Art

Characterization of the type of calcium channel primarily regulating GABA exocytosis from brain nerve endings. Maria Sitges and Luz Maria Chiu

Description
In an attempt to further characterize the type of Ca2+ channels primarily regulating GABA exocytosis, the effects of increasing concentrations of ωCTx MVIIC,-ω-Aga IVA and other Ca2+ channel blockers (nitrendipine, Cd2+ and Ni2+), commonly used for
Categories
Published
of 8
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Related Documents
Share
Transcript
  Neurochemical Research, VoL 20, No. 9, 1995, pp. 1073-1080 Characterization of the Type of Calcium Channel Primarily Regulating GABA Exocytosis from Brain Nerve Endings Maria Sitges ~ and Luz Maria Chiu ~ (Accepted July 12, 1995) In an attempt to further characterize the type of Ca + channels primarily regulating GABA exo- cytosis, the effects of increasing concentrations of ~oCTx MVI1C,-o~-Aga IVA and other Ca 2+ chan- nel blockers (nitrendipine, Cd 2+ and Ni2+), cornnaonly used for pharmacologically discerning among the various types of Ca 2+ channels, were tested on the dissected Ca 2+ dependent fraction of the depolarization evoked release of GABA from mouse brain synaptosomes. Our results show that o~-CTx MVIlC inhibits GABA exocytosis with a calculated ICs0 of 3 gM and to-Aga IVA with a calculated IC50 of 50 nM. The divalent cation Cd 2+ only diminishes GABA exocytosis at 70 gM, but does not modify this response at lower concentrations (i.e. 1 and 10 gM). Neither nitrendipine (10 gM) nor Ni 1+ (100/.tM and 500 gM) modified GABA exocytosis. The failure of nitrendipine at a high concentration to inhibit GABA exocytosis discards L-type Ca 2§ channels as the main regulators of this response; likewise that of Ni 2+ discards Ca 2+ channels of the N-type, and the failure of nM concentrations of o~-CTx MVIIC or 500 gM NF § also discards alphalA/Q-type Ca 2+ channels as the main regulators of the GABA response. On the basis of these results and in particular of the higher potency of o~-Aga IVA than oJ-CTx MVIIC, it is concluded that the type of Ca 2+ channels that primarily determine the exocytosis of GABA belong to a P-like type of Ca 2+ channels. KEY WORDS: ~o-Aga IVA; o~-CTx MVIIC; nitrendipine; Cd2+; Ni2+; synaptosomes. INTRODUCTION By opening in response to nerve terminal plasma membrane depolarization, voltage-gated Ca 2+ channels allow Ca + enter into the terminal playing a critical role in neurotransmitter exocytosis. Multiple types of volt- age-gated Ca 2+ channels have been however recently de- fined (1), and the depolarization evoked release of all neurotransmitters from mammalian brain isolated nerve terminals (synaptosomes) is not always regulated in the same manner (2,3). Thus, the aim of this study is to fitrther characterize the type of Ca 2+ channels primarily Instituto de Investigaciones Biom+dicas, Depto. de Biologia Molec- ular, UNAM, Apartado Postal 70228 Ciudad Universitaria 04510 Mrxice, D.F. Email sitges@servidor.unam.mx 1073 regulating the exocytosis of GABA, the main and more ubiquitous inhibitory nettrotransmitter in the mammalian brain. Classifications of voltage-gated Ca/+ channels are based on electrophysiological and pharmacological par- ameters mostly defined in the soma of cells suitable for electrophysiological studies (1,4-6). A preparation com- monly used for studying the depolarization evoked re- lease of a specific neurotransmitter is the synaptosomal preparation, as it is equivalent to dissect the brain bio- chemical machinery for neurotransmitter release, and the presence of neurotransmitter transporters permits to load discrete populations of synaptosomes with the neuro- transmitter (radioactively labeled) that they usually re- lease and then follow its subsequent release with high resolution. 