Neuroscience Letters, 114 (1990) 51 ~56 51 Elsevier Scientific Publishers Ireland Ltd.

NSL 06912

Spermine and philanthotoxin potentiate excitatory amino acid responses of Xenopus oocytes injected

with rat and chick brain RNA

P. Brackley l, R. G o o d n o w Jr. 2, K. Nakanish i 2, H.L. Sudan I and P .N.R. Ushe rwood 1

J Department o['Zoology, University ~[" Nottingham, Nottingham (U.K.) and eDepartment ~f Chemistry Columbia University in the City ~['New York, New York, NY (U.S.A.)

(Received 6 February 1990; Revised version received 19 February 1990; Accepted 19 February 1990)

Key words" Philanthotoxin; Spermine; Potentiation; Amino acid receptor; Xenopus oocyte

The effects of spermine and a synthetic analogue (PhTX-343) of the polyamine amide toxin. &phi- lanthotoxin, on the responses of Xenopus oocytes to application of amino acids were examined using vol- tage clamp. The oocytes were injected with either total rat brain RNA or chick cerebrum, poly(A + ) RNA. The responses to N-methyl-D-aspartate and L-kainate were potentiated by low concentrations (10 ~ 10 7M) of PhTX-343 and by l0 5 10 4M spermine. There was variability between oocytes in terms of their responsiveness to these compounds and recovery from their effects was slow and often incomplete. Prolonged or repeated applications of PhTX-343 and spermine eventually resulted in inhibition. Higher concentrations of these compounds always inhibited the responses to acidic amino acids. Low concentra- tions of PhTX-343 and spermine also potentiated the responses to nicotine and ~,-aminobutyric acid. These results are discussed in terms of the postulated polyamine binding site on the N-methyl-D-aspartate recep- tor.

L-Glutamate is a major excitatory transmitter in vertebrate central nervous systems where its actions are mediated by three classes of receptors, the N-methyl-D-aspartate receptors (NMDA-R), the kainate receptors and the quisqualate receptors [21]. Members of all 3 classes contain receptors which gate cation-selective ion channels, although some are coupled indirectly to channel proteins via secondary messengers. Biochemical studies have shown that mM concentrations of the polyamines, sper- mine and spermidine modulate co-operative binding of the non-competitive antago- nist [3H]MK-801 to NMDA-R of rat cortical membranes [12], possibly by binding to a polyamine site on the receptor and, thereby, increasing the affinity of the recep- tor for agonist [12]. In fact, Schaeffer et al. [14] have recently shown that [3H]-L-gluta- mate binding to rat brain membranes is potentiated by many natural polyamines, including spermine and spermidine. Electrophysiological support for these bio-

Correspondence." P.N.R. Usherwood, Department of Zoology, University of Nottingham, Nottingham NG7 2RD, U.K.

0304-3940/90/$ 03.50 ~, 1990 Elsevier Scientific Publishers Ireland Ltd.

52

chemical findings comes from studies by Thibault et al. [16] in which spermine pro- moted a long-term potentiation of the response to NMDA of rat hippocampal slices.

O

: A ~ NH2 A .,NH~ NH ~-~ NH2 v v ~.~ v

4.

HO ~ H N ~ P h i l a n t h o t o x i n ( P h T X - 3 4 3 ) O

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Spermine

Orb-web spider venom and the venom of the parasitic wasp Philanthus triangulum contain a number of polyamine-derived toxins which non-competitively antagonise glutamate receptors, including NMDA-R, in a variety of excitable systems [7]. One of these toxins, argiotoxin-636 [1, 3, 10] (argiopine [5]), at 10- I°M potentiates the response of the quisqualate-sensitive receptor of locust muscle to e-glutamate [9, 19] and increases the binding of [3H]e-glutamate to rat brain membranes [14]. These ob- servations raise the question of whether potentiation of one or more of the glutamate receptor types present in vertebrate central nervous systems can be observed electro- physiologically during application of low concentrations of argiotoxin-636 and other polyamine amide toxins. If the NMDA-R is potentiated, then does this result from interaction of these toxins with the postulated polyamine binding site on this recep- tor'? To answer these questions we have compared the effects of spermine and philan- thotoxin-343 (PhTX-343) (a synthetic polyamine amide derived from the wasp toxin, cS-philanthotoxin [4]) on the responses to NMDA, L-kainate and L-quisqualate of Xenopus oocytes injected with total rat brain RNA and chick brain mRNA. We have found that at concentrations below those which antagonise excitatory amino acid receptors, spermine and PhTX-343 potentiate the responses of the Xenopus oocyte to at least 2 of these amino acids; the toxin being at least 10 000-fold more effective than the polyamine in this respect. Spermine and PhTX-343 exert their potentiation via a mechanism which is independent of that underlying glycine potentiation of NMDA-R [8, 17].

