BY C. CLAIRE AICKIN, ALISON F. BRADING AND D. WALMSLEY From the University Department

22 Pages·2005·2.43 MB·English

J. Physiol. (1987), 391, pp. 325-346 325
With 12 text-figures
Printed inGreat Britain
AN INVESTIGATION OF SODIUM-CALCIUM EXCHANGE IN THE
SMOOTH MUSCLE OF GUINEA-PIG URETER
BY C. CLAIRE AICKIN, ALISON F. BRADING AND D. WALMSLEY
From the University Department ofPharmacology, South Parks Road,
Oxford OX] 3QT
(Received 25 November 1986)
SUMMARY
1. After application of ouabain (10-4 M), the intracellular Na+ activity (a'a) of
smooth muscle cells in the guinea-pig ureter stabilizes at arelatively low level which
can be rapidly lowered by reduction ofexternal Na+ (Na+) orelevation ofCa'+. Both
these procedures also elicit a transient contracture. These observations have pre-
viously beeninterpreted asevidence forNa+-Ca2+ exchange. Thepresence ofsuch an
exchange mechanism has now been further investigated by measurements of a'a,
tension, ion analysis and 22Na efflux.
2. Ion analysis demonstrated that tissues were able to maintain a high cellular
K+ content in the presence ofouabain, but slowly lost K+ and gained Na+ ifK+ was
alsoremoved, asexpectedforaninfiniteoutwardgradientforK+andafullyinhibited
Na+ pump.
3. Tissueswere only able to maintain alow cellular Na+ and high cellular K+ inthe
presence ofouabain ifCa2+ was present in the bathing solution. Reduction ofCa2+ to
very low levels also caused a continual slow rise in a'a in the presence of ouabain,
provided that theprolonged depolarization caused bythese low levelswasprevented
by elevation of Mg2+. Alteration of the membrane potential by changing K+ at
constant Na+ showed thataiadecreased byabout 1-2 mmfora 10 mVdepolarization,
within the range from -70 to -30 mV.
4. A small Ca2+-activated 22Na efflux was observed in ouabain-treated tissues in
the absence ofNa+. 40 mM-Ca2+ was not more effective at activating this efflux than
was 2-5 mM-Ca2+, while 40 mM_Mg2+ was ineffective. Restoration ofthe normal Na+
caused a large increase in the rate of 22Na loss.
5. Application ofMn2+ in the presence ofouabain caused a slow rise in a'a and a
small decline inresting tension. Thefall ina'aon reduction ofNa+ wasslowed by the
presence ofMn2+ (mean half-timeincreased from 1-7 to5*0 min) and the concomitant
contracture was almost abolished. These results are consistent with a Mn2+ -induced
inhibition of Na+-Ca2+ exchange. However, the fall in a' a induced by elevation of
Ca2+ was unaffected by the presence ofMn2+ and the attendant contracture was, if
anything, enhanced.
6. Observation of changes in ai and tension at various Mn2+ and Ca2+ concen-
trations demonstrated a competitive interaction between the two divalent cations.
7. The effects of other putative inhibitors of Na+-Ca2+ exchange (quinacrine,
326 C. C. AICKIN, A. F. BRADING AND D. WALMSLEY
dichlorobenzamil, dodecylamine and La3+) on the contractile responses to lowering
Na+ were examined. All were able to block the Na+-withdrawal contracture of
ouabain-treated tissues but at a concentration that was substantially higher than
that required to block the K+ contracture of normal tissues.
8. We conclude that Na+-Ca2+ exchange plays a major role in the regulation of
aia in the smooth muscle ofguinea-pig ureter when the Na+ pump is inhibited. Its
properties appear to be similar to those reported for the exchange mechanism in
other preparations.
