Neuroscience Letters, 115 (1990) 323-328 323 Elsevier Scientific Publishers Ireland Ltd.

NSL 07027

Failure of co-conotoxin to block L-channels associated with [3H]5-HT release in rat brain slices

I. Rijnhout, D.R. Hill and D.N. Middlemiss

Merck Sharp and Dohme Research Laboratories, Neuroscience Research Centre, Harlow, ( U.K.)

(Received 18 September 1989; Revised version received 29 March 1990; Accepted 11 April 1990)

Key words: ~o-Conotoxin; Bay K-8644; Serotonin release; Voltage-sensitive calcium channel

The effects of the mixed N- and L-type voltage-sensitive calcium channel (VSCC) antagonist, o)-con- otoxin GVIA and the L-type VSCC agonist Bay K-8644 on calcium-dependent, potassium evoked release of [3H]5-hydroxytryptamine ([3H]5-HT) were investigated in slices of rat hippocampus. Bay K-8644 (1 ,uM) enhanced, whilst o)-conotoxin (10-30 nM) attenuated, but did not abolish, evoked release of [3H]5-HT. The facilitatory actions of Bay K-8644 on evoked release were unaffected by concentrations of omega con- otoxin that significantly inhibited [3H]5-HT release. The experiments indicate that concentrations of e)- conotoxin which inhibit neurotransmitter release by blockade of N-type VSCC, may leave L-type calcium channel activity unaffected.

Voltage-sensitive calcium channels (VSCC) are present in all excitable tissues and allow passage o f calcium ions through the plasma membrane into the cytoplasm. Cal- cium channels in chick dorsal root ganglion cells, may be differentiated into 3 sub-

types, namely T-, L- and N-type depending upon their activation and inactivation

kinetics measured electrophysiologically [5, 9, 10, 15] and channels with similar prop- erties have been found in mammal ian neurons [9, 10, 21].

The 3 types o f calcium channel may also be distinguished pharmacological ly using

agents o f varying degrees o f selectivity. For example, the dihydropyridine 'agonist ' , Bay K-8644 [19], enhances L-channel activity in neurones [5, 13, 19, 20] and this

change may be reversed by dihydropyridine 'antagonis ts ' such as nitrendipine. Al though enhancement o f L-channel activity by Bay K-8644 is quite pronounced, blockade by antagonists per se is much less clear and the sensitivity o f L-channels

varies f rom cell type to cell type [9]. Fur thermore, dihydropyridine calcium antago- nists do not seem to affect neuronal function at pharmacological ly relevant concen- trations and, under normal circumstances, do not inhibit calcium-dependent neuro- transmitter release [6, 12, 13]. However, if neurotransmit ter release is measured under depolarising conditions, Bay K-8644 augments the release and this effect can be anta-

Correspondence: D.N. Middlemiss, Merck Sharp and Dohme Research Labs., Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex, CM20 2QR, U.K.

0304-3940/90/$ 03.50 45" 1990 Elsevier Scientific Publishers Ireland Ltd.

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gonised by low concentrations of calcium antagonists [12]. As such, this response represents a functional correlate of the increase in L-type calcium channel activity produced in neurones by Bay K-8644.

Another selective blocker of VSCC is ~o-conotoxin [4]. This is a 27 amino acid pep- tide that comes from the marine fish-eating snail Conus geographus, which in contrast to the dihydropyridines, does inhibit neurotransmitter release at many synapses [2, 3, 6, 7, 18]. This peptide blocks both the N- and L-type calcium currents in dorsal root ganglion cells [9, 10], thus suggesting N-type channels are closely linked with the neurotransmitter release process. This latter conclusion is supported by studies in rat pheochromocytoma (PCI2) cells and sympathetic neurones although the L- type channels in these preparations are not blocked by ~o- conotoxin [16].

Biochemical measurements of calcium fluxes into synaptosomes or intact cells measured using 45Ca or Fura-2 have confirmed the inhibitory effects of co-conotoxin on transmembrane calcium movement [17], but these results have also raised the pos- sibility that ~o-conotoxin does not block all neuronal L-type calcium channels. Thus, in a number of model systems, depolarisation-induced calcium fluxes were blocked by dihydropyridines but not by og-conotoxin, even at high concentrations [1, 11, 17]. Interestingly, it has also been reported that L-type calcium currents are selectively inhibited by co-conotoxin in isolated rat hippocampal neurones [23].