Total RNA was obtained from whole brains of 14-day-old male, Wistar rats and poly(A +) RNA was obtained from l-day-old chick cerebrum using standard tech- niques of extraction and purification by oligo(dt)-cellulose chromatography [2]. Qua- litatively similar results were obtained with the two messages. The responses of Xeno- pus oocytes to NMDA, e-kainate and n-quisqualate were tested electrophysiologi- cally, using a 2-electrode voltage clamp technique, 2-10 days after injection of the messages. Both folliculated and de-folliculated oocytes were used and gave qualitati- vely identical results. Responses to NMDA and L-kainate were characteristically

53

smooth and either non-desensitising or slowly desensitising, respectively [6, 20]. Some oocytes gave biphasic NMDA responses [19]. Oocytes injected with chick brain mRNA rarely expressed the NMDA-R. Responses to L-quisqualate comprised an initial smooth component on which was superimposed a fluctuating response [20], although in a minority of oocytes only the former was seen. The responses to NMDA were greatly potentiated in the presence of 10-6 M glycine [8].

When applied alone at concentrations ~ 10 -4 M, spermine usually failed to evoke a response. Higher concentrations elicited either inward or biphasic currents. When

10-4M spermine was applied either before or during the application of NMDA the response to the latter was potentiated (Fig. IA, B), a change which persisted for many minutes after the polyamine had been cleared from the perfusion bath. Poten- tiation occurred to the same extent in the absence of glycine. PhTX-343 also poten- tiated the NMDA response but at much lower concentrations (10-7M or less). With some oocytes, potentiation was seen with 10-11M PhTX-343, but with others a 100- fold higher concentration of toxin was required to elicit this phenomenon. Recovery from the effects of the polyamine and toxin was slow and incomplete. The potentia- tion of the NMDA responses by spermine is clearly seen in the dose-response rela- tionships illustrated in Fig. 2A. The parallel shift to lower concentrations indicates, perhaps, an increase in affinity of agonist, although the maximum response was also increased, suggesting, perhaps, inhibition of desensitization (Fig. 2B).

L-Kainate at 10 4M elicited inward currents of 100-300 nA at a holding potential of - 60 mY. 10- 5M spermine potentiated the kainate responses (Fig. 1 C, D). Figure 2 shows that PhTX-343 also potentiated the response to L-kainate (Fig. 2C, D), but this occurred at much lower concentrations (10-1°-10-7M) than with spermine; with repeated or prolonged applications of either spermine or toxin the potentiation was converted to an inhibition (Fig. 2D) (see also [9]). Recovery from either the potentiating or inhibitory effects of spermine and PhTX-343 was slow and incom- plete. After potentiating the kainate response with spermine it was sometimes possi- ble further to potentiate it with toxin (Fig. IG, H), but at other times the spermine- potentiated kainate response was inhibited by even a low concentration (10-l°M) of PhTX-343 (Fig. lI,J). However, the results of these and other experiments to deter- mine whether spermine and the toxin act at the same site on the kainate receptor were inconclusive. Neither spermine nor PhTX-343 markedly potentiate the L-quis- qualate response, but it was inhibited 50% by> 10-4M spermine (Fig. 1E, F) and

10-6M PhTX-343 (cf. [11]). A reduction in amplitude and duration of the response to L-quisqualate was accompanied by a reduction of its oscillatory component.

