BY C. CLAIRE AICKIN, ALISON F. BRADING AND TH. V. BURDYGA* Kiev 17, USSR declines

20 Pages·2005·2.26 MB·English

J. Physiol. (1984), 347, pp.411-430 411
With 10text-figure8
PrintedinGreatBritain
EVIDENCE FOR SODIUM-CALCIUM EXCHANGE IN THE
GUINEA-PIG URETER
BY C. CLAIRE AICKIN, ALISON F. BRADING AND TH. V. BURDYGA*
From the University Department ofPharmacology, South Parks Road, Oxford OXi
3QTand *the Research Institute ofPhysiology, Kiev State Shevchenko University,
Kiev 17, U.S.S.R.
(Received 5 May 1983)
SUMMARY
1. Theeffectsofhigh-Kandlow-Nasolutionsonthesmoothmuscleoftheguinea-pig
ureterhave beenexamined inboth normal tissues, andtissues inwhich theNapump
had been blocked by exposure to K-free solutions or oualain- (high_-Na tissues).
Tension recording, membrane potential measurements and ion analysis were used.
2. High-Ksolutionsdepolarizenormaltissues,leadingtoactionpotentialgeneration
and phasic contractions followed, at concentrations greater than 20-30 mm, by
cessation ofaction potentials and the development ofa biphasic contracture which
declines slowly during continuous exposure. The contracture is abolished by Ca-
antagonist drugs, procaine and Ca-free solutions.
3. Short exposures ofnormal tissues to Na-free solutions do not result in tension
development. Longerexposuresmayinitiatetension,dependingontheNasubstitute
used.Sucrosecausesdepolarizationofthecellsandspikedevelopmentassociatedwith
phasiccontractions, superimposedonasmallcontracture; Lidepolarizesthecellsbut
causes no tension generation; Tris hyperpolarizes the cells and a small increase in
basal tone may be seen.
4. On exposure to K-free solutions or ouabain, the tissues do not develop
significanttonebuttheirresponsetoshortapplicationofhigh-Ksolutionsgrowswith
time. Thetissues alsodevelopthe ability to contracton short applications oflow-Na
solutions. The low-Na contractures are resistant to concentrations ofCa antagonists
thatabolishthe Kresponses ofnormal tissues, butareabolishedinCa-free solutions.
5. The ability ofthe tissues to contract in Na-free solutions is accompanied by an
increase in intracellular Na and loss of intracellular K. Even after several hours'
exposure to ouabain, however, the tissues still contain significant amounts ofK and
the membranepotentialisthesame as, ormorenegativethanthat innormaltissues.
Thereforeitappearsthatanothermechanism, apartfrom theNapump, canregulate
intracellular Na. On continuous exposure to Na-free solutions, the contracture
declines rapidly. The decline is associated with a loss ofintracellular Na.
6. The Na-free contracture is larger when K rather than Tris is used as the
substitute. This difference persists in the presence of a concentration of Mn that
abolishesthe Kcontracture ofnormaltissues butisabolished byhighconcentrations
(10 mM) of procaine. Neither agent greatly modifies the depolarization caused by
412 C. C. AICKIN AND OTHERS
high-K solutions or the hyperpolarization caused by Na-free (Tris). These results
suggest that procaine eliminates the additional contracture associated with de-
polarization which is insensitive to Mn.
7. It is concluded that the tissues can maintain a low intracellular free Ca in the
absence ofan inwardly directed Na gradient, but that a Na-Ca exchange may play
a role in determining both intracellular Na and Ca levels when the Na pump is
inactivated. When intracellular Na is elevated and the Na gradient is reversed, the
exchange can mediate entry ofsufficient Ca to initiate a contracture.
INTRODUCTION
Since the demonstration ofa link between transmembrane Na and Ca movements
in heart (Reuter & Seitz, 1968) and squid giant axon (Baker, Blaustein, Hodgkin &
Steinhardt, 1969), work has been done in many tissues to investigate the existence
and role ofNa-Ca exchange.