INTRODUCTION
The finding of reciprocal transmembrane movements of Na+ and Ca2+ ions on a
reversible Na'-Ca2+ exchange mechanism (Reuter & Seitz, 1968; Baker, Blaustein,
Hodgkin & Steinhardt, 1969) excited much interest about therole ofthismechanism
in the regulation of intracellular Ca2+ and hence the contractile state of muscular
tissues. A considerable volume ofresearch has led to the firm establishment of the
existence and importance of Na+-Ca2+ exchange in cardiac muscle. Its role in the
positiveionotropyobservedonreductionofexternalNa+(Na+)orelevationofinternal
Na+ (Nat) is widely acknowledged (for recent review see Reeves, 1986) while its
electrogenic contribution to the cardiac action potential has been identified (see
Noble, 1986) and even its current recorded in isolated cardiac cells (Kimura, Noma
& Irisawa, 1986; Mechmann & Pott, 1986). The same success story, however, cannot
be told in smooth muscle. After initial enthusiasm which culminated in the pos-
tulation ofits involvement in the aetiology ofhypertension (Blaustein, 1977), there
has been increasing disenchantment with the concept of its existence, let alone
contribution to physiological or pathological behaviour. This stems from many
detailed studies of both radioisotope flux and tension which have been reviewed
elsewhere (van Breemen, Aaronson & Loutzenhiser, 1979; Casteels, Raeymaekers,
Droogmans & Wuytack, 1985; Brading & Lategan, 1985). In brief, genuine trans-
membrane Na+-dependent Ca2+ fluxes and Ca2+-dependent Na+ fluxes (as opposed
to Na+-Ca2+ competition at extracellular binding sites) have proved extremely dif-
ficult to demonstrate, while the ability ofmany smooth muscles toremain relaxed in
the absence ofNa+ and the finding ofa well-developed ATP-dependent Ca2+ pump
have clearly shown that Na+-Ca2+ exchange is not solely responsible for the regu-
lation ofinternalCa2+. Nevertheless itseemsalargeandunjustifiable steptodiscount
the presence ofNa+-Ca2+ exchange on these grounds.
In a study ofthe contractile responses ofthe guinea-pig ureter, we demonstrated
the development ofa transient Na+-withdrawal contracture following inhibition of
the Na+ pump (Aickin, Brading & Burdyga, 1984). This was dependent upon Nat
and Ca2+ and wasresistant to concentrations ofCa2+antagonists which abolished the
K+ contracture. Ion analysis, and later direct measurement of the intracellular
Na+activity(Aickin, 1987), showedthatNatstabilizedatasurprisinglylowlevelafter
Na+ pump inhibition and could still be extruded on reduction ofNa+ or elevation of
Ca2+, against the electrochemical gradient for Na+ ions. These findings seem strong
evidence for the existence of Na+-Ca2+ exchange.
We have now investigated these phenomena further using a combined approach
with Na+-sensitive micro-electrodes, ion analysis, measurement oftension andradio-
Na+-Ca2+ EXCHANGE IN SMOOTH MUSCLE 327
isotope fluxes. Results with the former three techniques have assured that the
phenomena cannot be explained by residual Na+ pump activity and have confirmed
that, in the presence of ouabain, Na+-Ca2+ exchange plays a major role in the
regulation of Nat. However, even under optimal conditions, it has been difficult to
obtain very convincing evidence for Na+-Ca2+ exchange with flux studies. We have
also tested the efficacy of various putative inhibitors of Na+ Ca2+ exchange on the
Na+-withdrawal contracture compared with the K+ contracture and have studied the
inhibition caused by the presence of Mn2+ ions.
Some of these results have been published in preliminary form (Aickin, 1984;
Brading & Aickin, 1985).
METHODS
Thesewereessentially thesame asdescribed previously (Aickin etal. 1984; Aickin, 1987). White
maleguinea-pigs were stunned and bled and both ureters removed. These were then maintained in
modified Krebs solution of the following composition (mM): NaCl, 120-3; KCl, 5-9; CaCl2, 2-5;
MgCl2, 1-2; Tris Cl, 16-6at pH 7-4; glucose, 11-5, equilibrated with 100% 02 at 35-37 'C. In many
experiments Na+ was reduced to 92 mm with an equimolar increase in Tris+ so that the concen-
tration ofdivalent cations could be raised without the complication ofa simultaneous reduction
afAncrease
in Na+ or in osmolarity. All other alterations to the solution were made by isosmotic
compensation with Tris+, with the exception ofreductions in Ca2+ to concentrations below 2-5 mm
when an equimolar increase in Mg2+ was made to maintain a minimum divalent cation concen-
tration of 3-7 mM. Ouabain (BDH, G strophanthin) was added directly to the modified Krebs
solutions immediately prior to use.
