Neuroscience Letters, 125 (1991) 9-11 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50

A DONIS 030439409100064A NSL 07655

Effect of ouabain on volume and chemosensitivity of Xenopus oocytes injected with rat brain mRNA V. Arvanov* and P.N.R. U s h e r w o o d Department of Zoology, University of Nottingham, Nottingham ( U.K. ) (Received 8 January 1991; Accepted 8 January 1991)

Key words." Ouabain; Xenopus oocyte; Chemosensitivity; Voltage clamp; L-Kainic acid: GABA; L-Glutamic acid Xenopus oocytes injected with rat brain RNA were voltage-clamped using a 2-electrode technique. At 4-10 days post-injection the oocytes responded to applications of 10 -6 M to 10 TM M y-aminobutyric acid (GABA), L-kainate and L-glutamate. All 3 compounds evoked inward currents. At 10-4 ouabain alone evoked an inward current of 445 nA at - 6 0 mV, but had no effect on membrane conductivity (ca. 5/tS). In ouabain-containing saline the responses to GABA, L-kainate and L-glutamate were increased by 80-120%, 20-30% and 20-40%, respectively, in both folliculated and defoUiculated oocytes. These changes were not accompanied by shifts in the reversal potentials for the agonist-induced membrane currents. At 10-4 M ouabain also increased oocyte weight and volume. It is proposed that ouabain, by increasing oocyte volume, increases the area of oocyte membrane containing receptors which is accessible to exogenously applied compounds.

It has been shown that when the electrogenic Na/K pump of mollusc neurones is inhibited by ouabain, the responses of these neurones to neurotransmitters, studied under voltage clamp, are enhanced [1, 2]. These changes in chemosensitivity are primarily not due to shifts in the reversal potentials for the currents evoked by the transmitters, which might be expected to accompany changes in ion distributions following inhibition of the Na/K pump. They are related, perhaps causally, to ouabain-induced changes in cell volume [2]. It is well known that the Xenopus oocyte has a well-developed Na/K pump and that its chemosensitivity can be readily manipulated by injection of appropriate RNA [3]. We have examined the effects of ouabain on this system and have found that inhibition of the Na/K pump is accompanied by increases in sensitivity of the oocyte to both excitatory and inhibitory receptor agonists and by changes in cell volume. Total RNA was obtained from whole brains of 14day-old Wistar rats using standard techniques of extraction and purification [3]. Terminal stage oocytes were obtained from adult Xenopus under sterile conditions Correspondence." P.N.R. Usherwood, Department of Zoology, University of Nottingham, Nottingham NG7 2RD, U.K. *Home address: Department of Biophysics, Institute of Experimental Biology, Academy of Sciences of Armenia, Hasratyak 7, Yerevan, 375044, U.S.S.R.

and maintained in sterile medium with daily changes in the latter [3]. The oocytes were injected with the rat brain RNA. Responses to 7-aminobutyric acid (GABA), Lkainate and L-glutamate were readily obtained from the RNA-injected oocytes 4-10 days post-injection. Doseresponse relationships for these compounds were similar to published data [4]. Agonist induced currents were recorded using a 2-elec-

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Fig. 1. Effect of ouabain on the response of a defolliculated Xenopus oocyte to brief applications of L-kainate (Sigma). When ouabain (10 -4 M) was applied alone (at arrow) a small inward current of about 5 nA was observed at a holding potential of - 6 0 mV. When 10 TM M L-kainate was applied as brief pulses at various times before, during and after continuous ouabain application, marked changes in response amplitude were observed. The response to L-kainate increased during ouabain application but declined after removal of this compound. The holding potential was - 6 0 mV.

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trode voltage clamp [4]. Application of ouabain alone induced 4-6 nA inward currents in both folliculated and defolliculated oocytes (Fig. 1), but had no effect on membrane conductivity (Fig. 2). Fig. 1 illustrates the effect of ouabain on L-kainate-evoked currents. The currents were unaltered 2-5 min after ouabain application, but after 15 min they were increased in amplitude by 3540%. Recovery to control amplitudes after ouabain washout took about 60 min. Similar results were obtained

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with GABA (Fig. 2) and L-glutamate. The potentiation of agonist-induced currents was 1.5-2 times greater in defolliculated oocytes than in folliculated cells. The reversal potentials for the agonist-induced currents were unaltered during ouabain application (see Fig. 2 for GABA I/V relationship), indicating that the increased response amplitudes seen with ouabain application are not due to ion redistributions following inhibition of the Na/K pump.