Itis interestingthatin cardiac muscles, physiologists have strongevidence for the
existenceofaNa-Caexchangemechanismandhavebecomepincreasinglyenthusiastic
aboutitsrolenotonlyinthe control ofintracellular free Ca, butalsoin manynormal
and experimentally induced behaviour patterns. The exchange is thought to be
electrogenic and voltage sensitive, and thus to affect not only tension development
(Vassort, 1973; Chapman, 1974; Chapman & Tunstall, 1980; Eisner, Lederer &
Vaughan-Jones, 1983) but also the membrane potential itself(Coraboeuf, Gautier &
Guiraudou, 1981; Mullins, 1981, 1983; but see Lederer, Sheu, Vaughan-Jones &
Eisner, 1983). In contrast, smooth muscle physiologists have less direct evidence for
the exchange, although the importance ofNa in determining the contractile state of
the tissues has been frequently investigated. Initially, such studies led research
workersto lookfavourablyonthe ideathataNa-Caexchange wasinvolved (see e.g.
Biamino & Johansson, 1970; Bohr, Seidel & Sobieski, 1973). This outlook was
reinforcedbythedemonstration(Reuter,Blaustein&Haeusler,1973)ofNa-dependent
Caeffluxinarterialsmoothmuscle,andculminatedinspeculationsontheinvolvement
ofNa-Caexchange in the aetiology ofhypertension (Blaustein, 1977). More recently
several workers, as a result of more detailed studies, have become increasingly
reluctant to assign much importance to a Na-Ca exchange, either in the regulation
ofintracellularCaorincontributingtothebehaviourofsmoothmuscle(Raeymaekers,
Wuytack & Casteels, 1974; Casteels & van Breemen, 1975; van Breemen, Aaronson
& Loutzenhiser, 1978; Droogmans & Casteels, 1979).
The smooth muscle ofthe ureter is rather unusual, and in many ways resembles
cardiacmuscle,thecontractionofthemusclebeingcontrolledalmosttotallybyaction
potential generation; the action potentials are prolonged, with a marked plateau
phase following the initial upstroke, and are dependent on both Na and Ca entry
(Kuriyama & Tomita, 1970; Shuba, 1977a); agonistdrugs suchasnoradrenaline and
histamine do not initiate changes in tonic tension as they do in most other smooth
muscles, butatlowconcentrationsalterthesizeanddurationofthephasiccontractile
responses through effects on the action potential configuration (Shuba, 1977b), and
in higher concentrations may initiate action potentials. Tonic tension can however
beinitiatedbydepolarizingthetissuewithhigh-Ksolutions (Washizu, 1967; Sunano,
Na-Ca EXCHANGE IN THE GUINEA-PIG URETER 413
1976; Johnishi & Sunano, 1978), and the contractures induced relax spontaneously,
as they do in heart muscle (Chapman, 1974).
Heart muscle, particularly from amphibia, is known to produce contractile
responses to low-Na solutions, an effect thought to reflect the activity of a Na-Ca
exchange mechanism (Chapman, 1974). Mammalian heart will show a similar
response under conditions of increased intracellular Na content (Coraboeuf et al.
1981). Because of the similarity in behaviour between heart and ureter, it seemed
possible that ureter might be a useful preparation to study with the hope ofgetting
more evidence as to the presence and properties ofa Na-Ca exchange mechanism in
asmooth muscle. WehavethereforestudiedtheabilityoflowNaandhighKtoinduce
contractions in guinea-pig ureter with normal and elevated intracellular Na, the
susceptibility of these responses to Ca-antagonist drugs and the changes in the
membrane potential that accompany them.
METHODS
Dissection. Male white guinea-pigs were stunned and bled. The ureters were cut close to the
bladderandkidneys, andremovedfromtheanimal. Theywerecarefullycleanedofconnectivetissue
underadissectionmicroscope. Forionanalysis, eachureterwasmountedonastainless-steel holder;
for tension recording, thin threads were tied round the ureter and each tissue was cut into three
pieces; for micro-electrode studies, the ureter was cut in halflengthwise and a short length ofthe
central portion used.
Solutions. A modified Krebs solution was used ofthe following composition (mM): Na, 120-3; K,
5-9; Tris, 16-6; Ca, 2-5; Mg, 1-2; Cl, 150-2; glucose, 11-5; equilibrated with 100% 02, pH 7-4.
Variations in the Na concentration were made by replacing NaCl isosmotically with KCl, Tris Cl,
LiCl, MgCl2 or sucrose.
