffered at pH 7.4.

Uptake of AIB Radioactive a-amino [3-''C] isobutyric acid (5.3 Cilmol;

1 Ci = 37 GBq) was supplied by New England Nuclear, Montreal, Canada, in HC1 solution, and was diluted to a final concentration of 2 x 10 " M by adding distilled water. Of this stock solution 100 pL was added to the uptake media (volurne = 20 mL) producing an AIB concentration of 1OPM. Higher concentrations were obtained by adding nonlabelled AIB. Following uptake, the tissues were digested overnight in 0.4 mL NCS tissue solubilizer (Amershaml Searle Corp.) at 50°C. Once tissue solubilization was complete, 4.5 mL of a toluene-based scintillant was added. The scintillant used had the following composition: 5 g 2,5-di- phenyloxazole; 200 mg 1,4-bis- (2-4-methyl-5-pheny loxa- zol yl ) benzene; 300 mL ethylene glycol monomethyl ether; toluene to I L. Glacial acetic acid (100 pL) was then added to neutralize the NCS and the tissues were counted on a Beckman scintillation counter. Following counting, 100 p L of the uptake solutions was added to the tissue samples, and

the counts in the uptake media were determined by subtraction. Quenching was corrected for using the channels ratio technique.

Uptake was expressed as either:

cprn/mng tissue the distributioil ratio (&/rng) -

~ p r n / ~ L solution or

nmolimg tissue = pL/mg X concentration (1nA4) of AIB in the solution

Eflux of AIB Tissues were loaded with radioactive AIB by incubation

in Krebs solution containing 50 {cM AIB for 4-5 h. They were then transferred at 10-min intervals through a series of plastic scintillation vials containing 3 mI- of the appropriate efnux solution. These vials were maintained at 37°C and oxygenated. At the end of the washout the muscle was removed from its holder and digested as described above. Aliquots of the uptake medium (100 pL) were added to both the efluent vials and to those containing digested muscles. The counts ilm the solution were obtained by subtraction. The slight difference ( 4 % ) in counting efficiency between emuent and muscle was corrected for.

Efflux was expressed as the fractional rate of loss of the tracer ( A ) .

A - R " (A + B)/2

where A and B are the cpm present in the tissue at the start and end of the 10-rnin efflux period.

Ileternzination of NuT alld K' Contents After removal from their holders the tissues were placed

in quartz test tubes, and 3-5 mL of 0.1 N HN03 was added. After overnight incubation the Na' and K' conteimts in the sample were determined using either an EEL flame photometer or a Pye Unicam SP 90 atomic absorption sgectro- photometer.

Tirne Course of Uptake Uptake of AIB showed no sign of saturating over

a 5-h period. One hour after addition of AIB, uptake from a medium containing 50 pM AIB was 0.31 k 0.03 nmol/mg wet weight; 2 h after it was 0.66 t 0.05 nmol/mg; 3 h after it was 1.02 t 0.06 nmol/n~g; and it was 1.94 t 0.07 nmol/mg after 5 h of incubation. Pn all subsequent experiments uptake was for 1 or 2 h. The linear dependence of uptake with time over this interval indicates that the uptake over this period is the same as the initial rate of influx.

Dependence of Uptake on the Concentration of AIB In the left-hand column of Table 1 arc shown the

distribution ratios achieved after 2 h incubation in media containing from 10 pM to 20 mM AIB. As the dry weight: wet weight ratio of this tissue is about 0.15 (Brading and Widdicombe 1977), values for the distribution ratio greater than 0.85 represent accumulation by the muscle. It is clear that there is

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1116 CAN. J. PHYSIOL. PHARMACOL. VOL. 57, 1979

TABLE 1. The effect of preincubation in K+-free or Na+-free in millilitres per gram. Instead, it is 0.704 -t- 0.056 (high-K+) media on the distribution ratio of AIB mL/g, significantly greater than any published value

for the extracellular space in this tissue. Thus, there Distribution ratio must be a component of nonspecific uptake within

~ ~ + - f ~ ~ ~ the cells themselves. Concentration, PLW Normal K+-free (high-Ki) Uptake of AIB by "Na+-loaded" and "Kf-loaded"

NOTE: Cells were Na+ loaded by incubation for 4 h in Kc-free medium, and Kf loaded by ir~cubation for 3 h in Na+-free (high-KC) medium. Uptake was for 2 h. Values are the meails + SEM of five to seven tissues, and are expressed as microlitres per milligram wet weight.

considerable accumulation of AIB by the tissue, this being most marked at the lowest concentrations used.