These studies show that spermine both potentiates and antagonises at least 2 of the 3 major classes of amino acid receptor of vertebrate brain. The same is true for the wasp toxin analogue, PhTX-343, which is orders of magnitude more potent than the polyamine in this respect. However, the effects of these compounds are not res- tricted to glutamate receptors. PhTX-343 potentiates binding of [3H]acetylcholine to the nicotinic acetylcholine receptor (nAChR) of Torpedo electroplax [3], and electro- physiological studies have shown that this toxin is also a non-competitive antagonist of nAChR of Torpedo electroplax and frog skeletal muscle [13]. In our studies,

54

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PhTX 343 i

Ka in + spermlne

V Ka ln Ka ln + spenmlne I ~ ' J • F

J, 200 s

Fig. 1. Effects of spermine on responses to N-methyl-o-aspartate (NMDA), L-kainate (Kain) and L-quis- qualate (Quis) of Xenopus oocytes injected with rat brain RNA. The 5 oocytes (A, B; C, D; E, F; G, H; I, J) were clamped at a holding potential of - 6 0 mV. A: response to N M D A (10 4M) alone, and (B) potentiation of this response during co-application of 10 4M spermine. C: response to 10-4M L-kainate alone, and (D) potentiation of this response during co-application of 10-SM spermine. E: response to 10- 4M L-quisqualate showing smooth and oscillatory components, and (F) loss of oscillations during co-ap- plication of 10 4M spermine. G: response to l0 4M L-kainate, which in (H) is potentiated by co-applica- tion of 10 5M spermine, l0 mM PhTX-343 applied during the response in (H) caused further potentia- tion, although there is also some evidence of antagonism also. I: response to 10-4M L-kainate, which in (J) was potentiated during co-application of 10-SM spermine. When 10 m PhTX-343 was applied during the response in (J) it caused a partial inhibition of the response.

oocytes injected with chick brain mRNA sometimes expressed nAChR and always expressed receptors for 7-aminobutyric acid (GABA). Application of 10-4M nicotine to these cells induced 10 nA inward currents at - 6 0 mV holding potential, which increased in amplitude by up to 50% after application of spermine. The responses to 10 5M GABA, which were about 40 nA at - 6 0 mV, were potentiated by 10-4M spermine in a manner suggesting inhibition of desensitization. However, further studies are necessary to test this suggestion.

Potentiation of ligand-gated receptors by polyamine amide Loxins, first reported by Usherwood and colleagues [9, 19] for an invertebrate system, is an unexpectedly general phenomenon which involves a variety of receptor types, including inhibitory (GABA) receptors. This lends some support to the proposal of Usherwood and Blag- brough [18] that through binding non-specifically to excitable cell membranes, sper- mine and PhTX-343 reduce membrane fluidity and, thereby, indirectly influence transmitter receptor function. However, our results do not exclude the possibility that these compounds also bind specifically to sites on the receptor proteins, but it remains unclear whether both PhTX-343 and spermine bind to the postulate polyam- ine site on the NMDA-R.

The polyamine amides exhibit some remarkable pharmacological properties [7], not least of which are their unusually high potencies and their markedly time-depen- dent actions. These newly-discovered molecules have considerable pharmaceutical

55

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Fig. 2. Dose response relationships for (A, B) N-methyl-D-aspartate (NMDA) and (C, D) L-kainate action on Xenopus oocytes injected with rat brain RNA. A: dose response relationship for NMDA in the absence (~)) and presence ( • ) of 10 4M spermine showing potentiation of the N MDA response by the polyamine. Mean data from 2 oocytes, with response to each concentration of NMDA expressed as a percentage of the maximum response (i.e. at 3.3 x 10 "M). B: similar data as in (A) but from another oocyte and plotted as response amplitude (nA) vs. NMDA concentration and showing an increase in the maximum response during co-application of spermine. C: dose response relationship for L-kainate in the absence (O) (mean data from 5 oocytes; SDs < 5%) and presence ( 0 ) (mean data from 4 oocytes; SDs < 5%) of 10-TM PhTX- 343 showing potentiation of the L-kainate response by the toxin with response to each concentration of u-kainate expressed as a percentage of the maximum response (i.e. at 10-3M). D: dose- response relation- ship for u-kainate before (©), after 15 min perfusion ( 0 ) and after 30 rain perfusion (Fq) with 10 8M PhTX-343. Data from 1 oocyte with current amplitude of response to L-kainate plotted against concentra- tion of agonist. Note potentiation of L-kainate response at 15 min, with increase in maximum response, followed by depression of e-kainate response after 30 rain. Curves fitted as in ref. 20.

p o t e n t i a l [15], w h i c h , as o u r r e su l t s c o n f i r m , m a y n o t be r e s t r i c t e d to g l u t a m a t e r g i c

sys tems .

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fice o f t he U S A r m y .

56

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