Ionanalysis. Tissues wereallowed toequilibrate forat least 1 h in Krebssolution at 37 TCbefore
being exposed to the experimental solution. Tissues were removed from the holders and blotted
to obtain the wet weight before analysis. For total ion analysis, the ureters were extracted in a
diluting fluid containing N-HNO3, 18 mM-La3+ and 5-5 mM-Li, and the solution analysed by flame
photometry. To estimate the cellular Na and K content, tissues were exposed to a Na-free, K-free
(Tris) solution for 5 min at 4 'C to remove most ofthe extracellular ions, before extraction in the
dilutingfluid. In theexperiment to measure the effect ofouabain on total tissue NaandK content
(Table 1), tissues were collected by colleagues from animals killed for other tissues and, in
consequence, were perhaps less carefully prepared than those specifically dissected (Table 2). This
may account for the lower K content ofthe control tissues in Table 1.
Tension recording. Sixpieces ofureterweresuperfusedsimultaneously intheapparatusdescribed
byBrading & Sneddon (1980) ataflow rateof1-2 ml/min andatemperature 32-37 'C. Contractile
responses were normally expressed as a percentage ofa control response to a 1-2 min application
of 126 mM-K.
Measurement ofmembranepotential. Pieces ofureterwere mounted well stretched over a silicone
rubber wedge with the outer surface uppermost (Aickin & Flaxman, 1983) and superfused with
solutions at 35 'C. Membrane potentials were recorded with micro-electrodes filled with a solution
of85% 0-6M-K2SO4 and 15% 1-5M-KCl, which had resistances of 30-50 MCI. They were dipped
in a solution of 1% tri-n-butylcholorosilane in 1-chloronaphthalene after they had been filled with
electrolyte. This procedure had no effect on the electrode resistance and greatly improved the
stability and duration ofintracellular recording (Aickin, 1981).
Drugs. Papaverine hydrochloride, nifedipine (a gift from Bayer, U.K.), D-600 and procaine
hydrochloride were used. Stock solutions were made upin distilled waterand added to the bathing
solution to give the desired concentration.
Statistics. Resultsareexpressedasthemean+S.E. ofthemean(numberofmeasurements), except
in Fig. 10, where the S.D. ofan observation is given.
414 C. C. AICKIN AND OTHERS
RESULTS
Much ofthe evidence put forward to suggest that Na-Ca exchange plays a role in
smooth muscle has been obtained from studying contractile responses to alterations
ofthe Na gradient across the cell membrane. This can be done either by reducing
externalNa, increasinginternalNa, oracombinationofthetwo. Oneoftheproblems
is that reducing external Na may have effects that are due to the substitute used,
rather than a change in the Na gradient. Therefore we have looked at the effects of
2min 10min
K K K
I ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~I
Na-free(sucrose)
Fig. 1. TheeffectofNa-free (sucrose) solution onnormal tissues. The ureterwasexposed
to 126mM-K for 1 min at the times indicated by the arrows. On application ofNa-free
(sucrose) solution, the tissue remained relaxed for about 15 min and then began to
produce phasic contractions. These were superimposed on a tonic tension which showed
aninitialtransientphase, andthenasmallsustainedincrease. Theresponseto 126 mM-K
was considerably decreased after exposure for more than 1 h to Na-free solution. Re-
addition of Na relaxed the tonic tension, but did not immediately restore the high-K
contracture.
several Na substitutes (including K, which clearly has its own effects due to
depolarization ofthe cell membrane). In this section we describe first the effects of
Na-free and high-K solutions on normal tissues, and the effects ofCa antagonists on
the responses. Thenwe describe the responses oftissues inwhichthe internal Na has
beenraised byincubation in ouabain orK-free solution (high-Natissues). Finallywe
havelookedinmoredetailattheeffectsofMnandalsostudiedtheeffectsofprocaine,
whichisknowntohaveactionsnotonlyontheplasmamembrane, butalsoonrelease
ofCa from intracellular stores (see e.g. Itoh, Kuriyama & Suzuki, 1981).
Normal tissues
(1) Effect ofNa-free (normal K) 8olution. Ifsmooth muscle from ureter is exposed
for a short period (1-2 min) to a Na-free solution in which Na is replaced by Tris,
Li or Mg, or NaCl is replaced by sucrose, it does not generate any tension.