In Fig. 1, the points from Table 1 for uptake from normal solution are plotted against the concentration of AIB. It is clear that there is little sign of saturation over the concentration range studied. Standard nonlinear least-squares regression analysis showed that the points were best fitted by a curve of the following form:

111 u = ess vm,,:/cs + KII + as where U is the uptake, V,,,, is the maximal uptake of a saturable component, K is the dissociation constant for saturable uptake, S is the concentration of AIB, and a is a constant. Thus uptake seems to consist of a saturable component and a nonspecific component, depending linearly on concentration. The best-fitting curve to the data, assuming only a saturable component of binding, produced a fit (as measured by the standard error about the curve) sonae five times less good than that produced by a curve in the form of Eq. 1.

The values for K, V,,,, and a from the computer fit were the following: K = 0.73 -t- 0.06 mM; V,,,, = 9.5 t 0.7 nmol/mg wet weight; and a =

0.704 =k 0.056 mL/g. The curve drawn through the uptake points in

Fig. 1 is predicted by Eq. 1 using these constants. It: is evident that there is an excellent fit between the observed and predicted values.

The extracellular space in this tissue has been determined frequently and almost all estimates fall between 35 and 45 % of the tissue volume (Brading and Jones 1969; Brading and Widdicombe 1977). If the linear component of uptake were purely extracellular, a would be equal to the extracellular space

Tissues Tissues were Na+ loaded by incubation for 4 h in

a K+-free medium; this period is sufficient to replace completely tissue K+ with Na+ (Axelsson and Holmberg 1971). K+ loading was achieved by exposure for 3 h to a solution in which K+ replaced Na+. Under these conditions K+ replaces tissue Na+ within minutes (Axelsson and Holanberg 197 1 ) . The uptake of tracer AIB from the loading solutions by these tissues is shown in the central and right-hand columns of Table 1. The uptake of AIB by both kinds of tissue was greatly reduced compared with tissues incubated in normal Krebs solution. There was a slight accumulation of AIB from K-free medium at the lower concentrations used, leading to significantly greater distribution ratios than from high-K (zero-Na) medium.

The Effects of Diflerent Na+ Substitutes In one series of experiments the NaCl of the

normal HC03--buffered Krebs solution was replaced by isosrnolar amounts of sucrose, MgCl,, KCI, RbC1, LiC1, CsCl, or choline C1. The solutions were other- wise unchanged. The [Na+] was thus 25 mM.

All solutions contained 10 p M AIB and the uptake of tracer over 2 h was determined. From each guinea pig eight strips of smooth muscle were dissected. One piece was placed in the normal Krebs solution and the others in each of the seven substituted uptake media. Inspection of Table 2 shows that all substitutions lowered the AIB uptake compared with that from normal Krebs solution. Using the paired t-test one can state that all uptakes were

TABLE 2. The effects of Na+ substitutes on uptake of AIB

Uptake Medium prnol/mg wet weight

NaCl Sucrose MgClz Choline Cl LiCl CsCl RbCl KC1

NOTE: Uptake was for 2 11 from normal HCOs-buffered Krebs solution, or from media in which the NaCl had been replaced by one of the substitutes listed. [AIB] = 10 p M . Values are meails i SEM of six tissues.

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WIDDICOMBE AND PATON

FIG. 1. The depencience of AIB uptake from normal medium on [AIB]. The points are taken from Table 1, and uptake is expressed as nanomoles per milligram wet weight. The inset shows the uptake values for AIB concentrations of 1 mM and less. The curve fitted through the points is the best statistical fit of Eq. 1 with a K , of 0.73 m M , maximal specific uptake of 9.5 nmolimg, and a no~lspecific uptake of 0.7 mLjg (see text for details). All points are the mean 3- SEM of six or seven tissues.

significantly different from one another (p < 0.05 ) , except for those from K or Rb media. Thus, the uptakes from the various substituted media were in the order sucrose > MgD > cholinef > Li+ > Cs+ > K+ = Rb+.

The Dependence of Uptake on [Na] , In two separate experiments, varying amounts of

Na+ in the uptake media were replaced by either choline+ or K+, and the uptake of tracer over 1 h was determined. The concentration of AIB was 10 pM throughout. The dependence of uptake on [Na],, as shown in Table 3, was approximately linear. Even with the highest Na+ levels used there was no sign of saturation. In the experiment with choline, NaCl was substituted for by choline C1, and to reach Na+ levels less than 25 mM, Na HC03 was replaced by KHC03. Over the range of Na+ concentrations from 25 to 100 mM the uptake in K+-substituted medium

is less than that in choline+-substituted medium, in agreement with the results of Table 2.