0364-3190/95/0900-1073507.50/0 9 1995 Plenum Publishing Corporation  1074 Sitges and Chiu The electrophysiological parameters of the Ca 2+ currents of synaptosomes have not been defined, prob- ably because of their small size (less than 1 gin). Nev- ertheless, a study of the Ca 2+ currents present in a heterogeneous population of synaptosomes will not clar- ify which specific type of voltage gated Ca 2+ channel is primarily involved in the exocytosis of a specific neu- rotransmitter. The other approach that has shown to be extremely valuable for characterizing which type of Ca 2+ channel is involved in a specific response is the pharmacological approach. Using this approach voltage gated Ca a+ chan- nels have been classified in the dihydropyridine (DHP) sensitive and the DHP insensitive group (1). The DHP Ca 2+ channel antagonists, which block L-type Ca 2+ cur- rents of cardiac and skeletal muscle with high potency (7-11) are required at gM concentrations to inhibit the evoked release of GABA from synaptosomes by mech- anisms not related to a blockade of L-type Ca 2+ channels (3,12,13). The DHP insensitive voltage gated Ca 2. currents pharmacologically distinguished include: the R type Ca 2+ currents, that are sensitive to Ni 2§ and Cd 2§ (1,14); the N-type, sensitive to to-CnTx GVIA (15,16) and the P- type, sensitive to m-Aga IVA (6). This toxin is however relatively specific on P-type Ca ~* channels, as it also blocks a recently described type of Ca 2+ channels named Q with less potency (17). CTx MVIIC is a new toxin now commercially available that conversely to eo-Aga IVA inhibits with higher potency the Q-type than the P- type Ca 2§ channels. The alpha~a current expressed in oo- cytes (1,4), which has been compared to the Q-type Ca 2+ current (18), likewise is potently (nM range) inhibited by o~-CTx MVIIC. In the low gM range o~-CTx MVIIC also inhibits P-type Ca 2+ channels (19). In the present study, several blockers of the DHP insensitive group of Ca 2+ channels were used for further characterizing the type of Ca 2+ channels primarily reg- ulating GABA exocytosis from mammalian brain nerve terminals. EXPERIMENTAL PROCEDURE Sources of Materials. [2,3 3HIGABA (spec. act. 34.7 Ci/mmoI), was obtained from New England Nuclear (Boston, M.A.). m-Aga IVA was kindly provided by Pfizer (see acknowledgments), co-CTx MVIIC was from Alamone Labs., nitrendipine and amino-oxyacetic acid were from Sigma Chemical Co. (St. Louis M.O.). gynaptosomal Preparation. In each experiment the brains, with- out the cerebellums, of 4 to 6 adult (I 1 weeks old) mate a~bino mice (CD1 strain) were used to obtain a purified synaptosomal fraction fol- lowing the method of Haj6s (20). Release Experiments. The method used to load synaptosomes with radioactive GABA and to study its release under continuous su- perfusiun has been described previously (21-23). Since for studying neurotransmitter release using the continuous superfusion system large volumes of buffer (some of them containing the toxins or drugs to be tested) are required, and the effect of ~o-CTx MVItC was tested at high concentrations, for obtaining the concentra- tion-respunse curves with o)-CTx MVIIC we used the following method: After loading synaptosomes with pH]GABA, the radioactivity not incorporated into the preparation was removed by centrifugatiun (instead of by a rapid perfusiun). Aliquots (500 el) of the pellet of [~HIGAt3A loaded synaptosomes were resuspended in Na~-free stan- dard Ringer's medium (SRM), as in the absence of external Na § the release of the preloaded [3H]GABA induced by high K + depolarization becomes completely Ca z+ dependent (23-25), and incubated for 6 min at 37~ in the absence (control) or presence of to-CTx MVIIC (0.5, 5, and 10 gM). After this time the synaptosomal suspension was de- polarized by the addition of an aliquot of a concentrated solution of KCI in order to obtain 25 mM K +fmaI in tile test tube, and the in- cubation continued for 4 more min. In some tubes the incubation was continued for the same period (4 rain) without the KC1 addition (in order to estimate the baseline release). The experiment was terminated by centrifugation in an IEC microcentrifuge at the highest speed. The radioactivity determined in the supematant plus that deter- mined in the respective pellet was taken as total radioactivity (100)%). The % of radioactivity released under resting conditions was sub- tracted from the response to high K +. As the peptidases released to the incubation medium can destroy the toxin present in it, 1 mg/ml bovine serum albumin (BSA) was added to the incubation buffers used in these experinaental