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Fig. 2. Effect of 10 4 M ouabain on the current-voltage characteristic of a folliculated oocyte in the presence (A) and absence (B) of GABA (10 4 M). Note the increased GABA response during ouabain application. Ouabain had no effect on membrane conductance (ca. 5/ts) in the absence of GABA. In C the responses to 10 -4 M GABA in the presence and absence of 10 -4 M ouabain (applied to the oocyte for 30 min) are compared. Below each current record is a video recording of the oocyte taken at the same times and showing that the increased GABA response of the ouabaintreated oocyte is accompanied by an increase in oocyte size (cross-sectional areas are 4247 mm 2 for the control and 4665 mm 2 for the ouabain-treated cell).

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Oocyte No.

Fig, 3. Effect of 10e4 M ouabain on oocyte weight. The oocytes were weighed before (left) and after (right) ouabain treatment for 40 min. Note that the largest oocytes did not increase in weight during ouabain application. Each column represents the mean of the averaged weight of 5 oocytes.

Ayrapetyan and Arvanov [2] have shown that ouabain increases the volume of snail neurones resulting in an increase in neurotransmitter induced currents. In Fig. 2 the size of a Xenopus oocyte is compared before and 20 min after application of 10m4 M ouabain. A 9.8% increase in maximum diameter was recorded. The responses of this oocyte to GABA are also illustrated to show that there is a clear correlation between oocyte size and sensitivity to the agonist. The ouabain-induced swelling of the oocyte could be compensated for by the addition of 5070 mM sucrose to the bathing medium. A more accurate determination of the effects of ouabain on the oocyte was obtained by weighing the cells before and at various times after ouabain treatment. Fig. 3 shows that increases in weight of up to 10% were obtained 40 min after ouabain application. However, there was an indication of an inverse correlation between control weight and the percentage increase in weight induced by ouabain. In preliminary studies, a ca. 15% increase in sensitivity to

L-kainate has been observed when oocytes were exposed for 1 h to hypotonic saline (2 parts saline: 1 part distilled water). When saline was made hypertonic by adding 100 mM sucrose the oocyte sensitivity to L-kainate fell by about 2096. Although these results support the contention that volume changes affect oocyte chemosensitivity, this conclusion must be viewed in the light of reduced ionic strength and cation content of the hypotonic saline. Overall the results reported herein demonstrate that ouabain, at concentrations which specifically inhibit the electrogenic Na/K pump of excitable cell membranes [5], potentiates the chemosensitivity of Xenopus oocytes, a change which is accompanied by an increase in cell volume. These results are very similar to those obtained previously by Ayrapetyan and Arvanov [l, 21 for snail neurones. The ouabain-induced increase in cell volume may ‘uncover’ receptors [2], possibly normally located in membrane clefts and, thereby, less accessible to exogenously applied compounds. However, other effects of ouabain such as changes in CAMP levels may account for the increased sensitivity of the oocyte to agonists. Whatever its mechanism, it follows that changes in the Na/K pump activity may contribute indirectly to the regulation of synaptic and extrasynaptic chemosensitivity of excitable cells in vivo. V.A. wishes to thank the Royal Society, London for financial support. We are grateful to Mr H. Sudan for technical assistance. 1 Ayrapetyan, S.N. and Arvanov, V.L., The sodium pump activity and ACh sensitivity of neuronal membrane, Comp. Biochem. Physiol., 58C (1977) 153-155. 2 Ayrapetyan, S.N. and Arvanov, V.L., The mechanism of the electrogenie Na-pump dependence of membrane chemosensitivity, Comp. Biochem. Physiol., 64A (1979) 601-604. 3 Barnard, E.A. and Bilbe, G., Functional expression in the Xenopus oocyte of mRNAs for receptors and ion channels. In A.J. Turner and H.S. Bachelard (Eds.), Neurochemistry: A Practical Approach, IRL Press, Oxford, 1989, pp. 243-270. 4 Brackley, P., Goodnow, Jr. R., Nakanishi, K., Sudan, H.L. and Usherwood, P.N.R., Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected with rat and chick brain RNA, Neurosci. Lett., I14 (1990) 51-56. 5 Thomas, K.G., Electrogenic Na pump in nerve and muscle cells, Physiol. Rev., 52 (1972) 5633594.

Effect of ouabain on volume and chemosensitivity of Xenopus oocytes injected with rat brain mRNA.

Xenopus oocytes injected with rat brain RNA were voltage-clamped using a 2-electrode technique. At 4-10 days post-injection the oocytes responded to a...
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