Ifthe tissue is continuously superfused with Na-free solution it normally remains
relatively relaxed except when NaCl is replaced with sucrose. In this case the tissue
goes into a contracture, after a lag of about 10-15 min, with phasic contractions
generated in response to spontaneous action potentials (see Figs. 1 and 2A). There
Na-Ca EXCHANGE IN THE GUINEA-PIG URETER 415
A
10min
-10 _ m
-20
-
E -30
-
E -40 -
-50
-60
-
Na-free(sucrose) Na-free(Tris) Na-free(Li)
8
10min
0
-10 _ Ouabain(10'M)
-20-
E-30-
E -40-
-50
-60 3mi
-70L
50%Na(Tris) 50%Na(Tris) Na-free(Tris) 33mM-K
Fig. 2. Effects ofalterations in the ionic composition ofthe superfusing solution on the
membrane potential (Em) ofureter smooth muscle cells. A, effects ofNa-free solutions
on normal tissues. Note the depolarization on exposure to Na-free (sucrose) solution,
which after 10min reaches the firing threshold of the cell. Na-free (Li) solution also
depolarizes the cell, but not to firing level, whereas Na-free (Tris) causes a transient
hyperpolarization. In all cases re-application ofthe normal Na concentration causes a
transient depolarization, followed by a longer lasting and more pronounced hyper-
polarization. B, arecordfromasinglepreparation. Inthenormaltissue, reductionofthe
Na content ofthe superfusing solution to 50% (Tris substitution) hyperpolarized the
membrane.Applicationofouabain(10-4 M,whichwaspresentinthesolutionsfortherest
ofthe experiment) caused a transient depolarization, after which there was a sustained
hyperpolarization ofthe membrane. After 80min in ouabain, the high-Na tissue shows
agreaterhyperpolarizationonreductionoftheexternalNa,andthereisnowapronounced
depolarization on re-addition ofthe normal Na concentration. Application of33mM-K
depolarizes the tissue.
is a small and slowly developing increase in the resting tone ofthe tissues in Na-free
(Tris) solution, whereas a decrease in the resting tone is often observed in Na-free
(Li) solution (see Fig. 3).
Fig. 2A shows examples of the change in resting potential (Em) that occurs on
replacement of Na with various substitutes. When sucrose is used, the membrane
depolarizesandafter 10 minactionpotentialsareseen. InNa-free (Tris) solution, the
membrane undergoes a transient hyperpolarization, and in Na-free (Li) solution the
membrane is depolarized after a briefhyperpolarization. In all cases there is a small
transient depolarization on re-admission of Na, which is followed by a hyper-
polarization before the initial resting potential is regained.
(2) Effectofhigh-KsolUtions Ashort (1-2 min) application of126 mM-K causesthe
generationofseveralphasiccontractions, followedbyabiphasiccontractureasshown
416 C. C. AICKINAND OTHERS
in Figs. 1 and 5a. The initial phasic contractions are initiated by action potentials
(Washizu, 1967). They are identical in size and shapetothephasic contractions that
can be initiated in response to depolarizing current pulses which have been shown
to produce action potentials in this tissue (Brading, Burdyga & Scripnyuk, 1983).
The dose-response curve ofthehighestpeakofthe contractureto 1 minapplications
ofincreasing Kconcentrations isshownincurvea, Fig. 5. Thiscanbecomparedwith
the relationship between the membrane potential and external K concentration
A B
20min
0 * * 0 0 0 *0 0 * 0 0 0 0 00 0 0 0 0*S
I ~~~,II g L
Na-free (Tris) Na-free Na-free Na-free(Li)
(Li) (Tris)
Fig. 3. A comparison ofthe effects of Na-free (Tris) and Na-free (Li) solutions on the
contractileresponseto 1 minapplicationsof126mM-K (0). A, inNa-free (Tris)solution,
there is a small increase in basal tone of the tissues, and the K response increases
transiently. Ifthe solution is changed to Na-free (Li), the response to K is immediately
abolishedandonlyreturnsslowlywhenNa-free(Tris)isre-applied.B,Na-free(Li)solution
causes a reduction in the basal tone ofthe tissues and rapidly suppresses the response
toK.
shown in Fig. 10. Three out ofsix tissues produced phasic contractions in response
to 11 mM-K (which depolarized the membrane by about 9 mV) but contracture was
only just beginning at 25 mm, and rose steeply from about 45 mM-K (which
depolarized the membrane by about 34 mV). Fig. 9 illustrates that small increases
inextracellular K initiated action potentials responsible forthe phasic contractions.
