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Bioscience Reports 2, 661-666 (1982) Printed in Great Britain 661 M o d u l a t i o n of p h o s p h a t e a c c u m u l a t i o n in i s o l a t e d c h i c k k i d n e y c e l l s by g l u c o n e o g e n i c s u b s t r a t e s Michael F. GRAHN and Peter J. BUTTERWORTH Department of Biochemistry, Chelsea College, University of London, Manresa Road, London 57#'3 6LX, U.K. (Received 26 July 1982) An i s o l a t e d proximal-tubule preparation is described t h a t accumulates Pi in a saturatable, Na+-dependent manner. The initial rate of Pi accumulation is greater in cells incubated with the gluconeogenic substrates p y r u v a t e and l a c t a t e t h a n in cells incubated with glucose. Glucose was produced from, the substrates under these conditions. Incubation with either NAD+ or NADH inhibits the initial rate of Pi accumulation. T h e s e d a t a provide e v i d e n c e t h a t the e f f e c t s of gluconeogenesis on Pi uptake are not mediated by the oxidation state ol the nicotinamide coenzymes. Much work by many groups utilizing a variety of experimental approaches has shown that phosphate absorption in intestine and kidney proceeds by Na+-dependent carriers and is under elaborate physiological control. The a g e n t s i m p o r t a n t for controlling kidney phosphate reabsorption seem to be parathyroid hormone (PTH), dietary phosphate status, and vitamin D compounds. These agents seem to act at the proximal-tubule region ol the nephron. The tubules seem to provide the major sites for Pi reabsorption (see various articles in Massry and Fleisch, 1980, for reviews of these subjects). In addition to their role in metabolite reabsorption, proximal-tubule cells are also important gluconeogenic sites particularly in birds where it has been proposed that the kidney is more important than the liver for glucose production from non-carbohydrate sources e.g. amino acids (Watford et al., 1981). Stimulation ol kidney gluconeogenesis by PTH has also been r e p o r t e d and the mechanism may involve elevated cellular cyclic AMP levels (Nagata & Rasmussen, 1970). The molecular mechanisms by which the various agents mentioned above modulate renal phosphate reabsorption have not been established, but it has been proposed that the inhibitory action of PTH on Pi transport is mediated by cyclic AMP. By analogy with other cyclicAMP-modulated systems, PTH action may involve a series of phosphorylation reactions of sensitive proteins inchJding a component of the Pi carrier system. It has been argued however that there is a lack of good evidence for such a 'direct' action of cyclic AMP (Dousa, 1980). Thus an a l t e r n a t i v e explanation for the hormone action has been proposed in which it is suggested that the raised cellular cyclic AMP stimulates the rate of gluconeogenesis. As a consequenc% the redox state of the N A I ~ ' / N A D H couple is altered and the changed ratio is assumed to have a direct inhibitory effect on the Pi carrier (Kempson 9 1982 The Biochemical Society 662 GRAHN & BUTTERWORTH e t al., 1981; Berndt et al., 1981). NAD + was shown to inhibit Na+-dependent Pi transport in vitro (Kempson et al., 19gl). Having demonstrated that renal brush border membranes hydrolyse NAD +, Tenenhouse and Chu (1982) suggest that the NAD + inhibition of transport is trivial in that Pi liberated from NAD +, and not NAD + itself, is the inhibitory agent. They question whether NAD + is likely to have any physiological role in controlling Pi transport. In attempts to establish the molecular basis for the control of kidney Pi reabsorption, and to investigate the link between transport and m e t a b o l i c a c t i v i t y , we have been studying the properties of isolated proximal-tubule cells prepared from chick kidney. These ceils can respond to treatments devised to promote gluconeogenesis. We r e p o r t the results of our preliminary experiments since they bear directly on the controversy generated by the proposals of Kempson et al. (1981). M a t e r i a l s and Methods Isolation of chick kidney tubule cells The method was based on that of Larkins et al. (197~). Male Light Sussex chicks were reared on a normal complete diet for 10-30 days before they were used for celt preparation. The isolation buffer was modified to the extent that phosphate