Measurement ofala
Theonly alteration to themethods recently described (Aickin, 1987) was in the filling procedure
of the double-barrelled electrodes. After silanization and at least 24h baking at approximately
180°C, a small amount ( 05,l) ofNa+-sensitive ligand (ETH 227, Fluka) was injected into the
back ofone barrel. The appearance ofthe ligand in the tip ofthe electrode was observed using a
light microscope and in some cases hastened by the use oflocalized heatingfrom amicro-forge. As
soon astheligandfilled theextreme tip, asimilar volume ofreferenceliquid ionexchanger (RLIE;
Thomas&Cohen, 1981) wasintroduced intothebackoftheotherbarrel. UsuallytheRLIErapidly
randown tothetipofitsbarrel butonoccasions localized heatingwasagainapplied. Theelectrode
was then placed in a desiccator which was evacuated and left overnight, during which time all
bubbles generally disappeared. Any remaining bubbles were extracted by localized heat from the
micro-forge. Thisnewprocedure markedly increasedtheyieldoffunctionalelectrodes, presumably
because ofthe much reduced time for the Na+ ligand to contaminate the tip ofthe other barrel.
Sensitivity ofthese electrodes to Mn2+ was found to be about one-fifth asgreat asthat to Ca2 .
Entry of Mn2+ into the intracellular space is likely to be small in these experiments and it is
probable that Mn2+ ions are then well buffered. Therefore it seems unlikely that application of
Mn2 would cause significant interference on the intracellular signal. In this respect it should be
noted that application of Mn2+ causes a maintained fall in ala in the presence of a functional
Na+ pump but aslow rise inala after pump inhibition (Aickin, 1987), indicating that interference
from intracellular Mn2+ is indeed insignificant.
Measurement oftension
Each ureterwascutintothreepiecesofapproximately equal length. Thepiecesweresuperfused
in the apparatus described by Brading & Sneddon (1980) and tension was recorded isometrically.
Ion analysis
Each ureter was cut into three pieces and equilibrated in modified Krebs solution for at least
1 h before being transferred sequentially into the appropriate experimental solution. Pieces were
removed after 1 or 3 h incubation, blotted and weighed (wet weight). They were then dried on
Teflon tape for 20 h at 50 °C, reweighed (dry weight), and extracted in 2 ml of a diluting fluid
328 C. C. AICKIN, A. F. BRADING AND D. WALMSLEY
containing 1 M-HNO3, 18mM-La3+ and 5-5mM-Li+. The solutions were then analysed by flame
photometry to estimate the total tissue content ofions.
Ionfluxes
Efflux of22Na was measured in a manner similar to that described for 36CI in the vas deferens
(Aickin & Brading, 1983). Whole ureters, mounted on stainless-steel holders, were loaded with
10min
I~~
Em
-50
Em -60
(mV) -70
-80L
200mg Tension
Ea
20
I~~~~~~~~~~~~~~~~N
aN8
(mm)
1
125mM-Ca2l 50% Na+
Fig.1. Pen recordings ofthe simultaneous measurement ofEm (shown at the top) and
aia (shown at the bottom) made with a double-barrelled Na+-sensitive micro-electrode,
and oftension (shown in the middle) recorded under the same conditions in a separate
experiment. Ouabain (10-4 M) was applied to the preparations for more than an hour
before the recordings were made and was present throughout the records shown. The
preparations were maintained in modified Krebs solution containing 106mM-Na+ and
25-mM-Ca2+ except for the intervals indicated when the changes in Ca2+ and Na+ were
madebyisosmoticalterationintheconcentration ofTris+.NotethatelevationofCa2+and
reduction of Na+ both cause a fall in a'a against the electrochemical gradient and a
transient contracture.
