Download Modulation of phosphate accumulation in isolated chick kidney cells

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.