Biochemical Society Transactions (1995) 23 293s
Trans-sarcolemmal Pi fluxes in perfused rat heart: a
quantitative analysis
GRAHAM J. KEMP, KIM E. POLGREEN, JAMES A. C.
HOPKINS, KIERAN CLARKE and GEORGE K. RADDA.
MRC Biochemical and Clinical Magnetic Resonance Unit,
Oxford Radcliffe Hospital, Oxford OX3 9DU.
The intracellular concentration of Pi depends in the long-term
on Na/Pi-cotransport across the cell membrane, and in the short
term on the permeability of the cell membrane to net fluxes of
Pi [I]. Both the steady-state relationship between intra- and
extracellular [Pi] and the rate and size of transient responses
depend on the kinetic properties of unidirectional Pi efflux,
which is difficult to study by classical radioisotope techniques
[I]. We have reported transient changes in cell [Pi] in the
perfused rat heart in response to perfusion without Pi and also
with insulin [2]. Here we use those data and the results of
perfusion with 2-deoxyglucose, an intracellular 'Pi-trap', to
deduce some properties of Pi efflux.
As described elsewhere [2], hearts isolated from Wistar rats
were perfused in the Langendorf mode with a Pi-containing
Krebs-Henseleit buffer in a 9.4 T vertical magnet. 31PMR
spectra were acquired each 3 min. Data were Fouriertransformed and spectral peak areas quantified using the NMRl
programme. Concentrations of Pi, phosphocreatine (PCr),
phosphornonoester (PME) and ATP were calculated using an
external phosphonate standard. Total phosphorus content (TP)
was calculated as Pi+PCr+3ATP+PME. and changes in TP were
used to estimate rates of net Pi influx or efflux. Data were
acquired under control conditions ( 1 or 2 mM extracellular Pi)
and during three manipulations: perfusion without Pi; perfusion
with 1 mM Pi and 20 nM insulin; and perfusion with 1 mM Pi
and 2 mM 2-deoxyglucose.
Data are interpreted as follows [ l , 21: at steady state,
unidirectional influx (basal I) is equal to unidirectional efflux
(basal E), and net efflux (J = E - I) is zero. During perfusion
without Pi, unidirectional influx is zero, so measured net efflux
is an estimate of E at each time point (J = E). Furthermore, the
initial value of J is an estimate of the pre-existing basal rate of
unidirectional Pi efflux (initial J = basal E ) . When an
intracellular P pool expands (e.g. PCr during insulin perfusion
[2], or deoxyglucose-6-phosphate during perfusion with
deoxyglucose), cell [Pi] tends to fall, and this drives net influx.
Given an estimate of unidirectional Pi influx, measured net
influx (= negative efflux) can be used to estimate unidirectional
efflux (estimated E = measured J + assumed I ) ; a suitable value
of I is obtained from the initial response to Pi-free perfusion
(see above). Our aim was to estimate unidirectional efflux E as
a function of [Pi].
During perfusion without Pi (Fig. A), cell [Pi] and [PCr]
decreased with time, and the associated fall in [TP] implied net
Pi efflux [2]. This had a linear relationship to [Pi] with slope
2M2 h-I, and the initial value extrapolated to t=O was 29f2
mMh (Fig. A). Surprisingly, net Pi efflux fell to zero when cell
[Pi] had decreased by only 1.5 mM (cf. control cell [Pi] = 4
mM). During perfusion with insulin (Fig. B), cell [Pi] decreased
and [PCr] increased with time, and the associated rise in [TP]
implies net Pi influx. For the first data point (Fig. B), the slope
of influx rate against the fall in cell [Pi] was 1M3 h-I. While
this influx continued, however, cell [Pi] returned to its basal
value [2]. During perfusio n with deoxplucosp (Fig. C), cell
[Pi] and [PCr] both decreased with time, while [2deoxyglucose-6-phosphate] increased at a rate of 3M2 mMh.
There was a steady rise in [TP], implying net Pi influx which
was proportional to the fall in cell [Pi], with a slope of 21f3 h-1
(Fig. C).
The best estimate [ l ] of the first-order rate constant of
unidirectional e f l u is the slope of net efflux against cell [Pi] in
Symbols: I,E = rates of unidirectional influx and efflux
J = rate of net efflux
the Pi-free perfusions, 20 h-I (Fig. A). This is quantitatively
consistent with the results of deoxyglucose perfusion (Fig. C).
The slope of net influx against fall in cell [Pi] in the insulin
experiment is also an estimate of the efflux rate constant (as in
the deoxyglucose perfusion), but this was only 10 h-l (Fig. B).
This discrepancy could arise if unidirectional Pi influx were
reduced about 60% by insulin. The basal Pi exchange rate
estimated from the Pi-free perfusion is 29 mMh. This should be
equal to the rate of Na-linked Pi uptake, which has a Km of 0.1 0.5 mM in vitro [3-51 and is therefore practically independent of
extracellular [Pi]. The exchange rate in vitro, measured using
32P-labelling experiments, is similar, around 25 mM/h [3-51.
This rate should also [I] be equal to the product of the efflux
rate constant and the exchangeable cell [Pi] (which is the
maximum fall observed in the Pi-free perfusion, i.e. 1.5 mM).
Calculation shows this is approximately so.
Fig. D combines all these inferences about the Pi
dependence of unidirectional Pi efflux. It shows the similar
slope in Pi-free perfusion and deoxyglucose experiments, as
well as the lower slope seen in perfusions with insulin. It
appears that unidirectional Pi efflux can be accounted for by a
single approximately first-order process during the responses to
perfusion without Pi (which causes net Pi efflux) and perfusion
with 2-deoxyglucose (which causes net Pi influx). However,
insulin alters Pi transport in some way unrelated to intracellular
Pi trapping by the expanding PCr pool.
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11-94
2. Polmeen. K. E.. Kemo. G. J.. Clarke. K. & Radda. G. K.
(19!%) J. Mol. Cell. Cardiol. 26,219-228
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256, C322-C328
4. Jack, M. G., Huang, W.-H. & Askari, A. (1989) J. Biol.
Chem. 264,3904-3908
5. Medina, G. & Illingworth, J. (1980) Biochem. J. 188,
297-3 1 1
"p#(
Net Pi efflux (mM/h)
Net Pi influx (rnM/h)
20
10
0
-1 0
Net Pi influx (mM/h)
-1.0
-0.5
0.0
Change in [pi] (mM)
Unidirectional efflux (mM/h)
-2
-
1
0
Change in [Pi] (mM)
EibTmLlmedSEM)
A. Net Pi efflux as a function of the fall in cell [Pi] during
Pi- free perfusion [2].
B.Net Pi influx as a function of the fall in cell [Pi] during
perfusion with 20 nM insulin [2].
C Net Pi influx during perfusion with 2 mM deoxyglucose.
D. Summary:Unidirectional efflux (inferred from Figs. A-C)
as a function of the fall in cell [Pi]. Circles = Pi-free
perfusion; triangles = insulin; squares = 2-deoxyglucose