series. Estimation of ICso Values. The ICs0 for w-CTx MVIIC on GABA exoeytosis was estimated from the incubation experiments as follows: in each independent experiment, the percentage of the total radioac- tivity released to the incubation medium by high K + in the absence of toxin for 4 min over baseline was taken as a 100% response to de- polarization (zero % inhibition). With this value the inhibition (in %) caused by the presence of 0.5, 5 and l0 ~tM to-CTx MVIIC on the response to high K § was calculated. For the case of ~-Aga IVA, the ICs0 on GABA exocytosis was estimated from the superfusion experiments. In each independent ex- periment the percentage of the total radioactivity released by high K § in the absence of toxin for 3 rain was was added and taken as a 100% response to depolarization (zero % inhibition). With this value the inhibition (in %) caused by the presence of 10, 30, 100, or 300 nM to-Aga IVA on the response to high K § was calculated. Buffers Composition. The composition of the SRM used to load synaptosomes with pH]GABA was (in raM); 127 NaCl, 1.18 KH2PO4, 3.73 KCI, 1.8 CaClz, 1.18 MgSO4, 11 glucose and 20 HEPES (N-[2- Hydroxyethyl]piperazine-N'-[2-ethanesutfonic acid]) (pH adjusted to 7.4 with NaOH). The solution was bubbled with OJCO a (95%/5% vol/vol). The effects of all the Ca z+ channel blockers used in this study were tested on the release of the Ca 2 dependent fraction of GABA induced by high (20 raM) K* in the absence of external Na § (referred from here as GABA exocytosis). The composition of the Na*-free SRM, used for the incubation or superfusion release experiments was the same to the SRM except that 127 mM n-methyl-gincamine chloride replaced NaCI. The com- position of the high K + buffer used for the release experiments per- formed with the continuous superfusion system was the same of the Na*-free SRM, except that 20 mM KCI replaced an equimolar con- centratiun of n-methyl-glucamine chloride. Where indicated, the above  A P-like-Type of Ca 2§ Channel Primarily Regulates GABA Exocytosis 1075 24 lad r 18 o ta.l O -..I ' ~ 12 O 113 r < 6 o / I -1- d tel (A) 100 - Z O i- 50 - l ..-r- z N 0 - iCs0= 5,u,M B) f~ ...... i' i i i i 4 0 0.5 5 10 0 2 4. 6 8 10 [w-CTx MVlIC] (/~M) [w-CTx MVIIC] (/~M) 2Fig. 1. Effect of increasing concentrations of ~o-CTx MVIIC on GABA exocytosis. (A) [3HIGABA oaded synaptosomes were incubated at 37~ in Na+-free SRM without (dark bar) or containing ~o-CTx MVIIC 0.5, 5, or 10 gM (dashed bars). After 10 rain of incubation an aliquot of a concentrated solution of KCI was added and the incubation continued for 4 more rain. The bars indicate the percentage of the total radioactivity released by depolarization (i.e. minus the radioactivity released for 10 rain in not depolarized synaptosomes). (B) The percentage of inhibitions was calculated from the data shown in (A) taking the response in the absence of toxin as the 100% (zero % inhibition). Data are the mean _+ SEM of four independent determinations. perfusion media also contained ~o-Aga IVA (30, 100 and 300 riM), ~o- Ctx MVIIC (0.5, 5, and 10 IxM), nitrendipine (10 gM), Ni z+ (100 and 500 gM) or Cd 2+ (1, 3, 10, and 70 gM). The GABA transaminase inhibitor, amino-oxyaeetic acid (0.1 raM) was present in all the buf- fers. RESULTS Effect of Increasing Concentrations of w-CTx MVIIC on GABA Exocytosis. Fig. 1A shows that at high concentrations (5 and 10 gM), o)-CTx MVIIC consid- erably decreases the release of the Ca 2+ dependent frac- tion of GABA induced by 25 mM K + for 4 rain. At 0.5 gM, o~-CTx MVIIC does not modify significantly this response. The baseline release of [3H]GABA determined in synaptosomes simultaneously incubated in non de- polarized conditions (i.e. in Na+-free SRM) is unmodi- fied by the presence of co-CTx MVIIC at the concentra- tions tested (data not shown). For estimating the ICs0 of ~o-CTx MVIIC on GABA exocytosis the inhibition caused by the increasing con- centrations of eo-CTx MVIIC was calculated in percent- age (Fig. 1B). At 5 and 10 gM, co-CTx MVIIC inhibits GABA exocytosis by 68 _+ 10 and 81 + 8%, respec- tively. An ICs0 of 3 gM oJ-CTx MVIIC was calculated from these data. Before obtaining the results presented in Fig. 1 we have used the