The Action of Ouabain and K + Medium on AIB Eflux

Tissues were loaded with tracer by 5 h incubation in Krebs solution containing 50 pM AIB. The loss of tracer was then followed. The results are illustrated in Fig. 2. After the first 30 min of washout the fractional rate of loss was constant at about 0.884 min-I. Exposure to K+-free solution led to an increase in the rate of loss of tracer, which became constant at 0.02 min-I after 3 h. Readmission of K+ during efflux caused the fractional rate of loss to re- turn to preexposure values (not illustrated).

Ouabain (2 )< M ) , which is sufficient to cause essentially complete inhibition of sodium pumping in this tissue (Brading and Widdicombe 19741, led to a dramatic increase in the efflux of

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11 18 CAN. J. PHYSIOL. PHAWkbACOL. VOL. 57, 1979

TABLE 3. The dependence of uptake on Na+ concentration I - -- - - - --

Uptake, pmol/sng wet weight

ma+], mlW Choline+ substitution K+ substitution

0 10 25 50 70

BOO 120 146

NOTE : Uptakes were for 1 h. [AIB] = 10 pA4. The values are the means f SEM of five to seven musciea.

AIB. This increase was more rapid and reached a higher level than that seen with K+-free medium. At exposure times greater than about 2 h the fractional rate of loss declined slowly. As Fig. 2 shows, tlae effect of ouabain was the same after 5 h of washout as after 1 h, and the effects of K+ ren~oval and otaabahl were not additive.

The Eflects of Ouabain and Kf-free Medi~dnz on Ion e0MteMt

Casteels et al. (1 973) have suggested that in K+-free medium the K+ leaking from the cells accumulates in the extracellular space, thercby ac- tivating the sodium pump and delaying the loss of K+ and uptake of Naf. It is possible that the difference in the effects of ouabain and K+-free solution on the efflux of AIB could be due to more rapid changes in tissue Na+ and K+ in the presence of ouabain. To test this hypothesis, tissues were placed in K-free ilaedium or in normal Krebs solution containing 2 X M ouuabain, and their Na+ and K+ contents were analyzed after 10-, 30-, or $0-min incubations. The control Na+ level was 67.4 * 15 mmol/kg wet weight and I h after addition of ouabain it had increased to 112.8 .i= 2.5 compared with 106.3 i- 4.8 mmol/kg wet weight in K-free medium. Over the I-h period ouabain caused a cor- responding fall in tissue K+ from 7'7.1 k 2.2 to 16.6 -t- 1.8 mmol/kg; in K-free medium the K level after 1 la was 24.5 k 3.1 mmol/kg. Thus ouabain- treated tissues gained more Na and lost more K+ than did tissues placed in K+-free medium. This was also true for the shorter exposure times of 10 and 3 0 min. However, the differences were slight, being far less than the difference in the rate of loss of AIB between the two treatments at the same incubation times. Thus the differences in the rates of change of tissue Na+ and K+ contents between ouabain- treated and K+ -free-treated cells may be inadequate

0 1 2 3 4 5 6

TIME (hours)

FIG. 2. The effects of ouabain (2 X M ) and K+-free solution on the efflux of AIB. The tissues were incubated for 5 h In a solution containing 50 plW AHB. One hour after the start of efflux the tissues were exposed to 2 >= M ouabain ( A ) , K4-free solution ( 0 ) , or both (A). The curve marked with open circles represents the control efflux into unmodified Krebs solution. After 5 h of washout tissues exposed to ouabain were also exposed to K-free medium, and tissues iincubated in K'-free medium or normal Krebs were in addition exposed to ouabain. The points are the means of five estimates z k SEM.

to account for the difference in tlae rate of loss of tracer.

The Action sf Lanthanum on the Eflects o f X-free Medium a d Ouabaisz on the Ef lux of AIB

The lanthanum ion (La3+) has been showia to prevent the uptake of Naf by tissues exposed to K+-free medium (Brading and Widdicombe 1977). Thus, if the action of Kg-free medium on AIB efflux is caused by an increase in [Na+],, tlaen this increase should be abolished by La3+. Figure 3 shows that 5 mM La3+ had no action of its own on AIB emux but almost completely annulled the action of K+-free medium. It also greatly reduced the effects of ouabain, but in the prcsence of La:%+, as in the abscnce, ouabain still caused a greater increase in the rate of tracer loss than omission of K+.

