Absence of (Na+, K+)-ATPase involvement in lactose production by lactating guinea pig mammary gland

12

Biochimica et Biophysica Acta, 392 ( 1 9 7 5 ) 1 2 - - 1 9 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 2 7 6 4 9

ABSENCE OF (Na ÷, K÷)-ATPASE INVOLVEMENT IN LACTOSE P R O D U C T I O N BY LACTATING GUINEA PIG MAMMARY GLAND*

J.H°A. V R E E S W I J K , J.J.H.H.M. DE P O N T a n d S.L. B O N T I N G

Department of Biochemistry, University of Nijmegen, Geert Grooteplein Noord 21, Nijmegen (The Netherlands) (Received O c t o b e r 21st, 1974}

Summary The role of the (Na ÷, K÷)-ATPase system in lactose production by the lactating guinea pig mammary gland has been studied in vitro with slices of the gland. In this system there is an initial fast lactose release, mainly representing secretion of preformed lactose, followed by a continuous slow lactose release, representing mainly lactose synthesis. The latter process occurs at a rate of 1.6 to 2.4 g lactose/kg wet wt/h, which value is a b o u t half of the lactose production in vivo (3.9 g/kg wet wt/h). Incubation of slices in the presence of 10 -4 M ouabain does not influence the rate of overall lactose production. When determined separately, it does not change either the rate of secretion or that of synthesis. This pleads against a role of the (Na ÷, K÷)-ATPase system in lactose secretion or synthesis, in particular it seems to rule out control of the rates of these processes by the intracellular potassium concentration. An explanation for the generally observed correlation between the lactose and potassium concentrations in milk, may be that both the maintenance of the intracellular potassium concentration and the lactose synthesis rate require the presence of ATP.

Introduction

In recent years several authors have emphasized that there is a relation between the lactose and potassium concentrations in milk. An inverse relationship between potassium and lactose content in milk from individual cows has been reported by R o o k and Wood [1] and Walsh and R o o k [2] and in milk from cattle, sheep and pig by Konar et al. [3]. On the other hand, a direct relationship has been shown by Barry and Rowland [4] during the lactation * Studies

on

(Na +, K+)-aetivated ATPase XXXIII.

13 period of cows. When certain conditions lead to a decrease in milk K ÷ concentration, the lactose content in milk is also decreased, whereas sodium and chloride concentrations increase at the same time. This effect has been described in milk of several species during the last part of the lactating period [3]. Linzell and Peaker [5,6] have observed this effect in milk of the goat during mastitis. Ouabain administration in vitro [7] or in vivo [8] causes the same result in milk of the goat. Silcock and Patton [8] suggest that this implies that lactose production is regulated by the intracellular ion concentration. Previously we have reported the presence and properties of an ouabainsensitive {Na ÷, K*)-ATPase system in guinea pig mammary glands [9]. We have also investigated the effect of (Na ÷, K÷)-ATPase inhibition on intracellular ion concentrations of the lactating guinea pig mammary gland [10]. In the present paper we have used mammary gland slices to investigate whether the lactose production is indeed regulated by intracellular ion concentrations or possibly even directly b y the (Na ÷, K+)-ATPase system. In this study we have tried to distinguish between the t w o c o m p o n e n t processes of lactose synthesis and secretion. Our results indicate that such a regulation is absent in either process. These results have previously been reported in abstract form [111. Materials and Methods Chemical reagents NAD ÷, fl galactosidase and fl galactose dehydrogenase have been obtained from Boehringer, Mannheim, G.F.R., ouabain from Merck, Darmstadt, G.F.R. and depolymerized dextran (Rheomacrodex) from Pharmacia, Uppsala, Sweden. All other reagents are of analytical grade. Preparation and incubation of mammary gland slices Mammary glands are obtained from female suckling albino guinea pigs (Central Institute for Breeding of Laboratory Animals, T.N.O., Zeist, The Netherlands), 5--10 days after giving birth. After killing the animal by cervical dislocation the mammary glands are removed within 5 min. Slices of 0.5 mm thickness are prepared with a Stadie-Riggs hand microtome. These slices are incubated at 37°C or 0°C in a modified Krebs-Ringer bicarbonate solution containing Na ÷, 150.6 mM; K ÷, 4.6 mM; Ca 2÷, 2.3 mM; Mg 2÷, 1.7 mM; CI-, 136.3 mM; HCO3-, 26.2 mM; H2 PO4-, 1.5 mM; acetate, 1.2 mM; glucose 1 g/1 and depolymerized dextran (mol. wt 40 000) 50 g/1. Dextran is added t o prevent swelling of the slices [10]. During incubation 95% 0 2 / 5 % CO2 or 95% N2/5% CO2 is continuously passed through the medium. The pH of the medium is between 7.3 and 7.6. After incubation the slices are removed, blotted lightly and weighed on preweighed pieces of aluminium foil. The slices are then homogenized and centrifuged for 15 min at 500 gm ax. In the supernatant the lactose content is determined. During the incubation period samples of the incubation medium are taken at various time intervals for lactose determinations. Lactose determination Lactose in mammary gland slices and in samples of the incubation me-