continuous superfusion system for testing the effect of co-CTx MVIIC within a lower concentration range (i.e. 60nM, 200nM and 600nM) on GABA exo- cytosis. Also using this experimental approach the low concentrations of ~o-CTx MVIIC did not modified GABA exocytosis, indicating that the results obtained following the incubation and the continuous superfusion procedures are comparable. Effect of Increasing Concentrations of w-Aga IVA on GABA Exocytosis. The effect of increasing concen- trations of eo-Aga IVA on the resting and depolarization evoked release of GABA was tested using the continu- ous superfusion system (Fig. 2A). At nM concentrations eo-Aga IVA does not modify the baseline release of [3H]GABA for 6 rain, whereas the increasing concentra- tions of eo-Aga IVA in the nM range progressively de- crease GABA exocytosis. Fig. 2B shows the percentage of inhibition exerted by the increasing concentrations of oJ-Aga IVA on the Ca ~+ dependent fraction of GABA release induced by high K + depolarization for 3 rain. At 10 nM ~o-Aga IVA inhibits this response by 23 + 4%, at 30 nM by 40 + 1%; at 100 nM by 64 + 9% and at 300 nM by 77 +_ 6%. The ICs0 of ~o-Aga IVA calculated from these data is 50 nM. Comparison of w-Aga IVA and ~o-CTx MVIIC Po- tency to Inhibit GABA Exocytosis. The log dose-response plots of the inhibition produced by m-Aga IVA and co-CTx MVIIC on GABA exocytosis were calculated from Figs. 1B and 2B for a direct comparison of the potencies of these toxins on this response (Fig. 3). o~- Aga IVA is a hundred times more potent than eo-CTx MVIIC for inhibiting GABA exocytosis. The Efficacy of w-Aga IVA on GABA Exoeytosis is Time Dependent. Fig. 4 shows that the degree of inhi- bition exerted by co-Aga IVA increases with the expo- sure time to the toxin before depolarization. In synaptosomes superfused in the presence of 100 nM to-  1076 Sitges and Chiu W .< tlJ m o -r 9 CTR n 30 nM 6 v 100 nM o 300 nM 4 K 3 6 CA) S I B) IC = 50nM 100 50 Z --- 50 ...... -~/ _z N 0 0 100 200 300 [w-Ago IVA] (nM) rain Fig. 2. Effect of increasing concentrations of ~-Aga IVA on GABA exocytosis. (A) Synaptosomes were loaded with [3H]GABA (300 nCi/mg protein) and perfused with Na+-free SRM without (e, control) or containing ~-Aga IVA 10 (not shown), 30 (tz), 100 (V) or 300 nM (o). Where indicated (arrow) these media were rapidly replaced with Na+-free, high K+-media without (e, control) or with o~-Aga IVA 10, 30 (tz), 100 (~7), or 300 nM (o). (B) The percentage of inhibition was calculated from the data shown in (A) taking the cumulative response (3 min) to high K + in the absence of toxin as the 100%. Data are the mean values _ SEM (bars) from three independent determinations. z 0 I-- -r- Z 100 w-CTx MVIIC m w-Aga 75 - 50 - 25 - -g 10 IVA / I_ s I_ 7 I_ 6 I_ 5 10 10 10 10 Toxin concentration (M) Fig. 3. Log Dose-Response plots of ~-CTx MVIIC and t~-Aga IVA on GABA exoeytosis. Results are calculated from the data in Figs. 1B and 2B. Aga IVA for 3 min before high K § depolarization, the toxin is less effective for inhibiting the evoked response (37% inhibition) than in synaptosomes superfused for a longer period (8 min) in the presence of the same con- centration of o~-Aga IVA before depolarization (58%). Effect of Nitrendipine, Cd ~+, and Ni 2§ on GABA Ex- ocytosis. Fig 5A shows that the Ca :+ channel antagonist of the DHP group, nitrendipine at a high concentration (10 I.tM) does not inhibit the release of the dissected Ca + dependent fraction of GABA induced by 20 mM K § in the absence of external Na +. The divalent cation, Cd 2+ at 9 No toxin 0 lOOnM w-Ago IVA a 2 m N 3 6 9 12 rain Fig. 4. The efficiency of o~-Aga IVA for inhibiting GABA exocytosis is time dependent. Synaptosomes loaded with [3H]GABA were per- fused with Na+-free SRM without (o) or containing 100 nM to-Aga IVA (o). After 3 min (first arrow) these media were rapidly replaced, in two perfusion chambers, with Na+-free, high K + medium without (e, control) or with 100 nM ~o-Aga IVA (o), or with the same media (with or without toxin) in the two remaining chambers, and the per- fusion continued. After 8 rain (second arrow) the baseline media of the two remaining chambers were replaced with the depolarizing me- dia (i.e. Na+-free high, K + medium without or with toxin) and the perfusion continued for 3 more min. This is a representative experi- ment of three independent determinations.  