The ouabain-insensitive sodium pump

The ouabain-insensitive sodium pump

Camp. Biochem. Physiol. Vol. 99A, No. 3, Printedin GreatBritain pp. 279-283, 1991 0300-9629/91$3.00+ 0.00 0 1991Pergamon Press plc MINI REVIEW THE ...

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Camp. Biochem. Physiol. Vol. 99A, No. 3, Printedin GreatBritain

pp. 279-283, 1991

0300-9629/91$3.00+ 0.00 0 1991Pergamon Press plc




FULGENCIO PROVERBIO,* REINALW M.&N and TERESAFQOVERBIO Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (I.V.I.C.), A.P. 21827, Caracas 102OA,Venezuela. Telephone: (582) 501 1395

(Received 31 August 1990)


Mammalian kidney cells can be loaded with Na+ and Cl- and depleted of their K+, by cooling the cells in a medium without K+. After the cooling period, rewarming of the cells in a Ringer medium with K+, leads them to extrude actively the gained Na+. The extrusion of Na+ occurs via two mechanisms: (1) in exchange for K+, which enters the cells and (2) together with Cl- and water (Mudge, 1951a,b; Whittam and Davies, 1953; Whittembury, 1965; Whittembury and Proverbio, 1970). If the cells are rewarmed in a medium without K+, or in the presence of K+ and high concentrations of cardiac glycosides, there is no more extrusion of Na+ in exchange for KC, however the cells still extrude Na+ together with Cland water (Kleinzeller, 1961; Kleinzeller and Knotkovii, 1964; Whittembury, 1965; Willis, 1966; Macknight, 1968; Whittembury, 1968; Whittembury and Fishman, 1969; Whittembury and Proverbio, 1970). On the other hand, if the cells are rewarmed in the medium with K+, in the presence of ethacrynic acid or furosemide, the cells extrude Na+ in exchange for K+, but are not able to extrude Na+ along with Cl- and water (W~tt~bu~ and Proverbio, 1970; Perez-Gonzalez et al., 1980). These results led us to postulate, for this tissue, the existence of two pumps responsible for the active Na’ extrusion from the cells: the ouabain-sensitive, K+-dependent, Na,K-pump, and the ouabain-insensitive, K+independent Na-pump. The existence of the two pumps was further corroborated in experiments carried-out with basolateral plasma membranes vesicles from rat kidney proximal tubular cells (Marin et al., 1985). ENERGY SOURCE FOR THE Na-PUMP

We have demonstrated that there is an oxygen consumption associated with each of the two modes of Na extrusion (Proverbio et al., 1985; Marin et al., 1989). Both modes of Na+ extrusion are curtailed by anoxia or by 2,4_dinitrophenol ~ittemb~, 1968; Whittembury and Proverbio, 1970), which are inhibitors of the synthesis of cellular ATP (Caldwell, 1956). Accordingly, the two Na-extruding mechanisms must *Address all correspondence to Dr F. F’roverbio, CBBIVIC, A.P. 21827, Caracas 102OA,Venezuela. CBP 99A/+--8

obtain their energy from hydrolysis of ATP, On this regard, we have demonstrated the existence of two Na-stimulated ATPases in basolateral plasma membranes from guinea-pig and rat kidney cells: the Na,K-ATPase and the Na-ATPase (Marin et al., 1985; Proverbio and de1 Castillo, 1981; Proverbio et al., 1983; del Castillo et al., 1982). The former, as widely accepted, responsible for the extrusion of Na + in exchange for K+, and the latter, as we will discuss in the next chapter, responsible for the extrusion of Na+, along with Cl- and water. R~~ONS~P BEAM THE K+-~DEPENDE~ Na-PUMP AND THE K+-~DEPENDENT Ns-ATPJw

Table 1 shows several characteristics of both the K+-independent, active extrusion of Na+ accompanied by Cl- and water from the kidney cells, and of the K+-independent, Na-stimulated ATPase activity of their basolateral plasma membranes. Notice that both expressions share similar characteristics. These common characteristics can be interpreted as an indication that the K+-independent active extrusion of Na+ accompanied by Cl- and water and the K+-independent, Na +-stimulated ATPase activity, are different expressions of the same system: the Na-pump or Na-ATPase. This interpretation has been supported by experiments carried out with basolateral plasma membrane vesicles from rat kidney proximal tubular cells (Marin et al., 1985), that unequivocally showed Nat-transport against its chemical gradient, catalysed by a K+-independent, ouabain-insensitive, Na-ATPase. FUNCTION OF THE ND-PUMP IN THE RENAL TISSUE