On continuous superfusion with high-K solutions the contracture slowly declined
with time, as previously reported by Washizu (1967) and Sunano (1976).
If tissues are exposed continuously to Na-free solutions and then tested with
126 mM-K, theresponse againdependsontheNasubstituteused. Inoneexperiment,
after 2 h in Na-free (Mg) solution the K response was totally absent, and in Na-free
(Li) solution the response was almost abolished, whereas in Na-free (Tris or sucrose)
solution theresponse waspresent butdiminishedinsize. Furtherexperimentson the
effect ofTris and Li as Nasubstitutes are illustrated in Fig. 3. The response to 1 min
application of 126 mM-K initially grows in size during the first hour ofexposure to
Na-free (Tris) solution before gradually diminishing, whereas it is progressively and
Na-Ca EXCHANGE IN THE GUINEA-PIG URETER 417
rapidly reduced in Na-free (Li) solution. When, after 1 h 20 min in Na-free (Tris)
solution, change was made to Na-free (Li) solution, the response to 126 mM-K was
immediatelyabolished, andonreturningtoNa-free(Tris)solutiontheresponsebegan
to recover. It appears that Li has some effect of its own in suppressing the K
contracture, an effect that has also been observed by Kishimoto & Urakawa (1980)
in the guinea-pig taenia.
Ofthevarious Nasubstitutesused, Trisseemedtobethe mostsuitableand, unless
otherwise stated, it was used to replace Na in the rest ofthe experiments described.
(3)EffectofCaantagonistsonhigh-Kcontractttres.TheeffectsofseveralCa-antagonist
drugs (including papavarine, which acts as a Ca antagonist in this tissue: Brading
et al. 1983) have been investigated on the contracture to a 1 min exposure to
126 mM-K. Nifedipine, D-600, papavarine and Mn are all capable of completely
abolishing the K-induced contracture, and have parallel log dose-inhibition curves
in a descending order ofpotency. The Ca-antagonist drugs have different effects on
the ability of the tissues to produce phasic contractions and contractures when
exposed to high-K solutions. Nifedipine and D-600 both abolish the contractures at
concentrationswhich have little effect onthephasiccontractions (I9-7 M and 10-6 M
respectively); papavarine abolishes both at a similar concentration (10-4M); Mn
abolishes the phasic contractions at 1 mm, which reduces but does not abolish the
Kcontracture. Anexample ofthisdifferentiation (bynifedipine andMn) canbe seen
in Fig. 6. Brading & Sneddon (1981) have also noted a differential effect of D-600
on spikes and depolarization-induced contractures in guinea-pig taenia.
TABLE 1.Theeffectof10-4M-ouabainonthetotaltissueKandNacontent.Thenumberofsamples
isgiven in parentheses. Values are means+s.E. ofmeans
Exposure Na K Na+K
to ouabain (mM/kg) (mM/kg) (mM/kg)
0 52-7+2-3 (4) 59-3±3-6 (4) 112-0
2h 75-3±4-1 (6) 32-5+1-6 (6) 107-8
3h 78-5+3-5 (8) 29-6±2A4 (8) 108-1
4h 73-3±20 (8) 26-4+1-4 (8) 99-7
5h 75-3±1-6 (6) 272+06 (6) 102-5
High-Na tissues
In the presence ofouabain 10-4 M or in the absence ofextracellular K (conditions
which block the Na pump) the ureter gains Na. Table 1 shows the total Na and K
content of tissues exposed for various intervals to 10-4 M-ouabain. Although the
tissues gain Na and lose K during the first 2 h, there is then little further change.
Even after 5 h in ouabain, there is still considerable K in the tissues. This behaviour
is reminiscent of heart muscle in which intracellular Na only rises a small amount
even after prolonged exposure to quabain (Ellis, 1977), and at variance with other
smooth muscles in which there is a striking elevation oftissue Na (Casteels, 1966).