was omitted and glucose was replaced by other substrates for some experiments (see 'Results' section). Phosphate accumulation The 'working incubations' containing cells and normally of 5 cm3 vol. were maintained at 37~ in a shaking water bath for 10 min before [32p]Na2HPO~ (50 pCi/mol) was added. Duplicate samples (0.5 cm3) were r e m o v e d at various t i m e s after the Pi addition and centrifuged through silicone oil to separate the ceils from the bathing medium. The cells w e r e ' c a p t u r e d ' in perchloric acid and the radioactivity in this fraction was determined by scintillation counting. Other methods R e s p i r a t i o n was studied using an oxygen-electrode assembly and p r o t e i n was d e t e r m i n e d by the method of Lowry et al. (1951). Glucose was measured by a glucose oxidase reagent kit (Boehringer Corp. London, Ltd). Results The influence of the pattern of carbohydrate metabolism on the i n i t i a l r a t e of Pi accumulation by the cells was investigated by i n c u b a t i o n of the cells with substrates that support predominantly g l y c o l y s i s ( g l u c o s e ) or which allow gluconeogenesis (pyruvate and lactate). The results are shown in Table 1. The 'isolation substrate' was t h a t p r e s e n t during the preparation of the cells. This was replaced by the 'incubation substrata' in which the ceils were then MODULATION OF Pi ACCUMULATION BY GLUCONEOGENESIS Table i. Effect of substrates on the initial rate of phosphate accumulation by chick proximal tubule cells Tubule cell suspensions were Prepared and incubated as ~[he Na + salts of pyruvic and lactic acids were used and accumulation was measured at 0. i mM concn, of Pi in The results shown were obtained in a single but typical isolation substrate (mM) 663 Incubation substrate (mM) Pre-incubation period (min) described in the text. the initial rate of Pi the incubation medium. experiment. Initial Pi accumulation pmol/min/mg protein None glucose (i0) 60 250 None pyruvate (I0) 60 558 None lactate (i0) 60 528 Pyruvate (i0) pyruvate (i0) 30 652 Pyruvate (i0) pyruvate (i0) + glucose (I0) 30 562 kept for the times shown prior to determination of the initial rate of Pi accumulation. Both pyruvate and l a c t a t e stimulated the initial rate of Pi uptake considerably when compared with glucose. Incubation of ceils for 30 min with pyruvate or l a c t a t e did not cause a significant change in the rate of respiration (data not shown)~ indicating that the increased transport rate cannot be ascribed to a g r e a t e r availability of ATP arising from a promotion of oxidative metabolism (see Mandel & Balaban, 1981). The presence of pyruvate during the cell isolation further stimulated the r a t e of Pi accumulation. The addition of glucose during pre-incubation antagonized this action of pyruvate. The stimulatory action of pyruvate on Pi uptake by cells prepared in the presence of glucose is shown in more detail in Fig. 1. The e f f e c t is half maximal at approx. 0.5 mM. The maximal stimulation of Pi accumulation was less th an t h a t observed with cells that had been p r e t r e a t e d with pyruvate alone (cf. Table 1). The glucose production of cells was determined under the same c o n d i t i o n s used in the transport studies and was found to be 1.6 nmol/min/mg protein for cells incubated with 10 mM pyruvate and 1.g nmol/min/mg for cells incubated with 10 mM l act at e. Thus these s u b s t r a t e s e l i c i t a s i g n i f i c a n t g l u c o n e o g e n i c response under the conditions in which transport was stimulated. Incubation of the cells with both NAD+ and NADH at relatively high concentrations markedly inhibited the initial rate of Pi accumulation (Fig. 2). The response was similar to that described for rat kidney brush border membrane vesicles (Kempson et a l , 1981). Discussion These results dem ons t r a t e that a direct inhibition of Pi uptake by NAD + and NADH occurs with c ompl et e cell preparations as well as with the membrane preparations used by Kempson et al. (1981). This inhibition may be a r t e f a c t u a l (Tenenhouse & Chu, 1979)~ and it is difficult to envisage a regulatory role for the coenzyme at these co n cen tr at i ons external to the cell. It seems unlikely that NAD(H) would cross the ceil membrane~ but plasma-membrane oxidoreductases m a y t r a n s f e r r e d u c i n g equivalents across the membrane and may 664 GRAHN & BUTTERWORTH 220 200 .1_ = 180 ug t.J 6.