22Na in the presence ofouabain (10-4M) for 2h and then washed out in a superfusion apparatus.
The superfusate was collected in 2 min samples and counted, together with the tissue, at the end
ofthe wash-out.
RESULTS
Evidencefor Na+-Ca2+ exchange
The results shown in Fig. 1 summarize the evidence for the existence ofNa+-Ca2+
exchange in the smooth muscle ofguinea-pig ureter, reported in ourprevious papers
(Aickin et al. 1984; Aickin, 1987). These recordings were made after several hours'
Na+-Ca2+ EXCHANGE IN SMOOTH MUSCLE 329
exposure to ouabain (10-' M), a concentration at which the Na+ pump would be
expected to be fully inhibited, and yet aia was stable at 16 mm (equivalent to a
Na+ equilibrium potential of +43 mV) while the membrane potential (Em) was
-60 mV. Clearly Na+ ions remain far from equilibrium across the sarcolemma and
thus must be actively extruded by some ouabain-insensitive mechanism. The rapid
decline in a'a against both the electrical and chemical gradients, and the concomit-
10min
-40
-50
Em -
E
(mV) -60 Em
-70
-80
-
8130-
(mm) 20ri-N
1
15
10
Ca2+ (mM): 1 05 02 01
Fig. 2. PenrecordingofpartofanexperimenttoinvestigatetheeffectofCa reductionon
2
Em and a'a after prolonged exposure to ouabain. Na+ was maintained at 92mm and
ouabain (10-4 M) was present throughout. Ca2+ was reduced from 2-5 mm, substituted by
Mg2+. Notethata' roseonreduction ofCa2+butthat theamplitude ofthisrisedeclined
with reductions to lower than 05mM.
tant transient contracture observed on elevation of Ca2+ or reduction of Na+
strongly suggest operation ofNa+-Ca2+ exchange. It is notable that the time course
ofthedecay intension issimilar tothatofthestabilization ofa'a, asexpected ifboth
were mediated by the same mechanism. It is also interesting that the accompanying
transient hyperpolarization is consistent with the reported electrogenic nature of
Na+-Ca2+ exchange, due to more than two Na+ ions being transported for each Ca2+
(see Mullins, 1983), although a change in membrane conductance, possibly resulting
from the change in Cai+, cannot be excluded (see Lederer, Sheu, Vaughan-Jones &
Eisner, 1983). The rapid recovery ofaia, transient relaxation and transient depola-
rization on return to normal Ca2+ or Na+ are consistent with operation of Na+-
Ca2+ exchange.
Is the Na+ pumpfully inhibited by application ofouabain?
Althoughtheseresultsseemgoodevidencefortheexistence ofNa+-Ca2+exchange,
the changes in a'a could be explained by residual Na+ pump activity. In this case,
the fall in a'a on reduction of Na+ or on elevation of Ca2+ could be caused by a
reduction in the inward Na+ leak. If, however, Na--Ca2+ exchange was responsible
330 C. C. AICKIN, A. F. BRADING AND D. WALMSLEY
for maintaining the relatively low a' a in the presence ofouabain, reduction ofCa2+
to very low levels should lead to a continued rise in a'a.