High-Na tissues remain relatively relaxed even after several hours in ouabain
(although a very small degree of resting tone is occasionally apparent) and the
membrane potential remains virtually unchanged (see Figs. 2 and 10) but there are
changes in the responses to high-K and Na-free solutions.
14 PHY 347
418 C. C. AICKIN AND OTHERS
(1)EffectofNa-free (normalK)andlow-Nasolutions. WhentheNapumpisblocked,
the tissues develop an ability to contract on briefapplications ofNa-free solutions.
Fig. 4 shows the time course of the development of the contractures to 1 min
application ofNa-free (Tris) solution in tissues exposed to either 1O-4 M-ouabain, or
toK-free solution. The response develops morerapidlywhenouabainisusedto block
theNapumpthanwhenextracellularKisremoved, probablybecauseKleakingfrom
140
20 (5)
-
g 100
.,_
80
C
0
60 (m)
C
40 cr s3)
I
20
0L
0 10 20 30 40 50 60 70 80 90 100
Time (min)
Fig.4.EffectofblockingtheNapumponthecontractileresponsesoftheureter.Theupper
curve (filled squares) shows the effect of ouabain (10-4 m, applied at time zero) on the
response to a 2 min application of 126 mM-K. The lower curves show the time course of
the development ofthe Na-free (Tris) contracture (applied for 1 min) after the addition
ofouabain(10-4 M,filledtriangles),andafterapplicationofK-freesolution(crosses).Each
curve shows the mean+S.E. ofmean; the number oftissues is shown in parentheses.
the cells can, in the absence ofouabain, partially activate the pump and thus slow
the gain oftissue Na. Forthe restoftheexperimentswith high-Natissues, thepump
was blocked with 1O-4 M-ouabain for at least 1 h to allow the maximum Na-free
response to be reached, before the experiment was begun.
Curve bofFig. 5shows the dose-response curve to reducingextracellular Nausing
Tris as a substitute. The maximum contracture that can be elicited in Na-free (Tris)
solution is less than the contracture ofnormal tissues to 126 mM-K solution.
High-Na tissueswill contract onexposure toNa-free solutions withanyofthefour
Na substitutes we have tested (sucrose, Tris, Li and Mg). The time course of
development ofthe ability ofNa-free solution to initiatea contracture afteraddition
ofouabain is the same with each, but the size and shape ofthe response to different
substitutes varies somewhat from tissue to tissue. In one series of experiments in
which only one Na substitute was investigated on each tissue, the size ofthe peak
response in Na-free solution after 50 min exposure to ouabain, expressed as a
percentage ofthe response ofthe high-Na tissues to 126 mM-K, was 31-1+4-8% for
Mg, 44-9+3-8% for sucrose, 47-0+6-1 % for Tris and 51-5+7-3% for Li (n = 5).
Na-Ca EXCHANGE IN THEGUINEA-PIG URETER 419
100 1
80-
C
0
a
C 60-
0
40-
20-
0 L
5 10 20 50 126 KorTris(mm) 1min
121 116 106 76 0 NaorTris(mM)
Fig.5.Dose-responsecurvesofthepeakofthecontractureelicitedinnormalandhigh-Na
tissues by 1 min applications ofsolutions containing altered K and Na concentrations.
a, normal tissues; *, response to increasing K at the expense of Na, expressed as a
percentage of the response to 126mM-K (Na-free). b, high-Na tissues; A, response to
decreasingNaattheexpenseofTris,expressedasapercentageoftheresponseofthehigh-Na
tissues to 126mM-K (Na-free). c, high-Na tissues; *, response to increasing K at the
expense ofNa (or to decreasing Na at the expense ofK), expressed as a percentage of
the response to 126mM-K (Na-free). d, high-Na tissues; A, response to increasing K at
the expense ofTris. In this experiment all the test solutions were Na-free (Tris), so the
response to increasing K will, in effect, be superimposed on a Na-free contracture.
Experiments ab andcshowthe means+s.E. ofmeansofsix tissues, anddofthree. The
high-Natissueshadbeenexposedtoouabain(10-4M)foratleast 1 hbeforethebeginning
ofthe experiment and ouabain was present for the duration ofthe experiment. On the
right are shown typical responses ofthe tissues to the corresponding conditions used in
thedose-responsecurves.Thecontractileresponseindwasverysimilartothatinb.Each
ofthese traces is the response to a2 min application ofthe relevant Na-free solution.