14 dium is determined by means of the enzymatic m e t h o d of Wallenfells and Kurtz [12], except that all volumes are reduced to one fifth. Results

Kinetics of lactose production When slices of the lactating mammary gland of the guinea pig are incubated in a Krebs Ringer bicarbonate solution at 37 °C under aerobic conditions, there is an increase in the lactose content of the incubation medium (Fig. 1, upper curve). The curve suggests that two processes can be distinguished. In the first few minutes there is a rapid release of lactose, which presumably was present in the slices before the start of incubation. After 60 min the lactose increase in the incubation medium is linear with time. This increase may reflect the secretion of lactose synthesized during incubation, assuming that synthesis rather than secretion is the rate-limiting step. In this period there is a lactose increase in the medium of 1.95 (SE: 0.07, n = 4) g/kg wet wt/h.

Effect of ouabain on overall lactose production Upon addition of 10 .4 M ouabain to the incubation medium (Fig. 1, lower

~o.o

8.0

g g ~ 2.0

L

i

40

i

i

80

t

i

i

120

t:i~e o~= i~c~b~io~ ( . ~

)

Fig. 1. L a c t o s e c o n t e n t in t h e i n c u b a t i o n m e d i u m , e x p r e s s e d in g / k g w e t w e i g h t o f t h e i n c u b a t e d slices, d u r i n g i n c u b a t i o n at 3 7 ° C u n d e r a e r o b i c c o n d i t i o n s (95% 0 2 ] 5 % CO 2) w i t h o u t o u a b a i n ( e e) and with 10 -4 M o u a b a i n (A A). T h e n u m b e r of i n v e s t i g a t e d glands is 4 for e a c h curve.

15 curve) the appearance of lactose in the medium follows a similar time course and the amounts of lactose appearing in the medium are n o t significantly different from the results obtained in the absence of ouabain. The lactose concentration in the medium is again linear with time after 60 min, indicating a lactose production of 1.56 (SE: 0.20, n = 4) g/kg wet wt/h. The influence of ouabain on lactose production in vitro has been further investigated by measuring not only the amount of lactose secreted in the incubation medium, b u t also the a m o u n t of lactose present in the slices before and after 150 min of incubation. From these values (Table I), all expressed as g lactose per kg wet wt, the overall lactose production can be calculated as the sum of the lactose contents in the incubation medium (Ct) and in the slices (Cr) after 150 min of incubation, minus the lactose content of the slices before incubation (Co). The calculation in Table I shows that there is no significant difference between the lactose production in the presence or in the absence of 10 -4 M ouabain in the medium. Effect o f ouabain on lactose secretion In the lactose production process, two steps can be distinguished; the synthesis of lactose and its secretion from the alveolar cells to the alveolar lumen. Hence, from the absence of an effect of ouabain on the overall lactose production it cannot y e t be concluded that the (Na ÷, K+)-ATPase system is not involved in either one of these t w o processes. Therefore, we have attempted to study the secretion process separately by incubating mammary gland slices under nitrogen atmosphere, so as to exclude synthesis of lactose. After 40 min there is no further increase in the lactose content in t h e incubation medium (Fig. 2). Together with the results given in Fig. 1 this indicates that no synthesis takes place. This is also shown by the following calculation. The total a m o u n t of lactose appearing in the incubation fluid under these conditions is TABLE

I

LACTOSE CONTENT AFTER INCUBATION

OF MAMMARY GLAND FOR 150 MIN AT 37°C

SLICES

AND INCUBATION

MEDIUM

BEFORE

M e a n v a l u e s w i t h s t a n d a r d e r r o r s o f t h e m e a n . N u m b e r o f a s s a y e d g l a n d s is 4 i n e a c h c o l u m n . production has been calculated by means of the formula: lactose production

-

C t +(C r-KCe)2.5

Co

AND

The lactose

g/kg wet wt. h

C o , l a c t o s e c o n t e n t i n t h e s l i c e s b e f o r e i n c u b a t i o n ; Ct, t o t a l l a c t o s e c o n t e n t i n t h e m e d i u m a f t e r i n c u b a tion; Cr, lactose content in the slices left after incubation; Ce, lactose concentration in the medium after incubation; K, % inulin space X % tissue water X 10 -4 (Vreeswijk [10] ; KCe, extracellular lactose content in slices after incubation.