As discussed in the previous section, ouabain may be a more potent inhibitor of sodium pumping than K+-free medium, for in the latter case K+ leaking from the cells may maintain a certain amount of sodium pumping. In K+-free nnedium containing lanthanum the dual action of residual sodium punnp- ing and the reduced P,, caused by lanthanum may be sufficient to prevent Na+ accumulation. However,

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WIDDICOMBE AND PATON

I I 1 I

30 60 90 12Q 150 TIME ( m i n )

FIG. 3. The modification of the effects of K+-free solution and ouabain on AIB efflux by 5 rnM La"'. Tissucs werc incubated with tracer for 3 h in a solution containing 50 pM AIR. At the down-pointed arrow 5 mM La"+ was added to certain of the washout media. The tissues were exposed to K--free medium, ouabain (2 x 1 0-4 M ) at the time indicated by the up-pointing arrow, or were untreated. A, ouabain; A, ouabain + Lx"; a, KT-free; 0, KT-free + La"; X, La"' alone. The points are the mcans + SEM of four tissues.

with the more nearly complete inhibition of the pump presumed to be caused by 2 x M ouabain, the action of Law on PP,, may be in- sufficient to prevent Na influx and a Na-linked increase in AIB efflux. That this may be the case is shown by the results in Fig. 4, where the changes in Na+ and K+ content are expressd as millirnoles per kilogram of fresh weight. In Tris-buffered K+- free medium there is a gain of Na+, and a loss of K+. The loss of K+ is greater than the gain of Na+ and is accompanied by water loss as reflected in a decline in the wet weight to fresh weight ratio of froin 0.91 k 0.05 to 0.79 t 0.01 over the 2-h ex- perimental period. In K+-free solution with added La3+ there was a rapid displacement of some 10-1 5 mmol of Na+/kg, which is thought to be associated with extracellular fixed negative sites (Brading and Widdicombe 1977). Loss of K occurred, and the

0 L , I J 0 TIME (hours) 1 2

FIG. 4. The changes in Na' (solid symbols) and K+ content (open synlbols) on exposure to Kt-free medium (A, A ) , K'-free + La"' solution (V, ), or ouabain (2 X 10 M) + La"' solution (0, B). The circles show the ion contents of tissues incubated in unmodified Krebs solution. Each point shows the mean 5 SEM of six muscles.

tissues shrank to 63 2 2% of their fresh weights. In the presence of La3+ and ouabain there was gradual K-t loss and a similar rapid displacement of Na+, which was however followed by a slow increase in the tissue Na+ content. The tissues shrank to 63 * 2% of their fresh weights. Thus, Las +om- pletcly blocks the uptake of Na+ in K+-free medium, but is less effcctive in ouabain-treated tissues.

Discussion Our results show that, with 50 pM AIB in the

bathing medium, uptake is linear ove~r a 5-h period. Uptake is also linear over this period in rat diaphragm (Peckham and Knobil 1962; London and Segal 1967; Elsas et al. 1968). In embryonic chicken heart (Guidotti et al. 1968; Manchester et a1. 197 1 ) , rat and rabbit uteri (Noall and Allen 1961; Roskoski and Steiner 1967; Riggs et al. 19681, and rabbit detrusor muscle (Osman and Paton 1971) uptake is also slow, only approaching

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1120 CAW. J . PHYSHOL. PHARMACBL. VOL. 57, 1979

a steady state after several hours. In most non- muscular tissues, such as kidney sliccs (Roscnberg et al. 1961; Scriver and Mohyuddi~n 1968), toad bladder (Thier 1968), and fat cells (Touabi and Jearmreneaud 1969), uptake reaches a steady value after about 90 min or less.

The K,, of 0.7 mlV for AIB uptake by taenia coli is similar to that found for other tissues (Wosei~berg et al. 1961; Wiggs et al. 1968; Guidotti et al 1968; Touabi and Jeanrenaud 1969; Manchester et al. 2971 ), which all have K,,,'s of about 1 mM. For rabbit detrusor nmuscle Osman and Paton ( 197 1) give a value of 1.1 nlM. The maximal uptake over 2 h of 9.5 k 0.7 nlnol/mg (Fig. 2 ) corresponds to about 10 mmol/E cell water per hour. The maximal uptakes for other nonepithelial tissues (Riggs et al. 1968; Elsas et al. 1968; Guidotti et a%. 1968; Scriver and Mohyuddin 1968; Bsrnan and Paton 1971) range from 2.6 to 28 nmmol j E per hour, thus blanketing this value. This range may reflect differences in tlae surface area to volume ratios of the tissues. Assuming an extracellular space of 40% (Brading and 'tViddicsmbe 1977) and a volume to surface area ratio of the cells of 0.8 p m (Goodford and Wostora 1976), this uptake is equivalent to a transrllembrane flux of 0.19 pnmol/cm"er second.