If Na-free solution is applied continuously to high-Na tissues, the resulting
contracture is of surprisingly short duration, the tissues relaxing within a few
minutes. If such relaxed tissues are then re-exposed to normal Na solution (in the
continuing presence of ouabain) their ability to contract again on removal of
extracellular Naalso recoversremarkably quickly. Thetime course oftherelaxation
in Na-free solution and the subsequent recovery ofthe ability oftissues to contract
again to Na-free solution was measured in a series oftissues. Both had half-times of
between 1-5 and 2 min.
Table 2 showsthe results ofanexperiment inwhich the cellularNaand Kcontent
oftissues wasanalysed. Afterexposure to ouabain for 1 h the tissues had lost K and
gained Na, but during a subsequent 10 min exposure to Na-free (Tris) solution at
14-2
424020 ~~~C. C. AICKIN AND OTHERS
TABLE 2. The effect of 10 min exposure to Na-free (Tris) solution on the Na content ofhigh-Na
tissues. High-Natissueswerepreparedbyexposingthemto 10-4 m-ouabainfor 1 h. Alltissueswere
rinsed for 5 mmn in Na-free (Tris) solution at 40C to remove most ofthe extracellular ions before
analysis. The number ofsamples is given in parentheses. Values are means+S.E. ofmeans
Na K Na+K
Treatment (mm/kg) (mm/kg) (mm/kg)
Normal tissues in Krebs 19-1± 1-1(8) 69-2±+36 (8) 88-3
High-Na tissues 44-1 ±22 (8) 40-9±+2-4 (8) 85-0
High-Na tissues after 10 min 14-1±1-1 (8) 35-3± 1-1 (8) 49-4
in Na-free (Tris) solution
37 'C (still in the presence of ouabain) the tissues lost all the Na they had gained,
with little change in the K content.
In the presence of ouabain, Na-free and low-Na solutions cause larger hyper-
polarizations than innormal tissues, even though the tissues contract. Fig. 2B shows
the effects of50% Na (Tris) on the membrane potential before and after the tissue
hadbeenexposedformorethan 1 htoouabain, andalsothe'effectsofNa-freesolution
on the high-Na tissue. Note the transient depolarization on application ofouabain,
and the fact that after 1 h in ouabain (and in other experiments, even after several
hours) the membrane potential is, ifanything, slightly more negative than in normal
Krebs solution.
(2) Effect ofhigh-Ksolutions. Fig. 4 shows how the response to a 1 min application
of high-K solution changes in size when the tissues are exposed to 10-4 m-ouabain.
During the first 40 min there is an increase in the size of the response which, with
some variation, then remains higher than the control for several hours (not shown
in Fig. 4). There is also a change in the shape of the response as the phasic
contractions, which precede the contracture in normal tissues, are abolished. Fig. 5
shows dose--response curves for the contractile response ofhigh-Na tissues to 1 min
application ofincreasing levels ofextracellular K. Since replacing Na with K could
be having an effect through both the increase in extracellular K and the reduction
ofextracellular Na, two curves are shown. In curve c ofFig. 5, K is increased at the
expense of Na, which means that both important variables are changing, whereas
in curve d all the test solutions are Na-free (Tris) and K is increased at the expense
ofTris. When K is increased at the expense ofNa, the curve is also a dose-response
curve to reducing extracellular Na, and it can be compared with the Na/Tris curve
(b)plottedonthesamegraph.WhenKisincreasedattheexpenseofTris,theK-induced
contracture will, ineffect, be superimposed on aNa-free contracture, andifthe curve
is scaled taking the Na-free, normal-K contracture as 0, it is similar in shape to the
K/Na dose-response curve (c).
Fig. 10 shows a dose--response curve ofthe membrane potential ofhigh-Na tissues
to extracellular K (Na replacement) which can be comparedwith Fig. 5, curve c. The
relationship between potential and extracellular K is slightly steeper in the high-Na
tissues than in normal tissues. An example ofa recording ofthe membrane potential
response ofa high-Na tissue to 33 mm-K is also shown in Fig. 2B.
(3) Effects ofCaantagonist8. The contractile responses ofhigh-Natissuesto Na-free
solutions (includingNa-free (K))aremuchmoreresistanttoCa-antagonistdrugsthan