C o (g/kgwet wt) C t (g/kgwet wt) C r (g]kgwetwt) C e (g/1 i n c u b a t i o n f l u i d ) K Lactose production (g/kg wet wt. h)

No ouabain

10 -4 M ouabain

6.52 10.85 1.62 0.27 0.083 2.4

6.52 9.43 1.65 0.27 0.084 1.8

± + + ± ± ±

1.14 1.22 0.20 0.04 0.013 0.67

± ± ± ± ± +

1.14 1.18 0.16 0.04 0.013 0.66

16

4..

6

~3 E4"s

15 Q;, t" ..p

2

2

o no

ouab=~i:n

• 1 0 - 4 V I o~alo~,,ir~

E (5

0 4a

o 0

I

0

I

I

I

40

20 title

of

I

60 i.c~b~t:io.

(K,/,)

Fig. 2. Lactose c o n t e n t in the i n c u b a t i o n m e d i u m , e x p r e s s e d in g / k g wet w e i g h t of the i n c u b a t e d slices. d u r i n g i n c u b a t i o n at 37°C u n d e r a n a e r o b i c c o n d i t i o n s (95% N 2 / 5 % CO2) w i t h o u t o u a b a i n (o c) a n d with 1 0 -4 M o u a b a i n (A A).

5.45 (SE: 0.31, n = 9) g lactose per kg wet wt. The sum of this a m o u n t and that remaining in the slices after anaerobic incubation (0.43 (SE: 0.13, n = 6) g/kg wet wt) is n o t significantly different from the a m o u n t of 6.0 (SE: 0.13, n = 4) g/kg wet wt derived for aerobic incubation by extrapolation of the straight part of the upper curve in Fig. 1 to t = 0. These findings prove that under anaerobic conditions we are indeed measuring only secretion of lactose. In fig. 2 the anaerobic efflux of lactose in the presence and absence of 10 -4 M ouabain is shown. The fact that no significant difference exists between these two conditions indicates that the (Na ÷, K+)-ATPase system has no effect on lactose secretion.

Effect of ouabain on lactose synthesis We have attempted to study the synthesis separately by first incubating the slices at 0°C for I h in order to remove preformed lactose, and thereafter at 37 °C in the presence and absence of 10 -4 M ouabain. The lactose content of the medium has become constant after 1 h incubation at 0°C, and amounts to 1.43 (SE: 0.19, n = 10) g/kg wet wt. The slices are then incubated for 2 h at 37°C in fresh medium in order to determine the rate of synthesis. The results in Fig. 3 indicate that during the incubation at 37°C there is a lactose production of 1.64 (SE: 0.18, n = 16) g/kg wet wt/h without ouabain and of 1.52 (SE: 0.19, n = 14) g/kg wet wt/h in the presence of 10 -4 M ouabain. These findings indicate that (Na ÷, K*)-ATPase is not involved in lactose synthesis, neither in a direct coupling to the (Na ÷, K+)-ATPase system, nor indirectly by means of the intracellular ion concentrations regulated by this enzyme.

17

nO ou4~bai~

b

10"4D1 o ~ a b ~ i ~

4.0 _

l--I

r~ed;~m in atlcea

t-

-~ z0 o

,,g 0 L)

.1::0 ~=60 f=120

~=0 "1:=60 t=120

~ime oF i~c~bat;ion (rni~) Fig. 3. L a c t o s e c o n t e n t o f slices ( s h a d e d b a r s ) a f t e r 1 h p r e i n c u b a t i o n at 0 ° C only a n d a f t e r p r e i n e u b a t i o n a n d 1 or 2 h i n c u b a t i o n at 3 7 ° C . L a c t o s e c o n t e n t o f i n c u b a t i o n m e d i u m ( o p e n b a r s ) a f t e r 1 or 2 h i n c u b a t i o n at 3 7 ° C o f slices, w h i c h h a v e b e e n p r e i n c u b a t e d f o r 1 h a t O°C.

L a c t o s e p r o d u c t i o n in vivo

From four lactating guinea pigs, milk is collected in three hourly intervals. The animals are anaesthetized, the nipple of the mammary gland is cannulated and the milk flow is initiated by injection of 0.1 I.U. oxytocin into the carotid artery. The average milk yield is 0.74 (SE: 0.12, n = 8) ml/h/gland. The lactose content of the milk is 41.8 (SE: 0.7, n = 8) g/1. The average gland wet weight is 7.93 (SE: 0.24, n = 10) g. Hence, the average hourly lactose production is 3.9 (SE: 0.64, n = 8) g/kg wet wt h. The in vitro production of lactose by guinea pig mammary gland slices in the above experiments ranges from 1.5--2.4 g/kg wet wt h which is a b o u t half of the in vivo production. Discussion

The production of lactose by slices of the guinea pig mammary gland can be divided into three steps; lactose synthesis, the release of lactose from the alveolar cell to the alveolar lumen and the diffusion of lactose from the alveolar lumen into the incubation medium. The last two steps, which we cannot measure separately, are therefore taken together under the term "secretion". Secretion can be measured by inhibiting the first step, the synthesis of lactose, through incubation in the absence of oxygen. In that case only preformed lactose is secreted into the medium. Synthesis is apparently the rate limiting step in the overall production process as shown by the following consideration. The rate of synthesis determined from the linear part of the slope in Fig. 1 is 1.56 (SE: 0.20, n = 4) g lactose/kg wet wt h. This is only slightly less than the total production rate of 2.4 (SE: 0.67, n = 4) g lactose/kg wet wt h (Table I). We have tried to study synthesis separately from secretion by incubation at 37°C after preincubation for 1 h at 0~C. In the subsequent incubation at 37°C lactose is synthesized again at the rate of 1.64 (SE: 0.18, n = 16) g lactose/kg wet wt h for at least 2 h, which is a b o u t the same as found without preincubation at 0 °C.

18 There is no significant effect of 10 -4 M ouabain on lactose production (Table I) or on lactose synthesis determined graphically (Fig. 1) or after preincubation at 0°C (Fig. 3). Concurrent determinations of the inhibitory effect of ouabain on the (Na ÷, K÷)-ATPase activity in guinea pig mammary gland homogenates indicate complete inhibition by 10 -4 M ouabain [9]. Under the conditions of the slice incubation experiments used for the study of lactose production addition of 10 -4 M ouabaln causes a large equimolar exchange of intracellular potassium for extracellular sodium [10], as commonly observed upon extensive inhibition of the (Na ÷, K÷)-ATPase system. In further experiments a complete inhibition of the [86] Rb uptake by the slices in the presence of 1 0 -4 M ouabain has been observed (de Pont et al., submitted for publication). Thus we conclude that while 1 0 - 4 M ouabain in the slice experiments must be fully inhibitory to the (Na ÷, K*)-ATPase system, it has no detectable effect on the lactose production. Although the secretion process is not rate limiting in the slices system, we have studied the effect of 10 -4 M ouabain on lactose secretion. Again there is no effect (Fig. 2), indicating that inhibition of the (Na *, K÷)-ATPase system has no influence on the secretion rate. Since our experiments indicate that (Na ~, K÷)-ATPase is not involved in lactose synthesis and secretion, the suggestion made by Silcock and Patton [8] that the intracellular potassium concentration regulates the lactose production can be rejected. The question then remains h o w to explain the positive correlation between potassium and lactose contents in milk [4--6,8]. Evidently, both the synthesis of lactose and the action of the (Na ÷, K~)-ATPase system require energy expenditure. We should like to suggest that the positive correlation between the lactose and potassium concentrations in milk is due to the fact, that both parameters are positively correlated with the energy expenditure of the mammary gland cells. When lactose secretion is indeed an exocytosis process as suggested by Brew [13] and Linzell and Peaker [14], and the ionic composition of the vesicles is a reflection of the intracellular potassium concentration, such an explanation seems likely. In the case of an energy deficiency both processes will slow down, leading to a parallel lowering of the lactose and potassium levels in milk. In order to maintain an adequate energy supply, the mammary gland must have an ample blood supply and an optimal metabolic activity. During involution and mastitis these conditions will not be fullfilled, hence both lactose and potassium levels in milk will decrease. The finding by Linzell and Peaker [7] and Silcock and Patton [8] that ouabain also causes a decrease in lactose and potassium levels in milk can be explained in a similar way. Ouabain in concentration below those necessary for full inhibition of the (Na ÷, K÷)-ATPase system causes vasoconstriction in various systems [15--17], and also in mammary gland according to our own experiments with the perfused goat udder [10]. Silcock and Patton [8] have described an increase in sodium and chloride contents of goat milk and a simultaneous decrease in lactose and potassium contents after intra-arterial injection of 14--80 pgouabain per kg body weight. This a m o u n t of ouabain will give a maximal blood concentration of 10 -6 M. This concentration is insufficient for full inhibition o f the (Na +, K÷)-ATPase system in mammary gland

19 (100% inhibition by 10 -4 M, 30% inhibition by 10 -6 M ref. 9), but is able to cause peripheral vasoconstriction as shown by several authors (see above}. Hence the effect of ouabain, described by Silcock and Patton [8] and by ourselves [10], will be due primarily to vasoconstriction in the udder and only secondarily to an inhibition of the (Na ÷, K÷)-ATPase system by ouabain.

Acknowledgements The skillful technical assistance of Mr H.P.G. Swarts is gratefully acknowledged. We wish to thank Mr A. Kusuma and Drs L. Brunninkhuis for their cooperation in parts of this study. This study was supported in part by a grant from the Netherlands Organization for Fundamental Research (ZWO)through the Netherlands Foundation for Biophysics.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

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