-E oJ E -,- ~ r 160 1/,0 o E 120 E m 10% --J~ i 01 3 Pyruvafe I~) (raM) Fig. i. The effect of pyruvate on phosphate uptake. Tubule cell suspensions (1.4 mg protein/cm 3) that had been isolated in glucose medium were pre-incubated for 30 m i n with sodium pyruvate (0-i0 mM) after which [32p]Na2HP04 (0.I raM) was added and the initial rate of phosphate accumulation was determined. 120 100 8O ~ ~ o 60 ~0 o ~ 20 "E 9 0 i I I I I I 2 3 ~ 5 NAD(H) (mH) Fig. 2. The actions of NAD + and NADH on phosphate uptake. Tubule cells were pre-incubated with NAD + ( O ) or N ~ H (O) at the concentrations s h o ~ for 30 min after ~ i c h [32p]Pi was added (0.i mM) and the initial uptake was determined. The results shown for NAD + are the means of two separate experiments of protein concns. of 0.6 and 1.6 mg/cm 3 and control Pi uptakes of 206 and 115 ~ o l / m i n / m g protein respectively. The NADH result s h m ~ is the mean • S.E.M. of 3 separate experiments. MODULATION OF Pi ACCUMULATION BY GLUCONEOGENESIS 665 p a r t i a l l y e n e r g i z e t r a n s p o r t processes (Christensen, 1977; Low & Crane, 1978). Without wishing to stress any physiological significance for the inhibition, it remains possible that its cause is less simple than T e n e n h o u s e and Chu (1982) propose. In this connection it is of in ter es t that NADH has been shown to stimulate organic acid transport in renal tubules (Nikiforov, 1982). In the original model proposing a direct link between Pi transport and gluconeogenesis, it was envisaged that increased gluconeogenesis is associated with a decreased rate of transport (Kempson et al.~ 1981). Obviously our results cannot be f it t ed by the model, since we find th at gluconeogenesis and Pi transport change in the same direction. A f e a t u r e of the original model was that gluconeogenesis was assumed to increase the cytosolic NAD+/NADH ratio, and because the oxidized form of the c o e n z y m e is less tightly bound to protein, the fraction os ' f r e e ' nicotinamide nucieotide would also rise. A direct interaction of the c o e n z y m e with the Pi transporter that a f f e c t e d its activity was proposed. We find t h a t both pyruvate and l a c t a t e stimulate Pi accumulation~ yet these substrates would be expected to alter the NAD+/NADH ratio in di f f er e nt directions. Although our results appear to invalidate the model as originally formulated, it does seem clear that there is a close linkage between the rate of Pi uptake and the pat t ern of cell metabolism, especially gluconeogenesis. Thus modulation of cellular metabolism is likely to be of considerable importance in the regulation of Pi reabsorption by renal proximal tubules. We are investigating the cause of the promotion o5 Pi uptake by gtuconeogenesis that seems to contrast with the known actions of PTH to both promote gluconeogenesis yet inhibit tubular Pi reabsorption. We have evidence that under conditions of Pi deprivation when the phosphaturic response to PTH is blunted, PTH can transiently stimulate Pi accumulation (Grahn & Butterworth, unpublished dat a). Acknowledgement The authors thank porting this work. the National Kidney Research Fund for sup- References Berndt TJ, Knox FG, Kempson SA & Dousa TP (1981) Endocrinology 108, 2005-2007. Christensen HN (1977) J. Supramol. Struct. 6, 205-213. Dousa TP (1980) Curt. Topics Membr. Transp. 13~ 401-413. Kempson SA, Colon-Otero G, Lise-Ou S-Y, Turner ST & Dousa TP (1981) J. Clin. Invest. 67, 1347-1360. Larkins RG, MacCauley SJ, Rapoport A, Martin TJ, Tulloch BR, Byfield PGH, Matthews EW & Maclntyre I (1974) Clin. Sci. Mol. Med. 46, 569-582. Low H & Crane FL (1978) Biochim. Biophys. Acta 515, 141-161. Lowry OH~ Rosebrough NJ, Farr AL & Randall RJ (1951) J. Biol. Chem. 193~ 265-275. Mandel LJ & Balaban RS (1981) Amer. J. Physiol. 240, F357-F371. 666 GRAHN & BUTTERWORTH Massry SG & Fleisch H (1980) Renal Handling of Phosphate, Plenum Medical Book Co., New York and London. Nagata N & Rasmussen H (1970) Proc. Natl. Acad. Sci. U.S.A. 65, 368-374. Nikiforov AA (1982) Biochim. Biophys. Acta 686, 36-46. Tenenhouse HS & Chu YL (1982) Biochem. J. 204, 635-638. Watford M, Hod Y9 Chiao Y-B, Utter MF & Hanson RW (1981) J. Biol. Chem. 256, 10023-10027.