To investigate whether oJ-conotoxin blocks L-channel as well as N-channel activi- ty, we have measured the ability of Bay K-8644 to augment [3H]5-HT release from rat brain slices in the absence and presence of e)-conotoxin at a concentration that significantly blocks depolarisation-evoked neurotransmitter release.

The methods used in this study were a modification of those described by Mid-

dlemiss and Spedding [12]. Briefly, male Sprague-Dawley rats were killed by decapitation, the brains removed

and the hippocampi dissected. The combined tissue from 2 rats was cross-chopped at 350/lm using a McIlwain tissue chopper. The tissue slices were carefully dispersed and equilibrated for 30 min at 20°C in 150 ml Krebs-bicarbonate solution (in mM: NaC1 125, KCI 2, MgSO4 1.2, CaC12 2, KHzPO4 1, NaHCO3 25, glucose 10). After this, they were transferred to 5 ml fresh buffer containing 10/tM pargyline and 0.1 /tM [3H]5-HT (NEN: 12.6 Ci/mmol) and incubated at 35°C for a further 20 min. The slices were then washed briefly with fresh medium and allowed to sediment under gravity. Aliquots containing approximately 40 mg packed slices were then randomly transferred to a series of perfusion chambers and perfused at 1 ml/min with Krebs- bicarbonate containing 10/,tM fluvoxamine to prevent the reuptake of [3H]5-HT. Af- ter 40 min, the medium was replaced with one containing control medium or co-con- otoxin GVIA (Peninsula Labs.) and the slices perfused for a further 20 min. Prelimin- ary experiments showed that such a period of preincubation was necessary to achieve consistent inhibition of evoked neurotransmitter release. Several 1 rain fractions of perfusate were then collected from each chamber to establish basal efflux before the potassium concentration of the medium was raised to 25 mM, at which it was main- tained until the end of the experiment. Where present, Bay K-8644 was introduced into the perfusate during the period of depolarisation. The radioactivity in the frac-

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tions, together with that remaining in the tissue slices at the end of the experiment, was determined and the fractional efflux release rate of each fraction was calculated.

Under control conditions, depolarisation of the tissue slices by elevated potassium (25 mM) increased the fractional release rate of [3H]5-HT by between 3 and 4 fold (Fig. 1, Table I) and this was maintained over a period of at least 10 min. Removal of calcium from the superfusion or addition of cadmium (100 #M) completely inhi- bited the evoked release of [3H]5-HT (not shown), indicating both the calcium sensiti- vity and the involvement of voltage-sensitive calcium channels in the release process. Addition of 1 #M Bay K-8644 to the slices under depolarising conditions resulted in an increase in the evoked release of [3H]5-HT when compared to control (Fig. 1, Table I) which was also maintained. The enhancement of evoked [3H]5-HT release produced by Bay K-8644 was blocked by the simultaneous application of nitrendi- pine (1 #M) (data not shown), although the L- channel antagonist failed to affect the evoked release of [3H]5-HT in the absence of Bay K-8644.

Preincubation of the tissue slices with co-conotoxin GVIA (10-30 nM) for 20 min prior to K +-stimulation, inhibited the evoked release by approximately 40% (Table I). to-Conotoxin produced a reduction in evoked [3H]5-HT release which was similar

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Fig. 1. Facilitation of evoked [3H]5-HT release from rat hippocampal slices by 1 #M Bay K-8644. Slices were perfused with Krebs-Henseleit solution to obtain a stable basal release of radioactivity before being stimulated by raising the K* concentration of the perfusate to 25 mM (at fraction 2). Bay K-8644 (1 #M final concentration) was introduced into the perfusate at fraction 6, and maintained there until the end of the experiment. The presence of Bay K-8644 enhanced the potassium evoked release of VH]5-HT from the tissue. The data are from a single experiment and each point is the mean +S.E.M. of 6 separate perfu- sion chambers.

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TABLE I

EFFECT OF ¢o-CONOTOXIN GVIA (CgTx) AND Bay K-8644 ON EVOKED RELEASE OF pH]5-HT FROM RAT HIPPOCAMPAL SLICES

Release of [3H]5-HT was evoked by 25 mM K + in the absence and presence of 10 or 30 nM o~-conotoxin, which was applied to the tissue 20 min before stimulation. Bay K-8644 was introduced into the perfusate at fraction 6 and maintained there until the end of the experiment. The effect of og-conotoxin on the frac- tional release of[3H]5-HT from the tissue was measured at fraction 6 (see Fig. 1). The increase in fractional release produced by Bay K-8644 was measured at fraction 11 as the difference in fractional release rate from control in the same experiment (see Fig. I).