(i) Effect of decreased Ca2+ on aia. Lowering Ca2+ to 0-5 mm in the presence of
ouabain has previously been shown to cause a rise in a'a to a higher stable level
(Aickin, 1987). Fig. 2 shows that when Ca2+ was reduced to lower concentrations
(substituted byMg2+ to maintainaconstantdivalentcation concentration of3-7 mM)
12min
-30 X
Em -40
(mV) -50EH
-60
-70L
aNa Na
(mM) 20
10
Ca2+(mM): 05 05 0.5 05 0.5 0.5
K+ (mM): 177 295
Fig. 3. Pen recording ofpart ofan experiment illustrating the effect ofdepolarization,
inducedbyelevationofK+,ontheriseina'acausedbyreductionofCa2+.Thepreparation
had been continually exposed to ouabain (10-4 M) for 6h prior to the beginning ofthis
recording. Na+was maintained at92 mm and ouabain was present throughout. Ca2+ was
reduced from 2-5 mm, substituted by Mg2+, and K+ was elevated with appropriate re-
ductions in Tris+. Depolarization alone caused a fall in a'8 and decreased the rise in a'
caused by the simultaneous reduction in Cao
there was, surprisingly, a smaller rise in aia. Exposure to 0-1 mM-Ca2+ even caused
a transient fall in a'a before a slow rise was observed. In the experiment shown,
10 min exposure to the low-Ca2+ solutions caused a 4-3 mm rise in a'a at 1 mM-Ca,2+
a 7-1 mm rise at 0-5 mM-Ca2+ but a 5-5 and 5.3 mm rise at 0-2 and 01 mm-Ca2+
respectively. It is, however, notable that the lower concentrations ofCa2+ caused a
larger and maintained depolarization. This may have limited the rise in a'a by
reducing the inward driving force for Na+ or by inhibiting the inward movement
of charge on Na+-CaF+ exchange. The effect of depolarization was therefore
investigated.
Alteration ofEm byvarying K+showed thatdepolarization causedafallinaia (see
Fig. 3) while hyperpolarization caused a rise in aiNa. In three experiments, an ap-
proximately linearrelationship wasfoundinwhicha'adecreasedbyabout 1-2 mmfor
a 10 mV depolarization within therange from -70 to -30 mV. The consequences of
thisrelationship arewellillustrated inFig. 3. Hereincreasingdepolarization, induced
by simultaneous elevation of K+, progressively reduced the rise in aia caused by
Na+-Ca2+ EXCHANGE IN SMOOTH MUSCLE 331
reduction ofCa2+ to 0.5 mM. In addition, there was an increasingly apparent initial
transient fall in a'a. Since reduction of Ca2+ in the continued presence of high K+
caused a rise in a4Na indistinguishable from that observed in the continued presence
ofnormal K+ (see Fig. 3), it can be concluded that the effects ofdepolarization and
ofreduction in Ca2+ are additive. It should be noted that the similarity ofthe rise in
aia on application of05 mM-Ca2+ in the continual presence ofnormal and elevated
10min
-40
-
-50
Em 60V-- Em
(mV)
-70 -
-80 L
30 -
Na20
(mm) -
10
Ca2W (mM): 05 0.2 0-2
12mM-Mg2+
Fig. 4. Penrecording ofpartofanexperimentshowingtheeffectofelevatedMg:+onthe
riseina'8 induced byalowCa2+afterprolonged exposure (3h) toouabain. Thefirsttwo
reductions in Ca2+ were made with equimolar substitution by Mg2+ while the third was
madewithisomoticsubstitutionbyTris+afterMg2+hadbeenelevatedto 12 mm.Na+was
maintained at92mMand ouabain (10-4M) waspresent throughout. Notethatwith high
Mg2+ a larger rise in a'a occurred on reduction ofCa2+ to 02mm than on reduction to
05mm, compared with the smaller rise observed in the presence of a normal external
divalent cation concentration.
K+ indicates that Na+-Cai2+ exchange is unaffected by changes in Em in the range
-65to -30 mV.Thisthensuggeststhatthedecreaseina'aondepolarizationwasdue
to the reduction in the passive inward leak ofNa+.
These results strongly indicate that the large and maintained depolarization ob-
served on reduction ofCa2+ to below 05 mmdoes limit the rise in a'a. In this respect
it seems particularly significant that the initial fall in a'a on application of01 mm-
Ca2+ (Fig. 2) was very similar to that observed on simultaneous application of05
mM-Ca2+ and high K+ (Fig. 3). We therefore attempted to alleviate the maintained
depolarization observed at low Ca2+ by application ofhigh Mg2+. Fig. 4 shows that
increasing the Mg2+ concentration to 12 mm of itself had no effect on aba. But the
presence ofhigh Mg2+ prevented the initial fall in a'a on reduction ofCa2+ to 0-2 mm
and revealed a more pronounced, slow rise in aia that was incomplete after 18 min.