A P-like-Type of Ca 2§ Channel Primarily Regulates GABA Exocytosis 1077 - 4 LG be) < ,., 3 _1 hi IZ < 2 m .< (9 I i I r,q = CTR (A) 0 NTR l Op, M 9 CTR 0 Cd 70~=M (B) I I 1 I I I 3 6 9 3 6 9 9 CTR o .15oo,M C) 3 6 9 12 rnin rnin rain Fig. 5. Effect of nitTendipine (NTR), Cd 2+ and Ni 2+ on GABA exocytosis. [3H]GABA loaded synaptosomes were perfused with Na+-free SRM without (e, in A, B and C) or containing 10 gM NTR (o, in A), 70 gM Cd 2+ (o, in B) or 500 gM N? + (o, in C). Where indicated (arrow) these media were rapidly replaced with Na+-free high K + media without (e, in A, B and C) or containing 10 [xM NTR (o, in A), 70 gM Cd 2+ (% in B) or 500 gM N? + (o, in C). Data are mean + SEM (bars) values from four or five independent experiments. *, p < 0.05. concentrations in the low gM range (i.e. 1 to 10 gM) does not modify at all this response (data not shown). At a higher concentration (70 gM) however, Cd 2+ mark- edly diminishes GABA exocytosis (Fig. 5B). The other inorganic Ca 2+ channel blocker tested, Ni 2§ at concentra- tions of a 100 and 500 gM was unable to modify GABA exocytosis. Fig. 5C shows GABA exocytosis from syn- aptosomes treated with 500 gM Ni 2+. The same profile was observed with 100 ~tM N? + (data not shown). DISCUSSION The aim of this study was to extend the pharma- cological characterization of the type of Ca 2+ channel primarily regulating GABA exocytosis from mammalian brain nerve terminals, by testing the potency of co-CTx MVIIC, o~-Aga IVA and other Ca 2+ channel blockers to inhibit the Ca 2+ dependent, high K + depolarization evoked release of GABA from mouse brain synapto- somes. Our results show that GABA exocytosis is much more sensitive to o~-Aga IVA than to any other Ca 2+ channel blocker tested, including o~-CTx MVIIC. There are several types of alpha1 Ca 2+ channel su- bunits that apparently define the pharmacological differ- ences between Ca 2+ channels (1). The P-type Ca 2+ current first described in cerebellar Purkinje neurons was often compared to the class A alpha1 current (6, 26-28). Comparison of the Ca 2+ channel phenotype generated by alpha~A in oocytes with its counterpart in Purkinje neu- rons has evidenced however some electrophysiological and pharmacological differences (1,4). In comparison with P type Ca 2+ channels of Purkinje neurons, alphaxA channels in oocytes are a hundred times less sensitive to eo-Aga IVA and about 10 fold more sensitive to CTx MVIIC. Moreover, the alphalA current expressed in oo- cytes, that is potently (ICso 150 nM) inhibited by ~o-CTx MVIIC, but is also sensitive to o~-Aga IVA (ICso 200 nM), strongly resembles a current of culture rat cere- bellar granule cells named Q (18). This new classifica- tion and our previous results (23) showing that when synaptosomes are pre-exposed to 300 nM eo-Aga IVA for 3 min before depolarization, eo-Aga IVA inhibits GABA exocytosis about 50%, prompt us to test the ef- fect of o~-CTx MVIIC on GABA exocytosis. We have found however, that in contrast with the high potency of o~-CTx MVIIC (ICs0, 150 nM) to inhibit the alphalA current expressed in oocytes, ~o-CTx MVIIC at nM concentrations failed to inhibit GABA exocytosis. The ICso of 3 gM calculated here for ~o-CTX MVIIC on GABA exocytosis is on the other hand, the same to that predicted (between 1 and 10 gM) for half block of P- type currents in Purkinje neurons (19). On the basis of this comparison it can be suggested that the P-type Ca 2+ channels in Purkinje neurons and the Ca 2+ channels reg- ulating GABA exocytosis share some pharmacological characteristics. When synaptosomes are exposed to m-Aga IVA for 6 min before depolarization, the calculated ICso for o~- Aga IVA on GABA exocytosis is 50 riM. o~-Aga IVA is not however as efficient for inhibiting GABA exo- cytosis as for inhibiting the Ca 2+ currents of Purkinje neurons. An ICs0 of 2 nM has been reported for a~-Aga IVA on Ca 2+ currents of Purkinje neurons (28). Although
Search
Similar documents
View more...
Tags
Related Search
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks