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Ü 0.3_i u) o I—I o control K - froo ouabain + K-frno 10 ₁₅ 20 25 30 5 minutes

Fig. 15. Effect of ouabain on the rate of loss of K from Fy3T5 cells. Cells were "loaded" for J> hours in an 2&Rb-

labelled Krebs solution. The radioactive solution was removed and the cells were washed rapidly with 3 changes

of cold (2®) inactive Krebs and rinsed once with Krebs at 37®. Tern ml of one of the following solutions were then added to a dish of cells : 1) control Krebs 2) Krebs + 10""^ M ouabain 3) K^-free Krebs 4) K^-free Krebs + 10"^ M

ouabain. After 3 min the solution v;as quickly poured into

a vial for Kb counting, and another lOml of the__ same solution was added to the dish. The washout of Rb during 6 successive 5 min periods is expressed as the fraction of

S&Rb lost/3 min. The total column height represents the

fractional loss from cells into Krebs + 10"^K ouabain.

Since the fractional loss into solution 1 , 3 and 4 wore not sir nif icantly different (lÿO.^) the mean ( - S.E,) is shown. The shaded column height thus represents the additional loss of S&Rb caused by ouabain. The increased efflux was blocked by the removal of extracellular K .

were measured by flame photometry (Fig. 16 ). Over a 60 minute period the ^ rose exponentially from 20 to 70 m-mole/1 and the j j ^ ^ f ell exponentially from 200 to l40 m-mole/1 indicating a continuing inhibition of the Na-K exchange over 60 minutes. Therefore, in the transformed cells, ouabain caused a simultaneous reduction of Na-K exchange and a stimulation of K-K exchange.

The dose-response of the two effects produced by

ouabain has been examined. . Fig. 17 shows the results of an experiment in which the effect of ouabain on the K"*" influx into Py3T3 cells was measured with or without ^0 minutes

— 8 “•3 .

pretreatment with the drug ( 10 to 10"" M)'.. The results show

that the inhibition of Na-K exchange and stimulation of K-K exchange exhibited a similar dependence on ouabain

concentration. There was neither inhibition (no .pr,etreatment) nor stimulation (pretreatment) of the K*^ influx at ouabain concentrations below 10” **'m, There was a small degree of inhibition and stimulation at 10~^M ouabain with larger effects at the maximum drug concentration of 10

The reversibility of these effects has been

invQs bi(’;aiod. -tV3'i’3 cells were IroaLod v/ibh oualsjin for CO minutes after which the cells were transferred to a control Krebs solution. The K* influx was measured at 13 minute intervals for 120 minutes after the removal of ouabain (Fig. IS ). The K^ influx decreased during the first 60 minutes, thereafter, the influx remained constant at a value some 40 to 30% below that of the untreated control. The

ouabain-induced K-K exchange was thus more readily reversible than ouabain inhibition of Na-K exchange.

The effect of a reduction in experimental

temperature to 20® on the response of the three cell lines to ouabain was examined. The results (Fig, 13 ) show that at

r4 0) 0

1

80

P y3T3

No

minutes of ouabain treatment

60

Fig, 16. Fffect of ouabain treatment time on the fhji and |Na^ i of Py3T3 cells.’ The and Na" of Py3T3 cells was measured by flame photometry after periods (0-60 min) of

treatment with 10"^M ouabain. Duplicate plates were used to measure mean cell number and cell volume/plate. The intracellular K"* (E!) and Na’*'(o) concentrations were cal­ culated and are expressed as m-mole/1. Each point

represents the value for Qc^i and i obtained from a single plate of cells. The lines were fitted by linear regression after logarithmic transformation

o ü g ü

I

200

Py3T3

Fip;. 17. Dose-response of the K influx in Pypïp cells to ounbain w1 th and without drug pro treatment, Tlic K influx was measured :).n the prose nee of various concentrations of ouabain in the range 10“® to 10"® M. The cells had pre­ viously been treated for J>0 min with either control Krebs

(o) or Krebs containing ouabain (o) at the indicated concentration. Each point represents the value for K influx obtained from a single plate of cells expressed as a % of the control K influx. The results show that

ouabain inhibition of Na-K exchange (control pretreatment) and ouabain stimulation of K-K exchange (drug pretreatment) exhibits a similar sensitivity to ouabain concentration.

150

_r

SV3T3

1

minutes after ouabain removed

j'bMS Hcver:;:i.bi.li Ly oT Lho rosponno of vi.ruo-

trarujforrned _ cells to ouabain. SV3T3 cells were treated with Krebs 4 ouabain for 60 min and then transferred to a control Krebs solution for periods ranf^ing from^O-120 min. The K influx was measured by incubation with

Krebs for 10 rain according to the procedure described in the legend to^Fig, 3. Each point represents a single value

for the K influx after ouabain treatment expressed as a

% of the values obtained for the untreated controls. The

10 rain influx measurement period has been added to the