At this time a'a had risen by 11-8 mm. The presence of high Mg2+also reduced the
amplitudeofthedepolarization onapplication of0-2 mm-Ca2+andrestoredtheability
332 C. C. AICKIN, A. F. BRADING AND D. WALMSLEY
of the cell to repolarize during the continued presence of low Ca". Application of
01 mM-Ca2+in thepresence of12 mM-Mg2+ caused asimilarprolongedrise ina'a (not
shown) that was incomplete in 30 min. a'a had risen by 15-4 mm and Em was
continuing to repolarize.
TABLE 1. Total tissue ion content (mmol/kg wet weight)
I h 3h
Na+ K+ Na+ K+
Control 525+3A1 71 5+P15 52-5+31 71-5+15
K+-free 64-7+5-4 60 1+4-5 76-3+557 39-6+558
Ouabain 74-8+1-7 49-2+19 82-4+12 38&6+009
K+-free+ouabain 87-4+2-1 35-6+0-6 102-6+1P8 12-0+30
Ca2+-free+ouabain 88-5+2-6 30-2+38 101P3+15 14-6+14
Ca2+-free,
K+-free+ouabain 111-5+2-2 41+04
Mean+S.E.ofmeanoffourtissues. Ouabainwasusedat 10-4 M.Ca2+-freesolutionsalsocontained
EGTA (05mM).
(ii) Effect ofremoval ofCa2+ on total ion content. Although these experiments with
Na+-sensitive micro-electrodes indicate a substantial rise in ai5 when Ca2+ was
reduced to 0-1 mm, prolonged exposure to very low Ca2+ or Ca2+-free conditions was
not attempted. This type ofexperiment is technically fareasier to perform using ion
analysis toestimate theintracellular Na+. Therefore thismethodwasusedto confirm
and extend the observations made with the Na+-sensitive electrode.
Qualitatively the results obtained with ion analysis agree with those from the
Na+-sensitive micro-electrodes. Table 1 shows that 3 h exposure to ouabain (10-i M)
or K+-free solution caused a similar rise in total tissue Na+ content and fall in K+
content. These alterations in tissue content were achieved more rapidly in the
presence ofouabain than in the absence ofK+. On exposure to ouabain, the cellular
K+ content fell to a level that is consistent with a passive distribution of K+ ions
across the cell membrane; as calculated previously, the K+ equilibrium potential
approximates to the measured Em (Aickin etal. 1984). On the other hand, even after
3 hexposure to K+-free solution, thetissuesstill contained alarge amountofK+; less
thanhalfthenormal content had beenlost. This could beexplained ifK+ions leaking
from the cells activated the Na+ pump and were therefore transported back into the
cells. Such an explanation is supported by the fact that a 3 h exposure to ouabain in
K+-free solution resulted in a very low tissue K+ content. This result also confirms
that ouabain (10-4 M) did fully inhibit the Na+ pump.
Under conditions in which both the Na+ pump and Nat-Ca2+ exchange were
inhibited (10-4 M-ouabain inCa2+-free solutionwith0'5 mM-EGTA), theNa+ content
progressively rose and the K+ content progressively fell towards the extracellular
level. Therise in tissue Na+andfallintissue K+ occurred mostrapidly in the absence
of K+ and Ca2+ and presence of 10-4 M-ouabain.
The effect ofCa2+ on 22Na efflux
Since the above results convincingly demonstrate that Na+-Ca2+ exchange exists
inthesmooth muscle ofguinea-pigureter, itshould bepossible toobtainradioisotope
Na'-Ca2 EXCHANGE IN SMOOTH MUSCLE 333
fluxes in this preparation that are unarguably mediated by Na'-Ca2" exchange.