Mean frac- tional n % Inhibition Increasein fractional n release rate of evoked release produced (mean x 10 3 release by 1 itM Bay K-8644 i S.E.M.) (mean x 10-3+S.E.M.)

Basal 7.73_+0.60 17 Stimulated control 25.58 +_ 1.39 17 4.20+0.38 18 Stimulated+ 10 nM CgTx 17.60+ 1.24" 7 45 4.09+0.35 7 Stimulated + 30 nM CgTx t9.90+0.78" 5 32 4.16_+0.64 5

*Significantly different from control.

at all t ime poin ts t h roughou t the du ra t ion o f the depola r i s ing st imulus, suggesting

that depo la r i sa t ion does not enhance the inh ib i tory effect o f eJ-conotoxin. The d ihyd-

ropyr id ine an tagonis t n i fendipine did not affect the residual c o m p o n e n t o f [3H]5-HT

release measured in the presence of 100 nM e~-conotoxin (not shown). The facili ta-

tory effect of Bay K-8644 on evoked [3H]5-HT release was unaffected by the presence

of ei ther 10 or 30 n M ~o-conotoxin (Table I) even though K + - e v o k e d release o f

[3H]5-HT itself was significantly inhibi ted.

These da ta provide suppor t for the not ion [12] tha t m o d u l a t i o n o f L- type calc ium

channels by d ihyd ropy r id ine agonis ts can, under depola r i s ing condi t ions , influence

ca lc ium-dependen t neu ro t r ansmi t t e r release f rom bra in slices. Fu r the rmore , our

findings paral le l o thers demons t r a t i ng d ihyd ropy r id ine agonis t effects on pers is tent

ca lc ium currents in neurons [9, 20, 21], tha t p re sumab ly under l ie the increase in neur-

o t r ansmi t t e r release observed in these exper iments . The da t a also clearly demons t r a t e

that low concen t ra t ions o f ~o-conotoxin can reduce, but do not comple te ly inhibit ,

the po ta s s ium-evoked release o f 5-HT f rom rat h i p p o c a m p a l slices.

The fai lure o f ~o-conotoxin to a t t enua te the effect o f Bay K-8644 on evoked release,

even though the evoked release o f neu ro t r ansmi t t e r i tself was m a r k e d l y reduced, sug-

gests tha t L- type calc ium channels were unaffected by the concen t ra t ion o f og-con-

o tox in used. This f inding con t ras t s with e lec t rophys io logica l da t a der ived f rom

exper iments using chick dorsa l r oo t gangl ia where low concen t ra t ions o f a~-con-

o tox in clearly inhibi t ca lc ium m o v e m e n t th rough N- and L- type channels [9, 10].

The use o f different species m a y prov ide a par t ia l exp lana t ion for these differences

327

as the effects of co-conotoxin on synaptic transmission are highly species dependent. For instance, transmission at the neuromuscular junction offish and amphibia is very sensitive to the toxin, yet the mammalian neuromuscular junction in unaffected [9, 18], suggesting that calcium channels in each species differ slightly yet subserve the same function. The recent finding that L-channels in rat pheochromocytoma cells and sympathetic neurones [16] and mouse motor nerves [1] are insensitive whilst those in rat hippocampal neurones are sensitive [23] to ~o-conotoxin also lends sup- port to the notion of subtypes of L-type calcium channel. Further, studies in human neuroblastoma, rat PC 12 and rat pituitary cells [11, 22] have each provided evidence for transmembrane calcium movement through dihydropyridine-sensitive L-channels that are not blocked by og-conotoxin.

Finally, it has been shown recently that excitatory synapses within the rat nucleus accumbens but not CA1 region of the hippocampus are blocked by og-conotoxin [7]. As both synaptic excitations in these regions are mediated by glutamate, this suggests that different classes of calcium channel may regulate the release of even the same transmitter within the central nervous system.

In summary, the present study shows that depolarisation-evoked release of [3H]5- HT from rat brain slices may be modulated by compounds acting at L- or N-type calcium channels. However, the ability of the L-channel agonist Bay K-8644 to en- hance evoked release is unaffected by concentrations of og-conotoxin which clearly block N-type calcium channels.

We would like to thank Mrs. E. Brawn and Mrs. S. Burton for typing the manus- cript.

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