When all but 25 mmol of Na+/E in the bathing Aujd was replaced by sucrose or Mg", the uptake of AIB fell to 62 and 49 7% of the value in normal Krebs solution, respectively (see Table 3 ) . With tnonovalent ions as the substitutes, uptake declined to values between 37 % (choline+ ) and B 8 96 (K+ ) of the control. One interpretation of this result is that the Na+-binding site is relatively nonspecific and all the various monovalent ions may bind and inhibit Na+ activation of AIB uptake. The affinity sequency for the Na+-binding site would then be K+ = Wb+ > $Is+ > Li+ > cholline+. Alternatively the difference in the uptakes with the various substitutes may not be caused by differences in affinity, but may be due to the fact that some or all of the ions are able to substitute for Na+ to variable ex- tents. Bihler and Adamic (1967) provided evidence that Li+ caused a small stimulation of sugar uptake by hamster small intestine, but state that it had no action on AIB uptake by the same tissue.

The linear dependence of AIB uptake om [Na+], has been described for fat cells (Touabi aisd Jeanrenaud 1969), rat diapl~ragm (Parrish and Kipnis 1964), and kidney slices (Fox et all. 1964). In the first two cases, choline+ was the substitute, in the last, either K+ or Tris+. The form of the curve reflects a number of factors other than the afinity of Na+ for the transport process. These may include

an inhibitory action of the replacing ion or changes in the affinity for the amino acid with changes in [Na+], (Curran et al. 1967).

La"+ abolishes or reduces the increases in [Na+Ii seen with ouabai~n or K-free medium. Similarly it inhibits the increase in AIB efflux seen with these treatments. These results suggest that AIB efflux, like influx, is a Na+-dependent process.

It should be pointed out, however, that La" not only prevents changes in the [NaAli but also the [K+Ii. Although in the presence of Lafi+, K+-free solution and suabain both cause large falls in tissue K+, the K+ lost is accompanied by 611- and water (Bradirmg and Widdicombe 1977). Thus the [K+li may be little changed. So, it is uncertain whether the increased efflux of AIB produced by ouabain and removal of K+ is due to a raised [Na+], or a lowered [K+li. f rocesses that abolish the transmembrane Na+

gradient, such as ouabain or removal of K+, also remove the K+ gradient, and it is possible that both these effects play a role in the increased eMux of AIB. That K+ can inhibit AIB uptake is indicated by the experiment shown in Table 2, where, when K+ is used to replace Na+, uptake is reduced more than with any other substitute. Thus, though the increase in tissue Na+ caused by ouabain is only slightly greater than that caused by K+-free solution, the combination of greater Na+ influx with greater K+ loss leading to reduced inhibition of Na+ activation may account for the greater effect of ouabain than K+ omission on AIB efflux. The efflux of noradrenaline from nerve terminals in rabbit atria is believed to be a Na+-dependent mechanism, and similarly is increased far more by ouabain ( 10-"-10-W) than omission of K+ (Paton 1973).

The work described here shows that the influx sf AIB is clearly dependent on the [Na+],. However, it is unclear whether the increases in emux seen on addition of ouabain or omission of K+ are primarily due to raised [Na+], or reduced [K+li. Suggestive evidence that the cl-nanges in emux are due to changes in [Na+li could be obtained with tissues in which K+i had been replaced by other ions. Incubation in a medium in which Li+ replaces Na+ is known to lead to nearly complete replacement of tissue K+ by Ei+ within arn hour (Goodford 1962). It would be in- tercsting to see how the efflux of AIB from such 66Ei+-loaded" tissues is altered by changes in [Na+Ii.

The influx of AIB is dependent on the [Na'],. Furthermore, La3+, by abolishing or reducing both the increases in [Na+Ii and ATB efflux seen with ouabain and K-free medium, suggests that efflux may also be Na+ dependent. Thus the accumulation

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WIDDICOMBE AND PATON 1121

of AIB by the smooth muscle of guinea pig taenia coli may depend on the transmembrane Na+ elec- trochemical gradient and, therefore, the energy for AIB accumulation would be provided by the sodium pump. Thic view is supported by the fact that when the Na+ gradient is abolished by incubation in Na+- free or K+-free media, then the distribution ratio of AIB is reduced to values little different from one, indicating greatly reduced active uptake (see Table 1 ) .

Acknowledgment

We thank Dr. Vojtech Licko for his assistance with the computer fitting of the nonlinear regressiotl curve in Fig. 1.

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