Optimal conditions for this are, however, difficult to achieve. First, the presence of
awell-developed Na+-Na+exchange (seeBrading, 1975) causesarapid lossoflabelled
intracellular Na+ in the presence of Na+ and tends to obscure Na+ movements
through other pathways. Secondly, once cells of the guinea-pig ureter have been
A
Rate
(min-')
010F
F
0-09
F
0-08
B
Rate
0-07 I (min-')
0-06 - 0-06 -
0.05
0-06 - 0-05
0.05
0-06 005
a
Na'
16 20 24 28 32 36 40 16 20 24 28 32 36 40
Time (min) Time (min)
Fig. 5. Stimulation of22Naefflux fromouabain-treated tissues bytheaddition ofexternal
cations. The tissues were initially washed in Na+-free, Ca2+-free solution and the cations
then added at the times indicated. Each curve is the mean of four tissues and repre-
sentative standard errors of means are given by the bars. A and B are from different
experiments. Note the small increase in the rate constant ofNa+ loss on addition ofCa2+
and large increase on restoration ofthe normal external Na+ concentration.
equilibrated with ouabain, in order to elevate intracellular Na+ and maximize Na+-
Ca2+ exchange, they also rapidly lose intracellular Na+ in the absence ofNa+ through
operation of Na+-Ca2+ exchange. Therefore, we loaded tissues with 22Na in the
presence ofouabain and looked for an effect ofadding Ca2+ to a Na+-free, Ca2+-free
superfusate. This is still not an ideal situation since even in the absence of Ca2+
intracellular Na+ is lost in Na+-free solution, perhaps through a conductive pathway
and/or through other transport processes, for example Na+-Cl- co-transport (Aickin
& Brading, 1985) and the Na+-dependent mechanism involved in extrusion of acid
equivalents (Aickin, 1986). Thus by the time a control efflux from the intracellular
space can be assured, the intracellular Na+ is low. Nevertheless, some evidence was
obtained that is consistent with the presence ofNa+-Ca2+ exchange. Fig. 5A shows
334 C. C. AICKIN, A. F. BRADING AND D. WALMSLEY
that addition of2-5 or 5 mM-Ca2+ caused a small increase in the instantaneous rate
constant of Na+ loss, although this was far less than the increase caused by Na+-
Na+ exchange on readdition of Na+. In another experiment (Fig. 5B), higher con-
centrations ofCa2+ were added and, as a control, a high concentration ofMg2+ was
10min
-50-
Em~~~~~~E
(mV) 70
-80
20 -
aha 10
(mM) 5
2_
1
10%Na+ 10%Na+
10mM-Mn2+
Fig. 6. Penrecordingofpartofanexperimentillustratingtheeffectof10mM-Mn2+onthe
changes ina'ainduced byreduction and readdition ofNa+ afterprolonged inhibition of
the Na+ pump. Na+ was 92 mm (77%) except where indicated and ouabain (10-4M) was
presentthroughout. Application of10mM-Mn2+causedaslowriseina'aandconsiderably
slowed the fall and subsequent recovery of a'a on reduction and readdition of Na+
respectively.
applied. All concentrations of Ca2+ tested (10-40 mM) increased the rate of loss of
Na+, but by no more than did 2-5 mM-Ca2+. Elevation of Mg2+ by 40 mm had no
effect.
Inhibition ofNa+-Ca2+ exchange by Mn2+
Mn2+ ions, known to interfere with a variety of Ca2+ movements (for review see
Reuter, 1973), have frequently been used to inhibit Na+-Ca2+ exchange (e.g. see
Baker, 1972; Reuter, 1973; Philipson, 1985). WehavereportedthatMn2+ionsinhibit
the Na+-withdrawal contracture of tissues previously exposed to ouabain (Aickin
et al. 1984) and cause a slow rise in a'a after equilibration in the presence ofouabain
(Aickin, 1987). We therefore further investigated the effects ofMn2+ on the changes
in aia and tension that we have interpreted to be mediated by Na+±-Ca2+ exchange.
(i) Effect ofMn2+ on thefall in aia induced byreduction ofNa+. Fig. 6 shows that in