The Na-ATPase, as well as the Na,K-ATPase are located at the basolateral plasma membranes of the kidney proximal tubular cells (Marin et al., 1985; Proverbio et at., 1985; Marin et al., 1989). Considering that the Na-pump extrudes Na+ together with Cl- and water from the cells, through their basolateral plasma membranes towards the interstitial space, and that an important fraction of the Na+ reabsorbed by the kidneys, is carried-out at the proximal tubules, it becomes clear that the Na-pump is involved in the process of reabsorption of Na+, Cland water, from the kidney lumen through the tubular cells, toward the interstitial space. The existence




Table 1. Common characteristics of the Na-pump and of the Na-ATPase of mammal kidney cells Common characteristics


K+-insensitivity Ouabain-insensitivity Ethacrynic acid sensitivity Furosemide sensitivity Lithium stimulation Anion insensitivity Location at the kidney cell basolateral plasma membranes

References Na-ATPase

1, 2, 3, 4, 5 3, 5, 9, 10, 11 5, 11 12, 14 (our data)* (our data)*

6, 6, 1, 7, 6, 6,

7, 8 8, 12, 13 13 12 15 15

5, 12

6, 12, 15, 16

References: (1) Whittembury, 1965. (2) Kleituelb, 1961. (3) Macknight, 1968. (4) Willis, 1966. (5) Wittembury and Proverbio, 1970. (6) Proverbio and de1 Castillo, 1981. (7) de1 Castillo e( al., 1982. (8) Proverbio er al., 1975. (9) Kleinzeller and Knotkova, 1964. (10) Whittembury, 1%8. (11) Proverbio er al., 1970. (12) Marin et al., 1985. (13) Marin et al., 1983. (14) Perez-Go&lez M. er al., 1980. (15) Marin el al., 1989. (16) Proverbio er al., 1985. *Unpublished.

of the ouabain-insensitive Na-pump, besides the ouabain-sensitive Na,K-pump, accounts for the observation (in dog and rats) that about half of the Na+ filtered by the kidney continues being readsorbed under conditions in which the Na,K-ATPase has been completely inhibited by large doses of cardiac glycosides (Torretti et al., 1972; Ross et al., 1974).



Ouabain-insensitive, K+-independent, active Na+ transport (Na-pump), and ouabain-insensitive, K+independent, Na+-stimulated A’lTase activity (NaAlTase), have been demonstrated in several tissues

Table 2. K+-independent, active Na+-transport and K+-independent, ATPase activity in different organisms and tissues Organism Guinea-pig Rat pig cow Chicken Duck Bullfrog Toad Iguana Sea bass Squid Trout Small mouth bass Sea bass Crab Shrimp Mussel Gilthead bream Guinea-pig Rat Tobacco hornworm Rat Rat LK sheep Planaria Acholeplasma laidlawii B Vibrio alginolyticw

Tissue kidney kidney kidney kidney kidney kidney kidney kidney kidney kidney gill gill gill gill gill gill gill gill small intestine small intestine midgut brain cortex liver red cells


References Transport ATPase 1, 2 5, 6, 7, 8


2, 3, 4 9, 10, * l

* l

. l



25 28, 29 -


t 11 12 13; 14; 15 16 17; 18 19 20; 21 22, 23 24 26, 27 29, 30, 31 34 t * t 32 33 35

(1) Whittembury and Proverbio, 1970. (2) Proverbio er al., 1970. (3) Proverbio and de1 Castillo, 1981. (4) Proverbio et al., 1975. (5) Marin er al., 1985. (6) Munday er al., 1971. (7) Proverbio er al., 1983. (8) Marin et al., 1989. (9) Marin et al., 1983. (10) Proverbio er al., 1986. (11) Pagharani ef al., 1988. (12) Proverbio er al., 1988. (13) Pfeiler and Kirschner, 1972. (14) Pfeiler, 1978. (15) Ventrella er al., 1989. (16) Pfeiler, 1976. (17) Borgatti et al., 1985. (18) Ventrella ef al., 1987. (19) Martelo er al., 1988. (20) Rodriguez et al., 1988. (21) Proverbio et a/., (in press).(22) Howland and Faus (1985). (23) Lagerspetz and Senius, 1979. (24) Ventrella et al., 1990. (25) de1 Castillo and Whittembury, 1987. (26) de1 Castillo and Robinson, 1985a. (27) de1 CastiBo and Robinson, 1985b.(28) Grsenigo er al., 1987. (29) Tosco er al., 1988.(30) Luppa and Miiller, 1982. (31) Grsenigo et al., 1988. (32) Venturini et al., 1981. (33) Lewis and McElhaney, 1983. (34) Wieczorek et al., 1986. (35) Dibrov et al., 1988. *Our data, unpublished.

Ouabain-insensitive Na-ATPase of different living organisms. Table 2 shows a summary of these findings. From the sag it is evident that the Na-ATPase is not restricted to the basolateral plasma membranes of mammalian kidney proximal tubular cells, but it is widely spread, being present in different tissues and organisms. The systems listed in Table 2 are not totally similar. Some of them present the same characteristics shown by the Na-ATPase or the Na-pump of the kidney cells indicated in Table 1; others differ in one or more features. Thus, the so-called Na-ATPase in all these papers, can be stimulated by Na+ and poorly by Li+ or even other monovalent cations, as in kidney and small intestine (de1 Castillo et al., 1982; Marin et al., 1983; de1 Castillo and Robinson, 1985b; Orsenigo et al., 1988); it can be stimulated by Na+, K+, NH: and Li+, to a similar extent, as in sea bass gill preparations fventrella el al., 1987) or it can be stimulated by Nat, K+, Rb’ and Li+, in decreasing magnitudes, as in shrimp gills (Proverbio et al., in press). It can be insensitive to K+, as in kidney and small intestine (de1 Castillo et al., 1982; Marin et al., 1983), or it can be inhibited by Kf, as in shrimp gills (Proverbio et al., in press). It hydrolyses preferentially ATP as in kidney (de1 Castillo et al., 1982), or it hydrolyses several nucelotides as in sea bass gills (Borgatti et al., 1985). However, all these systems share as a rule two characteristics: (1) They do not need K+ besides Na+ (or in some cases another monovalent cation), to reach their maximal activity and (2) they are insensitive to cardiac glycosides such as ouabain, even when used at very high concentrations. They generally also share the characteristic of being preferentially inhibited by ethacrynic acid over other mechanisms such as the Mg-, the Ca- and

the Na,K-ATPases. Up to now, even when there is a tendency to group all these sytems in an heterogeneous family, namely, the ouabain-insensitive Napump, we must be very careful on this respect. The existence of such different characteristics argues against the validity of the criteria utilized to group all these systems in one group. Are they different systems?, or are they just different expressions of one system working in different ways? Evidently, further work is required to answer these questions.


The function of the Na-pump in such tissues as kidney, small intestine and gills, seems to be evident, in a sense that it can produce a vectorial flux of Na+, from one side to the other of the epithelial cells, contributing in this way to the control of this ion within the organism. However, what is the Na-pump doing in cells not specifically involved in the body control of the Na’ ion? The experiment shown in Fig. 1 may answer this question. The figure shows the activity of the Na-ATPase of membranes prepared from kidney cells which were preincubated to let them gain water, i.e., to increase their volume. It can be seen that for any increase in the water content of the cells, there is a concomitant increase in the activity of the Na-ATPase of their membranes, until reaching maximal values. Considering that this effect is totally


Cell water content (g/g solids


Fig. 1. Na-ATPase activity of rat kidney cortex slices homogenates as a function of the eelI water content of the slices from where they were prepared. The indicated values aremeausfSEforn=6.

reversible (data not shown), and that it has been demonstrated to occur in other tissues such as gills, small intestine, liver, brain (data not shown), it can be concluded that, at least for these tissues, there is a clear and direct relationship between the cell volume and the activity of the Na-ATPase. These results indicate that the activity of the Na-ATPase is related to and modulated by the cell volume and they suggest that the main function of the Na-pump may be established through its association with the active cell volume regulation. This is relevant, as we found in the same experiments that the activity of the Na,KATPase is not altered by changes of the cell volume. The differential response of both Na- and Na,KATPase toward changes in the cell volume, gives strong support to the hypothesis of the existence of two distinct enzymes with presumably similar functions in the uphill extrusion of Na+ from the cell, but different function in the active cell volume regulation.


From the preceding discussion, it is attractive to speculate about the existence of a K-independent, Na- or another monovalent cation stimulated ATPase or pump, which, by extruding Na+ or another cation from the cells, along with Cl- and water, participates in the active regulation of the cell volume. This system, originally described for kidney cells and small intestine, seems to be widely spread. K+-independent, ouabain-insensitive, monovalent cation stimulated ATPase activities have been demonstrated to be present in the plasma membrane of cells of different tissues of very different organisms. Whether they constitute similar or different systems, has yet to be found out. Ack~owiedge~~ts-me authors wish to acknowledge Mrs Dhuwya Otero for her drawing.


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FULGENCIO Pru~muuo et al.


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